[1479683X - European Journal of Endocrinology] MECHANISMS IN ENDOCRINOLOGY_ Skeletal muscle lipotoxicity in insulin resistance and type 2 diabetes_ a causal mechanism or an innocent bystander_

Review

C Brøns and L G Grunnet Dysfunctional adipose tissue

and T2D

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MECHANISMS IN ENDOCRINOLOGY

Skeletal muscle lipotoxicity in insulin

resistance and type 2 diabetes: a causal

mechanism or an innocent bystander?

176:2

R67–R78

Charlotte Brøns and Louise Groth Grunnet

Department of Endocrinology (Diabetes and Metabolism), Rigshospitalet, Copenhagen, Denmark

Correspondence

should be addressed

to C Brøns

Email

charlotte.broens@regionh.dk

Abstract

European Journal of Endocrinology

Dysfunctional adipose tissue is associated with an increased risk of developing type 2 diabetes (T2D). One

characteristic of a dysfunctional adipose tissue is the reduced expandability of the subcutaneous adipose tissue

leading to ectopic storage of fat in organs and/or tissues involved in the pathogenesis of T2D that can cause

lipotoxicity. Accumulation of lipids in the skeletal muscle is associated with insulin resistance, but the majority of

previous studies do not prove any causality. Most studies agree that it is not the intramuscular lipids per se that causes

insulin resistance, but rather lipid intermediates such as diacylglycerols, fatty acyl-CoAs and ceramides and that it is

the localization, composition and turnover of these intermediates that play an important role in the development of

insulin resistance and T2D. Adipose tissue is a more active tissue than previously thought, and future research should

thus aim at examining the exact role of lipid composition, cellular localization and the dynamics of lipid turnover on

the development of insulin resistance. In addition, ectopic storage of fat has differential impact on various organs in

different phenotypes at risk of developing T2D; thus, understanding how adipogenesis is regulated, the interference

with metabolic outcomes and what determines the capacity of adipose tissue expandability in distinct population

groups is necessary. This study is a review of the current literature on the adipose tissue expandability hypothesis

and how the following ectopic lipid accumulation as a consequence of a limited adipose tissue expandability may be

associated with insulin resistance in muscle and liver.

European Journal of

Endocrinology

(2017) 176, R67–R78

Introduction

Obesity is a major global health problem with

increasing prevalence (1). Multiple metabolic disorders

and diseases including insulin resistance and T2D most

often accompany obesity. T2D pathophysiology is

associated with an increased fat mass (2), and insulin

resistance correlates with obesity even within the

Invited Author’s profile

Charlotte Brøns PhD, is currently head of the Department of Diabetes and Metabolism at Rigshospitalet, and

adjunct Associate Professor at the University of Copenhagen, Denmark. Her scientific expertise lies within the field

of metabolism and integrative physiology with an extensive focus on the physiologic and molecular mechanisms

underlying the etiology and pathophysiology of type 2 diabetes primarily in individuals exposed to intrauterine

over- (gestational diabetes) and under-nutrition (low birth weight).

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DOI: 10.1530/EJE-16-0488

© 2017 European Society of Endocrinology

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Review C Brøns and L G Grunnet Dysfunctional adipose tissue

and T2D

PROOF ONLY

normal range (3, 4). The adipose tissue plays an

important role in regulating energy balance via lipid

storage and secretion and by being an active endocrine

organ secreting adipokines that influence systemic

metabolism (5).

It is well known that obesity is associated with

increased plasma levels of free fatty acids (FFA) due

to increased lipolysis and thus FFA release from

the adipose tissue and/or due to a reduced FFA

clearance rate. Elevated plasma FFA levels inhibit the

antilipolytic effect of insulin resulting in a further

increased release of FFA into the circulation. However,

the relationship between circulating FFA and insulin

resistance is being debated. Studies have both provided

evidence of insulin resistance and T2D in the presence

of normal circulating levels of FFA (6) as well as

elevated FFA levels without concomitant insulin

resistance, thus questioning the causal role of FFA in

T2D pathophysiology (7).

Insulin resistance is a hallmark feature of T2D

preceding the onset of overt disease by decades.

