Proglucagon mRNA Levels Research Articles

Glucolipotoxicity and GLP-1 secretion

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide...

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells.

Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K+, and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA—mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K+-stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

P 158 - GLP-2 in the capacity of (-)-epicatechin and anthocyanidins to improve insulin sensitivity

Glucagon-like peptide-2 (GLP-2) is an incretin involved in energy balance through the regulation of postprandial glycaemia. GLP-2 is generated from the post-translational cleavage of proglucagon, being rapidly degraded in the bloodstream by dipeptidyl peptidase IV (DPP-IV). We currently investigated the effects of (-)-epicatechin (EC) and anthocyanidins (AC) to modulate GLP-2 using in vivo and in vitro approaches. To assess in vivo effects on proglucagon mRNA and plasma GLP-2 concentrations, male C57BL/6 J mice were fed for 14 w control (C) or high fat (HF) diets without or with 20 mg EC/kg or 40 mg AC/kg BW. Supplementation with EC and AC mitigated HF-induced insulin resistance (improved GTT, ITT, plasma insulin and glucose levels). Plasma GLP-2 levels were 148 and 200% higher in C mice supplemented with EC and AC compared to non-supplemented C mice, and 83 and 46% higher in HF mice supplemented with EC and AC compared to non-supplemented HF mice. Colon proglucagon mRNA levels were higher (84%) in AC-supplemented vs unsupplemented C mice, while EC had no effect. In vitro, cyanidin, but not EC, inhibited DPP-IV activity. The improvement of insulin sensitivity by EC and AC in a model of high fat diet-induced type 2 diabetes could be in part due to GLP-2 upregulation. The underlying mechanisms for AC involve an increase in proglucagon expression and decreased GLP-2 degradation.

Neuronostatin acts via GPR107 to increase cAMP-independent PKA phosphorylation and proglucagon mRNA accumulation in pancreatic α-cells.

Neuronostatin (NST) is a recently described peptide that is produced from the somatostatin preprohormone in pancreatic δ-cells. NST has been shown to increase glucagon secretion from primary rat pancreatic islets in low-glucose conditions. Here, we demonstrate that NST increases proglucagon message in α-cells and identify a potential mechanism for NST's cellular activities, including the phosphorylation of PKA following activation of the G protein-coupled receptor, GPR107. GPR107 is abundantly expressed in the pancreas, particularly, in rodent and human α-cells. Compromise of GPR107 in pancreatic α-cells results in failure of NST to increase PKA phosphorylation and proglucagon mRNA levels. We also demonstrate colocalization of GPR107 and NST on both mouse and human pancreatic α-cells. Taken together with our group's observation that NST infusion in conscious rats impairs glucose clearance in response to a glucose challenge and that plasma levels of the peptide are elevated in the fasted compared with the fed or fasted-refed state, these studies support the hypothesis that endogenous NST regulates islet cell function by interacting with GPR107 and initiating signaling in glucagon-producing α-cells.

Molecular cloning of a proglucagon in a cyprinid fish (Schizothorax prenanti): mRNA tissue distribution and quantification during periprandial changes and fasting

Abstract Proglucagon is the precursor for several peptides: glucagon, glicentin, oxyntomodulin and at least one glucagon-like peptide. Previous reports indicated that proglucagon-derived peptides are important in regulating feeding and energy homeostasis in both mammals and fish. In the present study, a 504 bp cDNA fragment encoding a highly conserved proglucagon in a cyprinid fish ( Schizothorax prenanti ) was amplified by homologous cloning. Tissue expression profiles of proglucagon showed that S. prenanti proglucagon had a broad tissue distribution, but was expressed primarily in the intestine and brain. Proglucagon mRNA levels significantly increased after feeding in the hypothalamus and intestine, while levels were dramatically reduced during a week of food deprivation and increased upon refeeding. Proglucagon mRNA expression changes in the hypothalamus and intestine responded to the altered feeding status and suggest that proglucagon is involved in the regulation of feeding and energy in S. prenanti . Statement of relevance In the present study, we deal with the molecular, distribution expressions and mRNA quantification during periprandial, fasting and refeeding in lower vertebrate Ya-fish ( S. prenanti ). S. prenanti is an endemic cold temperate fish in the southwest of China valuable in both aquaculture and research. However, slow growth rate has been recognized as a major problem in S. prenanti farming, increasing the final production cost. Studies concerning the expression of genes related to feeding and growth regulation have been conducted for this species in our laboratory. Recently, a growing number of homologues of mammalian appetite-regulating peptides and their receptors have been characterized in fish. Proglucagon is the precursor for several peptides: glucagon, glicentin, oxyntomodulin and at least one glucagon-like peptide. These peptides are derived in a tissue-specific manner via post-translational proteolytic processing. Previous reports in mammals indicated that proglucagon-derived peptides are important in regulating feeding and energy homeostasis. However, there is scarce information about their physiological roles in fish. Here, we cloned a 504 bp cDNA fragment of a single proglucagon gene, that is mainly expressed in the intestine and brain and responded to changes of feeding status. These suggest that proglucagon acts as a satiety factor involved in the regulation of food intake in S. prenanti . Our findings of this study provide a basis for future research to better understand the functions and mechanisms of actions of proglucagon and will be helpful in dealing with feeding and growth issues in fishes. We believe that something in this manuscript would be of interest to the journal's readers and hope that this paper is suitable for publication in Aquaculture (Physiology and Endocrinology: Fish).

