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1.  The Glycogenolytic Activity of Immunoreactive Pancreatic Glucagon in Plasma 
Journal of Clinical Investigation  1971;50(8):1650-1655.
Conclusions concerning the physiologic role of pancreatic glucagon in health and its contribution to disorders of carbohydrate metabolism, such as diabetes mellitus, are based entirely on measurements of plasma glucagon by radioimmunoassay. The changes in plasma immunoreactive glucagon can have the metabolic and clinical significance which has been implied, only if the glucagon detected by immunoassay has biological activity. The present study was designed to determine if a relationship between the immunoassayable glucagon and glycogenolytic activity of plasma could be demonstrated.
Plasma specimens obtained from normal and diabetic subjects under widely varying circumstances of alpha cell activity were extracted by a modification of the Kenny technique and the recovery of immunoreactive glucagon was calculated. Glycogenolytic activity of each extract was determined by perfusion in the Mortimore rat liver system, modified so as to detect as little as 1 ng of crystalline glucagon.
A significant correlation between the calculated quantity of immunoreactive glucagon and the glycogenolytic activity of plasma extracts was observed for both normal and diabetic subjects. Most of the glycogenolytic activity was abolished by incubating the extract with antiglucagon serum.
It was concluded that the glycogenolytic activity of extractable glucagon is proportional to its immunoreactivity as calculated from its original concentration in plasma. This would tend to support the view that all or most of the immunoreactive glucagon of plasma is biologically active.
PMCID: PMC442065  PMID: 5097572
2.  Effect of Prostaglandins on Hepatic Cyclic Nucleotide Concentration, Carbohydrate and Lipid Metabolism 
The effects of exogenous prostaglandin E1 (PGE1) or prostaglandin E2 (PGE2) were studied in the isolated perfused rat liver and in the intact canine liver in order to determine the possible physiological role of prostaglandins on hepatic carbohydrate and lipid metabolism. The data indicate that PGE1 and PGE2 did not stimulate cyclic AMP (cAMP) and cyclic GMP (cGMP) concentrations in intact dog liver and PGE1 failed to stimulate cAMP or cGMP in fed or fasted perfused rat liver. PGE1 did not promote hyperglycemia, glycogenolysis, lipolysis, or prevent epinephrine-induced hyperglycemia in the isolated perfused rat liver. Other known glycogenolytic agents including glucagon and epinephrine increased cAMP and glycogenolysis in the same perfusion system. This study does not support a physiologic role for PGE1 on hepatic glycogenolysis or lipolysis. If PGE1 subsequently is found to influence other metabolic parameters such as lipogenesis, gluconeogenesis, ureogenesis or amino acid transport in isolated perfused liver, such alterations would probably occur independent of changes in cyclic nucleotide activity.
PMCID: PMC2595716  PMID: 222076
3.  Role of KATP Channels in Glucose-Regulated Glucagon Secretion and Impaired Counterregulation in Type 2 Diabetes 
Cell Metabolism  2013;18(6):871-882.
Summary
Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na+ channels involved in action potential firing that, via reduced action potential height and Ca2+ entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.
Graphical Abstract
Highlights
•KATP channel closure stimulates insulin secretion but inhibits glucagon release•α cell depolarization reduces voltage-gated Ca2+ entry and glucagon release•An activating KATP channel mutation impairs glucagon release in mice•KATP channel closure corrects glucagon secretion defect in type 2 diabetic islets
doi:10.1016/j.cmet.2013.10.014
PMCID: PMC3851686  PMID: 24315372
4.  Effects of Acute Hyperglucagonemia on Hepatic and Intestinal Lipoprotein Production and Clearance in Healthy Humans 
Diabetes  2011;60(2):383-390.
OBJECTIVE
The metabolism of hepatic- and intestinally derived lipoproteins is regulated in a complex fashion by nutrients, hormones, and neurologic and other factors. Recent studies in animal models suggest an important role for glucagon acting via the glucagon receptor in regulating hepatic triglyceride (TG) secretion. Here we examined the direct effects of glucagon on regulation of hepatic and intestinal lipoprotein metabolism in humans.
RESEARCH DESIGN AND METHODS
Eight healthy men underwent two studies each, in random order, 4–6 weeks apart in which de novo lipogenesis, kinetics of larger VLDL1 TG, and kinetics of VLDL1 and smaller VLDL2 apolipoprotein (apo)B100 and B48 were studied using established stable isotope enrichment methods. Subjects were studied in the constant fed state under conditions of a pancreatic clamp (with infusion of somatostatin, insulin, and growth hormone) at either basal glucagon (BG study, 64.5 ± 2.1 pg/mL) or hyperglucagonemia (high glucagon [HG] study, 183.2 ± 5.1 pg/mL).