Since the studies by Sir Philip Randle documenting

substrate competition between fat and glucose

oxidation, the ‘glucose fatty acid cycle’ also known as

the ‘Randle Cycle’ has been a central concept explaining

the development of insulin resistance (8). Although

many studies have confirmed the mechanisms of

the ‘Randle Cycle’, new mechanisms of glucose and

fatty acids utilization have subsequently been discovered

suggesting various additional, parallel or corresponding

mechanisms by which fatty acids adversely affect

muscle insulin action and glucose uptake. Potentially

deleterious lipid intermediates such as diacylglycerol

(DAG) and ceramides accumulating within the

tissue have been associated with the development of

muscle insulin resistance (9). Also, increased adipose

tissue mass is related to an increased production of proinflammatory

cytokines and endoplasmic reticulum

stress which, together with elevated circulating

FFAs, are involved in the development of insulin

resistance (10).

In the present review, we will discuss how the

ectopic lipid accumulation may occur by the adipose

tissue expandability hypothesis and the impact of these

lipids on insulin resistance and mitochondrial function.

Furthermore, we will exemplify the link between

lipotoxicity and insulin resistance in individuals

exposed to intrauterine growth retardation because

these subjects have a prediabetic phenotype already at

a young age.

Adipose tissue expandability

176:2 R68

The underlying molecular mechanisms explaining the

link between obesity and T2D are not understood in

depth. One hypothesis is the ‘adipose tissue expandability’

hypothesis, proposing that it is the limited capacity or

dysregulation of the adipose tissue expansion, rather than

obesity per se which explains the link between a positive

energy balance and T2D (11). All human individuals

possess a maximum capacity for adipose expansion

that is determined by both genetic and environmental

factors. Once the adipose tissue expansion limit has been

reached, adipose tissue ceases to store energy efficiently

and lipids begin to accumulate in other tissues (Fig. 1).

Ectopic lipid accumulation in non-adipocyte cells causes

lipotoxic insults including insulin resistance, apoptosis

and inflammation (12).

There are two models of adipogenesis. One model is

from the pluripotent mesenchymal stem cell line that has

not undergone commitment to the adipocyte lineage.

Upon stimulation, the pluripotent stem cells can be

committed to a number of different lineages including

adipocytes, but once committed, the preadipocytes can

only differentiate into adipocytes. The commitment of

stem cells into the preadipocyte lineage and differentiation

of preadipocytes to adipocytes take place throughout life.

The other model is from the preadipocyte cell lines that

have been committed to the adipocyte lineage. When

stimulated, the preadipocytes undergo multiple rounds of

mitosis and then differentiate into adipocytes (13). The

two models together refer to hyperplasia of the adipose

tissue, whereas enlargement of adipocytes volume refers to

hypertrophy (Fig. 2). Together, the two abovementioned

processes are responsible for the increase in adiposity due

to excessive energy intake accompanied by low energy

expenditure, and it is the transcription factor PPARγ

(peroxisome-proliferator-activated receptor γ), which is

the main regulator of differentiation of the preadipocytes

intro mature adipocytes (14). Manipulation with PPARγ

suggests that treatment with PPARγ agonists results in

increased fat mass expansion, whereas the inactivation of

PPARγ decreases fat mass expansion (15).

When recruitment of new fat cells fails because of

impaired differentiation, it will eventually lead to a

decreased capacity of the adipose tissue to accumulate

excess lipids. Adipocytes that are filled to capacity are

extremely insulin resistant and therefore less efficient as

metabolic buffers (16, 17), and it has been shown that

adipocyte hypertrophy is a key phenotype of non-obese

T2D patients (18). Increased adipocyte size is also shown

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176:2 R69

Figure 1

The proposed hypothesis for limited

adipose tissue expandability. When the

body is in a positive energy balance, the

adipose tissue will expand to handle the

excess energy. If the adipose tissue is not

capable of expanding sufficiently, there

will be a spillover of FFA to non-adipose

tissue leading to harmful effects in liver,

muscle and pancreas.


in obese, prediabetic and T2D subjects (19, 20, 21, 22)

and is associated with insulin resistance independently

of BMI (23, 24). Also, patients with T2D have fewer total

subcutaneous adipocytes compared with BMI-matched

obese subjects (25).

