Transcript 20130321metformin_glucagon_itsaction

Journal Club
Leclerc I, Sun G, Morris C, Fernandez-Millan E, Nyirenda M, Rutter GA.
AMP-activated protein kinase regulates glucagon secretion from mouse
pancreatic alpha cells.
Diabetologia. 2011 Jan;54(1):125-34.
Miller RA, Chu Q, Xie J, Foretz M, Viollet B, Birnbaum MJ.
Biguanides suppress hepatic glucagon signalling by decreasing production
of cyclic AMP.
Nature. 2013 Feb 14;494(7436):256-60.
2013年3月14日 8:30-8:55
８階 医局
埼玉医科大学 総合医療センター 内分泌・糖尿病内科
Department of Endocrinology and Diabetes,
Saitama Medical Center, Saitama Medical University
松田 昌文
Matsuda, Masafumi
インスリン抵抗性
Liver
インスリン感受性低下
Muscle
肝インスリン抵抗性と筋インスリン感受性低下が
２型糖尿病の特徴である。
肝臓のインスリン抵抗性増大 グルカゴン作用増大
筋肉のインスリン感受性低下
Matsuda M: Measuring and estimating insulin resistance in clinical and
research settings Nutr Metab Cardiovasc Dis. 20:79-86, 2010.
FIG. 2. C: Comparisons of weekly nonfasting glucose
levels in Gcgr+/+ (●) and Gcgr-/- (□) after STZinduced b-cell destruction, and overnight fasting
glucose levels for Gcgr +/+ (◆) and Gcgr-/- (◇) at the
end of the study (n = 6). D: Glucose values for oral
glucose tolerance test (OGTT) (2 g/kg) performed
after a 16-h fast in normal Gcgr +/+ (●), Gcgr-/- (☐),
and STZ-treated Gcgr-/- (▲) mice (n = 4). E: Insulin
levels for OGTT in normal Gcgr +/+ (●), Gcgr-/- (☐),
and STZ-treated Gcgr-/- (▲) mice (n = 4).
グルカゴン受容体のノックアウトでβ細胞破壊で血糖上昇せず
Diabetes 60:391–397, 2011
(n=9)
韓国人２型糖尿病患者
の膵α細胞，膵β細胞
(n=10)
(n=25)
Control 1
Normal pancreas donors (control group 1, n = 9). Whole
pancreases were obtained from organ donors (six men
and three women) between 19 and 64 yr of age (average,
41.3 ± 14.2 yr). The main causes of death were cerebral
hemorrhage, traffic accident, and myocardial infarction.
Control 2
Patients with a pancreatic neoplasm but without
diabetes (control group 2, n = 10).
DM
Patients with type 2 DM.
The 25 type 2 diabetic patients (15 men and 10 women)
were of mean age 60.0 ± 8.5 yr (range, 40–70 yr) and
had a mean diabetes duration of 4.9yr (range 0-20).
Their mean BMI was 22.2 ± 3.8 kg/m2 (17.8–29.1 kg/m2).
A1c was 7.3 ± 2.8%
Yoon, K. H. et al. J Clin Endocrinol Metab
2003;88:2300-2308
Copyright ©2003 The Endocrine Society
２型糖尿病患者の
食後グルカゴン抑制の喪失
Plasma glucagon, insulin, and
glucose levels in response to a
large carbohydrate meal in subjects
with NGT and in patients with
T2DM. Plasma glucagon (A), insulin
(B), and glucose (C) in 14 subjects
with NGT and 12 patients with
T2DM during ingestion of a
highcarbohydrate meal.
Mean ± SEM.
Muller WA, Faloona GR, Aguilar-Parada
E, Unger RH: Abnormal a-cell function in
diabetes. Response to carbohydrate and
protein ingestion. N Engl J Med 283:109–
115, 1970.
