Leptin’s Role in Lipodystrophic and Nonlipodystrophic Insulin-Resistant and Diabetic Individuals

REVIEW

Leptin’s Role in Lipodystrophic and Nonlipodystrophic

Insulin-Resistant and Diabetic Individuals

Hyun-Seuk Moon,* Maria Dalamaga,* Sang-Yong Kim,* Stergios A. Polyzos,

Ole-Petter Hamnvik, Faidon Magkos, Jason Paruthi, and Christos S. Mantzoros

Division of Endocrinology, Diabetes, and Metabolism (H.-S.M., S.-Y.K., O.-P.H., F.M., J.P., C.S.M.), Beth Israel

Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; Department of Clinical

Biochemistry (M.D.), Attikon General University Hospital, Athens University Medical School, Athens 15784, Greece;

Division of Endocrinology and Metabolism (S.-Y.K.), School of Medicine, Chosun University, Gwang-Ju 501-717, South

Korea; Second Medical Clinic (S.A.P.), Aristotle University of Thessaloniki, Ippokration Hospital, Thessaloniki 11527,

Greece; Division of Endocrinology, Diabetes, and Hypertension (O.-P.H.), Brigham and Women’s Hospital, Harvard

Medical School, Boston, Massachusetts 02115; and Section of Endocrinology (C.S.M.), Boston VA Healthcare System,

Harvard Medical School, Boston, Massachusetts 02118

Leptin is an adipocyte-secreted hormone that has been proposed to regulate energy homeostasis as well as metabolic,reproductive,neuroendocrine,andimmunefunctions.Inthecontextofopen-labeluncontrolledstudies,leptin

administration has demonstrated insulin-sensitizing effects in patients with congenital lipodystrophy associated

with relative leptin deficiency. Leptin administration has also been shown to decrease central fat mass and improve

insulin sensitivity and fasting insulin and glucose levels in HIV-infected patients with highly active antiretroviral

therapy (HAART)-induced lipodystrophy, insulin resistance, and leptin deficiency. On the contrary, the effects of

leptin treatment in leptin-replete or hyperleptinemic obese individuals with glucose intolerance and diabetes mellitus

have been minimal or null, presumably due to leptin tolerance or resistance that impairs leptin action. Similarly,

experimental evidence suggests a null or a possibly adverse role of leptin treatment in nonlipodystrophic patients

with nonalcoholic fatty liver disease. In this review, we present a description of leptin biology and signaling; we

summarize leptin’s contribution to glucose metabolism in animals and humans in vitro, ex vivo, and in vivo; and we

provide insights into the emerging clinical applications and therapeutic uses of leptin in humans with lipodystrophy

and/or diabetes. (Endocrine Reviews 34: 377–412, 2013)

I. Introduction

II. Leptin Biology

A. Leptin discovery

B. Leptin production and circulation

C. Leptin receptors

D. Leptin’s antisteatotic and antilipotoxic role

E. Leptin’s role in glucose homeostasis

F. Leptin’s role in immune modulation

III. Leptin Signaling

A. Leptin and JAK2/STAT3/STAT5 signaling

B. Leptin and AMPK signaling

C. Leptin and PI3K/Akt/mTOR signaling

D. Leptin and FoxO1 signaling

E. Leptin and SHP2/MAPK signaling

F. Leptin and JAK2-independent signaling

IV. Leptin and Insulin Signaling

V. Leptin Resistance or Tolerance

A. Impaired activation of leptin receptor signaling/

inhibitors of leptin signaling

ISSN Print 0163-769X ISSN Online 1945-7189

Printed in U.S.A.

Copyright © 2013 by The Endocrine Society

Received August 22, 2012. Accepted January 2, 2013.

First Published Online March 8, 2013

B. Impaired leptin transport across the BBB

C. Impaired ObRb trafficking

D. ER stress

E. Saturable nature of leptin signaling pathways

F. Impaired leptin neural circuitry

VI. Effects of Leptin on Glucose Metabolism in Animals

and Humans

* H.-S.M., M.D., and S.-Y.K. contributed equally to this work.

Abbreviations: ACC, acetyl-coenzyme A carboxylase; AgRP, agouti-related protein; ARC, arcuate

nucleus; AMPK, AMP-activated protein kinase; BDNF, brain-derived neurotrophic factor;

BBB, blood-brain barrier; BMI, body mass index; CaMKK2, Ca 2 /calmodulin-dependent protein

kinase; CC, colorectal cancer; CNS, central nervous system; CoA, coenzyme A; DIO, dietinduced

obesity; ER, endoplasmic reticulum; FoxO1, Forkhead box O1; GTT, glucose tolerance

test; HALS, HIV-associated lipodystrophy syndrome; hAT, human adipose tissue; HbA 1C , glycated

hemoglobin; HAART, highly active antiretroviral therapy; HFD, high-fat diet; HOMA,

homeostatic model assessment; HA, hypothalamic amenorrhea; HSC, hepatic stellate cell; icv,

intracerebroventricular; IKK, inhibitor of nuclear factor-B kinase subunit ; IRS, insulin receptor

substrate; JAK2, Janus kinase 2; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein;

MC3R, melanocortin-3 receptor; mTOR, mammalian target of rapamycin; NAFLD,

nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; NF-B, nuclear factor-B;

NPY, neuropeptide Y; ObR, obese receptor; P13K, phosphatidylinositol 3-kinase; PBMC, peripheral

blood mononuclear cell; POMC, proopiomelanocortin; PPAR, peroxisome proliferator-activator

receptor-; PTP, protein tyrosine phosphatase; SH2, Src homology-2; SH2B1,

SH2B adaptor protein 1; SHP2, SH2-containing protein tyrosine phosphatase 2; sOB-R, soluble

leptin receptor; SOCS3, suppressor of cytokine signaling 3; SS, simple steatosis; STAT3, signal

transducer and activator of transcription 3; STZ, streptozotocin; TG, triglyceride; Th, T helper;

UPR, unfolded protein response; VMH, ventromedial hypothalamus.

doi: 10.1210/er.2012-1053 Endocrine Reviews, June 2013, 34(3):377–412 edrv.endojournals.org 377

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378 Moon et al Effects of Leptin on Glucose Metabolism Endocrine Reviews, June 2013, 34(3):377–412

A. States of leptin deficiency

B. States of leptin excess

C. Benefits, potential adverse effects, and challenges

in relation to leptin administration

VII. Future Directions

I. Introduction

Leptin has a crucial role in the regulation of energy homeostasis,

insulin action and lipid metabolism (1). As

a hormone secreted by adipocytes in quantities mainly

correlated with fat cell mass, leptin serves as an important

signal of body energy stores. Its importance is well illustrated

by the physiological consequences of leptin deficiency

in mice homozygous for a mutation in the obese

(ob) gene, which prevents leptin production or leads to

secretion of a truncated inactive leptin molecule (1–3).

Ob/ob mice exhibit hyperphagia, early-onset obesity, insulin

resistance, diabetes (1, 2), and several neuroendocrine

abnormalities (3, 4). All these abnormalities can be

corrected by exogenous leptin administration (1, 2, 5, 6),

suggesting that leptin plays a role in glucose homeostasis

and possibly in the pathogenesis of other obesity-related

metabolic complications. Interestingly, leptin-induced

normalization of hyperglycemia and hyperinsulinemia

in ob/ob mice is observed even before body weight reduction,

suggesting that leptin’s effects on glucose homeostasis

are, in part, independent of its weight-reducing

effects (5, 6). Similar to ob/ob mice, other mouse

models of obesity and leptin resistance or tolerance

(7–9) present abnormalities of leptin deficiency due to

subnormal leptin action (7, 10, 11).

The importance of leptin is also evident in human physiology

(3, 4, 7–18). Leptin administration has been demonstrated

to successfully treat obesity and its complications

in individuals with congenital leptin deficiency, and

thus, leptin is available on a compassionate basis for these

patients (5–7). Moreover, leptin is effective in correcting

neuroendocrine abnormalities and insulin resistance in

patients with HIV-associated lipodystrophy (11–13) and

congenital lipodystrophy (8–10). Therefore, an application

has been submitted to the U.S. Food and Drug Administration

(FDA) for approval of leptin in replacement

doses for the treatment of congenital lipodystrophy. In

contrast, in subjects with garden-variety obesity or diabetes

(who exhibit hyperleptinemia presumably due to leptin

tolerance), treatment with additional exogenous leptin

has not been associated with significant weight loss or

reduction in metabolic complications (19–21). This suggests

that leptin tolerance or resistance exists in these subjects

(22, 23). Hence, although great progress has been

made in understanding the role of leptin in many physiological

systems, much research is currently being directed

toward elucidating the mechanisms and pathophysiology

of leptin’s effects on glucose metabolism. In this review, we

describe the effects of leptin biology and signaling on glucose

metabolism in animals and humans in vitro, ex vivo,

and in vivo and provide novel insights into emerging clinical

applications and therapeutic uses of recombinant leptin

in the area of lipodystrophy, insulin resistance, and

diabetes in humans.

II. Leptin Biology

A. Leptin discovery

In 1994, the mouse ob gene was found to encode a

16-kDa 167–amino-acid secreted protein with a 4-helix

bundle motif similar to that of a cytokine, which was

named leptin (from the Greek word leptos, meaning thin)

(10, 24). Leptin signals primarily the status of body energy

reserves in fat to the brain and other tissues so that appropriate

changes in food intake, energy expenditure, and

nutrient partitioning can occur to maintain whole-body

energy balance (24). This system is especially sensitive to

energy deprivation. The discovery of leptin in 1995 also

defined a novel endocrine role for adipose tissue and thus

subsequently increased our understanding of how food

intake and energy metabolism are regulated (10).

B. Leptin production and circulation

Leptin is primarily produced in adipose tissue but is also

expressed in many other tissues, including the placenta,

mammary gland, testes, ovary, endometrium, stomach,

hypothalamus, pituitary, and others (25). Leptin concentrations

in blood are pulsatile and follow a circadian

rhythm, with lowest levels in the early to midafternoon

and highest levels between midnight and early morning

(26). The pulsatile characteristics of leptin secretion are

similar in lean and obese individuals with the exception

that the obese exhibit higher pulse amplitudes of leptin

(26). Circulating leptin levels are directly proportional to

the amount of body fat (23) and fluctuate with acute

changes in caloric intake (27). Women tend to have higher

leptin concentrations than men, although a significant reduction

in the amount of circulating leptin occurs after

menopause (28). This sexual dimorphism, which is independent

of body mass index (BMI), is partly attributed to

differences in fat mass, body fat distribution, and sex hormones

(29). Subcutaneous fat expresses more leptin

mRNA than omental (visceral) fat, and this may partially

contribute to higher leptin levels in women compared with

men (29). Thus, compared with visceral fat, accumulation

of sc fat is associated with higher leptin levels (26) and

steatotic and lipotoxic complications appear more fre-


doi: 10.1210/er.2012-1053 edrv.endojournals.org 379

quentlyinvisceralobesity(27).Table1depictsfactorsthat

affect circulating leptin levels in humans.

C. Leptin receptors

Leptin exerts its effects via binding to specific leptin

receptors (obese receptors [ObRs]) located throughout the

central nervous system (CNS), including the hypothalamus,

and several peripheral organs (4, 10, 28). The leptin

receptor is a receptor in the class 1 cytokine receptor family,

with a single membrane-spanning domain (10, 24, 28,

29). At present, 6 ObR isoforms, produced by alternative

splicing of the leptin receptor gene, have been identified in

rats (ObRa–ObRf) (30). In mice and humans, only 5

(ObRa–ObRe) and 4 (ObRa–ObRd) alternative spliced

isoforms have been described, respectively (30, 31). These

isoforms present homologous extracellular leptin-binding

domains but distinct intracellular domains varying in

length and sequence due to alternative mRNA splicing

(32). The short isoforms ObRa and ObRc may play an

important role in transporting leptin across the bloodbrain

barrier (BBB) (32).

The long leptin receptor isoform, ObRb, which contains

a full-length intracellular domain required for cell

signal transduction, is primarily responsible for leptin signaling

(30, 31, 33) (Figure 1). ObRb is strongly expressed

throughout the CNS and particularly in the hypothalamus,

where it regulates energy homeostasis (food intake

and energy expenditure) and neuroendocrine function

(30, 32–35). In the db/db mouse model, the ObRb is dysfunctional,

resulting in obesity and the metabolic syndrome

(36). ObRb mRNA is highly expressed in the hypothalamus,

particularly in the arcuate nucleus (ARC), the

ventromedial hypothalamus (VMH), the lateral hypothalamic

area, the dorsomedial hypothalamus (DMH), and

the ventral premammillary nucleus (32). Via its receptor,

leptin directly targets hypothalamic neurons, particularly

ARC neurons (proopiomelanocortin [POMC] and agoutirelated

protein [AgRP] neurons), which are critical mediators

of leptin action (37). POMC neurons are anorexigenic

(appetite suppressor) neurons coexpressing POMC

and cocaine- and amphetamine-regulated transcript. After

proteolytic cleavage, POMC generates -MSH, which

stimulates melanocortin-3 and -4 receptors (MC3R and

MC4R), which are important in energy homeostasis (37).

Genetic defects in the melanocortin system (POMC or

MCR genes) are related to obesity in humans (38). Conversely,

AgRP neurons are orexigenic (appetite stimulator)

neurons that inhibit POMC activity by releasing inhibitory

-aminobutyric acid and coexpress the orexigenic

AgRP and neuropeptide Y (NPY), antagonists of both

MC3R and MC4R (39). Leptin inhibits AgRP/NPY expression

and AgRP neuronal excitability as well as -aminobutyricacid

release from AgRP neurons (39). Moreover,

in the VMH, leptin stimulates the expression of anorexigenic

brain-derived neurotrophic factor (BDNF) through

MC4R signaling (40). Finally, in the hypothalamus, leptin

acts on neurons that directly or indirectly regulate levels of

other circulating hormones (eg, thyroid hormone, sex steroids,

and GH) (30, 32, 33, 41). Leptin action on these

hypothalamic neurons also controls the activity of the autonomic

nervous system; direct effects of leptin on ObRbcontaining

neurons in the brainstem and elsewhere may

also play an important role (31, 33).

ObRb is also expressed in multiple peripheral tissues,

including pancreatic islets, adipose tissue, skeletal muscle,

liver, and immune cells (36, 37, 42). In the pancreatic

islets, leptin directly inhibits insulin expression and secretion

(42). In liver and white adipose tissue, leptin inhibits

lipogenesis and stimulates lipolysis (36, 37) and adipocyte-specific

overexpression of ObRb prevents dietinduced

obesity (DIO) in mice (38). Leptin directly promotes

fatty acid oxidation in isolated adipocytes and skeletal

muscle (39, 40) and decreases lipid levels in isolated

livers (43). Liver-specific overexpression of ObRb prevents

hepatic steatosis in ObR-deficient fa/fa rats (44, 45).

However, deletion of the ObRb in these peripheral tissues

has no effect on energy balance, body weight, or glucose

homeostasis in mice, indicating that these processes are

Table 1.