Paradoxically, the metabolic consequences of having

too much fat are surprisingly similar to those of having

an abnormal lack of fat i.e. lipodystrophy, and it has

Figure 2

Models of adipose tissue enlargement. Obesity is an

enlargement of adipose tissue to store energy surplus, and

the adipose tissue grows by two mechanisms namely

hyperplasia (increase in cell number) and hypertrophy

(increase in cell size).

been demonstrated that lipodystrophy in both animal

and humans results in severe insulin resistance and T2D

(26, 27). Furthermore, there are several similarities between

the biochemical and clinical profiles of these two groups

of individuals including dyslipidemia and hepatic steatosis.

The physiological importance of adipose tissue

expandability has been demonstrated in multiple studies.

For example, evidence from animal models has shown

that severe insulin resistance was present in an obese

mouse model with a limited capacity of adipose tissue

expandability (14). On the contrary, a transgenic mouse

model of morbid obesity was associated with significantly

greater amounts of adipose tissue – mainly non-visceral

depots, reduced triglyceride levels in liver and muscle

and an improved metabolic profile (28). A polymorphism

of the lipid sensor G-protein-coupled receptor 120

(GPR120) has been associated with a reduced capacity to

proliferate subcutaneous tissue in response to a high-fat

diet, subsequently resulting in ectopic accumulation of

fat in liver and muscle (29). A recent study showed that

regulation of lipid storage-related genes (DGAT2, SREBP1c

and CIDEA) was defective in the subcutaneous adipose

tissue of subjects exhibiting the largest fat accumulation

in the visceral adipose tissue of non-obese subjects

when overfed for 56 days, supporting the adipose tissue

expandability theory (30). Apart from adipogenesis and

lipogenesis, appropriate plasticity ensured by remodeling

of the extracellular matrix and the vasculature are required

for the maintenance of adipose tissue expandability (31).

A suboptimal early-life environment due to poor

nutrition and/or stress during pregnancy can influence

the phenotype of the offspring (32), and low birthweight

is consistently associated with increased risk of T2D

(33, 34). Young, healthy men born with a low birthweight

have significant changes in body fat content and

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distribution, increased rate of lipolysis and impaired

preadipocyte maturation (35, 36), which may contribute

to the development of whole-body as well as adipose

tissue and hepatic insulin resistance later in life. De Zegher

et al. found that fetal growth restriction was associated

with increased circulating levels of preadipocyte factor 1

(Pref-1), which inhibits adipocyte differentiation (37).

These data suggest that Pref-1 may be a mediator of reduced

adipocyte differentiation in growth-restrained fetuses,

and thus of a reduction in life-long lipid storage capacity

and thereby of adult vulnerability to metabolic disease,

once lipid storage becomes an issue. Following this line of

thinking, Sebastiani et al. showed that SGA children have

more hepatic fat than matched appropriate for gestational

age (AGA) children (38). Supporting the adipose tissue

expandability hypothesis as a reason for lipotoxicity,

the authors propose that a postnatal reduction of

subcutaneous adipogenesis, followed by hyperexpansion

and subsequent overfilling of subcutaneous adipocytes

due to post-natal catch-up growth, may be among the

mechanisms predisposing SGA children to excessive lipid

storage in other organs, including in the liver (38).

We have shown, in a study of human subjects and

rats exposed to suboptimal nutrition during fetal life, that

increased in vivo expression of miR-483-3p, programmed

by early-life nutrition, limits the storage of lipids in

adipose tissue via translational repression of the growth

differentiation factor-3 (GDF3) (39). Dysregulation

of miR-483-3p expression could possibly affect the

whole body physiology by constraining adipose tissue

expandability and therefore lipid storage, promoting

ectopic triglyceride storage and lipotoxicity in other tissues

and thus increasing susceptibility to metabolic disease.

Furthermore, SGA subjects have immature preadipocyte

stem cells as reflected by impaired leptin mRNA

176:2 R70

expression and protein synthesis in these cells, which

was shown to be associated with a significantly increased

degree of DNA methylation of the leptin promoter in

the preadipocytes from SGA subjects (36). This provides

evidence of a prime role for immature adipose tissue stem

cell function in developmental programming of T2D

possibly mediated by early defects in the adipose tissue

and a reduced expandability resulting in ectopic lipid

storage, lipotoxicity and insulin resistance (Fig. 3).