２型糖尿病における膵島の異常
グルカゴン
過剰分泌
促進が過剰になる
インスリン抵抗性
α細胞
膵島
肝糖産生
抑制が
弱まる
血糖上昇
β細胞（減少）
インスリン感受性低下
インスリン
分泌低下
促進が弱まる
糖取り込み
6
Ohneda A, et al: J Clin Endocrinol Metab 46, 504-510, 1978
Gomis R, et al: Diabetes Res Clin Pract 6, 191-198, 1989より作成
ビグアナイド薬とチアゾリジン薬
インスリン抵抗性改善効果
標的臓器
血糖降下作用
肥満に対する作用
動脈硬化症のリスクファクター改善効果
脂質、血圧、炎症反応
アディポネクチン上昇
動脈硬化症のイベント改善
EBM
動物実験
糖尿病発症抑制・膵β細胞保護
EBM
動物実験
ビグアナイド薬
チアゾリジン薬
肝臓＞骨格筋
（＋）
骨格筋、肝臓
（ ）
促進しない
促進することあり
（＋）
（－）
（
（
（＋）UKPDS
（＋）
（＋）DPP（-31%）
（＋）
（
）
）
）PROactive
（ ）
（＋）DPP（-78%）
（ ）
The Experiment & Therapy 2004,674,33
肝糖産生
筋糖利用
Mean (SE) Percent Changes within Subjects in Endogenous Glucose Production and the
Glucose Disposal Rate under Hyperinsulinemic-Clamp Conditions after Three Months of
Therapy with Metformin or Troglitazone. NS denotes not significant.
Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI.: efficacy and metabolic effects of metformin
and troglitazone in type II diabetes mellitus. N Engl J Med. 1998 Mar 26;338(13):867-72.
AMP-activated protein kinase
encoded by Prkaa genes
protein kinase, AMP-activated,
alpha 2 catalytic subunit
It should not be
confused with cyclic
AMP-activated
protein kinase
(protein kinase A),
which, although
being of similar
nature, may have
opposite effects.
http://flipper.diff.org/app/pathways/info/2064
I. Leclerc : G. Sun : E. Fernandez-Millan : M. Nyirenda : G. A. Rutter (*) Section of
Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Faculty of
Medicine, Imperial College, London,
C. Morris Department of Electrical and Electronic Engineering, Faculty of Engineering,
Imperial College, London, UK
*:UK e-mail: [email protected]
Aim/hypothesis
AMP-activated protein kinase (AMPK),
encoded by Prkaa genes, is emerging as a
key regulator of overall energy homeostasis
and the control of insulin secretion and
action. We sought here to investigate the
role of AMPK in controlling glucagon
secretion from pancreatic islet alpha cells.
Methods
AMPK activity was modulated in vitro in clonal
alphaTC1-9 cells and isolated mouse pancreatic
islets using pharmacological agents and
adenoviruses encoding constitutively active or
dominant negative forms of AMPK. Glucagon
secretion was measured during static incubation
by radioimmunoassay. AMPK activity was
assessed by both direct phosphotransfer assay
and by western (immuno-) blotting of the
phosphorylated AMPK α subunits and the
downstream target acetyl-CoA carboxylase 1.
Intracellular free [Ca2+] was measured using
Fura-Red.
Abbreviations
ACC Acetyl-CoA carboxylase
AICAR 5-Aminoimidazole-4-carboxamide
a cell permeant analogue of AMP
1-β-D-ribofuranoside
AMPK AMP-activated protein kinase
[Ca2+]i Free intracellular calcium concentration
GFP Green fluorescent protein
KBH Krebs’ Ringer bicarbonate HEPES buffer
MOI Multiplicity of infection
PPG Preproglucagon
PVDF Polyvinylidene fluoride
Glucose effect
AICAR効果なし
Glucose 1mM=18mg/dl, 17mM=306mg/dl
Surprisingly, however, AICAR was ineffective in activating AMPK in this cell type?!
A-769662, is a thienopyridone
drug that selectively activates
AMPK allosterically, by targeting
β1-containing complexes
□：ブドウ糖 0ｍM
■：ブドウ糖 17mM
An adenovirus encoding constitutionally active AMPK under the
preproglucagon promoter (PPG-AMPK-CA) , DN: dominant negative
infected with Null- GFP (white bars),
AMPK-CA (grey bars) or
AMPK-DN (black bars) viruses
white bars, 0.1 mmol/l glucose,
black bars, 17 mmol/l glucose
GFP Green fluorescent protein
Fig. 3 Effects of molecular modulation of
AMPK activity on glucagon secretion in
alphaTC1-9 cells. a AMPK activity in
alphaTC1-9 cells overexpressing AMPK-CA
(α1312 T172D) and AMPK-DN (α1 D157A).