Factors That Regulate Serum Leptin Levels

Factors Promoting Leptin Secretion

Excess energy stored as fat (obesity) a

Overfeeding a

Glucose

Amino acids

Insulin

Glucocorticoids

Estrogens

Inflammatory cytokines TNF- and IL-6 (acute effect)

Factors Inhibiting Leptin Secretion

Low energy states with decreased fat stored (leanness) a

Fasting a

Thyroid hormones

Free fatty acids and other lipid metabolites

Catecholamines and adrenergic agonists

Androgens

PPAR agonists b

Inflammatory cytokine TNF- (prolonged effect)

a Denotes major factor influencing leptin levels.

b Unlike animals, PPAR agonists in humans decrease leptin gene expression but increase sc fat mass, thus presenting a null effect.



Figure 1.

Figure 1. Intracellular signaling pathways recruited by leptin. The full-length isoform, ObRb, contains intracellular motifs required for activation of

the JAK2/STAT3 signal transduction pathway. ObRb contains 4 important tyrosine residues that provide docking sites for signaling proteins with

SHP2 domains. Activation of JAK2 occurs by transphosphorylation and subsequent phosphorylation of tyrosine residues in the cytoplasmic region

of the receptor. Phosphorylation of STAT3 leads to their dissociation from the receptor and the formation of active dimers, which translocate to

the nucleus to regulate gene expression, binding to the promoter regions of target genes. The leptin-regulated STAT3 signaling may interact with

STAT5, which may regulate STAT3-dependent gene expression. The ability of SOCS3 to inhibit leptin-stimulated phosphorylation of JAK2 provides

a negative-feedback mechanism on the leptin signaling system. The ERK members of the MAPK family are components of the well-defined AMPK-

Ras/Raf/MAPK signaling cascade and have become activated by leptin. Leptin activates PI3K signaling via IRS-1 and/or IRS-2 phosphorylation. The

PTP1B inhibits leptin-stimulated phosphorylation of IRS-1/2 and/or JAK2. Leptin administration results further in Akt/mTOR phosphorylation

followed by regulation of FOXO and S6 expression.

mainly regulated by the central effects of leptin in the

brain (46).

D. Leptin’s antisteatotic and antilipotoxicity role

Whenever leptin action is not present either due to hypoleptinemia

or leptin resistance, overfeeding could lead

to fat deposition to nonadipose tissues characterized by

generalized steatosis, lipotoxicity, and lipoapoptosis.

Generalized steatosis leading to a dysfunction of nonadipose

tissues, ie, lipotoxicity, may affect pancreatic -cells

(47, 48), myocardium (49), liver, kidneys, skeletal muscle

(50), and other nonadipose tissues (27). When there is

ectopic lipid accumulation during overfeeding, the surplus

of fatty acids follows the nonoxidative metabolic pathway

resulting in metabolic trauma characterized by lipotoxicity

and lipoapoptosis (51). Also, apoptosis results from

excessive ceramide synthesis coupled with underexpression

of the antiapoptotic factor bcl-2 (52). Ceramidotoxicity,

which is not clearly identified in metabolic syndrome,

has been shown to occur spontaneously in the

pancreatic islets of Zucker diabetic fatty (ZDF) rodents

with type 2 diabetes and metabolic syndrome (53). In humans,

the constellation of lipotoxic abnorrmalities that

leads to insulin resistance, type 2 diabetes mellitus, and

fatty liver is referred to as the metabolic syndrome, suggesting

that this syndrome includes lipotoxicity of pancreatic

-cells, liver, myocardium, and skeletal muscle in a manner

similar to lipotoxicity observed in rodents (27, 54, 55).



Leptin constitutes a liporegulatory hormone that controls

lipid homeostasis in nonadipose tissues particularly

during periods of overfeeding (27). It has been suggested

that leptin regulates intracellular fatty acid homeostasis

and prevents tissues from lipotoxicity, similar to insulin,

which regulates intracellular glucose homeostasis and prevents

glucotoxicity (56). Leptin activates AMP-activated

protein kinase (AMPK), arresting lipogenesis and promoting

fatty acid oxidation by inactivating acetyl coenzyme A

(CoA) carboxylase (ACC) (99). In nonobese diabetic mice

with uncontrolled diabetes type 1, leptin therapy normalizes

the levels of a wide array of hepatic intermediary metabolites

comprising acylcarnitines, tricarboxyl acid cycle

intermediates, amino acids, and acyl CoAs (58). Leptin

reduces both lipogenic and cholesterologenic transcription

factors and enzymes and lowers plasma and tissues

lipids (58).

E. Leptin’s role in glucose homeostasis

Leptin may also directly regulate glucose homeostasis

independently of its effects on adiposity; leptin regulates

glycemia at least in part via the CNS, but it may also directly

regulate the physiology of pancreatic -cells (59, 60)

and peripheral insulin-sensitive tissues (61, 62). Low

doses of leptin that do not reduce food intake and body

weight almost normalize the severe hyperglycemia observed

in ob/ob mice, demonstrating that leptin actions in

glucose homeostasis are independent from its actions in

food intake and body weight (2). In the context of severe

obesity and insulin resistance, the potent leptin actions in

glucose homeostasis are predominantly mediated by

POMC-expressing neurons within the arcuate nucleus of

the hypothalamus (64), although the AgRP-expressing

neurons and other hypothalamic and extrahypothalamic

neurons may also have important roles (65).

Recent studies have shown remarkable antidiabetic

properties of leptin as a powerful suppressor of glucagon

(58, 66, 67). Leptin suppresses glucagon hypersecretion in

rodents with diabetes type 1 at least as effectively as somatostatin

and insulin, without producing undesirable

adverse effects (58, 67, 68). Administration of recombinant

leptin to mice with uncontrolled diabetes type 1 reversed

the catabolic syndrome in a similar manner as insulin

did. Moreover, when leptin was added to low-dose

insulin therapy at 10% of the optimal dose, the volatility

of insulin monotherapy disappeared (58). This leptin

action was ascribed to the tonic suppression of hyperglucagonemia,

affecting hyperglycemia (66). The antihyperglycemic,

glucagon-suppressing action of peripherally administered

leptin has been duplicated via leptin infusion into the

intracerebral ventricle (68). Therefore, leptin suppresses

-cells directly or via a leptin-responsive hypothalamic pathway

for glucagon production or both (66, 69).

Diabetes type 1 is characterized by loss of the endocrine

and paracrine functions of insulin. -Cells lack constant

regulation of high insulin levels from juxtaposed -cells

and hypersecrete glucagon, which causes directly diabetic

symptomatology (70). It has been proposed that insulin

monotherapy corrects the endocrine insulin deficiency but

may not fully correct the paracrine insulin deficiency that

is responsible for the glycemic volatility and the catabolic

syndrome of diabetes type 1 (66). If glucagon hypersecretion

is the major cause of metabolic aberrations in human

diabetes, glucagon inactivation or suppression could provide

an attractive therapeutic vista for managing diabetic

symptomatology compared with insulin monotherapy.

F. Leptin’s role in immune modulation

Leptin presents a pivotal role in the modulation of immune

function. In normal subjects, leptin’s role is associated

with a balance between the number of T helper (Th)1

cells and Th2/Treg (regulatory T cells), which are able to

suppress immune and autoimmune responses (71). On one

hand, leptin contributes to protection from infectious diseases

whereas on the other hand to loss of tolerance and

autoimmunity. Studies in mice have shown that effects of

leptin on the immune system are implemented via central

or peripheral pathways (72, 73). Leptin plays a role in the

cellsbelongingtoboththeinnateandtheadaptiveimmune

responses. In fact, as a cytokine, leptin affects thymic homeostasis

and promotes Th1-cell immune responses by

increasing interferon-, tumor necrosis factor (TNF)-,

and IgG2a production from B cells (74). Leptin is involved

in all processes of NK cell development, differentiation,

proliferation, activation and cytotoxicity (75). Leptin constitutes

a negative signal for the expansion of naturally

occurring human Foxp3 CD4 CD25 regulatory T cells

(Treg), a cellular subset suppressing autoreactive responses

mediated by CD4 CD25 T cells (76). Hypo- and

aleptinemia results in impaired Th1 response and induction

of Treg cells; reducing thus the immunocompetence

and autoimmunity in mice and humans and increasing

susceptibilitytoinfectionssuchastuberculosis(TB),pneumonia,

candidiasis, and bacterial and viral diarrhea (77–

79). Conversely, hyperleptinemia and leptin resistance are

associated with a low proportion and proliferation of Treg

cells, an expansion of Th1 cells and increased secretion of

proinflammatory cytokines that sustain and enhance the

development of immunoinflammatory responses and autoimmune

diseases in subjects with predisposition to autoimmunity

(72).



III. Leptin Signaling

A. Leptin and JAK2/STAT3/STAT5 signaling

It has been shown that ObRb activates a cascade of

several signal transduction pathways, including Janus kinase2(JAK2)/signaltransducerandactivatoroftranscription

3 (STAT3) (58) (Table 2 and Figure 1). The STAT

family comprises Src homology-2 (SH2) domain-containing

transcription factors situated in the cytoplasm in quiescent

cells. STAT activation results in homo- or heterodimerization,

nuclear translocation, and transcriptional activation.

STAT3isatranscriptionfactorthatisencodedbytheSTAT3

gene in humans (80). It mediates the expression of a variety

of genes in response to cell stimuli and thus plays a key role

in many cellular processes such as cell growth and apoptosis

(80, 81). Constitutive STAT3 activation is associated with

various human cancers and indicates poor prognosis, suggesting

that STAT3 has antiapoptotic and proliferative effects

(82–84). In contrast, loss-of-function mutations in the

STAT3 gene result in hyper-IgE syndrome, associated with

recurrent infections as well as disordered bone and tooth

development (85, 86).

The JAK2/STAT3 pathway is the first identified signaling

mechanism associated with the leptin receptor and

plays a pivotal role in energy homeostasis and neuroendocrine

function (80, 81, 84) (Table 2 and Figure 1). It

has been well established that leptin stimulates ObRb

dimerization, which results in JAK2 activation, autophosphorylation,

and phosphorylation of Tyr 985 , Tyr 1077 , and

Tyr 1138 , which act as docking sites for key downstream

signaling molecules (59). Also, in response to leptin,

STAT3 binds to phospho-Tyr 1138 , allowing JAK2 to phosphorylate

and stimulate STAT3. Leptin binds to its ObRb

receptor and activates the JAK2/STAT3 pathway in the

ARC of the hypothalamus, inducing the transcription of

the anorexigenic POMC and suppressing the orexigenic

AgRP/NPY (65). Moreover, leptin administration induces

phosphorylation of JAK2/STAT3 in several cell lines including

keratinocytes, endometrial cancer cells, murine

adipocytes, and mouse adipose tissue (87). We have recently

demonstrated for the first time that leptin activates

STAT3 signaling in mouse GT1-7 hypothalamic, C2C12

muscle, and AML12 liver cell lines (19). Also, we have

Table 2.

Main Signaling Pathways, Main Sites of Action, and Main Effects of Activation by Leptin a

Signaling

Pathway Primary Site of Action Effects

JAK2/STAT3 Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite, energy balance, and body weight

Regulates neuroendocrine function

Regulates cell proliferation

Regulates cell growth and apoptosis

Regulates bone and tooth development

STAT5 Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates energy balance and body weight

Regulates oncogenesis

MAPK Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite and body weight

Regulates fatty acid oxidation

Regulates cell hypertrophy

Regulates cell proliferation and differentiation

AMPK Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite, energy balance, and body weight

Regulates gluconeogenesis and energy homeostasis

Regulates fatty acid oxidation

Regulates insulin resistance

Regulates cell proliferation and survival

Regulates oncogenesis

PI3K/Akt/mTOR Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite, fuel balance, and body weight

Regulates insulin/leptin resistance

Regulates sympathetic outflow


Regulates adiposity and glucose homeostasis

Regulates cell proliferation

FoxO1 Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite and body weight

Regulates energy expenditure

Regulates cell proliferation and differentiation

Regulates insulin signaling

SHP2/MAPK Hypothalamus, adipocytes, PBMCs, cancer cells, muscle cells, liver cells, etc Regulates appetite and body weight

Regulates STAT3 signaling with SOCS3

Regulates mitosis, proliferation, and differentiation

Regulates cell apoptosis and survival


a Adapted from Refs. 3 and 19–21.



demonstrated that leptin activates STAT3 signaling in human

adipose tissue (hAT) in vivo and ex vivo and in human

primary adipocytes in vitro (20, 21). Conversely, disrupting

the ability of the leptin receptor to activate the STAT3

pathway in mice leads to severe obesity and several neuroendocrine

abnormalities including decreased linear

growth and infertility (88).

Leptin may also activate phosphorylation of ObRb on

Tyr 1077 , which binds to STAT5 and stimulates STAT5

phosphorylation (32) (Figure 1). STAT5 deletion in the

brain causes hyperphagia and obesity to a lesser extent

than STAT3 deletion, suggesting that the ObRb/JAK2/

STAT5 pathway may also play a role in the regulation

of body weight and energy balance by leptin (70) (Table

2). It is important to study in depth the effects of leptin

on human hypothalamic or other CNS cells; leptinincreased

STAT3/STAT5 signaling in several tissues

and cells may have important clinical implications including

oncogenesis.

B. Leptin and AMPK signaling

AMPK is an evolutionarily conserved serine/threonine

protein kinase central to the regulation of cell

growth, proliferation, differentiation, motility, and survival

(71) (Table 2 and Figure 1). AMPK is considered

a cellular energy sensor that is stimulated by an increase

in the intracellular AMP to ATP ratio (71). In its classical

role as an intracellular metabolic stress-sensing

kinase, AMPK switches on fatty acid oxidation and glucose

uptake while switching off hepatic gluconeogenesis.

It also exerts a broader role in metabolism by controlling

appetite (89, 90). AMPK is also involved in

cellular energy homeostasis through AMPK phosphorylation

and inhibition of ACC, thus stimulating fatty

acid oxidation (91–93).

Leptin’s actions in peripheral tissues are partly due to its

effects on AMPK, which contribute to leptin’s effects on

fatty acid oxidation and glucose uptake (94). In mouse

skeletal muscle, leptin activates AMPK, which leads to

phosphorylation and inhibition of ACC and subsequent

stimulation of fatty acid oxidation. There appear to be 2

mechanisms through which leptin activates AMPK in

muscle (95, 96). One pathway involves the hypothalamus

including the -adrenergic pathway, resulting in a prolonged

effect, and as shown in our previous ex vivo and in

vitro experiments (84), the second mechanism encompasses

leptin’s direct effect on skeletal muscle. The ability

of leptin to stimulate fatty acid oxidation in skeletal muscle,

which plays a pivotal role in the pathophysiology of

insulin resistance (97, 98), may prevent lipotoxicity and

subsequent insulin resistance and may underlie leptin’s

ability to improve insulin resistance and metabolic syndrome

in hypoleptinemic humans (99, 100), thus supporting

the potential use of leptin in treating lipodystrophy

and, possibly, obesity and type 2 diabetes.

In contrast to its actions in muscle, leptin has been

shown to exert an inhibitory effect on AMPK in the hypothalamus

that results in stimulation of the hypothalamic

ACC and subsequent reduction of food intake and

weight gain (86). Leptin’s hypothalamic effects on AMPK

are in part mediated by the MC4R pathway, which promotes

anorexia (86). Moreover, the Ca 2 /calmodulindependent

protein kinase (CaMKK2) is an upstream activator

of AMPK in the hypothalamus, whereas CAMKK2

inhibition reduces appetite and body weight (88). These

observations indicate that the hypothalamic CaMKK2/

AMPK/ACC pathway may also mediate leptin’s anorexigenic

action. Further in vivo studies in humans are needed

to elucidate whether leptin administration may alter in

vivo signaling by altering a third intermediate factor that

possibly could not be studied in vitro.