During pregnancy, the adipose tissue must expand

rapidly not only to meet the needs of the growing fetus

but also to support the future needs of the offspring

through lactation. Rojas-Rodriguez et al. showed that

women with gestational diabetes had greater adipocyte

size and decreased capillary density in the omental fat

compared with women with a normal glucose tolerance

test during pregnancy, indicating an impaired adipose

tissue expandability in gestational diabetes (40). In

addition, it was recently shown that the progenitor density

(and thus the source of new adipocytes) was lower and

accompanied by limited expansion of adipocyte number

when challenged with a high-fat diet in a 3-month-old

rat offspring of maternal obese dams compared with that

in the offspring of control dams. This was associated

with lower DNA methylation of the zinc finger protein

423 (involved in adipogenesis) in the epididymal fat

in the offspring of obese dams (41), suggesting a focus

on epigenetic changes in adipocyte progenitors when

looking for possible mechanisms in fetal programming of

an obesogenic maternal environment.

Altogether, the importance of normal adipose tissue

development, and that disturbances of adipose tissue

function can result in features of T2D is well documented,

supporting the adipose tissue expandability hypothesis.

The importance of the adipose tissue and especially the

Figure 3

Metabolic consequences of an immature

preadipocyte. Decreased expression of the

differentiation markers such as FABP4,

PPARγ and GLUT4 mRNA suggested

impaired human adipocyte maturation. In

addition, reduced leptin mRNA expression

and a reduced leptin release from these

preadipocytes suggest diminished

endocrine function that may lead to a

decreased ability to store lipids in the

adipose tissue and eventually insulin

resistance (adapted from (36)).

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dynamics of adipose tissue turnover have furthermore

been illustrated by Arner et al. They showed that the

lipid removal rate was positively associated with the

capacity of adipocytes to break down triglycerides and

inversely associated with insulin resistance in human

subjects independent of the degree of obesity (42).

Nevertheless, knowledge on programming of adipose

tissue expandability in humans is still limited.

Lipotoxicity and insulin action in

skeletal muscle

Lipid overload

Infusion of lipids in various doses has been used in

many studies to imitate the state of lipid oversupply as

can be seen in obesity and T2D. In healthy individuals,

acute elevation of plasma FFA by lipid infusion produces

acute insulin resistance of the skeletal muscle tissue (43).

It has been demonstrated that increased levels of plasma

FFAs induce insulin resistance by initial inhibition of

glucose transport/phosphorylation and thereby by the

impairment of the proximal insulin signaling pathway,

followed by a reduction in both the rate of muscle

glycogen synthesis and glucose oxidation (44, 45).

However, the molecular mechanisms underlying this

impairment is presently not fully understood, and data

are somewhat contradicting. For example, Belfort et al.

showed that an increase in plasma FFA caused a dosedependent

inhibition of insulin-stimulated glucose

disposal and insulin signaling in skeletal muscle of lean,

healthy individuals. The inhibitory effect of plasma

FFA was already significant after a modest increase in

plasma FFA and developed at concentrations within the

physiological range (46). Kruszynska et al. have reported

similar results on insulin signaling (47). In contrast,

when lipids are infused to already insulin-resistant obese

individuals, physiological elevation of plasma FFA levels

resulted in lipid-induced metabolic changes, which could

not be explained by reduced proximal insulin signaling in

skeletal muscle (48). Lipid infusion studies can obviously

induce responses different from what would have been

observed if a dietary intervention was applied because

ingestion of food evidently affects different hormones and

pathways, which are not activated during lipid infusion.

Thus, lipid infusion studies may be artificial and needs to

be interpreted with that in mind.

Studies on the effects of high-fat feeding on insulin

signaling in human subjects are lacking; however, rat

studies have reported defective insulin-induced activation

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of glucose transport in the high-fat-feeding model of

insulin resistance (49, 50).