AlphaTC1-9 cells were infected with NullGFP (white bars), AMPK-CA (grey bars) or
AMPK-DN (black bars) viruses at an MOI
of 100 units/cell 48 h before glucose
stimulation at 0.1 and 17 mmol/l for 2 h in
Dulbecco’s modified Eagle’s medium.
After being washed in PBS containing 0.1
and 17 mmol/l glucose three times, as
indicated, cells were lysed and 20 μg whole
cell lysates were used for AMPK
measurements, as described in Methods.
Data are means±SEM of three separate
experiments. *p<0.05, ***p<0.001. b, c
Glucagon secretion assay in alphaTC1-9
cells following adenoviral overexpression of
AMPK-CA (b) and AMPK-DN (c).
AlphaTC1-9 cells were cultured in 12-well
plates and infected with null (expressing
GFP only), AMPK-CA (b, white bars, 0.1
mmol/l glucose, black bars, 17 mmol/l
glucose) or AMPK-DN (c, white bars, 0
mmol/ l glucose, black bars, 17 mmol/l
glucose) adenoviruses at an MOI of 100
units/cell for 48 h before glucagon assays
were performed at low or high glucose
concentrations as described in Methods.
Data are means±SEM of at least three
separate experiments. *p<0.05, †p=0.063,
‡p=0.089
We subsequently modulated AMPK activity molecularly by using
adenoviruses encoding constitutively active or dominant negative
forms of the kinase. AlphaTC1-9 cells were transduced for 48 h at
an MOI of 100 units/cell with either Null-GFP, AMPK-CA or AMPKDN adenoviruses before incubation in 0.1 or 17 mmol/l glucose
and subsequent cell lysis, for measurement of AMPK activity (Fig.
3a), or assay of glucagon secretion (Fig. 3b,c).
Strikingly, forced activation of AMPK activity at 17 mmol/l glucose
was sufficient to stimulate glucagon secretion, whereas forced
inhibition of AMPK activity at 0 mmol/l glucose blunted glucagon
secretion, confirming an essential role of AMPK in controlling
glucagon secretion from alpha cells.
Null-GFP
AMPK-CA
AMPK-DN
in contrast to the effects of AMPK-CA, AMPK-DN overexpression
significantly lowered apparent basal free [Ca2+]i
Even with this relatively low transduction of the alpha cells, we observed a significant
increase in glucagon secretion, selectively at high (inhibitory) glucose concentrations,
in islets infected with PPG-AMPK-CA adenovirus compared with those infected with
null-GFP virus
no significant difference was apparent in the release of insulin
Results
Increasing glucose concentrations strongly inhibited
AMPK activity in clonal pancreatic alpha cells.
Forced increases in AMPK activity in alphaTC1-9
cells, achieved through the use of pharmacological
agents including metformin, phenformin and A769662, or via adenoviral transduction, resulted in
stimulation of glucagon secretion at both low and
high glucose concentrations, whereas AMPK
inactivation inhibited both [Ca2+]i increases and
glucagon secretion at low glucose. Transduction of
isolated mouse islets with an adenovirus encoding
AMPK-CA under the control of the preproglucagon
promoter increased glucagon secretion selectively at
elevated glucose concentrations.
Conclusions/interpretation
AMPK is strongly regulated by glucose
in pancreatic alpha cells, and increases
in AMPK activity are sufficient and
necessary for the stimulation of
glucagon release in vitro. Modulation of
AMPK activity in alpha cells may
therefore provide a novel approach to
controlling blood glucose
concentrations.
