Glucagon Diabetes mellitus Islet microcirculation

Glucagon
Isobel Franklin 1 , Jesper Gromada 2 , Asllan Gjinovci 1 ,
Sten Theander 1 , and Claes B. Wollheim 1
1
Department of Cell Physiology and Metabolism,
University Medical Centre, Geneva, Switzerland
2
Lilly Research Laboratories, Hamburg, Germany
Diabetes 54:1808-1815,June 2005
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2
Blood glucose homeostasis
Diabetes mellitus
• Type 1 (lacking ß-cells)
• Type 2 (impaired insulin secretion)
Normal inhibition of glucagon release
Hyperglucagonemia
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Worsens diabetes
Jiang G& Zhang, 2003
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Hyperglycemia
Islet microcirculation
α-cell glucagon release
Supported by..
1) Functional ß-cells are required in rats and dogs for
high glucose inhibition of glucagon secretion
2) Type 1 diabetic patients exhibit hyperglucagonemia
3) Overactive ß-cells can prevent the glucagon response
to hypoglycemia in humans or other secretagogues in
rat
Ishihara et al., 2003; Greenbaum et al., 2002; Unger, 1985
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"Core to mantle"
Samols et al.,1988
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Isolation of islet cells
Quantitative RT-PCR
Fluorescence-activated
cell sorter (FACS)
purifications
1 st sort, by FAD (flavine
adenine dinucleotide)
content
ß- & α-cells
2 nd sort, by NAD(P)H
content of the α-cell
fraction
pure α- & non α-cells
• Total RNA was extracted from α- and ß-
cell fractions using RNeasy Mini kit ®
• Converted into cDNA using Superscript
reverse transcriptase
• Primers were designed to amplify the
insulin receptor, Kir6.2, and
sulfonylurea receptor (SUR)1; glucagon,
insulin and cyclophilin transcripts
Pipeleers et al.,1985
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• At least 4 independent experiments
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• Zinc secretion assay
– atomic absorption spectrophotometer
• Hormone secretion assays
– static incubation (triplicate)
• FACS-isolated cells : 15-30 min
• Dispersed islet : 60 min
– radioimmunoassay
• Glucagon : anti-glucagon
• Insulin : anti-insulin
• C-peptide : commercial kit
Electrophysiology
Isolated α-cell
Perforated-patch whole-cell configuration
Patch perforation Amphotericin B
Pipette solution
(in mM): K 2
SO 4
76,
NaCl 10, KCl 10,
MgCl 2
1, HEPES 5
(pH 7.35 with KOH)
Extracellular solution
(in mM): NaCl 138, KCl
5.6, MgCl 2
1, CaCl 2
2.6,
HEPES 5 (pH 7.40 with
NaOH), glucose 0
+ additional agents
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Electrophysiology
Isolated α-cell
whole-cell patch-clamp recordings of K ATP channel current activity
Pipette resistance 2-6 MOhm
Statistical analyses
• Two-tailed, Student’s t test
Pipette solution
(in mM):KCl 125,
KOH 30, EGTA 10,
HEPES 5, MgCl 2
1,
Mg-ATP 0.3,
K-ADP 0.3
(pH 7.15)
Extracellular solution
(in mM):NaCl 138, KCl
5.6, MgCl 2
1, CaCl 2
2.6, HEPES 5 (pH 7.40
with NaOH), glucose
0 + additional agents
-60
-70
-80
200 ms
2 s
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Islet cell
High-glucose conditions
Zn
Isolated α-cell
Zn
Zn & insulin
secretion
Glucagon &
Insulin secretion
α-Cell electrical
activity
• α-Cells are markedly zinc sensitive
• ? Zinc may inhibit glucagon secretion by preventing
calcium influx
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Glucagon secretion from FACS-isolated adherent α-cells
dispersed
islet
Membrane potential recordings from isolated α-cells
using the perforated-patch whole-cell configuration
Isobutylmethylxanthine (IBMX)
Monomethylsuccinate Foskolin(forsk) Tolbutamide (mmsucc) Pyruvate (tolbut)(pyruv)
Mean ± SE, n = 3, *P < 0.01, **P < 0.005
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Membrane potential recordings from isolated α-cells
using the perforated-patch whole-cell configuration
Secreted glucagon relative to basal (100%)
n = 7
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n = 7 *P < 0.01
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ß-cells
Isolated α-cell
glucagon
glucagon
glucagon
glucagon
glucagon
Tolbutamide
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Glucagon secretion from isolated α-cells
Whole-cell patch-clamp recordings of
K ATP channel current activity in isolated α-cells
Mean ± SE, n = 3, *P < 0.05
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Relative increases in K + current amplitude
in response to Zn
Relative transcript abundance of K ATP channel subunits
Kir6.2 and SUR1 in FACS-isolated α- and ß-cells
Mean ± SE, n = 7 for each point
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Data are presented relative to ß-cells , n = 3, *P < 0.05
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α-cells
Zinc
opened K ATP channels
inhibited glucose and pyruvate-stimulated
glucagon secretion