C. Leptin and PI3K/Akt/mTOR signaling

Phosphatidylinositol-3 kinase (PI3K) has been linked

to an extraordinarily diverse group of cellular functions,

including cell growth, proliferation, differentiation, motility,

survival, and intracellular trafficking (101–103)

(Table 2 and Figure 1). These functions are related to the

ability of class I PI3Ks, which are responsible for the production

of phosphatidylinositol 3-phosphate, phosphatidylinositol

(3,4)-bisphosphate, and phosphatidylinositol

(3,4,5)-trisphosphate, to modulate protein kinase B, suggesting

that PI3K/Akt/mammalian target of rapamycin

(mTOR) signaling pathway play an important role in diabetes

mellitus (103, 104). Leptin activates ribosomal S6

kinase, an important physiological substrate of mTOR

kinase in the hypothalamus (92). The PI3K/Akt/mTOR

signaling pathway is required for an extremely diverse

array of cellular activities including proliferation and survival

(105–107).

Leptin increases cell proliferation in mouse GT1-7 hypothalamic,

C2C12 muscle, and AML12 liver cell lines

through mTOR and Akt signaling pathways (19). Moreover,

in vitro and in vivo studies have suggested that leptin

stimulates cell proliferation through PI3K signaling in

myocytes (19, 108) and hepatocytes (109). Leptin may

also stimulate this pathway in human cancer cells (110,

111) and in human adipose (21), liver (112), and muscle

tissues (19, 113). Similar to the JAK2/STAT3 pathway, the

PI3K pathway is also important in central leptin action.

This pathway activates POMC-expressing neurons by activating

ATP-sensitive potassium channels and voltagegated

calcium channels (114). Also, similar to insulin, the

appetite-suppressing effects of leptin are attenuated by in-



tracerebroventricular (icv) administration of PI3K inhibitors

(116, 286). Interestingly, the insulin-PI3K pathway

hyperpolarizes POMC neurons, which makes them less

sensitive to leptin, and may be one common mechanism

for both leptin and insulin resistance in obesity (117, 118).

Chronic activation of the PI3K pathway in POMC neurons

may cause leptin resistance and hyperphagia in mice

with POMC neuron-specific deletion of phosphatase and

tensin homolog (PTEN) (105). Hence, activation of the

PI3K signaling pathway in neuronal cells may influence

energy homeostasis and neuroendocrine function,

whereas activation of this pathway in the peripheral tissues

may mediate leptin’s effect on insulin resistance.

D. Leptin and FoxO1 signaling

Forkhead box protein O1 (FoxO1) belongs to the forkhead

family of transcription factors that are characterized

by a distinct forkhead domain (119) (Table 2 and Figure

1).Thespecificfunctionofthistranscriptionfactorhasnot

yet been determined; however, it may contribute to myogenic

growth and differentiation (119). FoxO1 is a downstream

effector of insulin signaling with an important role

in the regulation of metabolism in various organs including

liver (120), pancreas (121), muscle (122), adipose tissue

(122), and hypothalamus (123). Also, FoxO1, as a

transcription factor, is one of the substrates for Akt phosphorylation

resulting in cytoplasmic shuttling from the

nucleus, thereby inactivating FoxO1 (124, 125). FoxO1,

which stimulates the expression of AgRP and NPY and

inhibits POMC expression, is an important downstream

mediator of the PI3K pathway (286). It has been demonstrated

that the activation of hypothalamic FoxO1 is inhibited

by leptin via the PI3K pathway (123). In contrast,

overexpression of constitutively active FoxO1 in the

mediobasal hypothalamus of rats by adenoviral microinjection

leads to a loss of the inhibitory effect of leptin

on feeding and results in body weight gain (126). Moreover,

hypothalamus-specific FoxO1 knock-in mice

have increased food intake and decreased energy expenditure

(127).

E. Leptin and SHP2/MAPK signaling

MAPKs are serine/threonine-specific protein kinases

that respond to extracellular stimuli (mitogens, osmotic

stress, heat-shock, and proinflammatory cytokines) and

regulate various cellular activities, such as gene expression,

mitosis, differentiation, proliferation, and cell survival/apoptosis

(19, 21, 128, 129). MAPKs encompass a

number of signaling molecules that include ERK1/2, p38

and c-Jun N-terminal kinase (JNK) (21). ERK1/2 represents

the major MAPK involved in leptin’s central effects

contributing to the regulation of food intake, energy expenditure,

and body weight (3).

Leptin stimulates ERK1/2 activation via SH2-containing

protein tyrosine phosphatase 2 (SHP2) (Table 2 and

Figure 1). SHP2 is a protein tyrosine phosphatase (PTP)

that contains 2 SH2 domains located in its NH 2 terminus

as well as a COOH-terminal PTP domain (130). This specific

structure suggests that SHP2 is involved in regulating

signals initiated by receptor tyrosine kinases (130). These

phosphotyrosines act as docking sites for recruitment of

SH2-containing proteins, including SHP2, to activate

downstream signaling cascades (131). SHP2 promotes

ERK1/2 activation in response to insulin and epidermal

growth factor binding to their receptors (132, 133).

Leptin induces phosphorylation of the Tyr 985 amino

acid residue of the ObRb after JAK2 activation, thereby

creating a binding site for the carboxyl-terminal SH2 domain

of SHP2 (134). SHP2 itself becomes phosphorylated,

recruits the adaptor protein growth factor receptor-bound

protein-2 and induces JAK2-dependent activation of

ERK1/2 (135). Leptin also activates the MAPK pathway

independently of SHP2, probably via receptor activation

leading to direct binding of growth factor receptor-bound

protein-2 to JAK2 (136). Recently, it has been shown that

young mice homozygous for a mutation at the site of the

leptin receptor phosphorylation were slightly leaner than

wild-type mice, although they still developed adult-onset

or DIO (137). These phenotypes probably do not reflect

the effect of this mutation on leptin-induced ERK activation

but are consistent with the role of the mutation site as

a binding site for the suppressor of cytokine signaling 3

(SOCS3), which is a negative regulator of leptin receptor

signaling (137). In vitro studies have also demonstrated

that ERK is required for the phosphorylation of S6 by the

S6 kinase, which enhances cap-dependent translation and

protein synthesis (138).

Other members of the MAPK family, p38 and JNK, are

also activated by leptin in several cell types and present

mainly peripheral actions (139–141), although the associated

pathways have not been well characterized. In rat

cardiomyocytes, leptin administration stimulates p38,

which results in an increase in fatty acid oxidation,

whereas inhibition of p38 activation prevents

leptin-induced fatty acid oxidation (134). Similar to p38,

JNK is not activated in response to leptin treatment in

neuronal cells (135, 136) but confers an oncogenic potential

to leptin’s action after its activation by promoting cancer

cell survival and invasion (137).

F. Leptin and JAK2-independent signaling

You et al (137) observed that leptin may stimulate the

MAPK and STAT3 pathways in cultured cells that are



genetically JAK2 deficient; however, the JAK2-independent

signaling and its pathophysiological importance have

not been verified in animals or humans. The src tyrosine

kinase family members appear to be responsible for mediating

leptin JAK2-independent signaling pathway

(138). The JAK2-dependent and independent signaling

pathways may synergistically mediate responses to leptin

(286). Because in vivo leptin actions may differ from in

vitro actions, studies of in vivo leptin signaling in humans

are needed to study the JAK2-independent leptin signaling

and implications.

IV. Leptin and Insulin Signaling

Insulin is a central hormone regulating carbohydrate and

fat metabolism in the body (142). Insulin causes cells in the

liver, muscle, and fat tissues to take up glucose from the

blood, storing it as glycogen in the liver and muscle (142–

144). Insulin binds to the extracellular portion of the

-subunits of the insulin receptor (143, 144). This, in turn,

causes a conformational change in the insulin receptor

that activates the kinase domain residing on the intracellular

portion of the -subunits (143, 144). The activated

kinase domain autophosphorylates tyrosine residues on

the C terminus of the receptor as well as tyrosine residues

in the insulin-receptor substrate (IRS)-1 protein (144). The

actions of insulin include 1) increasing or decreasing cellular

intake of certain substances, most prominently increasing

glucose uptake in muscle and adipose tissue (comprising

about two-thirds of total body cells) (142, 144), 2)

increasing DNA replication and protein synthesis via control

of amino acid uptake (145), and 3) modification of the

activity of numerous enzymes (146).

Evidence from in vivo and in vitro studies supports the

hypothesis that leptin and insulin signaling networks may

overlap on several levels. Intravenous infusion of leptin in

mice has several effects on insulin-regulated processes, including

increased glucose turnover, increased glucose uptake

in skeletal muscle and brown adipose tissue, and decreased

hepatic glycogen content (147–149). In vivo leptin

administration has also been reported to stimulate insulin’s

inhibitory effects on hepatic glucose output, while

antagonizing insulin’s effect on glucokinase and phosphoenolpyruvate

carboxykinase (PEPCK) expression, 2

key metabolic enzymes (150–152). In HepG2 human hepatoma

cells, leptin has been shown to antagonize insulininduced

down-regulation of PEPCK expression, decrease

insulin-stimulated tyrosine phosphorylation of IRS-1, and

enhance IRS-1-associated PI3K activity (153). Also, in

C2C12 muscle cells, leptin stimulates a non-IRS-1–associated

PI3K and mimics insulin action on glucose transport

and glycogen synthesis. In contrast, in OB-R L -transfected

HepG2 cells, leptin treatment resulted in the

recruitment of p85 to IRS-2 but did not modulate the response

to insulin (154). Interestingly, in Fao cells, leptin

alone had no effects on the insulin signaling pathway, but

leptin pretreatment transiently enhanced insulin-induced

tyrosine phosphorylation and PI3K binding to IRS-1,

while inhibiting these effects on IRS-2 (155). Also, this

study demonstrated that treatment by leptin alone induces

serine phosphorylation of Akt and glycogen synthase kinase

3 but to a lesser extent than treatment by insulin

alone, and that the combination of these hormones did not

result in an additive effect (155). These observations suggest

complex interactions between the leptin and insulin

signaling pathways that can potentially lead to differential

modification of the metabolic and mitotic effects of insulin

exerted through IRS-1 and IRS-2 and the downstream kinases

that are activated. Together, these data point toward

cell- and/or tissue-specific interactions between leptin and

insulin signaling that are quite diverse and complex. Although

the details of this interaction remain to be elucidated,

we propose that leptin may contribute to some of

the alterations in the metabolic and mitotic effects of

insulin action that are involved in the development

of insulin resistance, a characteristic manifestation of

type 2 diabetes.

An example of the overlap between leptin and insulin

resistance has been reported elsewhere (156). The JAK2/

STAT3 pathway leads to increased transcription and expression

of SOCS3. In fact, SOCS3 acts as a feedback

inhibitor by attenuating ObR signaling, thereby playing a

key role in leptin resistance, whereas it also attenuates

insulin signaling, thereby playing a key role in insulin resistance

(157). Apart from leptin, SOCS3 may be induced

by other adipocytokines, including resistin (158), TNF-

(159), and IL-6 (160), which may act as amplifiers of

SOCS3 expression, thereby further increasing both leptin

resistance and insulin resistance.

In summary, it has been shown that a complex network

of interacting signaling pathways appears to regulate food

intake, energy balance, and body weight (161), suggesting

that, in addition to signaling in the CNS, leptin exerts its

metabolic effects by acting directly in peripheral tissues.

The relative importance of this signaling for regulating

adiposity and glucose homeostasis in the periphery remains

to be explored. Furthermore, other important challenges

include 1) the identification of different signaling

pathways downstream of leptin that are integrated in the

regulation of body weight, 2) the leptin-insulin cross talk

in the CNS, 3) the elucidation of the molecular pathways

responsible for leptin resistance/tolerance in obesity, and

4) the elucidation of the underlying mechanisms respon-



sible for the overlapping leptin and insulin resistance occurring

in the brain in pathological states such as obesity

and diabetes that are associated with insulin resistance in

the periphery.

V. Leptin Resistance or Tolerance

The poor efficacy of endogenous leptin to induce weight

reduction in obese individuals has given rise to the term of

functional leptin resistance, which is based on the concept

of insulin resistance in diabetes type 2, where reduced cellular

and metabolic insulin responsiveness coexist with

insulin hypersecretion. The lack of a precise definition of

the term leptin resistance in a universal and quantifiable

manner led the National Institutes of Health to conduct a

workshop, Toward a Clinical Definition of Leptin Resistance

(162). Leptin resistance or, as in our opinion is more

correctly stated as tolerance, could be generally defined as

the failure of endogenous or exogenous leptin to promote

anticipated salutary metabolic outcomes, such as suppression

of appetite and weight gain and stimulation of energy

expenditure, although leptin’s inability to induce desired

responses in specific situations results from multiple molecular,

neural, environmental, and behavioral mechanisms

(162).

The main physiological cause for leptin hypersecretion

is diet-induced expansion of adipocytes. However, in

DIO, the physiological function of progressive hyperleptinemia

remains to be fully defined. It has been shown,

however, that lipotoxicity in nonadipose tissues of congenitally

aleptinemic obese rodents is far greater than in

hyperleptinemic DIO rodents, leading to ectopic lipid deposition

in liver, pancreatic islets, and heart and skeletal

muscle and causing severe organ dysfunction and cell

death with a disease cluster similar to metabolic syndrome

(52, 163, 164). Increasing leptin levels in this condition

appear to prevent lipotoxicity by minimizing ectopic lipid

accumulation into nonadipocytes via leptin-induced fatty

acid oxidation. In obesity, an expanding adipose compartment

may initially improve whole-body insulin sensitivity,

at least temporarily (165, 166). However, later in life, as

energy storage capacity in adipocytes is exceeded during

the period between the onset of overweight/obesity and

the start of the metabolic syndrome, which is considered

to be the result of failure to prevent organ damage inflicted

by ectopic fatty acids and metabolic trauma (163, 167),

leptin resistance develops.

The onset of leptin resistance varies among ObRbexpressing

neurons. Diet-induced leptin resistance appears

to manifest first in the ARC of the hypothalamus and later

in other hypothalamic areas such as the VMH and dorsomedial

hypothalamus (153). Interestingly, the impairment

in the ObRb/PI3K signaling pathway precedes that

of the ObRb/JAK2/STAT3 pathway (154).

First, leptin resistance was thought to be due to leptin

receptor mutations or mutations of other genes downstream

of leptin, such as POMC and MC4R, which also

produce an obese phenotype with associated neuroendocrine

dysfunction (3). The instance of genetic mutation

abrogating leptin receptor function is similar to classical

hormone resistance/insensitivity syndromes (type A insulin

resistance, GH insensitivity, etc,), in which genetic alterations

of the hormone receptor prevent hormone action.

Nonetheless, only a few cases of obesity in humans

are due to monogenic syndromes; instead, most cases are

multifactorial (155). Hence, the underlying mechanisms

Table 3.