Message
メトホルミンで膵α細胞からグルカゴンが
分泌される可能性がある。（実際にピオグ
リタゾンほどインスリンが低下しない！）
グルカゴン作用については抑制しそうな論
文もある。(次のNatureの論文)
Laboratory efficacy and safety variables with pioglitazone versus metformin
0
40
2.0
3.2
0
～
～
26.5
25.0
27.5
***
19.0
15.0
-1
0
5.0
-1.5
P<0.015
90
-25
～
～
～
Baseline week16
-8
21.0
20.5
P<0.0001
20.0
18.5*
～
～
Baseline week16
-2
-2.05
35.0
20.0
15.0
-0.5
-1
10.0
-1.5
5.0
-2
γGT
(U/L)
35.5
28.0
32.0
0
-2
***
19.5
-4
-6
-8
-10
0.0
P<0.003
P<0.014
Baseline week16
40.0
Metformin
-1.95
0.0
25.0
0
-1.9
***
6.3
2.0
1
19.0
-1.85
8.1
4.0
30.0
0.5
Pioglitazone
8.3
1.5
19.5
17.0
0
8.0
AST
21.0
17.5
10.0
P<0.01
21.5
18.0
10.0
6.0
-30
Baseline week16
18.5
-6
Metformin
Pioglitazone
Metformin
-2
-4
10.0
0.5
Baseline week16
-20
(U/L)
0
20.0
1.5
0.0
100
P<0.01
ALT
28.0
-15
Insulin
12.0
0
110
0～
80
-15
Baseline week16
30.0
135
***-5
126
***
-10
20.0
2.3
***
2.0
-0.5
1.0
Pioglitazone
-10
(U/L)
Metformin
Pioglitazone
3.5
144
120
-5
P<0.04
140
(mU/L)
2/21抄読会
Metformin
-0.5
HOMA index
2.5
**
57.8
Metformin
45
153
150
130
0
50
-0.4
Baseline week16
3.0
5
65.1
55
-0.3
0.0
68.5
60
1.0
0.5
70.2
65
-0.1
1.4 *
-0.2
1.5
70
160
(mg/dL)
Metformin
0
1.8
75
(μg/mL)
Pioglitazone
2.0
1.8
Metformin
Pioglitazone
2.5
2.0
FPG
E-selectin
(mg/L)
Pioglitazone
CRP
metformin:850-2500mg (n=26)
Pioglitazone
pioglitazne:30-45mg (n=24)
Baseline week16
P<0.0001
* P < 0.01 vs. baseline; ** P < 0.05 vs. baseline; *** P < 0.001 vs. baseline
Genovese S, Ceriello A, et al.:Effect of Pioglitazone Versus Metformin on Cardiovascular Risk Markers in Type 2 Diabetes. Adv Ther. Adv Ther. 2013 Feb;30(2):190-202.
1Institute
for Diabetes, Obesity, and Metabolism, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 2Cell
Signaling Technology, Inc., 3Trask Lane, Danvers, Massachusetts 01923, USA.
3Inserm, U1016, Institut Cochin, Paris 75014, France. 4Cnrs, UMR8104, Paris
75014, France. 5Universite´ Paris Descartes, Sorbonne Paris cite´ , Paris 75006,
France.
Nature. 2013 Feb 14;494(7436):256-60.
Glucose production by the liver is essential for providing a
substrate for the brain during fasting. The inability of insulin
to suppress hepatic glucose output is a major aetiological
factor in the hyperglycaemia of type-2 diabetes mellitus and
other diseases of insulin resistance. For fifty years, one of
the few classes of therapeutics effective in reducing glucose
production has been the biguanides, which include
phenformin and metformin, the latter the most frequently
prescribed drug for type-2 diabetes. Nonetheless, the
mechanism of action of biguanides remains imperfectly
understood. The suggestion a decade ago that metformin
reduces glucose synthesis through activation of the enzyme
AMP-activated protein kinase (AMPK) has recently been
challenged by genetic loss-of-function experiments. Here we
provide a novel mechanism by which metformin antagonizes
the action of glucagon, thus reducing fasting glucose levels.
METHODS SUMMARY
Primary hepatocytes were isolated by collagenase perfusion as described
previously29. Adenine nucleotides were extracted from cells and liver with
perchloric acid and measured by ion-pair reversed-phase (RP)-HPLC. cAMP in
primary hepatocytes and frozen liver tissue was measured by ELISA (GE
Healthcare) using the manufacturer’s lysis buffer. PKA activity was assayed in
cell lysates as PKI-sensitive Kemptide phosphorylation. PKA FRET-activity
probes were used to examine intracellular PKA activity on a spinning-disc
confocal microscope16. Adenylyl cyclase assays were performed using
adenosine-59- triphosphate [a-32P] (American Radiolabelled Chemicals), and
cAMP was quantified as previously described30. Glucose output studies in
primary hepatocytes from fasted mice were carried out in Krebs buffer
containing gluconeogenic substrates (20mM lactate, 2mM pyruvate, 10mM
glutamine) and were quantified using hexokinase-based glucose assays
(Sigma). For in vivo experiments, metformin was gavaged at the indicated
dosage and glucagon was injected intraperitoneally at the indicated dosages.