Intramyocellular lipids

Accumulation of toxic lipids species as intramyocellular

lipid (IMCL) may occur as a result of increased fatty acids

uptake due to sustained lipid overload and/or as a result of

a reduced rate of fatty acids oxidation (51) and might be

a primary factor in the development of insulin resistance

(52, 53). IMCL is the common denominator for any form

of lipid species that is located within the myocyte and

stored in lipid droplets, mostly in the form of triglyceride

(TG) but in some cases, as lipid intermediates such as longchain

fatty acyl-CoAs, DAG and ceramides. It is well known

that IMCLs have a physiological function as an important

energy source that drives muscle fat oxidation, and data

from early studies suggested that IMCL could be used as

a source of fuel during exercise as muscle TG was found

to be depleted after prolonged exercise (54), and during

exercise, large amounts of circulating FFAs are directed

into muscle cells for energy (54). Studies have shown

a strong association between high IMCL content and

skeletal muscle insulin resistance in obese subjects (55),

in lean non-diabetic offspring of T2D subjects (56) and in

T2D subjects (53). Furthermore, a study has shown that,

a diet high in fat and intravenous lipid infusion increase

the amount of IMCL and simultaneously decrease insulin

sensitivity (20). The association between IMCL and insulin

resistance is further supported by studies showing decreased

IMCL concentrations and corresponding improved insulin

sensitivity in response to weight loss (20, 21). It has been

shown that under conditions of lipid oversupply the

content of signaling molecules (intermediates), including

long-chain acetyl CoA, DAG and ceramides is increased

in skeletal muscle, which can activate protein kinase

C (PKC), resulting in a serine phosphorylation of the

insulin receptor substrate-1 (IRS-1), impairing its ability

to associate with the insulin receptor and interfering with

PI3K activation and insulin signaling (57).

However, the causal relationship between skeletal

muscle lipotoxicity in insulin resistance and T2D has

been challenged, suggesting that accumulation of IMCL

per se is not the direct cause of the development of insulin

resistance, but merely a marker for the presence of lipid

intermediates within the cell interfering with insulin

signaling (58, 59). Indeed, there are data to support a

key role of both DAG and ceramide content in the

pathogenesis of lipid-induced muscle insulin resistance

in obese and T2D individuals (60, 61, 62). This notion

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was also confirmed by Bergman et al. who showed that

both the cellular localization and composition of DAG

influence the relationship with insulin sensitivity,

and their data suggested that only saturated DAGs in

skeletal muscle membranes were related to insulin

resistance in obese and T2D subjects (63). Even though

there is strong evidence to support a role of DAG and

ceramides in the development of insulin resistance,

recent studies also question the casual influence of

these lipid intermediates that may among other factors

depend on their intracellular distribution. Recently, it

was shown that especially the membrane and cytosolic

C18:2 DAGs were associated with both acute lipidinduced

and chronic insulin resistance in humans (64).

However, some studies found no association between

ceramides and insulin resistance (65) or between DAGs

and insulin resistance (66) indicating that the exact role

of both these lipid intermediates is still debated. The

phenomenon named the athlete’s paradox has revealed

that athletes who possess a high oxidative capacity and

enhanced insulin sensitivity also have higher skeletal

muscle IMCL content (67), and do likewise challenge

the causality between increased muscle IMCL content

per se and insulin resistance. Acute exercise, endurance

training as well as moderate aerobic exercise training

increase the rate of IMCL synthesis (68, 69), suggesting

that lipid accumulation in itself is not sufficient to

explain the lipid-induced muscle insulin resistance.

Perilipin 5 (PLIN5) affects the size of the lipid droplets

and has been suggested as a unifying factor in conditions

in which insulin sensitivity is maintained despite

the presence of high levels of IMCL. A very recent

study proposes that PLIN5 is driving the association

between promotion of the capacity to store lipids as

IMCL and amelioration of fasting-induced insulin

resistance (70). In addition, it has been demonstrated

that IMCL turnover is high during submaximal exercise

without causing changes in the total IMCL pool (71),

whereas in healthy males, a single session of aerobic

exercise decreases IMCL levels, indicating that ectopic

lipids are flexible lipid depots (72). The IMCL could

therefore be considered a reservoir for fatty acids that

can be used for readily available substrate delivery, for

example during exercise (54), and the harmful effects of

IMCL may be limited when accompanied by an increased

oxidative capacity (73).

Altogether, these studies suggests that the extent to

which increased availability of plasma FFA levels and

intracellular fat accumulation in itself impairs insulin

signaling transduction and thereby causes muscle insulin

176:2 R72

resistance remains controversial indicating that the

relationship is more complex than first anticipated.

Lipid accumulation in skeletal muscle

vs liver

Ectopic lipid accumulation in the liver, named

nonalcoholic fatty liver disease (NAFLD) affects up to 30%

of the general population and is thus the most common

liver disease (74). NAFLD is characterized by steatosis,

which is caused by hepatic triglyceride accumulation

possibly serving as a protective mechanism against

lipotoxicity (75). Insulin resistance and excessive fatty

acid influx into the liver are the major driving forces

for hepatic steatosis, and NAFLD is considered to be the

hepatic component of the metabolic syndrome (76).