Potential Mechanisms of Underlying Leptin Resistance or Tolerance

Gene mutations

Impaired leptin signaling

Impaired leptin neural circuitry

Impaired BBB transport

Impaired ObRb trafficking

ER stress

Saturable nature of leptin pathways

Leptin Resistance or Tolerance

Leptin receptor

Molecules downstream of leptin receptor (POMC, MC4R)

SH2B12

SOCS3, PTP1B, SHP21

Altered synaptic plasticity

Axon guidance

ObRe–ObRa Antagonism

Triglycerides1

Bardet-Biedl syndrome protein2

UPR response3impaired STAT3 phosphorylation

At a free leptin level 40–50 ng/ml

Leptin resistance occurs rarely due to monogenic mutations of leptin receptor or other genes downstream of leptin (POMC and MC4R). Leptin tolerance is usually

multifactorial due to 1) impaired activation (down-regulation of SH2B1) or inhibition (up-regulation of SOCS3 or PTP1B) of leptin signaling; 2) impaired leptin-induced

neural plasticity/circuitry, given that defects in MC4R and BDNF receptor signaling in the hypothalamus may modulate synaptic plasticity and axon guidance; 3)

impaired leptin transport across the BBB because soluble leptin receptor isoform (ObRe) may antagonize the action of ObRa (which is abundantly expressed in brain

microvessels constituting the BBB), thereby resulting in leptin’s endocytosis, whereas hypertriglyceridemia may also impair leptin transport; 4) impaired ObRb trafficking,

as has been reported in relation to Bardet-Biedl syndrome proteins (responsible for promoting ObRb trafficking to the plasma membrane) impairment; 5) ER stress,

which promotes the UPR, thereby inhibiting STAT3 phosphorylation; and 6) saturable nature of leptin signaling pathways.



responsible for leptin resistance are apparently multiple

and probably include the following (Table 3): 1) impaired

leptin receptor signaling (11, 127, 156–161,

163–167), 2) defective leptin transport across the BBB

(168–172), 3) impaired ObRb trafficking (173, 174), 4)

endoplasmic reticulum (ER) stress (21, 175–177), 5)

saturable nature of leptin signaling pathways (19, 20,

178, 179), and 6) impaired leptin-induced neural plasticity/circuitry

(180–183).

A. Impaired activation of leptin receptor signaling/

inhibitors of leptin signaling

Leptin signaling is controlled by both positive (SH2B

adaptor protein 1, SH2B1) and negative (SOCS3 and

PTP1B) regulators. SH2B1 is an SH2 and pleckstrin homology

domain-containing adaptor implicated in cell signal

transduction and represents an important positive regulator

of leptin sensitivity (115). SH2B1 binds to Tyr 813

and enhances JAK2 stimulation, promoting leptin signaling

pathways downstream of JAK2 (111). Moreover,

SH2B1 binds directly to IRS-1 and IRS-2, inhibiting tyrosine

dephosphorylation of IRS-1/2 and increasing PI3K

pathway activation (115).

A large contributor to leptin resistance is the defect in

negative feedback mechanisms such as SOCS3 and

PTP1B, which dampen leptin receptor signaling and prevent

overactivation of leptin signaling pathways (168,

169) (Figure 1). The JAK/STAT pathway is negatively regulated

by members of the SOCS family, and leptin signaling

via Tyr 1138 and STAT3 induces SOCS3 expression

(169, 170), which in turn switches off leptin signaling by

binding to tyrosine residue Tyr 985 and inhibiting phosphorylation/activation

of JAK2 (171). Therefore, SOCS3

represents a pivotal feedback mechanism preventing the

overactivation of leptin-signaling pathways. Overexpression

of SOCS3 has been proposed as one of the main mechanisms

for the onset of leptin resistance (169). Indeed,

hypothalamic SOCS3 expression is significantly increased

in several leptin-resistant animal models (169). Immunohistochemical

studies suggest that the ARC is selectively

leptin resistant in DIO mice, and this may be caused by

elevated SOCS3 in this hypothalamic nucleus (172). Mutation

of Tyr 985 to leucine (l/l) increases leptin sensitivity

in female mice (173). However, in contrast to female l/l

mice, both male and female Tyr 985 mice showed increased

susceptibility to high-fat diet (HFD)-induced obesity, supporting

a positive action of signaling via Tyr 985 in attenuating

diet-induced impairment of energy metabolism.

Also, because PTP1B is increased in leptin-resistant animals,

a role for PTP1B in the onset of leptin resistance has

been suggested (174). PTP1B is a ubiquitously expressed

tyrosine phosphatase implicated in the negative regulation

of leptin and insulin receptor signaling (156, 157, 163)

(Figure 1). It has been shown that PTP1B deficiency in

LepRb-expressing neurons results in reduced body weight

and adiposity compared with wild-type controls and likely

underlies the improved metabolic phenotype of global and

brain-specific PTP1B-deficient models, suggesting that

PTP1B may, independent of leptin signaling, contribute to

the regulation of energy balance in mice (184). PTP1B

represents another signaling molecule that may inhibit leptin

signaling via JAK2 dephosphorylation (164). PTP1Bknockout

mice are lean and have increased leptin sensitivity

(164). Overexpression of the endogenous leptin

enhancer SH2B1 has been shown to counteract PTP1Bmediated

inhibition of leptin signaling in cultured cells

(175). PTP1B expression is increased by HFD feeding and

inflammation (176), but it is still unclear how PTP1B expression

and activity are regulated after leptin stimulation

or in case of DIO.

SHP2 has also recently been shown to play a critical role

in leptin signaling by functioning in the hypothalamic control

of energy balance and metabolism (177) (Table 2 and

Figure 1). SHP2 appears to down-regulate the ObRb-

JAK2/STAT3 pathway while promoting ERK1/2 activation

by leptin (177, 178). It has been shown that a prominent

phenotype of the mutant (CaMKII-Cre:Shp2flox/

flox or CaSKO) mice was the development of early-onset

obesity, with increased serum levels of leptin, insulin, glucose,

and triglycerides (178). This study demonstrated

that SHP2 down-regulates JAK2/STAT3 activation by

leptin in the hypothalamus but that JAK2/STAT3 downregulation

is offset by the dominant SHP2 promotion of

the leptin-stimulated ERK1/2 pathway, leading to induction

rather than suppression of leptin resistance upon

SHP2 deletion in the brain. These results suggest that a

primary function of SHP2 in postmitotic forebrain neurons

is to control energy balance and metabolism, representing

a critical signaling component of leptin receptor

ObRb in the hypothalamus (178). Hence, pharmaceutical

augmentation of SHP2 activity in the brain might prove to

be an efficient therapeutic strategy for alleviation of leptin

resistance in obese patients.

B. Impaired leptin transport across the BBB

Leptin enters the brain by a saturable transport system

(179). To reach the ObRb-expressing neurons in the brain,

leptin has to cross the BBB (180). Leptin levels in the cerebrospinal

fluid are decreased in cases of obesity, suggesting

that defective leptin transport may contribute to

leptin resistance (180). The short isoform of the leptin

receptor, ObRa, abundantly expressed in brain microvessels

constituting the BBB, plays a key role in leptin transport

across the BBB (181). The soluble leptin receptor



isoform ObRe has been shown to antagonize ObRa action

and leptin transport through inhibition of leptin surface

binding and endocytosis (171). However, a distinct subpopulation

of ARC neurons, which can be labeled by BBBimpermeable

fluorescent tracers, has been shown to respond

more rapidly and sensitively to circulating leptin

compared with other hypothalamic ObRb neurons. These

neurons might therefore make direct contact with the

blood circulation by projections through the BBB (179,

180). In addition to hyperleptinemia, hypertriglyceridemia

may also impair leptin transport (171). The exact

clinical significance of these findings and/or any differential

effects of icv leptin administration in humans remain

to be elucidated.

Further studies are needed to clarify the contribution of

impaired leptin transport to the pathogenesis of leptin resistance.

Because leptin does not appear to be able to cross

the BBB easily, it may be possible to develop leptin agonists

that cross the BBB more freely or, possibly, antagonists

acting only peripherally. Such agents could possibly have

beneficial effects on immune responses in autoimmune

disease and/or on cardiovascular disease, without causing

excess weight gain as would be expected from blocking

central leptin action (182).

C. Impaired ObRb trafficking

Few ObRbs are located on the plasma membrane mediating

leptin effects; most ObRbs are present in the trans-

Golgi network and in small vesicles (173). It has been

shown that specific proteins, named Bardet-Biedl syndrome

proteins, are responsible in promoting ObRb trafficking

to the plasma membrane (173). In mouse models,

deletion of Bardet-Biedl syndrome proteins results in leptin

resistance and obesity (174). Nonetheless, the importance

of these proteins in humans with obesity has not

been determined yet.

D. ER stress

ER homeostasis is regulated by balancing ER loading of

nascent proteins with ER ability to fold these proteins. An

imbalance may result in an overload of unfolded/misfolded

proteins in the ER lumen, inducing ER stress (175).

ER stress promotes the unfolded protein response (UPR)

involving the activation of several UPR intracellular signaling

pathways (176).

ER stress has recently been shown to play a role in the

development of leptin resistance. Obese, diabetic humans

and animals present higher levels of ER stress in the liver,

adipose tissue, and pancreatic -cells (175), suggesting

that ER capacity is directly related to leptin sensitivity

(183, 184). Hence, ER stress reversal via treatment with

chemical chaperones that decrease ER stress (176) could

be a strategy to sensitize obese mice, and by extension

humans, to leptin. Studies have shown that reducing ER

function in mice creates ER stress, leading to blocked leptin

action and hence leptin resistance, with a significant

augmentation of obesity on a HFD (183). This suggests

that ER stress inhibits STAT3 phosphorylation at an upstream

step and provides a potential mechanism by which

increased ER stress antagonizes STAT3-mediated leptin

signaling. We have demonstrated that pretreatment with

the ER stress inducers, tunicamycin and dithiothreitol,

completely blocks leptin-stimulated STAT3 activation in

human primary adipocytes and human peripheral blood

mononuclear cells (PBMCs) in vitro, suggesting that increased

ER stress in obese people may induce leptin resistance

(21). ER stress may also act via other pathways. For

example, overnutrition atypically activates the inhibitor

of nuclear factor B (NF-B) kinase subunit (IKK)/

NF-B, at least in part through elevated ER stress in the

hypothalamus (185). Although forced activation of hypothalamic

IKK/NF-B interrupts central leptin signaling

and actions, suppression of IKK significantly protects

against obesity and leptin resistance (185). Because in vivo

leptin actions may differ from in vitro actions, studies of

in vivo leptin signaling in humans are needed to explore

this hypothesis. Also, the cross talk between leptin and

UPR signaling pathways needs to be fully clarified.

E. Saturable nature of leptin signaling pathways

To study leptin signaling in humans, we have recently

performed for the first time in vivo experiments involving

metreleptin administration in humans in doses that result

in an increase of circulating levels of free leptin up to a peak

level of 40 to 50 ng/mL (20). In long-term leptin administration

trials in humans, we found that circulating free

leptin levels did not result in weight loss, which indicates

that free leptin levels in or above the 40- to 50-ng/mL

range may be clinically ineffective (20). Also, we observed

that in vivo metreleptin administration (0.01 mg/kg for 30

minutes) induces phospho-STAT3 in both hAT and hPB-

MCs, but this signaling is saturable at a level of 50 ng/mL

metreleptin (20). These levels are higher than the putative

threshold for saturating the BBB leptin transport system,

which may explain the absence of clinical effect of high

leptin levels in obese humans despite the dramatic effects

seen when replacing leptin in subjects with very low concentrations

of leptin (178, 179).

Consistent with this result, we have also observed in

hAT ex vivo and human primary adipocytes in vitro and

in mouse hypothalamic, muscle, and liver cell lines in vitro

that leptin signaling pathways were saturable at a level of

50 ng/mL (19), suggesting that no additional signaling

effect may be observed at higher doses. Overall, we suggest



that the saturable nature of leptin signaling pathways may

play a key role in the development of leptin tolerance in

obese humans.

F. Impaired leptin neural circuitry

It has been shown that leptin-deficient (ob/ob) mice

differ from wild-type mice in terms of numbers of excitatory

and inhibitory synapses and in terms of postsynaptic

currents onto NPY and POMC neurons (181). Hence,

when leptin was delivered systemically to ob/ob mice, the

synaptic density rapidly normalized, an effect detectable

within 6 hours, several hours before leptin’s effect on food

intake (181). This study suggested that leptin-mediated

plasticity in the ob/ob hypothalamus may underlie some of

the hormone’s behavioral effects. Also, it has been reported

that leptin changes brain structure, neuron excitability,

and synaptic plasticity and regulates feeding circuits,

suggesting that leptin may play a role in the

development of the CNS (182). Indeed, leptin has been

proposed to be a crucial regulator of both synaptic plasticity

and axon guidance within the hypothalamus, suggesting

fresh links between nutrition and neurodevelopment

mediated by leptin, with potentially important

implications for the physiology of energy balance and

body weight homeostasis (183). Leptin’s anorexigenic and

metabolic effects are mediated by a sophisticated network

of neurons in the hypothalamus as well as other areas in

the brain. Leptin resistance, hyperphagia and obesity are

characteristic manifestations after defects in MC4R and

BDNF receptor signaling in the paraventricular hypothalamus

and the VMH, respectively (180). Therefore, leptin

sensitivity may be impaired by genetic or environmental

factors that modulate leptin neural circuitry. More studies

are required to study in depth the leptin neurocircuity by

analyzing its anatomic, biochemical, and electrical

components.

VI. Effects of Leptin on Glucose Metabolism in

Animals and Humans

Leptin replacement is a promising therapeutic approach in

several disease states characterized by aleptinemia or hypoleptinemia,

including congenital complete leptin deficiency,

and states of energy deprivation, comprising

anorexia nervosa or milder forms of hypothalamic amenorrhea,

as well as syndromes of insulin resistance observed

in conditions such as congenital or acquired lipodystrophy.

Conversely, states of energy excess such as gardenvariety

obesity and diabetes type 2, which represent the

vast majority of the obese and diabetic population, are

associated with hyperleptinemia and are characterized by

the absence of clinical effects of leptin, reflecting leptin

tolerance or leptin resistance. Table 4 displays leptin administration

effects in different disease states based on

initial leptinemia. If the leptin resistance or tolerance obstacle

is overcome, leptin treatment would become an effective

therapeutic approach against common obesity and

diabetes type 2.

A. States of leptin deficiency

1. Congenital leptin deficiency

Mice homozygous for a nonsense mutation in the ob

gene, the ob/ob mice, have been studied for a long time and

represent a commonly used model of obesity. The first

demonstration of an effect of leptin on glucose homeostasis

was reported in 1995 when these mice were treated

with ip injections of leptin (2, 5, 6). Administration of

leptin effectively decreased serum glucose and insulin concentrations

in a dose-dependent manner, even at the lowest

dose where no significant body weight loss was observed.

Similarly, a single icv injection of leptin improved

glucosetoleranceasassessedbyanipglucosetolerancetest

(GTT) (186). In contrast, db/db mice, which have a mutation

in the leptin receptor gene resulting in expression of

a truncated and dysfunctional receptor, show no reduction

in body weight or improvement in metabolic abnormalities

in response to treatment with leptin (187). These

observations collectively suggest that leptin may play an

important role in glucose metabolism and that leptin

administration may correct the metabolic disturbance

in leptin-deficient states.