did not inhibit arginine-induced glucagon release
… when K ATP channel activity is bypassed, Zn is unable to
block hormone secretion
Pancreatic ß-cell K + -ATP sensitive channel: SUR1/Kir6.2
SUR1
Kir6.2
Transcripts encoding K ATP subunits were more
abundant in α- than ß-cells (FACS-isolated)
Zinc action probably results from direct activation of
α-cell K ATP channels rather than inhibition of
Ca 2+ channels
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Site of Zn action may be located on the SUR1 subunit
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Relative abundance of insulin receptor and GLP-1
receptor transcripts in FACS-isolated α- and ß-cells
and compared with liver
Data are presented relative to ß-cells , n =3, *P < 0.05, **P < 0.01
39 40
Analysis of islet hormone secretion
Analysis of islet hormone secretion
absence
Insulin antiserum
presence
absence
presence
“Zn-free”
exogenous insulin
absence
presence
exogenous insulin
mean ± SE **P < 0.01
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means+SE **P < 0.01
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Isolated α-cell
• Insulin receptor transcript was relatively abundant in α-
cells, similar to liver
α-Cells are also important sites of insulin action
• Transcript for GLP-1 receptor was not detected in α-cells
nor did GLP-1 stimulate α-cell glucagon release
in agreement with earlier studies:
– cAMP levels in α-cells were unaffected by GLP-1
– type 1 DM : GLP-1 had no effect on plasma glucagon levels
during hyperinsulinemic-euglycemic clamp
• Apparent direct inhibition by GLP-1 on glucagon secretion
: paracrine factors may have mediated the suppression
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Glucagon secretion from in situ–perfused rat pancreas
Membrane potential recording from an isolated α-cell
using the perforated-patch whole-cell configuration
Data are mean+SE of six independent perfusions
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The recording is typical of six cells.
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Electrical activity in response to “Zn-free" insulin was
analyzed and the effect on spike frequency calculated
Glucagon secretion from isolated α-cells
Data are mean+ SE of six experiments
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mean+SE *P < 0.05, **P < 0.005
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Loss of β-cell function
α-Cell hyperactivity
Glucose responsiveness in
neighboring α-cells
Hyperglucagonemia
Worsens diabetes
Greenbaum et al.,2002
Other secretory products capable of
influencing glucagon release in this assay
would have included
- Somatostatin
- GABA (γ-aminobutyric acid)
released from the synaptic-like
microvesicles of ß-cells
Design of drugs aiming at reducing
postprandial α-cell activity
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Brunicardi et al., 2001; Cejvan et al., 2003;
Wendt et al.,2004; Franklin et al.,2004
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Isolated α-cell
Glucose
Subjects with
type 1 diabetes
Stimulus-secretion coupling in
the α-cell mirrors that of the ß-cell
glucose
Monomethylsuccinate
Tolbutamide
Glucagon secretion
Rat pancreas perfused
in retrograde direction
glucose
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• α-Cells express both glucokinase and GLUT1, a
lower capacity isoform than GLUT2, expressed
in ß-cells
• Glucose oxidation in α-cells is only 30% of that
in ß-cells (although steady-state glucose
utilization is the same).
glucose is a relatively poor substrate for
mitochondrial ATP generation in α-cells.
Heimberg et al.,1996, Schuit et al.,1997
• An earlier study reported glucose inhibition of
arginine-stimulated glucagon release from
isolated α-cells
Technical disparities
(culture and assay conditions) Pipeleers et al.1986
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Isolated rat α-cells
High glucose
As in ß-cells
Initial ATP consumption by
glucokinase
Spontaneous
electrical activity
Tolbutamide
SUR1
Opening of K ATP channel
transient hyperpolarization
In α-cells, this could take longer (minutes rather
than seconds) due to the relatively slow rate of glucose
oxidation
Bokvist et al.,1999; Arkhammar et al.,1987
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• Tolbutamide triggers α-cell electrical activity and
hormone secretion
• Tolbutamide also increased circulating glucagon
levels in patients with advanced type 1 diabetes
Bohannon et al.,1982
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Conclusion
This study demonstrates that only by
removing α-cells from the repressive
environment of the islet micro-organ can
we begin to identify direct effectors of
glucagon secretion and characterize the
stimulus-secretion coupling pathways that
lead to glucagon release
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