There are studies suggesting that hepatic insulin

resistance may precede skeletal muscle insulin resistance,

and it is therefore suggested that early ectopic lipid

accumulation in the liver and concurrent hepatic

insulin resistance are major contributing factors in

the development of T2D. In elderly twins, total muscle

triglyceride content was not associated with peripheral

insulin sensitivity but was associated only with hepatic

insulin resistance, proposing that the muscle triglyceride

content may reflect the general ectopic accumulation of

triglycerides including fat in the liver, that unfortunately

was not measured in the study (77). This theory is further

supported by a study showing that liver fat content

measured by 1 H magnetic resonance spectroscopy was

negatively correlated with insulin sensitivity in overweight

men (78). Mice exhibiting a genetic inactivation of

adipose triglyceride lipase disproved the hypothesis that

triglyceride accumulation per se causes lipotoxicity and

insulin resistance as these mice have both increased

insulin sensitivity and glucose tolerance despite increased

triglyceride levels in muscle and liver (79). However, these

genetic manipulated mice models are not physiological

and therefore the results should be interpreted with

caution. Dufour et al. showed that the healthy young lean

individuals born with a low birth weight, and therefore

at increased risk of developing T2D compared with the

background population, exhibited both hepatic and

muscle insulin resistance independent of ectopic lipid

accumulation in these organs (80). In addition, when

young men are overfed for 5 days with a high-fat diet,

their liver becomes insulin resistant, whereas peripheral

tissues as mainly reflected by the muscles do not become

insulin resistant (81), thus providing new evidence to

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imply that increased hepatic lipid infiltration possibly

occurs early in the pathogenesis of insulin resistance.

Altogether, the role of ectopic fat in the development

of insulin resistance and T2D points toward a central role

of the liver as opposed to the muscle in particularly the

early pathogenesis of T2D.

Lipotoxicity and mitochondrial function in

skeletal muscle tissue

T2D is characterized by disturbances in fatty acid

metabolism, and mitochondrial dysfunction has been

associated with muscle insulin resistance in multiple

studies and has therefore been suggested to play an

important role in the pathogenesis of insulin resistance

and T2D (82, 83, 84). Mitochondrial function has been

associated with deficient oxidative capacity resulting in

increased amounts of IMCL species and hence lipotoxicity

interfering with insulin signaling causing insulin

resistance (82, 85). However, the cause of decreased

oxidative capacity is not fully elucidated.

The idea that mitochondrial deficiency occurs in

close association with impaired insulin action, and hence

could be a general mechanism contributing to a variety

of known metabolic defects in T2D, has been widely

accepted although data are not fully convincing and have

been challenged by recent studies. For example, only few

studies have linked reduced oxidative enzyme activity –

often caused by decreased mitochondrial content – to an

evidence of functional impairment of the mitochondria.

Furthermore, most of the studies on the function of

the mitochondria have been performed using indirect

measures of oxidative capacity i.e. ATP production in

the resting state as determined by nuclear magnetic

resonance (NMR), and several studies have shown a

dissociation between mitochondrial function and insulin

sensitivity (86, 87). The most obvious metabolic defect of

impaired mitochondrial function is reduced capacity for

aerobic ATP synthesis, either due to a lowered number of

otherwise normal mitochondria or decreased capacity of

the individual mitochondria. Although such changes are

unlikely to cause complete loss of ATP synthesis, it may be

expected that significant changes will become unmasked

under demanding conditions, such as in response to

energy-depleting exercise (88).

We recently assessed in vivo mitochondrial function

by 31 P-MRS after energy-depleting exercise in young

healthy men born with a low birth weight (LBW) and at

increased risk of developing T2D, and since diets high

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in fat and calories have been linked to mitochondrial

dysfunction (89, 90), we investigated the potential effect

of 5-day high-fat overfeeding. We found that in vivo

mitochondrial function was not affected by overfeeding

LBW individuals compared with that in controls (81),

thereby not supporting the proposed hypothesis that

high-fat diet induces alterations in mitochondrial

function – not even in subjects at risk of developing insulin

resistance and T2D. Indeed, peripheral insulin resistance

was induced by high-fat overfeeding in the LBW subjects

only, but still there were no signs of dissociation between

insulin resistance and mitochondrial dysfunction (81).