Congenital leptin deficiency is a rare autosomal recessive

disorder, caused by mutations in the gene encoding for

leptin. In 1997, 2 severely obese children who were members

of the same highly consanguineous pedigree of Pakistani

origin were reported to exhibit nearly undetectable

concentrations of serum leptin (188). They had a homozygous

frame-shift mutation involving the deletion of a single

nucleotide in codon 133 of the leptin gene. Clinically,

they exhibited profound obesity, hyperphagia, hyperinsulinemia,

and hyperlipidemia as well as reproductive and

immune dysfunction (189–191). Leptin replacement in 1

of these congenital leptin-deficient children decreased appetite,

food intake, body weight, and serum insulin and

cholesterol concentrations (192), and similar effects have

been noted with leptin replacement in other unrelated individuals

with leptin deficiency (193, 194). Since then,

several other Pakistani children with leptin deficiency

have been identified (195). We have studied another group

of 4 adult patients of Turkish origin who carry a homozygous

missense mutation (78, 191, 196, 197). Hyperinsulinemia

was a common feature in these patients, and 1 of



Table 4.

Baseline a

Effects of Leptin Administration in Different Disease State on the Basis of Circulating Leptin Levels at

Disease States Associated With Different Initial Leptin

Levels Therapeutic Approach Main Results

Disease states associated with undetectable initial leptin levels

(0.05 ng/mL)

Congenital leptin deficiency Leptin Significantly reduced food intake and body weight

and improved metabolic derangements

Disease states associated with very low initial leptinemia

(5 ng/mL)

Congenital lipodystrophy syndromes Leptin Significantly improved insulin sensitivity and

dyslipidemia

HALS Leptin Significantly decreased central fat mass and

improved insulin sensitivity but not dyslipidemia

HA Leptin Recuperation of menstruation; correction in the

gonadal, thyroid, GH, and adrenal axes;

improvements in bone metabolism and skeletal

health

Diabetes mellitus type 1 Leptin and reduced insulin Improved insulin sensitivity, glycemic control, and

dyslipidemia in 2 patients

Disease states associated with moderately low initial

leptinemia (5 ng/mL)

Lipodystrophy-associated diabetes type 2 Leptin Ameliorated lipidemic profile but not glucose

abnormalities

Disease states associated with initial hyperleptinemia

(15 ng/mL)

Garden-variety obesity Leptin monotherapy No effect on weight reduction and glycemic

control

Garden-variety obesity Leptin and pramlintide Moderately diminished body weight

Obesity and diabetes type 2 Leptin Did not alter body weight, marginal or no

amelioration in glycemic control

a Adapted from Refs. 3, 4, 10, and 11.

them had diabetes mellitus (196). Leptin replacement dramatically

reduced body weight and food intake in all patients.

Leptin effectively decreased fasting and postprandial

glycemia and normalized glycated hemoglobin

(HbA 1C ) levels within 2 months of treatment in the diabetic

patient (191). The other patients, who had normal

glucose and insulin levels at baseline, maintained normal

fasting glucose levels, but their insulin and C-peptide levels

decreased significantly to less than half of their baseline

values (191). Among them, only 1 adult male patient was

submitted to meal tolerance tests before and after 1 week,

18 months, and 24 months of leptin replacement (198).

The authors used deconvolution analysis of C-peptide kinetics,

modified insulin kinetics modeling, and minimal

glucose modeling to determine pancreatic insulin secretion,

hepatic insulin extraction, and peripheral insulin sensitivity.

After 1 week, leptin replacement increased hepatic

insulin extraction by 2.4-fold (resulting in a 1.8-fold decrease

of posthepatic insulin delivery), without significant

changes in insulin secretion, and also increased insulin

sensitivity by 10%. After 24 months, leptin replacement

increased insulin sensitivity by at least 10-fold, with additional

decreases in insulin secretion and hepatic insulin

extraction (198).

In 1998, Clément et al (199) reported a homozygous

mutation in the human leptin receptor gene that results in

a truncated leptin receptor lacking both the transmembrane

and the intracellular domains. Patients homozygous

for this mutation showed early-onset morbid obesity,

whereas their serum leptin concentrations were much

higher than normal. These patients presented normal fasting

glycemia and normal glucose tolerance despite their

extreme obesity (199). More recently, Farooqi et al (200)

sequenced the leptin receptor gene from subjects with

early, severe obesity to determine the prevalence of pathologic

mutations. Of the 300 subjects, 8 (3%) had nonsense

or missense leptin receptor gene mutations; 7 were homozygous

and 1 was heterozygous. Most subjects with the

mutation presented normal glucose concentrations, although

the 2 oldest adults had type 2 diabetes managed

with oral hypoglycemic medications. All subjects had hyperinsulinemia

to a degree consistent with their obesity

(200). These data demonstrate that, in humans, several

phenotypic features seen in subjects with leptin receptor

mutations are not as severe as those in subjects with leptin

deficiency. This is different from the situation observed in

mice, where mice homozygous for a mutation in the leptin

receptor gene (the db/db mouse) develop more evident dia-



betes than obesity when compared with ob/ob mice (201).

The discrepancy between humans and mice is not yet explained,

although mouse studies have shown that genetic

background is an important modifier of the effects of leptin

deficiency on insulin resistance and diabetes (202).

Leptin is currently available for life-long treatment of

subjects with congenital leptin deficiency through a manufacturer-sponsored

expanded-access program (203,

204). Figure 2 depicts the effect of recombinant leptin

administration in congenital leptin deficiency.

2. Lipodystrophy

Lipodystrophy is a group of clinically heterogeneous

acquired or inherited disorders characterized by complete

or partial absence of sc fat (lipoatrophy) that can occur in

conjunction with the pathological accumulation of adipose

tissue (lipohypertrophy) in other distinct regions of

the body (10, 204). Although congenital lipodystrophies

are exceedingly rare, acquired lipodystrophies (especially

those associated with HIV infection and its treatment) are

more common. Interestingly, lipodystrophic patients exhibit

insulin resistance, hyperglycemia, dyslipidemia, and

hepatic steatosis (205). The severity of these metabolic

derangements typically correlates with the degree of adipose

tissue loss.

Several mouse models of lipodystrophy exist, and the

metabolic profiles of these models are similar to those of

human lipodystrophy (206). Among them, the lipodystrophic

adipocyte fatty acid binding protein 2 sterol reg-

Figure 2.

Figure 2. The effect of recombinant leptin administration in leptin deficiency or leptin excess states. Recombinant leptin may have beneficial effects

when administered in leptin-deficiency states, but minimal benefit and/or mostly adverse effects when administered in leptin-excess states. In

congenital leptin deficiency and congenital lipodystrophy, recombinant leptin administration results in metabolic and neuroendocrine

improvement; improvement seems to be more limited in HIV-associated acquired lipodystrophy syndrome, in which coadministration of

pioglitazone, which acts by up-regulating endogenous adiponectin, may increase the metabolic benefit. On the other hand, recombinant leptin

administration in type 2 diabetes mellitus and/or common obesity and normal or high circulating leptin levels has minimal or null metabolic effect,

whereas it increases circulating leptin-binding protein and antileptin antibodies. Coadministration of pramlintide, an amylin analog targeting leptin

tolerance, or leptin administration after weight loss, when leptin’s declined levels increase appetite, might prove to be of some benefit. Similarly,

leptin up-regulation in animal models with NAFLD resulted in increased inflammation and fibrosis, thereby progression to NASH. Data on adverse

effects, including renal impairrment or T-cell lymphoma, observed mainly in a minority of leptin-deficiency patients, are currently inconclusive. Data

on carcinogenic effect of leptin, mainly observed in hyperleptinemic individuals, are also inconclusive but may be tissue-specific; however,

hypoleptinemia itself may be also implicated in carcinogenesis in hypoleptinemic individuals, and thus this aspect of leptin physiology remains to

be fully elucidated. *, Only experimental data.



ulatoryelement-bindingprotein1c(aP2-SREBP-1c)transgenic

mice exhibit many typical features of human

generalized lipodystrophy (207). These animals have

marked insulin resistance, hyperglycemia, and dyslipidemia

and very low serum concentrations of leptin due to the

lack of sc adipose tissue. When these mice were treated

with leptin, their metabolic abnormalities and diabetic

phenotypes were almost normalized (207). Leptin treatment

was associated with reduced food intake and weight

loss, but inducing weight loss by restricting access to food

did not lead to changes in insulin or glucose concentrations

in transgenic mice, suggesting that the beneficial effects

of leptin are separate from its effects on body weight

(207). The icv administration of leptin was as effective as

peripheral administration (208).

Another mouse model of lipodystrophy is the lipoatrophic

A-ZIP/F-1 mouse model (209), which has a more

severe phenotype with almost complete lack of adipose

tissue, insulin resistance and diabetes, and low leptin levels.

In this mouse model, administration of a high dose of

leptin (30 g/d) led to only moderate improvements in

glucose and insulin concentrations (209). In contrast,

transgenic overexpression of leptin in these mice leads to

a lipoatrophic mouse model that has elevated leptin concentrations

(LepTg/:A-ZIPTg/). These mice have significantly

fewer metabolic abnormalities when compared

with the low-leptin A-ZIP/F-1 mice (210). The somewhat

discrepant findings may be related to the difference in the

achieved leptin concentrations with exogenous administration

versus transgenic overexpression of leptin. In contrast

to the former study in which plasma leptin concentration

increased to 5 ng/mL (209), plasma leptin

concentrations in the latter study were markedly elevated

to 50 ng/mL (210). Collectively, these animal experiments

demonstrated that exogenous leptin administration may

overcome the diabetic abnormalities characteristic of lipodystrophy,

and thus, these studies prompted therapeutic

trials of leptin replacement in patients with lipodystrophy.

In humans, congenital lipodystrophy syndromes are

rare; there have been fewer than 1000 described cases in

the literature (203, 211). Lipodystrophic subjects have

partial or total leptin deficiency (222). Leptin replacement

dramatically improves dyslipidemia and insulin sensitivity

and reduced HbA 1C levels and intrahepatic fat content in

various types of human lipodystrophy, notably those associated

with severe hypoleptinemia (fasting serum leptin

less than 4–5 ng/mL in our laboratory) (213–216, 218–

220, 319). Fasting blood glucose and HbA 1C levels decreased

markedly even in patients who are not fully responsive

to other antihyperglycemic medications or high

doses of insulin. Javor et al (216) also reported that 15

patients with lipoatrophic diabetes were able to decrease

or even discontinue diabetic therapy and maintain normoglycemia

after 12 months of leptin replacement. Petersen

et al (214) performed a hyperinsulinemic-euglycemic

clamp in 3 patients with lipoatrophic diabetes and

showed remarkable improvement in insulin sensitivity after

leptin treatment. This beneficial effect was seen both

with hepatic insulin sensitivity and with whole-body insulin

sensitivity. In conjunction with this, there was an

86% reduction in intrahepatic triglyceride content and a

33% reduction in muscle triglyceride content (214).

Chong et al (218) recently published an open-label, uncontrolled

prospective study in 48 patients with various

acquired and inherited forms of lipodystrophy. Leptin replacement

effectively decreased serum triglyceride concentrations

by 59% and HbA 1C levels by 1.5 percentage

points within 1 year in these patients. The benefits of leptin

replacement were sustained for up to 8 years of follow-up

(218). Oral and Chan (220) collected several small, nonrandomized,

open-label trials in a composite study reporting

on a total of more than 100 patients with severe lipodystrophy.

Based on these studies, they reported that

leptin treatment resulted in improvement in several metabolic

parameters, including glycemic control, insulin sensitivity,

plasma triglycerides, caloric intake, liver volume

and lipid content, and intramyocellular lipid content.

These beneficial responses were durable after 3 years of

therapy (221).

However, lipodystrophy comprises a heterogeneous

group of disorders with diverse circulating levels of leptin

(222). Indeed, most patients with lipodystrophy may exhibit

moderate hypoleptinemia (fasting serum leptin 5

ng/mL). Recent data have shown that although leptin

treatment is effective in ameliorating lipid profiles, notably

hypertriglyceridemia, it does not restore metabolic homeostasis

in lipodystrophic subjects with moderate hypoleptinemia

(223), hence questioning the antidiabetic

potential of leptin in lipodystrophic patients. Therefore,

lipodystrophic subjects with moderate hypoleptinemia are

expected to respond poorly to leptin’s effect on glycemic

control but may benefit from its hypolipidemic effect. This

limitation of leptin treatment in the context of lipodystrophy

associated with moderately low levels of circulating

leptin highlights the need of developing better therapeutic

approaches including combination therapy to treat

metabolic derangements in these patients.

Leptin administration in replacement doses constitutes

an important step forward in the understanding and treatment

of various lipodystrophic syndromes, conditions

characterized by a hypoleptinemic state. Particularly, recombinant

methionyl leptin administration in patients

with lipodystrophy exhibiting severe hypoleptinemia

ameliorated hyperinsulinemia, insulin resistance, hyper-



glycemia, hypertriglyceridemia, and neuroendocrine abnormalities

without significant adverse effects and provided

the rationale for metreleptin treatment in these

patients. It is important to underscore that the antidiabetic

effect of leptin observed in these patients was achieved

even after discontinuation of previously administered antidiabetic

treatment, therefore excluding the possibility of

a synergistic positive effect of antidiabetic medication and

leptin on glucose and lipid abnormalities (213). However,

there are several caveats. Existing studies are all open-label

and uncontrolled; hence, a placebo-controlled trial is warranted,

although the small number of patients and the lack

of firm diagnostic criteria present hurdles to this endeavor

(203). Well-designed, probably crossover, randomized,

placebo-controlled trials would be needed to accurately

assess and quantify efficacy as well as to allow for a comparison

with the current standard of care. Furthermore,

the side-effect profile of leptin has not yet been fully characterized.

Deterioration of renal function, development of

lymphomas, and other side effects have been reported in

lipodystrophic subjects (223–225), but because these

studies were not placebo controlled, it remains unknown

whether the observed side effects are leptin-specific or random

and/or represent the natural history of the underlying

disease. Thus, although the optimal treatment regimen for

this condition has not yet been fully determined, metreleptin

is currently available for these subjects as part of an

expanded access program, while an application for approval

for this indication is under review by the FDA.

Figure 2 depicts the effect of recombinant leptin administration

in lipodystrophy.

3. HIV-associated lipodystrophy syndrome

Currently, the most prevalent type of lipodystrophy is

an acquired form of the syndrome that occurs in HIVinfected

individuals treated with highly active antiretroviral

therapy (HAART) (224). HIV-associated lipodystrophy

syndrome (HALS) affects an estimated 15% to 36%

of HIV-infected patients and is associated with an increased

risk of insulin resistance, diabetes mellitus, dyslipidemia,

and cardiovascular disease (225). HALS has

been associated with dyslipidemia including elevated serum

low-density lipoprotein (LDL) cholesterol and triglyceride

(TG) levels and lower high-density lipoprotein

(HDL) cholesterol (226). HALS patients usually exhibit sc

fat loss and increased abdominal fat. The impact of lipodystrophy

on the psychosocial health in HIV-positive individuals,

ranging from somatic discomfort to low selfesteem,

stigmatization, and depression can be significant

(227). The exact physiological mechanisms that lead to

HALS are still not fully defined, but it has been suggested

that protease inhibitors interfere with peroxisome proliferator-activated

receptor- (PPAR) action leading to an

impairment of normal metabolic processes in the adipocyte

(228). Nucleoside analogs are also associated with

HALS, which is thought to be secondary to mitochondrial

injury. There may also be direct effects of the HIV itself

(224, 228).