Tissues were collected rapidly from anaesthetized mice and frozen in precooled metal clamps. All results are expressed as the mean6s.e.m. All twogroup comparisons were deemed statistically significant by unpaired two-tailed
Student’s t-test if P<0.05.
Figure 1 | Biguanides inhibit cAMP accumulation. a, Primary hepatocytes were incubated with the indicated
phenformin concentrations for 2 h, 5nM glucagon or no treatment (NT) for 15 min, lysed, and assayed for total
cellular cAMP and protein. N54 for each point. b, Primary hepatocytes incubated with the indicated concentration
of phenformin for 2 h were extracted with perchloric acid and cellular nucleotides quantified by high-performance
liquid chromatography (HPLC). N54 for each point. c, d, Primary hepatocytes were incubated with the indicated
concentration of phenformin (c) or metformin (d) for 24 h, treated with 5nM glucagon, lysed, and assayed for total
cellular cAMP. N54 for each point. Error bars represent standard error of the mean (s.e.m.).
Figure 1 | Biguanides inhibit cAMP accumulation.
e, Primary hepatocytes were incubated with the indicated concentrations of phenformin for 2 h, treated with 5nM
glucagon, lysed, and PKA kinase activity determined. N56 for 0 and 1,000 mM phenformin groups, N=4 for 100
and 300 mM phenformin groups.
f, Primary hepatocytes were incubated with phenformin for 2 h, then with glucagon, and protein was analysed by
western blot with the phospho- (p)PKA substrate motif antibody, total (t) and phospho- (p)PFKFB1 antibodies, and
total and phospho-IP3R antibodies. Error bars represent standard error of the mean (s.e.m.).
Phenformin antagonized phosphorylation of the PKA substrates PFKFB1 and the inositol1,4,5-trisphosphate receptor IP3R (also known as ITPR1), as revealed by western blots using
phospho-specific antibodies against these proteins
Figure 2
Biguanides inhibit glucagon signalling.
a, b, Primary hepatocytes were cultured
for 18 h in the presence or absence of 65
mM phenformin and for 15 min with the
indicated concentrations of glucagon (a)
or the cell-permeable PKA agonist SP8Br-cAMPS-AM (b). Western blot
analysis of total (t) and phosphorylated
(p) PFKFB1,CREB, IP3R and AMPK.
c, Cells were treated with the indicated
concentration of metformin and either 1
nM glucagon or 3 mM SP-8Br-cAMPSAM, or were left untreated (NT), for 14 h
and then glucose output measured for 5
h. Data represent the means of three
experiments, N=6 for each experiment.
Error bars represent s.e.m.
As the biguanides and other drugs we
used activated AMPK in parallel to the
reduction in cAMP, we asked whether
the kinase was required for the effects
of biguanides. Mice homozygous for the
floxed alleles of both catalytic a1 and a2
subunits of the AMPKcomplex were
infected with adeno-associated virus
expressing Cre recombinase, and
western blots confirmed deletion of
AMPK a protein and loss of phenformindependent phosphorylation of the
AMPK substrate acetyl-CoA
carboxylase (ACC) (Fig. 3a). In
hepatocytes lacking any detectable
AMPK activity, phenformin blocked
glucagon-dependent cAMP
accumulation in a manner
indistinguishable from that in control
cells (Fig. 3b).
Figure 3 | Mechanism of biguanide effect on cAMP production. a, Ampka1/ a2lox/lox mice were infected with AAVTBG-GFP or AAV-TBG-Cre virus and 14 days later primary hepatocytes were isolated. Cells were treated with the
indicated concentrations of phenformin for 2 h followed by 5nM glucagon or no treatment (NT) for 15 min. a, Total
cellular protein was analysed by western blot for total (t) and phosphorylated (p) T172 AMPK and total and
phospho- S79 ACC. b, Hepatocytes were lysed and total cellular cAMP levels were quantified by ELISA. N=4 for
all points. c, Primary hepatocytes were incubated with the indicated concentrations of phenformin for 2 h and 50
mM RO-20-1724 (PDE4 inhibitor; PDE4i) for the final 30 min. Cells were then treated with 5nM glucagon for 15
min, lysed, and total cellular cAMP was assayed. N=4 for all points. d, The membrane fraction of primary
hepatocytes was isolated by differential centrifugation and used in assays for adenylyl cyclase activity in the
presence of the indicated AMP and ATP concentrations, 100nM glucagon and 100 mM GTP. N=6 for all points.