RESULTS
There were no significant differences in plasma concentration of VLDL1 or VLDL2 TG, apoB100 or apoB48 between BG and HG studies. There was, however, lower (P < 0.05) VLDL1 apoB100 fractional catabolic rate (−39%) and production rate (−30%) in HG versus BG, but no difference in de novo lipogenesis or TG turnover, and glucagon had no effect on intestinal (B48-containing) lipoprotein metabolism.
CONCLUSIONS
Glucagon acutely regulates hepatic but not intestinal lipoprotein particle metabolism in humans both by decreasing hepatic lipoprotein particle production as well as by inhibiting particle clearance, with no net effect on particle concentration.
doi:10.2337/db10-0763
PMCID: PMC3028336  PMID: 20980459
5.  Brain glucagon-like peptide–1 increases insulin secretion and muscle insulin resistance to favor hepatic glycogen storage 
Journal of Clinical Investigation  2005;115(12):3554-3563.
Intestinal glucagon-like peptide–1 (GLP-1) is a hormone released into the hepatoportal circulation that stimulates pancreatic insulin secretion. GLP-1 also acts as a neuropeptide to control food intake and cardiovascular functions, but its neural role in glucose homeostasis is unknown. We show that brain GLP-1 controlled whole-body glucose fate during hyperglycemic conditions. In mice undergoing a hyperglycemic hyperinsulinemic clamp, icv administration of the specific GLP-1 receptor antagonist exendin 9–39 (Ex9) increased muscle glucose utilization and glycogen content. This effect did not require muscle insulin action, as it also occurred in muscle insulin receptor KO mice. Conversely, icv infusion of the GLP-1 receptor agonist exendin 4 (Ex4) reduced insulin-stimulated muscle glucose utilization. In hyperglycemia achieved by i.v. infusion of glucose, icv Ex4, but not Ex9, caused a 4-fold increase in insulin secretion and enhanced liver glycogen storage. However, when glucose was infused intragastrically, icv Ex9 infusion lowered insulin secretion and hepatic glycogen levels, whereas no effects of icv Ex4 were observed. In diabetic mice fed a high-fat diet, a 1-month chronic i.p. Ex9 treatment improved glucose tolerance and fasting glycemia. Our data show that during hyperglycemia, brain GLP-1 inhibited muscle glucose utilization and increased insulin secretion to favor hepatic glycogen stores, preparing efficiently for the next fasting state.
doi:10.1172/JCI25764
PMCID: PMC1297248  PMID: 16322793
6.  The effect of experimental insulin deficiency on glucagon secretion 
Journal of Clinical Investigation  1971;50(9):1992-1999.
Suppression of pancreatic glucagon secretion by hyperglycemia is a characteristic of normal alpha cell function. However, in diabetic subjects, plasma glucagon is normal or high despite hyperglycemia. It seemed possible that the presence of glucose or its metabolites within the alpha cell might be essential for suppression of glucagon secretion, and that in diabetes an intracellular deficiency of glucose secondary to insulin lack might be responsible for the nonsuppressibility. The present study was designed to determine the effect upon glucagon secretion of blockade of glucose metabolism and of experimental insulin deficiency.
Blockade of glucose metabolism was induced in dogs by administration of 2-deoxyglucose or mannoheptulose. A striking rise in glucagon was observed despite accompanying hyperglycemia and hyperinsulinemia, which, in the case of mannoheptulose, was induced by infusing crystalline insulin.
To determine if insulin lack also causes paradoxical hyperglucagonemia, dogs were made severely diabetic by alloxan. Fasting glucagon levels ranged from 3 to 22 times normal despite severe hyperglycemia, and were quickly restored to normal by infusing insulin. Diabetes induced in rats by anti-insulin serum was also associated with significant elevation in plasma glucagon. However, diazoxide-induced insulin lack did not increase glucagon in dogs.
It is concluded that normal suppression of glucagon secretion by hyperglycemia does not occur when glucose metabolism is blocked or when severe insulin deficiency is produced. It is suggested that normal glucose metabolism within the alpha cell may be an insulin-requiring process without which hyperglycemic suppression of glucagon release cannot occur.