In patients with HIV who are on HAART, leptin levels

are mostly related to their overall fat mass, as also seen in

individuals without HIV infection (229–231). Adiponectin

levels, which are also low in patients with HIV, are

mainly related to abnormal fat distribution rather than

overall fat mass (232). Approximately 20% to 30% of

individuals who have HALS manifest hypoadiponectinemia

and hypoleptinemia, but these manifestations are less

dramatic than those seen in individuals with congenital

lipodystrophies (231). Nevertheless, both lower adiponectin

and leptin levels have been associated with worse

insulin resistance (231). Hence, we and others have investigated

the role of leptin replacement in these patients.

Nonetheless, generally it will be fair to expect a less dramatic

response to leptin treatment in individuals suffering

from HALS given that these patients do not manifest an

absolute leptin deficiency comparable to congenital lipodystrophy

(203).

In our initial randomized, placebo-controlled, crossover

2-month interventional study, we found that leptin in

physiological doses improved metabolic disturbances in

these patients (233). Leptin treatment was associated with

a 15% decrease in central fat mass as well as significant

improvements in insulin sensitivity and fasting insulin and

glucose levels, albeit having no effects on LDL and TG

(233). These results were confirmed by a longer independent

study of open-label leptin treatment for 6 months

(234). Leptin treatment was associated with a 32% decrease

in visceral fat as well as significant improvements in

fasting insulin, glucose levels, and lipid parameters including

a decrease in LDL and non-HDL cholesterol and an

increase in HDL by the sixth month (234). This study

showed that whole-body insulin sensitivity was markedly

improved and found that the leptin-induced increase in

insulin sensitivity was especially pronounced in the liver

but not skeletal muscle (234). These observations suggest

that the benefits of leptin replacement are sustained, and

even increased, for as long as treatment continues in patients

with HALS. Although there are no head to head

comparisons, studies performed to date indicate that leptin’s

ability to improve insulin sensitivity is comparable to

or better than that of other available medications, including

metformin and thiazolidinediones (235, 236). However,

the more pronounced effect of leptin on lipid metabolism

in the study by Mulligan et al (234) needs to be

interpreted cautiously given that this study was nonran-



domized and not placebo-controlled and its study participants

continued to use their lipid-lowering regimens

during leptin treatment. Recent results from a small randomized,

double-blinded, placebo-controlled clinical trial

indicate that leptin administration improves glycemia and

non-HDL cholesterol levels to some extent but cannot

ameliorate lipid kinetics in adults with HIV-associated

dyslipidemic lipodystrophy, providing a mechanistic reasoning

behind the inconsistent effect of leptin on lipid profiles

(237). Specifically, metreleptin administration to hypoleptinemic

patients with HIV-associated dyslipidemic

lipodystrophy at doses that significantly elevate plasma

leptin levels does not ameliorate the markedly abnormal

fasting lipid kinetic defects characteristic of this condition,

ie, accelerated lipolysis and adipocyte and hepatic reesterification,

with blunted oxidative disposal of free fatty

acids (237). Conversely, recombinant human GH and human

GHRH, which present beneficial effects on lipid metabolism,

have been shown to improve visceral fat, TG,

and cholesterol levels but not glucose parameters in patients

with HALS, presenting also an unclear impact on

cardiovascular endpoint (238, 239).

Similar to leptin, hypoadiponectinemia is associated

with insulin resistance, hypertriglyceridemia, and adipose

tissue redistribution in patients with HALS (240–242).

Adiponectin administration has been demonstrated in animals

to improve insulin sensitivity and dyslipidemia (243,

244). Although adiponectin or leptin alone only partly

alleviate insulin resistance in mouse models of lipodystrophy,

the combined administration of both hormones fully

normalizes insulin sensitivity (244). Given that no synthetic

form of adiponectin is yet available for human use,

animal studies as well as studies on medications that indirectly

increase endogenous levels of adiponectin such as

thiazolidinediones, have highlighted adiponectin’s therapeutic

potential (245). We have found that pioglitazone, a

thiazolidinedione that increases adiponectin levels, may

have beneficial effects on HALS-associated insulin resistance.

Recently, we performed a randomized, doubleblinded

pilot study on the combined administration of

leptin and pioglitazone for 3 months in 9 HALS patients

(246). Compared with pioglitazone alone, combined

treatment reduced fasting serum insulin concentrations,

increased adiponectin concentrations, improved insulin

resistance (homeostasis model assessment [HOMA]), and

attenuated postprandial glycemia in response to a mixed

meal (246). These preliminary findings indicate that this

combination could further improve glycemic control and

needs to be replicated in larger and longer-term studies. If

confirmed and proven to be superior to the current standard

of care, the treatment of HALS in the future may

involve leptin replacement in conjunction with pioglitazone

and/or adiponectin. Figure 2 depicts the effect of

recombinant leptin administration in HALS.

4. Hypothalamic amenorrhea

Hypothalamic amenorrhea (HA) is a disorder characterized

by the cessation of menstrual cycles with anovulatory

infertility and moderate to severe hypoleptinemia,

usually caused by reduced food intake or chronic energy

deficiency secondary to strenuous exercise (204). Leptin

administration to lean women who are affected by this

disorder and undertake strenuous exercise resulted in

recuperation of reproductive outcomes, correction in

the gonadal, thyroid, GH, and adrenal axes, and improvements

in markers of bone formation (247–249).

Nonetheless, larger clinical trials are needed to confirm

the previous studies.

B. States of leptin excess

1. Common obese state

In contrast to the few obese subjects with congenital

leptin deficiency, most obese individuals exhibit greater

leptin concentrations than lean subjects due to their

greater amount of fat mass and are unresponsive to exogenous

leptin in terms of anorectic effects, body weight

reduction, and glycemic control, due to leptin resistance as

discussed in Introduction (22, 23) (Table 5). These results

reduced expectations from leptin-based antiobesity therapeutic

approaches.

Despite their shortcomings, mouse models with HFDinduced

obesity may provide important insights into understanding

the effects of leptin on glucose metabolism in

obese humans because these mouse models are the closest

to human obesity. Chronic infusion of leptin in C57BL/6J

mice on HFD did not improve the glucose response to

insulin during an insulin tolerance test, even though the

body weight of mice effectively decreased compared with

control mice (250). Moderate hyperleptinemia achieved

by adenovirus gene therapy somewhat improved glucose

tolerance in animals that had 4-fold greater leptin concentrations

compared with control rats (251). Therefore,

serum concentrations of leptin may be an important factor

responsible for the restoration of aberrant glucose metabolism

in HFD-induced obese animal models. Leptin administration

in other mouse models of obesity has shown

mixed results. Harris et al (252) found that leptin treatment

improved insulin resistance and hyperglycemia in

some strains of db/db mice, but the effects depended on sex

and mode of administration (chronic or bolus ip administration).

Other authors found no benefits of leptin administration

in db/db mice (5). Leptin treatment in a mouse model



Table 5.

Disease States Associated With Leptin Deficiency and Leptin Resistance a

Disease State Estimated Prevalence Clinical Characteristics

Fat loss associated with leptin deficiency

Congenital generalized lipoatrophy Rare Generalized fat wasting, impaired glucose tolerance,

insulin resistance or type 2 diabetes, dyslipidemia,

hepatic steatosis, acanthosis nigricans

HAART-induced lipoatrophy 15%–36% of HIV-infected patients Fat wasting of the face, arms, legs, and buttocks;

impaired glucose tolerance, insulin resistance, or

type 2 diabetes; hypertriglyceridemia, hepatic

steatosis

HA (functional) 3%–8.5% in women aged 13–44 y Strenuous exercise, psychogenic stress, energy

Up to 69% of trained female athletes deficit, osteoporosis, neuroendocrine dysfunction

with decreased GnRH pulsatility and estradiol

levels, decreased thyroid and IGF-I levels, and

increased GH levels

Anorexia nervosa 1%–3% of college-age girls Disturbed body image, severe restriction of food

intake, body weight loss, neuroendocrine

dysfunction

Obesity as a manifestation of leptin deficiency

Complete congenital leptin deficiency Rare Hyperphagia, early-onset morbid obesity

hypogonadotropic hypogonadism, advanced bone

age, hyperinsulinemia and type 2 diabetes,

immune dysfunction

Heterozygous leptin deficiency 5%–6% in obese subjects Garden-variety obesity with hypoleptinemia relative

to fat mass, normal neuroendocrine function

Obesity associated with leptin resistance (involving

leptin and molecular signaling pathways

downstream of the leptin receptor)

Leptin receptor gene mutation Rare Phenotype similar to congenital leptin deficiency,

hyperphagia but less remarkable

hyperinsulinemia, hypogonadotropic

hypogonadism, mild growth retardation,

hypothalamic hypothyroidism, abnormal GH

secretion

POMC mutations Rare Hyperphagia, early-onset obesity, ACTH deficiency

with adrenal insufficiency/crisis, lack of MSH

function at MC1Rs resulting in pale skin and red

hair

Prohormone convertase deficiency Rare Hyperphagia, early-onset obesity, hypogonadotropic

hypogonadism, abnormal glucose homeostasis,

hypoinsulinemia, hypocortisolemia

MC4R mutations 5%–8% of childhood obesity Hyperphagia, early-onset obesity, increased fat and

lean body mass, increased linear growth and bone

density, severe hyperinsulinemia

Melanin-concentrating hormone receptor-1

mutations

Rare

Dysregulation of energy homeostasis with onset in

childhood

Neurotropin receptor tropomyosin-related kinase

B mutations

Rare

Mutations of other molecules downstream of Rare

leptin receptor

Mechanism to be discovered 90% of obese individuals Garden-variety obesity

a Adapted from Refs. 3, 4, 10, and 11.

BDNF deficiency resulting in severe obesity,

hyperphagia, developmental delay, and cognitive

dysfunction

Obesity with onset in childhood

of obesity that is deficient in brown adipose tissue resulted in

no improvement in glucose metabolism (253).

Subsequently, Heymsfield et al (22) conducted a randomized,

double-blind, placebo-controlled trial to determine

the relationship between increasing doses of exogenous

leptin administration and weight loss in both lean

and obese humans. The results of this trial indicated that

daily administration of recombinant human methionyl

leptin induced dose-dependent but modest weight loss in

most but not all subjects. Only at pharmacological doses

didleptininducesomeconsistentdegreeofweightloss,but

even then, the effect was relatively mild (average weight

loss was 7.1 kg after 24 weeks in the group receiving the

highest dose of leptin). In addition, these investigators performed

a standard oral GTT at baseline and after 24 weeks

and found no indication that leptin administration af-



fected glycemic control (22). Another study also demonstrated

that weekly injection of 60 mg of pegylated recombinant

human leptin did not lead to clinically

significant weight loss after 8 weeks of treatment, despite

serum leptin concentrations being as high as 3000 ng/mL,

ie, 100-fold higher than physiological levels. Insulin sensitivity,

assessed with the HOMA score, was also not significantly

affected by leptin treatment (254). This has been

confirmed in a recent study using the hyperinsulinemiceuglycemic

clamp, where leptin treatment for 14 days did

not have any effect on insulin sensitivity (281). These results

were confirmed by others and illustrate that leptin

administration is not an effective treatment for metabolic

disturbances in common obesity, at least not unless leptin

resistance can be overcome (256, 257). Nevertheless, in a

manner equivalent to administration of insulin for the

control of glycemia in type 2 diabetes with insulin resistance,

the possibility exists that leptin administration

might be able to overcome leptin resistance.

In leptin-based antiobesity approaches, another important

issue to consider is that leptin levels drop dramatically

with weight loss and remain low for up to 1 year after

weight loss (258–260). This has been proposed to be associated

with activation of mechanisms inducing weight

regain, such as increased food intake and reduced energy

expenditure (259) as seen in the case of lean subjects (261).

Hence, studies have looked at the role of leptin replacement

to physiological levels after weight loss. Small studies

have shown an improvement in weight loss maintenance

as well as a reversal of changes in thyroid hormones, energy

expenditure, and neural activity centers involved in

the regulatory, emotional, and cognitive control of food

intake (259, 262). However, the fact that these studies

were small and of sequential study design indicates that

future randomized and controlled studies are required to

define this potential use of leptin in more detail. This may

be related to saturability of the leptin pathways at higher

leptin concentrations, leading to absent response to increases

beyond the saturation level but marked responses

after weight loss when leptin levels are low (20). In this

respect, studies of leptin administration in lean women

showed a further reduction in fat mass without a reduction

in lean body mass, indicating that lean subjects with low

physiological leptin levels at baseline are sensitive to

exogenously administered leptin, which decreases primarily

fat mass while sparing lean mass and increasing

bone mass (12).

Recent clinical data have shown that combination

treatment with leptin and pramlintide, an amylin analog,

modestly decreased body weight (12%) in leptin-resistant

obese individuals more than leptin or amylin alone,

but the effect of the combination was rather additive not

synergistic as had previously been suggested by experiments

in rodents (264, 283). However, due to the relatively

small period of treatment (20 weeks), it remains to

be seen whether a longer-term clinical trial would result in

further reduction or maintenance of body weight or may

cause unaticipated beneficial or adverse effects. To address

these issues, longer-term clinical trials have been initiated,

and results are awaited with interest.

In summary, leptin monotherapy is not an effective

therapeutic approach for obesity in most obese subjects

(representing 90% of the obese population) with hyperleptinemia

(circulating leptin levels above 15 ng/mL).

However, the efficacy of leptin monotherapy or combination

therapy in obese subjects exhibiting hypoleptinemia

or normoleptinemia remains unknown. Figure 2 depicts

the effect of recombinant leptin administration in the

common obese state.

2. Diabetes mellitus

a. Type 1 diabetes mellitus. Many patients with type 1 diabetes

have low levels of leptin (265), and this has also been

seen in animal models of type 1 diabetes mellitus (266). A

decrease in leptin secretion may be related to a reduced

adipose mass and reduced assimilation and storage of energy

substrates in adipose tissue in insulin deficiency

(266).