Supporting our findings, other recent studies have also

observed normal mitochondrial function during of highfat

induced insulin resistance (86, 87).

The role of PGC1α in lipid overload

Peroxisome proliferator-activated receptor γ

coactivator-1 alpha (PGC1α) plays a central role as a

metabolic sensor and regulator by regulating glucose

homeostasis and promoting fatty acid oxidation via

increasing mitochondrial function and activity (91).

PGC1α has therefore been suggested to play a key role

in the pathogenesis of T2D by preventing lipotoxicity.

Indeed, severe caloric restriction, which improves insulin

sensitivity, increases PGC1α expression in skeletal muscle

of obese subjects (92). Conversely, infusion of lipids

decreases expression of PGC1α and nuclear-encoded

mitochondrial genes (93), and experimental high-fat

feeding in healthy humans also results in decreased

PGC1α and mitochondrial gene expression in skeletal

muscle only after 3 days (89). Interestingly, high-fat

and/or hypercaloric diets have been shown to promote

alterations in mitochondrial oxidative phosphorylation

(OXPHOS) activity and to decrease PGC1α gene

expression (90, 94). Impaired OXPHOS gene expression

is anticipated to lower the capacity of the cell to handle

substrate in excess, which could also contribute to

exhaustion of mitochondria during longer periods of

high energy intake. Nevertheless, when studying the

expression of the OXPHOS and PGC1α genes in the young

healthy men, no significant effect of high-fat overfeeding

on mRNA levels was observed (95). These findings were

further confirmed by the indifferent effect of highfat

overfeeding on pathways associated with insulin

resistance and OXPHOS performed by whole-genome

microarray analyses (95). This finding is in contrast to

the study by Sparks and colleagues that found a diet high

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in fat downregulated genes necessary for OXPHOS and

mitochondrial biogenesis (89). In a separate experiment,

they fed mice a high-fat diet for 3 weeks and verified their

findings in humans as they found the same OXPHOS and

PGC1α mRNA to be downregulated by approximately

90% (89). However, the subjects included in this study

were selected according to a high preference for dietary

fat, as indicated by a food preference questionnaire. This

could possibly explain the lowered gene expression as

IMCL could already have been accumulated in muscle

of these subjects, and thereby alter their sensibility to

overfeeding. The discrepancies between the studies could

be further explained by differences in the composition of

the intervention diet and duration of the intervention as

well as of the size of the study population. Furthermore,

short-term elevation of lipid availability, by lipid infusion

reduces insulin-stimulated increase of ATP synthase flux

in skeletal muscle and decrease PGC1α expression in

healthy male subjects (94, 96). This is also in contrast to

our findings; however, lipid infusions may yield different

metabolic responses due to the non-physiological nature

of intravenous infusion.

Interestingly, LBW (a prediabetic phenotype) in

twins has previously been shown to be associated with

reduced expression of PGC1α in skeletal muscle (97).

When exposed to high-fat overfeeding, the mRNA

levels of the OXPHOS genes, NDUFB6, UQCRB, ATP5O

and PGC1α, were decreased by approximately 25% in

LBW subjects with a previously documented range of

metabolic abnormalities in insulin-sensitive tissues

compared with healthy control subjects, and a similar

trend was observed for COX7A1. The significant

difference in ATP5O could indicate a prediabetic trait of

the LBW subjects, as Mootha et al. found this particular

gene to be most significantly reduced in skeletal muscle

of T2D patients (98). UQCRB status has also been found

to be a strong predictor of T2D, which likewise could

indicate an early prediabetic trait in LBW subjects (98).

During insulin stimulation, PGC1α gene expression was

significantly decreased in LBW compared with NBW

subjects during overfeeding, and a similar trend was

observed in the remaining OXPHOS genes (99).

The indifferent response observed between NBW

and LBW subjects during control diet suggests that

the difference in gene expression between NBW and

LBW subjects is only evident during metabolic stress

accompanying overfeeding. Assuming that altered

transcriptional regulation will lead to physiologically

relevant changes in mitochondrial function, we should

be able to measure alterations in in vivo estimations of,

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for instance, respiratory changes and ATP production.