PMCID: PMC292125  PMID: 4935445
7.  A MECHANISM OF THE GLYCOGENOLYTIC ACTION OF BACTERIAL ENDOTOXIN 
These experiments have demonstrated that liver glycogen may rise or fall after endotoxin administration, depending upon the antecedent diet and that total adrenalectomy followed by corticosteroid replacement abolishes the glycogenolytic effect of sublethal doses of endotoxin. It is concluded that the derangements of carbohydrate metabolism observed following the administration of sublethal quantities of bacterial endotoxin represent, not a direct hepatotoxic effect of endotoxin, but rather the passive consequence of epinephrine release.
PMCID: PMC2137213  PMID: 13746229
8.  Glucagon Receptor–Mediated Extracellular Signal–Regulated Kinase 1/2 Phosphorylation in Rat Mesangial Cells 
Hypertension  2006;47(3):580-585.
Glucagon, a major insulin counterregulatory hormone, binds to specific Gs protein–coupled receptors to activate glycogenolytic and gluconeogenic pathways, causing blood glucose levels to increase. Inappropriate increases in serum glucagon play a critical role in the development of insulin resistance and target organ damage in type 2 diabetes. We tested the hypotheses that: (1) glucagon induces proliferation of rat glomerular mesangial cells through glucagon receptor–activated phosphorylation of mitogen-activated protein kinase extracellular signal–regulated kinase 1/2 (p-ERK 1/2); and (2) this phosphorylation involves activation of cAMP-dependent protein kinase A (PKA) and phospholipase C (PLC)/[Ca2+]i signaling pathways. In rat mesangial cells, glucagon (1 nM) stimulated [3H]-thymidine incorporation by 96% (P < 0.01). This proliferative effect was blocked by the specific glucagon receptor antagonist [Des-His1-Glu9] glucagon (1 μmol/L; P < 0.01), a mitogen-activated protein kinase/ERK kinase inhibitor PD98059 (10 μmol/L; P < 0.01), a PLC inhibitor U73122 (1 μmol/L; P < 0.01), or a PKA inhibitor H-89 (1 μmol/L; P < 0.01). The proliferation was associated with a 2-fold increase in p-ERK 1/2 that peaked 5 minutes after glucagon stimulation (P < 0.01) and also was blocked by [Des-His1-Glu9] glucagon. Total ERK 1/2 was not affected by glucagon. Pretreating of mesangial cells with U73122 or H89 significantly attenuated ERK 1/2 phosphorylation induced by glucagon. We believe that these are the first data showing that glucagon activates specific receptors to induce ERK 1/2 phosphorylation and thereby increase mesangial cell proliferation and that this effect of glucagon involves both PLC/[Ca2+]i- and cAMP-dependent PKA-activated signaling cascades.
doi:10.1161/01.HYP.0000197946.81754.0a
PMCID: PMC2367309  PMID: 16391176
kidney; cyclic AMP; calcium; diabetes mellitus; glomerulosclerosis; insulin resistance
9.  Glucagon-like peptide-1 decreases intracerebral glucose content by activating hexokinase and changing glucose clearance during hyperglycemia 
Type 2 diabetes and hyperglycemia with the resulting increase of glucose concentrations in the brain impair the outcome of ischemic stroke, and may increase the risk of developing Alzheimer's disease (AD). Reports indicate that glucagon-like peptide-1 (GLP-1) may be neuroprotective in models of AD and stroke: Although the mechanism is unclear, glucose homeostasis appears to be important. We conducted a randomized, double-blinded, placebo-controlled crossover study in nine healthy males. Positron emission tomography was used to determine the effect of GLP-1 on cerebral glucose transport and metabolism during a hyperglycemic clamp with 18fluoro-deoxy-glucose as tracer. Glucagon-like peptide-1 lowered brain glucose (P=0.023) in all regions. The cerebral metabolic rate for glucose was increased everywhere (P=0.039) but not to the same extent in all regions (P=0.022). The unidirectional glucose transfer across the blood–brain barrier remained unchanged (P=0.099) in all regions, while the unidirectional clearance and the phosphorylation rate increased (P=0.013 and 0.017), leading to increased net clearance of the glucose tracer (P=0.006). We show that GLP-1 plays a role in a regulatory mechanism involved in the actions of GLUT1 and glucose metabolism: GLP-1 ensures less fluctuation of brain glucose levels in response to alterations in plasma glucose, which may prove to be neuroprotective during hyperglycemia.
doi:10.1038/jcbfm.2012.118
PMCID: PMC3519409  PMID: 22929437
blood–brain barrier; 2-deoxy-glucose; diabetes; energy metabolism; GLP-1; glucagon-like peptide-1; glucose; pharmacology
10.  The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression 
Journal of Clinical Investigation  2001;108(9):1359-1367.