Hence, several studies have examined the effects of leptin

administration on glucose metabolism in insulin-deficient

animal models of diabetes mellitus. Subcutaneous

infusion of leptin restored euglycemia and normalized peripheral

insulin sensitivity in streptozotocin (STZ)-induced

type 1 diabetic animals (267, 272). Central leptin

infusion also restored euglycemia in STZ-induced diabetic

animals (269–271). Similar effects of leptin were also

demonstrated in other studies by using various animal

models of type 1 diabetes (58, 272–274). Pair-feeding

studies indicate that decreased food intake does not account

by itself for the restoration of normoglycemia in

leptin-treated type 1 diabetic animals (273). These effects

of leptin were achieved through an insulinlike or insulinsensitizing

but insulin-independent mechanism mediated

by the CNS (68). It has been suggested that leptin may act

via activating intracellular signaling pathways that overlap

with those of insulin and/or by other mechanisms

(mainly suppression of glucagon) that remain to be fully

elucidated. Indeed, leptin monotherapy or combination

with low-dose insulin reversed the lethal catabolic state via

suppression of hyperglucagonemia in nonobese diabetic

mice with uncontrolled type 1 diabetes (58, 67). Moreover,

leptin normalized HbA 1C levels with far less glucose

variability compared with insulin monotherapy (58). This

recent promising work from the Unger laboratory (54, 55)



suggests that leptin administration in humans may present

many advantages over insulin monotherapy for the treatment

of diabetes type 1, characterized as a condition with

relative leptin deficiency. In humans, significant reductions

in circulating leptin levels have been documented in

the newly diagnosed untreated human type 1 diabetic patients,

suggesting that reduced leptin secretion might be a

common feature in insulin-deficient diabetes (265). Insulin

deficiency in type 1 diabetes leads to a state of increased

lipolysis from adipocytes elevating circulating levels of

free fatty acids and ketonemia. These metabolic products

may reduce adipocyte’s ability to secrete leptin, which signals

an energy deficit. In view of its beneficial effects in

animals, it has been suggested that leptin replacement

therapy may prove to be a useful adjunct therapy to restore

euglycemia in type 1 diabetes (277). Indeed, 1 year of leptin

therapy was effective in improving glycemic control

and lipid profiles in 2 patients with type 1 diabetes and

acquired generalized lipodystrophy (276). In these individuals,

leptin therapy improved insulin sensitivity, although

insulin was not completely discontinued but was

significantly reduced (276). Leptin’s main therapeutic

benefits in patients with type 1 diabetes may include its

slow-acting glycemia-lowering effect preferred over the

fast-acting glycemia-lowering effect that can trigger hypoglycemic

events, its antisteatotic and antilipotoxic action

in peripheral tissues preferred over the steatosis promoted

by insulin therapy that may cause cardiovascular

disease, its suppressive effect on glucagon that could reduce

the need for insulin, and its ability to restore insulin

secretion in -islets by reversing lipotoxicity (277). Nonetheless,

larger clinical trials are needed to be performed to

determine leptin’s safety and efficacy in reducing insulin

requirements, hyperglycemia, glycemic fluctuations, and

dyslipidemia in subjects with diabetes type 1.

b. Type 2 diabetes mellitus. The use of leptin in type 2 diabeteshasgeneratedinterestinviewofthepotentialinsulinindependent

activation of intracellular pathways involved

in glucose metabolism. Hence, several studies have investigated

the use of leptin in animal models of type 2 diabetes

mellitus, and more recently, we and others published human

studies (in this Section). Despite results from several

animal studies demonstrating that leptin ameliorated insulin

resistance, glycemic control, and lipid abnormalities

in mice with diabetes type 2, the results of 2 clinical trials

have shown that leptin treatment is largely ineffective in

improving insulin resistance and diabetes in obese subjects

with type 2 diabetes (20, 281). Given the fact that a nonnegligible

proportion of subjects with diabetes type 2 are

not obese, it will be important to evaluate the antidiabetic

effect of leptin in those subjects who exhibit normal or low

levels of leptin.

Toyoshima et al (278) investigated the use of leptin in

the MKR mouse, a diabetic mouse model with normal

leptin levels and defects in IGF-I and insulin receptor signaling.

They found an improvement in diabetes that was

independent of the reduced food intake and was associated

with reduced lipid stores in liver and muscle (278).

Similarly, Kusakabe et al (279) generated a mouse model

mimicking a combination of human type 1 and type 2

diabetes using low-dose STZ and HFD and examined the

effect of leptin treatment. These animals exhibited hyperglycemia

and hyperleptinemia and only mildly elevated

serum insulin levels, suggestive of both insulin deficiency

and insulin resistance. After leptin infusion, food intake

and body weight decreased, glucose tolerance improved,

and serum insulin levels decreased during an ip GTT. Pair

feeding showed that the improved glycemic control was

independent of the reduction in food intake (279). Finally,

our group has investigated leptin therapy in a mouse

model deficient in brown fat, which is hyperleptinemic

and very sensitive to DIO (253). In these mice, treatment

with ip leptin was not associated with any changes in insulin

or glucose concentrations (253).

Human studies have also evaluated the use of leptin in

obese diabetic patients. Recently, we conducted a doubleblinded,

placebo-controlled, randomized clinical trial to

evaluate the effects of leptin in obese type 2 diabetic patients

(20). Seventy-one obese subjects with diet-controlled type 2

diabetes mellitus were recruited and randomized to receive

either metreleptin (20 mg/d) or placebo for 16 weeks. Metreleptin

administration did not alter body weight or circulating

inflammatory markers, but marginally reduced

HbA 1C (8.01 0.93 to 7.96 1.12, P .03) (20). Furthermore,

we have found that levels of leptin-binding protein

and antibodies against metreleptin increased in response

to metreleptin treatment, limiting circulating free

leptin to 50 ng/mL despite mean total leptin levels of

1000 ng/mL in these subjects. Although these levels are

higher than the putative threshold for saturating the BBB

leptin transport system (280), circulating free leptin levels

did not correlate with weight loss, indicating clinical ineffectiveness

of free leptin levels in the 40- to 50-ng/mL

range. Mittendorfer et al (281) also performed a study to

evaluate the effects of exogenous leptin in obese type 2

diabetic patients. They conducted a randomized, placebocontrolled

trial in obese subjects with newly diagnosed

type 2 diabetes assigned to placebo or low-dose (30 mg/d)

or high-dose (80 mg/d) metreleptin for 14 days and performed

a hyperinsulinemic-euglycemic clamp in conjunction

with stable isotope-labeled tracer infusions to evaluate

multiorgan insulin sensitivity before and after leptin



treatment. There were no significant differences between

groups in body weight and body composition. Insulin sensitivity

of the liver (suppression of glucose production),

skeletal muscle (stimulation of glucose uptake), and adipose

tissue (suppression of lipolysis) and basal substrate

kinetics were not affected by leptin treatment, even though

serum leptin concentrations increased 150-fold compared

with baseline in the high-dose group (281). This is very

different from leptin replacement in lipodystrophic patients,

where the obtained leptin levels were near-physiological

(214).

These findings provide more evidence of leptin resistance

in the population of obese diabetic subjects. Because

most patients with type 2 diabetes are overweight/obese

and present hyperleptinemia with leptin resistance, leptin

treatment is considered ineffective (20, 281). Targeting the

molecular mechanisms of leptin resistance will be important

in the treatment for obesity and diabetes type 2. For

instance, it has been suggested that the combination of

leptin treatment with leptin sensitizers could be a potentially

feasible way to overcome leptin resistance. Amylin

has been suggested to be one such sensitizer on the basis of

animal experiments (282). Amylin may act with leptin to

induce fat-specific weight reduction (274), and the amylin

analog pramlintide has been used in conjunction with leptin

in clinical trials presenting modest effects (277) and

potential safety concerns (276). In a phase II clinical trial,

the combination of an amylin analog and recombinant

leptin produced significantly more weight loss than either

treatment alone in obese and overweight subjects (283).

This effect was accompanied by trends for improved metabolic

parameters such as total cholesterol (9%), LDL

cholesterol (8%), glycemia (4 mg/dl), insulinemia

(22%), and insulin resistance assessed by HOMA

(25%) (283). However, these results seem to be additive

rather than synergistic, which suggests that amylin may

have independent effects on weight loss but does not likely

improve leptin sensitivity. We have recently shown that

leptin administration does not influence circulating amylin

levels (284), and subsequently, we have performed signaling

studies in human adipose tissue ex vivo and human

primary adipocytes and peripheral blood mononuclear

cells in vitro (21). We demonstrated that leptin and amylin

alone and in combination activate STAT3, AMPK, Akt,

and ERK1/2 signaling pathways (21). These data indicate

that leptin and amylin activate overlapping intracellular

signaling pathways in humans and have additive, but not

synergistic, effects (21).

However, leptin resistance or tolerance may or may not

represent an insurmountable obstacle regarding the antidiabetic

actions of leptin. Leptin resistance could selectively

affect leptin signaling pathways related to appetite

and body weight reduction and not those related to glycemic

control. Although the ObRb/JAK2/STAT3 pathway

is important for leptin-mediated reduction of appetite

and body weight, it plays a minor role in leptin’s ability to

improve euglycemia (285). Moreover, pharmacological

inhibition of the hypothalamic PI3K signaling pathway

impairs the insulin-sensitizing effects of leptin (286). Research

aiming at identifying molecular mechanisms and

medications that could up-regulate insulin-sensitizing signaling

pathways downstream of leptin is awaited. Data

from rodents have also indicated that low doses of leptin

can ameliorate hyperglycemia without affecting food intake

or body weight, underscoring that the pathways underlying

the glucose-lowering actions of leptin are more

sensitive than the ones responsible for its weight-reducing

effects. Future clinical trials are needed to uncover the

clear antidiabetic action of leptin in nonobese patients

with diabetes type 2 who exhibit normo- or hypoleptinemia

due to low adipose tissue mass in contrast to previous

studies examining obese patients with type 2 diabetes. Results

of larger studies on combination treatments or the

development of specific leptin sensitizers that would enhance

leptin’s efficacy are awaited with great interest. Figure

2 depicts the effect of recombinant leptin administration

in type 2 diabetes.

3. Nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD), which is

considered to be the hepatic manifestation of insulin resistance

syndrome, has emerged as a significant public

health problem with worldwide distribution. The prevalence

of NAFLD in the general population is 10% to 46%

in the United States and 6% to 35% in the rest of the

world, with a median of approximately 20% (287). The

incidence of NAFLD in both adults and children is rising,

in conjunction with the growing epidemics of obesity and

type 2 diabetes mellitus. The histologic spectrum of

NAFLD encompasses a wide spectrum of liver damage

ranging from nonalcoholic simple steatosis (SS) to nonalcoholic

steatohepatitis (NASH) and NASH-related cirrhosis

with its complications. SS progresses to NASH in about

15% to 30% and in cirrhosis in less than 5% of cases,

whereas NASH progresses to cirrhosis in 10% to 15% of

cases over 10 years and in 25% to 30% of cases in the

presence of advanced fibrosis (288).

As described in the section of leptin signaling (Section

III), under normal conditions, leptin suppresses hepatic

glucose production and de novo lipogenesis, whereas it

induces fatty acid oxidation in hepatocytes, thereby providing

an antisteatotic and insulin-sensitizing effect. On

the other hand, hyperleptinemia has been proposed to play

a role in the pathogenesis of NAFLD (156, 289). There is



evidence that leptin acts as a proinflammatory and profibrogenic

cytokine, thereby affecting liver fibrosis, a key

feature of NASH. Xenobiotics-induced liver fibrosis was

extremely diminished in ob/ob mice and Zucker (fa/fa)

rats (290). Activated hepatic stellate cells (HSCs) express

ObRb and possibly other ObR isoforms, whose activation

leads to increased expression of proinflammatory and

proangiogenic cytokines and growth factors, such as angiopoietin-1

and vascular endothelial growth factor,

which affect hepatic inflammation and fibrosis (291).

Moreover, leptin was shown to up-regulate collagen 1in

HSCs through the sequence p38 MAPK activation and

SREPB-1c down-regulation (292). Furthermore, leptin

may induce hepatic fibrogenesis by stimulating the production

of tissue inhibitor of metalloproteinase-1 (293)

and repressing matrix metalloproteinase-1 gene expression

(294) in activated HSCs. Both these processes are

mediated by the JAK/STAT and the JAK-mediated H 2 O 2 -

dependant MAPK pathways. Inflammatory, fibrogenic,

and proliferative leptin actions on HSCs may also be mediated

via nicotinamide adenine dinucleotide phosphate

oxidase, which acts downstream of JAK activation, but

independently of STAT3; inhibition of nicotinamide adenine

dinucleotide phosphate oxidase in HSCs resulted in

a reduction of leptin-mediated up-regulation of fibrogenic

markers (collagen 1 and -smooth muscle actin) and of

inflammatory mediators (monocyte chemotactic protein-1

and macrophage inflammatory proteins 1 and 2)

and in a reduction of leptin-mediated HSC proliferation

(295). Additionally, leptin facilitates proliferation and

prevents apoptosis of HSCs (296), and it modulates all

features of the activated phenotype of HSCs in a profibrogenic

manner (myofibroblastic phenotype) (297).

Once activated, HSCs contribute to further leptin expression

(298), thereby possibly establishing a vicious

cycle (156).

Data regarding the role of leptin on hepatic fibrosis via

an effect on Kupffer or sinusoidal endothelial cells are

currently limited. Leptin may promote hepatic fibrogenesis

through up-regulation of TGF- and connective tissue

growth factor in isolated Kupffer cells; based on this finding,

Kupffer cells-HSCs cross talk has been proposed for

hepatic fibrosis (299). Leptin has also been reported to

affect hepatic fibrosis through up-regulation of TGF- in

sinusoidal endothelial cells (290).

More recently, an up-regulation of CD14 (an endotoxin

receptor recognizing bacterial lipopolysaccharide)

in Kupffer cells was followed by hyperreactivity toward

low-dose lipopolysaccharide in HFD steatotic mice but

not in chow-fed control mice, which resulted in NASH

progression (300). When recombinant leptin was administered

in chow-fed mice, an up-regulation of CD14 in

Kupffer cell was similarly observed resulting in hyperreactivity

toward lipopolysaccharide, thereby inducing hepatic

inflammation and fibrosis without steatosis. On the

contrary, recombinant leptin administration to ob/ob

mice did not up-regulate CD14 or induce inflammation

and fibrosis, despite severe steatosis (300); these results

demonstrated that leptin deficiency, apparently due to

lack of its lipolytic effects, may lead to hepatic steatosis,

but it is protective toward the progression of SS to NASH,

whereas excess of this proinflammatory adipocytokine

may favor hepatic inflammation and fibrosis.

In line with the aforementioned data, it was previously

speculated that leptin under normal conditions may prevent

liver steatosis, whereas its effect on quiescent HSCs

and Kupffer and sinusoidal cells is minimal. However,

prolonged hyperleptinemia, as observed in leptin tolerance

states (ie, common obesity), may ultimately result in

overexpression of SOCS3 and in activation of HSCs and

Kupffer and sinusoidal cells. Overexpressed SOCS3 may

aggravate both leptin tolerance and insulin resistance in

hepatocytes and outweigh the primarily beneficial effect of

leptin on hepatocytes. Activated HSCs and Kupffer and

sinusoidal cells, which seem to display minimal or no leptin

tolerance, may trigger the proinflammatory and profibrogenic

cascade (156).

Although evidence for the inflammatory and fibrogenic

role of leptin in NAFLD from in vivo and animal models

is strong, relevant data from clinical studies in NAFLD

patientsarebasedmainlyoncross-sectionalstudies,which

cannot establish a causative relationship. Most authors

have reported higher leptin levels in NAFLD (301, 302) or

NASH (303, 304) patients than controls, in parallel with

higher insulin resistance. However, other authors have

reported similar leptin levels between NASH patients and

controls (305, 306). Regarding liver histology, some authors

have also reported that leptin levels were positively

associated with hepatic fibrosis (303, 307, 308) or inflammation

(303, 308). However, other authors reported no

association between serum leptin levels and hepatic fibrosis

or inflammation (304, 306).

Soluble leptin receptor (sOB-R) has been reported to be

lower in the NAFLD patients than controls, as well as

negatively associated with circulating leptin in one study,

which might be attributed to counteracting sOB-R downregulation

(301). However, other authors have reported

higher sOB-R levels in morbidly obese patients with diabetes,

which are positively associated with the stage of

hepatic fibrosis (309), whereas others found no association

of sOB-R levels with hepatic histology in children

with NAFLD (308). More studies are needed to elucidate

the interaction of leptin with its soluble receptor and its

role, if any, in the pathogenesis of NAFLD. The cross-



sectional nature of the aforementioned clinical studies together

with the heterogeneity within them render the

drawing of any conclusion risky. Larger studies with better

characterized and/or homogenous populations and

carefully matched controls are of importance.