Error bars represent s.e.m.
Figure 4 | Biguanides antagonize glucagon
signalling in vivo.
a, Mice were gavaged with 500mgkg-1
metformin and 1 h later were injected
intraperitoneally with 200 mg kg-1 glucagon,
and glucose levels were measured at the
indicated times.N=6 for water/PBS and
metformin/glucagon, N=7 for water/glucagon
and metformin/PBS. b, c, Fed mice were
fasted for 1 h and gavaged with water or
500mgkg-1 body weight of metformin. Onehour later mice were injected
intraperitoneally with 2 mgkg-1 glucagon, and
liver tissue was collected 5 min later. Liver
was analysed for total hepatic cAMP by
ELISA (b; N=3 for each group) and total
hepatic PKA activity (c; N=7, 8, 6 and 7 for
water/PBS, water/glucagon, metformin/PBS
and metformin/glucagon, respectively).
*P<0.05 compared to PBS. d–f, 18-h fasted
mice were gavaged with water or 250mgkg-1
metformin, 1 h later liver tissue was
collected, hepatic metabolites were extracted
with perchloric acid and total hepatic AMP
(d) and cAMP (e) levels were assayed. N=12
and 9 for the water and metformin groups,
respectively. g–i, Mice fed HFD for 10 weeks
were fasted overnight, gavaged with either
water or 250mgkg-1 metformin, and after 1 h
liver tissue was collected for western blot
analysis of the phosphorylation status of
AMPK (g), PFKFB1 (h) and IP3R (i). N=3 for
each group. Error bars represent s.e.m.
Summary
Inmouse hepatocytes,metformin leads
to the accumulation of AMP and related
nucleotides, which inhibit adenylate
cyclase, reduce levels of cyclic AMP
and protein kinase A (PKA) activity,
abrogate phosphorylation of critical
protein targets of PKA, and block
glucagon-dependent glucose output
from hepatocytes.
Conclusion
These data support a mechanism of
action for metformin involving
antagonism of glucagon, and suggest
an approach for the development of
antidiabetic drugs.
Message
ペンシルバニア大学の教授らは、食事をしない
と血中グルコースが減少するが、膵臓からグル
カゴンは分泌され肝臓でのグルコース産生が増
加する所に注目しマウスで研究を進めたとこ
ろ、メトホルミンはグルカゴンのカスケード内
でAMPの蓄積を促し、これによりアデニル酸シ
クラーゼの作用が抑制されることが判明。これ
はさらにcAMP濃度とプロテインキナーゼの機能
を減少させます｡結果としてメトホルミンは肝
臓でのグルカゴン依存的なグルコース産生を抑
制し、血糖降下作用を示す。
．．． グルカゴン自体が上昇したら？
肝細胞からの糖産生
Biguanides inhibit glucagon signalling.
Primary hepatocytes were treated with the indicated concentration of metformin and either 1 nM
glucagon or 3 mM SP-8Br-cAMPS-AM, or were left untreated (NT), for 14 h and then glucose
output measured for 5 h. Data represent the means of three experiments, N=6 for each experiment.
Error bars represent s.e.m.
Miller RA, Chu Q, Xie J, Foretz M, Viollet B, Birnbaum MJ.: Biguanides suppress hepatic glucagon signalling by
decreasing production of cyclic AMP. Nature. 2013 Feb 14;494(7436):256-60. doi:10.1038/nature11808.
Biguanides antagonize glucagon
signalling in vivo.
Mice were gavaged with 500mg/kg
metformin and 1 h later were injected
intraperitoneally with 200 mg/kg
glucagon, and glucose levels were
measured at the indicated times.N=6
for water/PBS and
metformin/glucagon, N=7 for
water/glucagon and metformin/PBS.
Miller RA, Chu Q, Xie J, Foretz M, Viollet B, Birnbaum MJ.: Biguanides suppress hepatic glucagon signalling by
decreasing production of cyclic AMP. Nature. 2013 Feb 14;494(7436):256-60. doi:10.1038/nature11808.