Type 2 diabetes is characterized by the inability of insulin to suppress glucose production in the liver and kidney. Insulin inhibits glucose production by indirect and direct mechanisms. The latter result in transcriptional suppression of key gluconeogenetic and glycogenolytic enzymes, phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6p). The transcription factors required for this effect are incompletely characterized. We report that in glucogenetic kidney epithelial cells, Pepck and G6p expression are induced by dexamethasone (dex) and cAMP, but fail to be inhibited by insulin. The inability to respond to insulin is associated with reduced expression of the forkhead transcription factor Foxo1, a substrate of the Akt kinase that is inhibited by insulin through phosphorylation. Transduction of kidney cells with recombinant adenovirus encoding Foxo1 results in insulin inhibition of dex/cAMP–induced G6p expression. Moreover, expression of dominant negative Foxo1 mutant results in partial inhibition of dex/cAMP–induced G6p and Pepck expression in primary cultures of mouse hepatocyes and kidney LLC-PK1-FBPase+ cells. These findings are consistent with the possibility that Foxo1 is involved in insulin regulation of glucose production by mediating the ability of insulin to decrease the glucocorticoid/cAMP response of G6p.
PMCID: PMC209440  PMID: 11696581
11.  The role of the Wnt signaling pathway in incretin hormone production and function 
Glucose metabolism is tightly controlled by multiple hormones and neurotransmitters in response to nutritional, environmental, and emotional changes. In addition to insulin and glucagon produced by pancreatic islets, two incretin hormones, namely glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP, also known as glucose-dependent insulinotropic peptide), also play important roles in blood glucose homeostasis. The incretin hormones mainly exert their regulatory effects via their corresponding receptors, which are expressed in pancreatic islets as well as many other extra-pancreatic organs. Recent studies have shown that the genes which encode these two incretin hormones can be regulated by the effectors of the Wnt signaling pathway, including TCF7L2, a transcription factor identified recently by extensive genome wide association studies as an important type 2 diabetes risk gene. Interestingly, TCF7L2 and β-catenin (β-cat), another effector of Wnt signaling pathway, may also mediate the function of the incretin hormones as well as the expression of their receptors in pancreatic β-cells. In this review, we have introduced the incretin hormones and the Wnt signaling pathway, summarized recent findings in the field, and provided our perspectives.
doi:10.3389/fphys.2012.00273
PMCID: PMC3429047  PMID: 22934027
Wnt signaling pathway; GLP-1; GIP; TCF7L2; insulin; β-catenin
12.  cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene 
Background
The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE).
Results
The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene.
Conclusions
Using a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB.
doi:10.1186/1471-2199-6-2
PMCID: PMC548273  PMID: 15659240
13.  The role of aminogenic glucagon secretion in blood glucose homeostasis 
Journal of Clinical Investigation  1969;48(5):810-822.
Hyperaminoacidemia is a powerful stimulus of pancreatic glucagon secretion. These studies were designed to elucidate the role of aminogenic hyperglucagonemia in glucoregulation. Conscious dogs with previously implanted indwelling venous catheters were employed. The results support the view that a role of glucagon is to limit blood glucose decline during hyperaminoacidemia.
First, a significant negative correlation between the area of glucagon increment during the 1st 20 min of a 10 amino acid infusion and the maximum fall in glucose concentration was observed. Second, when endogenous glucagon secretion was suppressed by means of a continuous glucose infusion, hyperaminoacidemia induced a maximal glucose decline which averaged 35 mg/100 ml, differing significantly from mean maximal fall of 3 mg/100 ml, which normally occurs in the presence of endogenous hyperglucagonemia. Third, when, during hyperglycemic suppression of endogenous glucagon secretion, 50 mμg of exogenous glucagon/min was infused via the mesenteric vein with the amino acids, the fall in glucose was reduced to an average of 5 mg/100 ml. Similarly when pancreozymin, administered during the combined infusion of glucose and amino acids, overcame glucose suppression of endogenous glucagon secretion, plasma glucose did not fall.