Prospective data regarding circulating leptin levels in

NAFLD are limited. In a 3-year prospective study with

paired biopsies, serum leptin levels were decreased more in

patients with stable or improved disease compared with

those with disease worsening; however, leptin could not

independently predict disease or fibrosis progression

(310). In a 7-year prospective study, leptin levels were

higher in patients with than without NAFLD at baseline;

however, leptin change was similar in those with disease

remission or sustaining disease (311). The limitation of

this study was its noninvasive nature (NAFLD was not

biopsy-proven). Further prospective studies with paired

biopsies and long-term follow-up are needed.

The effect of various therapeutic interventions on circulating

leptin levels in patients with NAFLD are summarized

in Table 6. Briefly, leptin levels remained essentially

unaffected after treatment with pioglitazone (312),

rosiglitazone (312), atorvastatin (313), ezetimibe (314),

cysteamine (315), and ursodeoxycholic acid with or

without vitamin E (316) but increased after short-term

melatonin treatment (317) or decreased equally after

metformin and lifestyle modifications or only lifestyle

modifications (57).

Importantly, no study to date has investigated or is

planned to investigate the effects of recombinant leptin

administration on NAFLD patients with normal or high

leptin levels. On the other hand, metreleptin administered

in 10 NAFLD patients (8 with NASH) with lipodystrophy

for a mean duration of 6.6 months resulted in improvement

of hepatic steatosis and ballooning but not portal

inflammation or fibrosis (63). These results are similar to

Table 6. Effect of Various Therapeutic Interventions

on Circulating Leptin Levels in Patients With NAFLD

Ref. Intervention Duration

Circulating Leptin

Levels

317 Melatonin 10 mg/d 28 d Increased

275 Rosiglitazone 4 mg /d 6 mo Unchanged

315 Enteric-coated cysteamine 6 mo Unchanged

600–2000 mg/d

314 Ezetimibe 10 mg/d 2 y Unchanged

316 Ursodeoxycholic acid 12–15 2 y Unchanged

mg/kg/d Vitamin E

800 IU/d

313 Atorvastatin 10 mg/d 2 y Unchanged

57 Metformin 1700 mg/d

diet/exercise

6 mo Decreased in either

group

312 Pioglitazone 30 mg/d 1 y Unchanged

Data are presented in publication date order.

Imajo et al (300) experimental model, indicating that recombinant

leptin administration in NAFLD patients and

lipodistrophy may improve steatosis but not fibrosis. Furthermore,

there are 2 ongoing clinical trials having as primary

endpoint the effect of recombinant leptin administration

in hepatic histology in NAFLD patients with low

leptin levels. In one of them (single-group, prospective,

open-label), metreleptin (0.1 mg/kg/d once a day sc) in 10

adult male patients with NASH and low circulating leptin

levels, but not lipodystrophy, was to be administered for

1 year (NCT00596934). In the other study, (single-group,

prospective, open-label), metreleptin (0.1 mg/kg/d once a

day sc) was planned to be administered for 1 year in 20

male or female patients with SS or NASH and lipodystrophy

(NCT01679197).

Despite the lack of direct clinical evidence, leptin administration

in NAFLD patients with hyperleptinemia

might have adverse effect on liver fibrosis, thereby possibly

worsening the prognosis of liver disease. If the results

of the Imajo et al study (300) were translated into human

pathophysiology, they might have 2 main implications: 1)

leptin excess, which is usually observed in common forms

of obesity, may be contributing toward NASH progression

and 2) recombinant leptin administration for severe

obesity may adversely affect the liver of obese individuals,

because it may promote proinflammatory responses,

whereas it has no lipolytic effects above a certain level due

to its permissive role in controlling obesity (20). Considering

that the prevalence of NAFLD in obese individuals

is 50% to 95%, this becomes of crucial importance. Figure

2 depicts the effect of recombinant leptin administration in

NAFLD.

C. Benefits, potential adverse effects, and challenges in

relation to leptin replacement therapy

The benefits and potential adverse effects of recombinant

leptin administration in leptin deficiency or leptin

excess states are summarized in Table 3. Generally, leptin

replacement therapy using an analog of leptin, metreleptin,

also known as recombinant methionyl human leptin,

has been proven effective for the treatment of congenital

leptin deficiency, congenital and non-HIV–related acquired

generalized lipodystrophy associated with severe

hypoleptinemia, and HA (219, 220).

Metreleptin offers metabolic benefits such as improvements

in insulin sensitivity, glucose tolerance, fasting glucose,

glycosylated hemoglobin, hypertrigyceridemia, and

liver function tests (219, 220). Patients with partial lipodystrophy,

including those with PPAR mutations (216),

also gain metabolic benefits from leptin administration. In

these subjects, leptin administration has been extensively

studied in the context of open-label clinical trials (221).



HIV patients with HALS also exhibit significant improvements

in their metabolic profile that are comparable to

other treatment options such as metformin and thiazolidinediones

(234). Leptin replacement therapy provides

benefits beyond metabolic improvements: it ameliorates

glomerular injury in generalized lipodystrophy (223), it

has immunomodulatory effects in hypoleptinemic patients

with severe lipodystrophy (222), it normalizes menstrual

abnormalities in young women with lipodystrophy

and polycystic ovary syndrome (224), and it improves the

thyroid, GH, and adrenal axis abnormalities seen in

women with HA (224).

Although open-label trials have shown benefits of metreleptin

use, larger, randomized, placebo-controlled clinical

trials are needed to conclusively demonstrate its efficacy

and safety, particularly because severe adverse effects

have been noted, including deterioration of renal function

and proteinuric nephropathy (224) and occurrence of T-

cell lymphomas (223) in a small number of patients with

lipodystrophy. It is important to underscore that the openlabel

nature of the study design did not allow the investigators

to establish or refute a causal link between metreleptin

treatment and reported adverse effects. Similar

adverse effects would not be entirely unexpected on the

basis of the natural history of the disease, particularly in

patients suffering from acquired lipodystrophy, which is

traditionally thought to be due to autoimmune factors.

Approximately 25% of cases with acquired generalized

lipodystrophy are caused by autoimmune disease such as

juvenile-onset dermatomyositis, rheumatoid arthritis, systemic

lupus erythematosus, and Sjögren syndrome (219).

Given the immunomodulatory effect of metreleptin,

which clearly regulates the Th1/Th2 imbalance in leptindeficient

individuals (224), large, crossover, randomized,

placebo-controlled clinical trials of sufficient duration are

needed to study efficacy and adverse effects more tightly.

Potential disadvantages of leptin therapy may include increased

immune system function and inflammation and

the generation of neutralizing autoantibodies against exogenous

leptin (115, 190, 212, 219). Moreover, it has

been reported that leptin accelerates autoimmune diabetes

in the nonobese diabetic mouse model of diabetes type 1,

probably by inducing proinflammatory cell responses

(217).

It remains unknown whether similar adverse effects in

response to leptin administration could potentially also be

observed in disease states accompanied by hyperleptinemia

due to leptin resistance such as garden-variety obesity.

It also remains unknown whether similar side effects could

be seen in response to higher than normal leptin levels,

which are needed to effectively lower glycemia in conditions

such as type 1 diabetes. In these conditions, potential

side effects of leptin may become really prevalent due to

the increased prevalence of the underlying disease. Common

adverse events seen particularly in combination treatment

with pramlintide/metreleptin in obese subjects included

nausea and injection site erythema and were mild

to moderate and decreasing over time (263). It has been

proposed that leptin could possibly cause hypertension,

impair endothelial function, promote platelet aggregation

leading to thrombosis, increase immune function, foster

inflammation and angiogenesis, and worsen diabetic complications

and other concomitant diseases on the basis of

preclinical studies, but no such side effects have been seen

in the clinic (283). We have failed to observe effects in

hypertension (255, 283), angiogenesis (263), or inflammation

(20). Although it cannot be excluded that prolonged

administration of leptin may cause hypertension,

particularly in predisposed obese diabetic individuals in

contrast to obese hypotensive subjects who present with

leptin deficiency (255), we and others have not observed

such side effects in our trials (20, 268). Moreover, leptin’s

action in suppressing glucagon may place patients suffering

from type 1 diabetes at increased risk for severe hypoglycemic

episodes by impairing the counterregulatory

response necessary to restore glycemia (283). Until now,

these particular side effects have not been reported in leptin-deficient

lipodystrophic or other patients treated with

replacement doses of leptin. Other probable adverse effects

may theoretically include sleep disorders, an early

onset of puberty in children, and an increased risk of unplanned

pregnancy, but again, they have not yet been observed

in the clinic, whereas the potential teratogenic effects

of leptin treatment need to be explored.

It is of paramount importance to discuss the potential

carcinogenic adverse effects of leptin administration in

patients with garden-variety obesity. It has been proposed

that leptin may exert neoplastic effects via 2 mechanisms.

First, leptin can act directly on cancer cells by stimulating

receptor-mediated signaling pathways leading to tumor

cell growth, migration, and invasion (193). Notably, this

activity of leptin is commonly reinforced through entangled

cross talk with multiple oncogenes, cytokines, and

growth factors. Second, leptin may act indirectly by reducing

tissue sensitivity to insulin, causing hyperinsulinemia

and by regulating inflammatory responses with overproduction

of cytokines such as IL-6, IL-12, and TNF-

(284) and influencing tumor angiogenesis, but we have not

observed such effects of leptin in vivo (285). Leptin has

also been proposed to play a role in hormone-dependent

malignancies, such as breast and endometrial cancers, by

activating the enzyme aromatase, an enzyme responsible

for the transformation of androgens to estrogens (81). Expression

of leptin receptors has been reported in numerous



cancer cell types including breast, colon, prostate, and

thyroid (80). Leptin, through its receptor ObRb, may

modulate growth and proliferation of cancer cells via activation

of various growth and survival signaling pathways

including canonical (JAK2/STAT3, PI3K/Akt, and

MAPK/ERK1/2) and noncanonical (protein kinase C,

JNK, and p38 MAPK) signaling pathways (80). For example,

leptin has been demonstrated in vitro to stimulate

JNK in human breast cancer cells in both a time- and a

dose-dependent manner, with greater phosphorylated

JNK levels after long-term exposure (80). In breast cancer,

JNK activation by leptin leads to an up-regulation of matrix

metalloproteinase-2 activity, which promotes cancer

cell invasion (80). Leptin exerts proliferative and antiapoptotic

effects on human colon cancer cells, which are mediated

in part though JNK activation (80). It should be

noted, however, that most in vitro studies have used extremely

high leptin levels.

However, in contrast to many in vitro studies, epidemiological

studies report inconsistent associations between

serum leptin levels and risk of several malignancies.

For example, in the case of colorectal cancer (CC), an

obesity-associated cancer, some prospective studies found

a positive association between serum leptin levels and CC

risk (291), whereas others did not show any relationship.

Moreover, some studies revealed that even hypoleptinemia

was associated with CC risk, independently from BMI

(293). Our group, which has focused on adipokines and

cancer, has recently shown that hypoleptinemia, and not

hyperleptinemia, was linked to pancreatic cancer independently

from BMI and weight loss, to B cell chronic lymphocytic

leukemia, and to low-risk myelodysplastic syndrome

after adjustment for BMI and other risk factors

(297) but these associations may reflect reverse causality.

In addition, increasing serum leptin levels did not appear

to increase substantially the risk of premenopausal breast

cancer in situ, invasive pre- and postmenopausal breast

cancer, endometrial cancer, prostate cancer, multiple myeloma,

melanoma, and lung cancer (318–321). Thus, the

possible association of leptin and malignancies in vitro

appears to be not supported by published studies in humans.

A possible association with leptin needs to be studied

further with larger prospective, longitudinal, and

mechanistic studies, which are needed to prove causality,

provide further insights into both the mechanisms underlying

the actions of this hormone and its potential role in

cancer, and analyze its action in tumor progression if leptin

is prescribed as an alternative or adjunct approach in

patients with diabetes and malignancies in the future.

In conclusion, future larger trials using metreleptin are

needed to systematically determine 1) the diagnostic criteria

to be used as indicating hypoleptinemia and thus

serve as inclusion criteria in ongoing and future trials, 2)

the appropriate initial metreleptin dose, 3) how high the

dose of metreleptin should be raised (based upon which

algorithm) for the best metabolic response to be achieved

or what dose would be needed to safely break through a

possible resistance to metreleptin in certain individuals, 4)

how patients on metreleptin should be monitored, and 5)

whether the concomitant use of other leptin-sensitizing

treatments could be useful therapeutic options in the treatment

of obesity or to prevent metabolic adaptation induced

by caloric restriction and to promote weight maintenance.

Garden-variety obesity is a chronic disease with

currently limited successful long-term therapy options,

apart from bariatric surgery, which is both costly and

risky. Combination therapy for obesity represents an encouraging

step forward in the pharmacologic armamentarium.

Finally, large randomized leptin administration

studies are needed to obtain accurate information on potential

adverse effects and long-term outcomes.

VII. Future Directions

Leptin is an adipocyte-secreted hormone that interacts

with several organs that are involved in the regulation of

energy homeostasis and metabolism. Although great

progress has been made in terms of understanding leptin’s

pathophysiological role, several fundamental questions,

particularly regarding the effects of leptin on glucose metabolism,

still remain to be answered. Much progress has

been made with leptin treatment in patients with leptin

deficiency and lipodystrophy, and such treatment does

correct metabolic abnormalities. Results from these studies

suggest that leptin could prove to be a potential antidiabetic

agent in leptin-deficient states. However, formal

double-blinded placebo-controlled phase III clinical trials

are required to accurately determine placebo-subtracted

efficacy and to assess comparative efficacy in relation to

other effective therapies, and dose-finding studies are necessary

to help guide appropriate treatment for these individuals.

It is also unclear whether all patients with lipodystrophy

would benefit from leptin therapy or whether

only those with the most profound leptin deficiency are

more likely to benefit. In contrast, the vast majority of

obese individuals are hyperleptinemic and resistant or tolerant

to exogenous leptin. Therefore, it is questionable

whether leptin alone can be used to favorably modify glucose

metabolism in garden-variety obesity and/or diabetes.

Hence, findings from ongoing and upcoming studies

combining leptin with other antiobesity medications or

sensitizers will be of particular interest.



Acknowledgments

Address correspondence and requests for reprints to: Christos S. Mantzoros,

MD, DSc, Professor of Medicine, Harvard Medical School,

JP9B52A, 150 South Huntington Avenue, Boston, Massachusetts

02130. E-mail: cmantzor@bidmc.harvard.edu.

The Mantzoros laboratory is supported by National Institute of Diabetes

and Digestive and Kidney Diseases Grants 58785, 79929, 81913,

and AG032030 and a VA Merit award (Grant 10684957). The Mantzoros

Laboratory is also supported by a discretionary grant from Beth

Israel Deaconess Medical Center.

All authors contributed to writing the manuscript. All authors read

and approved the final manuscript.

Disclosure Summary: All authors state that there is no conflict of

interest related to this paper.

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Leptin’s Role in Lipodystrophic and Nonlipodystrophic Insulin-Resistant and Diabetic Individuals

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