However, there were no significant differences in V max

measured either in the forearm flexor muscles or in the

tibialis anterior muscle of the leg and insulin-stimulated

glucose uptake between NBW and LBW subjects during

control and overfeeding diets.

Concluding remarks

Many studies have examined the interaction between

lipotoxicity and insulin resistance, but whether skeletal

muscle lipotoxicity is a casual mechanism or an innocent

bystander in insulin resistance and T2D is still not fully

elucidated. The results are somewhat conflicting, and the

human data linking skeletal muscle lipotoxicity to insulin

resistance are mostly correlative and limited by the lack

of consistency and therefore do not provide any proof of

causality. The inconsistent findings are further amplified

by the use of different methodology to determine IMCL as

well as the selection of study participants. In addition, the

dietary standardization of study participants seems to be

of importance as composition of the diet influences fatty

acid composition to a great extent, and thus is a limitation

of human studies investigating IMCL composition,

possibly explaining the large variations between studies.

The cellular and molecular mechanisms causing

muscle insulin resistance are not fully understood.

However, the majority of studies agree that it is not the

total amount of IMCL per se that causes insulin resistance,

but rather the accumulation of the lipid intermediates

as well as the cellular localization of the lipids. Finally,

many studies do agree that a dysfunctional or overloaded

adipose tissue and metabolic consequences hereof

have been seen in prediabetic phenotypes including

individuals subjected to suboptimal fetal environment,

suggesting that lipotoxicity could be potentially more

harmful to certain ‘at-risk’ groups of the population. This

harmful effect can possibly be prevented and/or delayed

if the subcutaneous adipose tissue can expand sufficiently.

Focus should therefore be on improved understanding of

the developmental origins and functions of adipose tissue

depots throughout the body.

Future areas of research

The white adipose tissue has a central role in regulating

energy homeostasis and insulin sensitivity, and

impaired expandability of the subcutaneous adipose

tissue may be a leading cause for storage of ectopic fat

www.eje-online.org





and T2D

PROOF ONLY

in ‘diabetogenic tissues’ including liver, muscle and

pancreatic beta-cells, and thus future research needs to

focus on the developmental origin of adipose tissue.

How is adipogenesis regulated and what determines

the capacity of adipose tissue expandability in different

phenotypes predisposed to insulin resistance and T2D?

Are the mechanisms similar in prediabetic phenotypes

such as individuals subjected to intrauterine growth

restriction, in offspring of women with gestational

diabetes i.e. intrauterine overnutrition and in obese

subjects with and without impaired glucose tolerance?

Answering these questions could possibly explain why

some prediabetic phenotypes develop insulin resistance

and T2D, whereas others do not.

To elucidate to what extent abnormal triglyceride

turnover influences skeletal muscle lipid accumulation

and insulin resistance, it would be interesting to examine

the interactions further as a possible pharmacological or

non-pharmacological intervention target for prevention

of development of T2D. Research in the last decade has

shown that IMCL is not just a depot for fat storage but

is a sophisticated functional tissue. Thus, future research

needs to take into account not only the total amount of

IMCLs but also the composition and localization of the

lipid droplets within the IMCLs. Lately, attention has

also been drawn toward the influence and functional

role of intermuscular adipose tissue (IMAT) in metabolic

disease; however, further studies are needed to elucidate

if IMAT plays a causal role in the development of insulin

resistance (100, 101).

Declaration of interest

The authors declare that there is no conflict of interest that could be

perceived as prejudicing the impartiality of this review.

Funding

This work was supported by The Danish Council for Independent Research

– Medical Sciences (FSS), the Danish Council for Strategic Research, the

Programme Commission on Food and Health (FØSU), the Danish Diabetes

Association, the European Foundation for the Study of Diabetes (EFSD),

Copenhagen University Hospital, the EU 6th Framework EXGENESIS Grant

and the Aase and Ejnar Danielsen Foundation.

Author contribution statement

C B and L G G researched data, wrote the manuscript, reviewed and edited

the manuscript.

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Received 10 June 2016

Revised version received 19 August 2016

Accepted 14 September 2016

www.eje-online.org



[1479683X - European Journal of Endocrinology] MECHANISMS IN ENDOCRINOLOGY_ Skeletal muscle lipotoxicity in insulin resistance and type 2 diabetes_ a causal mechanism or an innocent bystander_

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