Similar results were obtained when aminogenic hyperglucagonemia was prevented by other means. Hyperlipacidemia, induced by infusing a triglyceride emulsion and giving heparin injections, also suppressed aminogenic hyperglucagonemia in two of four experiments; in these two dogs glucose fell 15 and 11 mg/100 ml. In a final group of experiments, the canine pancreas was resected except for the uncinate process, which is virtually devoid of α-cells. In two dogs, in which this procedure resulted in zero portal venous glucagon levels, the administration of amino acids and/or pancreozymin resulted in a glucose decline of 14 and 16 mg/100 ml, despite the reduced β-cell population resulting from the subtotal pancreotectomy.
It thus appears that the secretion of pancreatic glucagon during hyperaminoacidemia in association with insulin secretion, serves to limit the decline of glucose concentration.
PMCID: PMC322289  PMID: 5780193
14.  Antidiabetic, Antihyperlipidemic and Antioxidant Activities of a Novel Proteoglycan from Ganoderma Lucidum Fruiting Bodies on db/db Mice and the Possible Mechanism 
PLoS ONE  2013;8(7):e68332.
Previously, we screened a proteoglycan for anti-hyperglycemic, named FYGL, from Ganoderma Lucidum. For further research of the antidiabetic mechanisms of FYGL in vivo, the glucose homeostasis, activities of insulin-sensitive enzymes, glucose transporter expression and pancreatic function were analyzed using db/db mice as diabetic models in the present work. FYGL not only lead to a reduction in glycated hemoglobin level, but also an increase in insulin and C-peptide level, whereas a decrease in glucagons level and showed a potential for the remediation of pancreatic islets. FYGL also increased the glucokinase activities, and simultaneously lowered the phosphoenol pyruvate carboxykinase activities, accompanied by a reduction in the expression of hepatic glucose transporter protein 2, while the expression of adipose and skeletal glucose transporter protein 4 was increased. Moreover, the antioxidant enzyme activities were also increased by FYGL treatment. Thus, FYGL was an effective antidiabetic agent by enhancing insulin secretion and decreasing hepatic glucose output along with increase of adipose and skeletal muscle glucose disposal in the late stage of diabetes. Furthermore, FYGL is beneficial against oxidative stress, thereby being helpful in preventing the diabetic complications.
doi:10.1371/journal.pone.0068332
PMCID: PMC3708940  PMID: 23874589
15.  A computational systems analysis of factors regulating α cell glucagon secretion 
Islets  2012;4(4):262-283.
Glucagon, a peptide hormone secreted from the α-cells of the pancreatic islets, is critical for blood glucose homeostasis. We reviewed the literature and employed a computational systems analysis of intracellular metabolic and electrical regulation of glucagon secretion to better understand these processes. The mathematical model of α-cell metabolic parameters is based on our previous model for pancreatic β-cells. We also formulated an ionic model for action potentials that incorporates Ca2+, K+, Na+ and Cl- currents. Metabolic and ionic models are coupled to the equations describing Ca2+ homeostasis and glucagon secretion that depends on activation of specific voltage-gated Ca2+ channels. Paracrine and endocrine regulations were analyzed with an emphasis on their effects on a hyperpolarization of membrane potential. This general model simulates and gives insight into the mechanisms of regulation of glucagon secretion under a wide range of experimental conditions. We also reviewed and analyzed dysfunctional mechanisms in α-cells to determine key pharmacological targets for modulating glucagon secretion in type 1 and 2 diabetes.
doi:10.4161/isl.22193
PMCID: PMC3496652  PMID: 23093806
diabetes; calcium; computational model; ion channels; insulin; islets; pancreas
16.  Characterization of a novel protein kinase C response element in the glucagon gene. 
Molecular and Cellular Biology  1997;17(4):1805-1816.
To maintain glucose levels in blood within narrow limits, the synthesis and secretion of pancreatic islet hormones are controlled by a variety of neural, hormonal, and metabolic messengers that act through multiple signal transduction pathways. Glucagon gene transcription is stimulated by cyclic AMP and depolarization-induced calcium influx. In this study, the effect of protein kinase C on glucagon gene transcription was investigated. After transient transfection of a glucagon-reporter fusion gene into the glucagon-producing islet cell line alphaTC2, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated glucagon gene transcription. By 5' deletions, 3' deletions, internal deletion, and oligonucleotide cassette insertion, the TPA-responsive element was mapped to the G2 element (from -165 to -200). Like TPA, overexpression of oncogenic Ras (V-12 Ras) stimulated G2-mediated transcription whereas overexpression of a dominant negative Ras mutant (N-17 Ras) blocked the effect of TPA. A mutational analysis of G2 function and nuclear protein binding indicated that protein kinase C and Ras responsiveness is conferred to the glucagon gene by HNF-3beta functionally interacting with a protein that binds to a closely associated site with sequence similarity to binding sites of Ets family proteins. HNF-3beta belongs to the winged-helix family of transcription factors and has been implicated in the control of cell-specific and developmental gene expression. The results of the present study show that the cell lineage-specific transcription factor HNF-3beta is an essential component of a novel protein kinase C response element in the glucagon gene.
PMCID: PMC232027  PMID: 9121428
17.  Insulin and Glucagon Impairments in Relation with Islet Cells Morphological Modifications Following Long Term Pancreatic Duct Ligation in the Rabbit – A Model of Non-insulin-dependent Diabete 
Plasma levels of glucose, insulin and glucagon were measured at various time intervals after pancreatic duct ligation (PDL) in rabbits. Two hyperglycemic periods were observed: one between 15–90 days (peak at 30 days of 15.1 ± 1.2mmol/l, p < 0.01), and the other at 450 days (11.2 ± 0.5 mmol/l, p < 0.02). The first hyperglycemic episode was significantly correlated with both hypoinsulinemia (41.8 ± 8pmol/l, r= –0.94, p < 0.01) and hyperglucagonemia (232 ± 21ng/l, r=0.95, p < 0.01). However, the late hyperglycemic phase (450 days), which was not accompanied by hypoinsulinemia, was observed after the hyperglucagonemia (390 days) produced by abundant immunostained A-cells giving rise to a 3-fold increase in pancreatic glucagon stores. The insulin and glucagon responses to glucose loading at 180, 270 and 450 days reflected the insensitivity of B- and A-cells to glucose. The PDL rabbit model with chronic and severe glycemic disorders due to the predominant role of glucagon mimicked key features of the NIDDM syndrome secondary to exocrine disease.
doi:10.1155/EDR.2001.101
PMCID: PMC2478542  PMID: 12369713
18.  The effects of corn silk on glycaemic metabolism 
Background
Corn silk contains proteins, vitamins, carbohydrates, Ca, K, Mg and Na salts, fixed and volatile oils, steroids such as sitosterol and stigmasterol, alkaloids, saponins, tannins, and flavonoids. Base on folk remedies, corn silk has been used as an oral antidiabetic agent in China for decades. However, the hypoglycemic activity of it has not yet been understood in terms of modern pharmacological concepts. The purpose of this study is to investigate the effects of corn silk on glycaemic metabolism.
Methods
Alloxan and adrenalin induced hyperglycemic mice were used in the study. The effects of corn silk on blood glucose, glycohemoglobin (HbA1c), insulin secretion, damaged pancreatic β-cells, hepatic glycogen and gluconeogenesis in hyperglycemic mice were studied respectively.
Results
After the mice were orally administered with corn silk extract, the blood glucose and the HbA1c were significantly decreased in alloxan-induced hyperglycemic mice (p < 0.05, p < 0.01, respectively), while the level of insulin secretionn was markedly elevated in alloxa-induced hyperglycemic mice (p < 0.05). The alloxan-damaged pancreatic β-cells of the mice were partly recovered gradually after the mice were administered with corn silk extract 15 days later. Also, the body weight of the alloxan-induced hyperglycemic mice was increased gradually. However, ascension of blood glucose induced by adrenalin and gluconeogenesis induced by L-alanine were not inhibited by corn silk extract treatment (p > 0.05). Although corn silk extract increased the level of hepatic glycogen in the alloxan-induced hyperglycemic mice, there was no significant difference between them and that of the control group(p > 0.05).
Conclusion
Corn silk extract markedly reduced hyperglycemia in alloxan-induced diabetic mice. The action of corn silk extract on glycaemic metabolism is not via increasing glycogen and inhibiting gluconeogenesis but through increasing insulin level as well as recovering the injured β-cells. The results suggest that corn silk extract may be used as a hypoglycemic food or medicine for hyperglycemic people in terms of this modern pharmacological study.
doi:10.1186/1743-7075-6-47
PMCID: PMC2785813  PMID: 19930631
19.  Diabetes mellitus and the exocrine pancreas. 
Diabetes and carbohydrate intolerance can occur in pancreatitis. Although one-half of patients with acute pancreatitis will have some evidence of glucose intolerance during their acute illness, few will require insulin administration on either a short- or long-term basis. The diabetes seen in acute pancreatitis is likely due to a combination of factors, including alerted insulin secretion, increased glucagon release, and decreased glucose utilization by the liver and peripheral tissue. Chronic pancreatitis is often associated with diabetes mellitus, with the incidence as high as 70 percent when pancreatic calcification is present. These patients tend to be very sensitive to the effects of insulin and hypoglycemia. This is probably secondary to concurrent hepatic disease, malnutrition, and a relative decrease in glucagon reserves. The diabetes seen in chronic pancreatitis is associated with decreased insulin production. Finally, although the endocrine pancreas may influence the exocrine gland through a portal system, primary diabetes mellitus probably does not result in clinically significant alterations in pancreatic exocrine function.
PMCID: PMC2589620  PMID: 6367237
20.  Effect of insulin-glucose infusions on plasma glucagon levels in fasting diabetics and nondiabetics. 
Journal of Clinical Investigation  1975;56(5):1132-1138.
The effect of the intravenous infusion of insulin plus glucose on plasma glucagon levels was studied in hyperglycemic fasting adult-type and juvenile-type diabetics and compared with fasting nondiabetics. Adult-type diabetics were given insulin for 2 h at a rate of 0.03 U/kg-min, raising their mean insulin to between 25 and 36 muU/ml; glucagon declined from a base-line value of 71+/-2 (SEM) to 56+/-1 pg/ml at 120 min (P less than 0.001). In juvenile-type diabetics given the same insulin-glucose infusion, glucagon declined from a base-line level of 74+/-8 to 55+/-5 pg/ml at 120 min (P less than 0.05). The absolute glucagon values in the diabetic groups did not differ significantly at any point from the mean glucagon levels in nondiabetics given insulin at the same rate plus enough glucose to maintain normoglycemia. When glucagon was expressed as percent of baseline, however, the normoglycemic nondiabetics exhibited significantly lower values than adult-type diabetics at 90 and 120 min and juvenile-type diabetics at 60 min. In nondiabetics given insulin plus glucose at a rate that caused hyperglycemia averaging between 134 and 160 mg/dl, glucagon fell to 41+/-7 pg/ml at 120 min, significantly below the adult diabetics at 90 and 120 min (P less than 0.01 and less than 0.05) and the juvenile group at 60 min (P less than 0.01). The mean minimal level of 39+/-2 pg/ml was significantly below the adult (P less than 0.001) and juvenile groups (P less than 0.05). When insulin was infused in the diabetic groups at a rate of 0.4 U/kg-min together with glucose, raising mean plasma insulin to between 300 and 600 muU/ml, differences from the hyperglycemic nondiabetics were no longer statistically significant. It is concluded that, contrary to the previously reported lack of insulin effect in diabetics during carbohydrate meals, intravenous administration for 2 h of physiologic amounts of insulin plus glucose is accompanied in unfed diabetics by a substantial decline in plasma glucagon. These levels are significantly above hyperglycemic nondiabetics at certain points but differ from normoglycemic nondiabetics only when expressed as percent of the baseline. At a supraphysiologic rate of insulin infusion in diabetics, these differences disappear.
PMCID: PMC301975  PMID: 1184740
21.  Sitagliptin Reduces Cardiac Apoptosis, Hypertrophy and Fibrosis Primarily by Insulin-Dependent Mechanisms in Experimental type-II Diabetes. Potential Roles of GLP-1 Isoforms 
PLoS ONE  2013;8(10):e78330.
Background
Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart.
Methods
Goto-Kakizaki (GK) rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin) or vehicle (n=10, each). After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays.
Results
Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36), alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells.
Conclusions
Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36) promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actions.
doi:10.1371/journal.pone.0078330
PMCID: PMC3840053  PMID: 24302978
22.  Glucagon gene transcription is negatively regulated by insulin in a hamster islet cell line. 
Journal of Clinical Investigation  1989;84(2):672-677.
Complex interrelationships exist between the four pancreatic islet cell types and their respective secretory products, insulin, glucagon, somatostatin, and pancreatic polypeptide. These hormones are known to interact with the different islet cells and modulate their functions. Insulin inhibits glucagon secretion from the A cell both in vivo and in vitro and, in states of insulin deficiency, high glucagon levels are observed that are normalized by insulin replacement. To determine if insulin also regulates glucagon biosynthesis, we studied its effects on glucagon gene expression. Our studies indicate that insulin, in a dose-dependent fashion decreases steady-state glucagon mRNA levels in a clonal hamster islet cell line, In-R1-G9; this decrease is secondary to an inhibition of glucagon gene transcription as assessed by transcriptional run-on assays and does not involve detectable changes in mRNA stability. Inhibition of glucagon gene transcription is accompanied by corresponding decreases in glucagon immunoreactivity in both cell extracts and medium. We conclude that insulin may not only regulate glucagon secretion but also glucagon gene expression.
Images
PMCID: PMC548931  PMID: 2668337
23.  Evidence for a catabolic role of glucagon during an amino acid load. 
Despite the strong association between protein catabolic conditions and hyperglucagonemia, and enhanced glucagon secretion by amino acids (AA), glucagon's effects on protein metabolism remain less clear than on glucose metabolism. To clearly define glucagon's catabolic effect on protein metabolism during AA load, we studied the effects of glucagon on circulating AA and protein dynamics in six healthy subjects. Five protocols were performed in each subject using somatostatin to inhibit the secretion of insulin, glucagon, and growth hormone (GH) and selectively replacing these hormones in different protocols. Total AA concentration was the highest when glucagon, insulin, and GH were low. Selective increase of glucagon levels prevented this increment in AA. Addition of high levels of insulin and GH to high glucagon had no effect on total AA levels, although branched chain AA levels declined. Glucagon mostly decreased glucogenic AA and enhanced glucose production. Endogenous leucine flux, reflecting proteolysis, decreased while leucine oxidation increased in protocols where AA were infused and these changes were unaffected by the hormones. Nonoxidative leucine flux reflecting protein synthesis was stimulated by AA, but high glucagon attenuated this effect. Addition of GH and insulin partially reversed the inhibitory effect of glucagon on protein synthesis. We conclude that glucagon is the pivotal hormone in amino acid disposal during an AA load and, by reducing the availability of AA, glucagon inhibits protein synthesis stimulated by AA. These data provide further support for a catabolic role of glucagon at physiological concentrations.
PMCID: PMC507404  PMID: 8690809
24.  Generation of Glucose-Responsive Functional Islets with a Three-Dimensional Structure from Mouse Fetal Pancreatic Cells and iPS Cells In Vitro 
PLoS ONE  2011;6(12):e28209.
Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D) structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.
doi:10.1371/journal.pone.0028209
PMCID: PMC3228734  PMID: 22145030
25.  Glucagon-Like Peptide-1 Induced Signaling and Insulin Secretion Do Not Drive Fuel and Energy Metabolism in Primary Rodent Pancreatic β-Cells 
PLoS ONE  2009;4(7):e6221.
Background
Glucagon like peptide-1 (GLP-1) and its analogue exendin-4 (Ex-4) enhance glucose stimulated insulin secretion (GSIS) and activate various signaling pathways in pancreatic β-cells, in particular cAMP, Ca2+ and protein kinase-B (PKB/Akt). In many cells these signals activate intermediary metabolism. However, it is not clear whether the acute amplification of GSIS by GLP-1 involves in part metabolic alterations and the production of metabolic coupling factors.
Methodology/Prinicipal Findings
GLP-1 or Ex-4 at high glucose caused release (∼20%) of the total rat islet insulin content over 1 h. While both GLP-1 and Ex-4 markedly potentiated GSIS in isolated rat and mouse islets, neither had an effect on β-cell fuel and energy metabolism over a 5 min to 3 h time period. GLP-1 activated PKB without changing glucose usage and oxidation, fatty acid oxidation, lipolysis or esterification into various lipids in rat islets. Ex-4 caused a rise in [Ca2+]i and cAMP but did not enhance energy utilization, as neither oxygen consumption nor mitochondrial ATP levels were altered.
Conclusions/Significance
The results indicate that GLP-1 barely affects β-cell intermediary metabolism and that metabolic signaling does not significantly contribute to GLP-1 potentiation of GSIS. The data also indicate that insulin secretion is a minor energy consuming process in the β-cell, and that the β-cell is different from most cell types in that its metabolic activation appears to be primarily governed by a “push” (fuel substrate driven) process, rather than a “pull” mechanism secondary to enhanced insulin release as well as to Ca2+, cAMP and PKB signaling.
doi:10.1371/journal.pone.0006221
PMCID: PMC2704866  PMID: 19593440

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