Resistant maltodextrin promotes fasting glucagon-like peptide-1 secretion and production together with glucose tolerance in rats.

Glucagon-like peptide-1 (GLP-1), which is produced and released from enteroendocrine L cells, plays pivotal roles in postprandial glycaemia. The ingestion of resistant maltodextrin (RMD), a water-soluble non-digestible saccharide, improves the glycaemic response. In the present study, we examined whether the continuous feeding of RMD to rats affected GLP-1 levels and glycaemic control. Male Sprague-Dawley rats (6 weeks of age) were fed an American Institute of Nutrition (AIN)-93G-based diet containing either cellulose (5 %) as a control, RMD (2.5 or 5 %), or fructo-oligosaccharides (FOS, 2.5 or 5 %) for 7 weeks. During the test period, an intraperitoneal glucose tolerance test (IPGTT) was performed after 6 weeks. Fasting GLP-1 levels were significantly higher in the 5 % RMD group than in the control group after 6 weeks. The IPGTT results showed that the glycaemic response was lower in the 5 % RMD group than in the control group. Lower caecal pH, higher caecal tissue and content weights were observed in the RMD and FOS groups. Proglucagon mRNA levels were increased in the caecum and colon of both RMD and FOS groups, whereas caecal GLP-1 content was increased in the 5 % RMD group. In addition, a 1 h RMD exposure induced GLP-1 secretion in an enteroendocrine L-cell model, and single oral administration of RMD increased plasma GLP-1 levels in conscious rats. The present study demonstrates that continuous ingestion of RMD increased GLP-1 secretion and production in normal rats, which could be stimulated by its direct and indirect (enhanced gut fermentation) effects on GLP-1-producing cells, and contribute to improving glucose tolerance.

Probing the intra‐islet control of beta cell function (1108.3)

Neuronostatin (NST) is produced in pancreatic delta cells from the somatostatin preprohormone, and has been shown to inhibit glucose‐stimulated insulin secretion from beta cells of primary islets, but not from isolated beta cell cultures. We have demonstrated that NST stimulates glucagon release and have hypothesized that this is necessary for the peptide’s in vivo effect to impair glucose tolerance. Here we demonstrate the action of NST to increase proglucagon message in alpha cells and identify a potential mechanism to be via phosphorylation of protein kinase A. The NST receptor, GPR107, is abundantly expressed in the pancreas, in particular in the alpha cell population and both the effect on proglucagon mRNA levels and PKA phosphrylation are absent in alpha cells in which the expression of the NST receptor has been compromised using siRNA. Taken together with our observation that NST infusion in conscious rats impairs glucose tolerance and that plasma levels of the peptide are elevated in the fasted compared to the fed or fasted‐refed state, our studies support the hypothesis that endogenous NST is a physiologically relevant determinant of islet cell function and may contribute to the hyperglucagonemia of the prediabetic state.Grant Funding Source: NIH HL66023

The Correlation Between Intestinal Gonadotropin-Releasing Hormone (GnRH) and Proglucagon in Hyperlipidemic Rats and Goto-Kakizaki (GK) Rats

Our experiment investigated the mRNA expression of intestinal gonadotropin-releasing hormone (GnRH), proglucagon (PG), and glucagon-like peptide 1 receptor (GLP-1R) in the jejunum, ileum, and colon of rats fed with high-fat diet and Goto-Kakizaki (GK) rats and revealed the physiological role of intestinal GnRH. We found that the GnRH and PG mRNA levels in high-cholesterol (HCh) diet were higher than in the control. However, the GnRH receptor (GnRHR) and GLP-1R mRNA levels did not differ significantly between HCh and control. The GnRH, PG, and GLP-1R mRNA levels in GK rats were lower, respectively, than those in control rats, while the GnRHR levels did not differ significantly between GK rats and control rats. There were no difference in GnRH, PG, GnRHR, and GLP-1R mRNA levels in the ileum and colon tissue between HCh and control rats. The GnRH mRNA levels of GK rats were lower than those in control rats; however, the PG, GLP-1R, and GnRHR levels did not differ significantly between GK and control rats. The GLP-1R mRNA levels of GK rats were lower than those in control rats. The GnRH mRNA expression showed positive correlation with PG mRNA expression in different intestinal sections. The GnRH level in the jejunum showed a significant effect on blood glucose level, while the PG level in the jejunum showed a significant effect on insulin level. This may imply that, compared with the ileum and colon, the jejunum had greater impact on glucose metabolism; furthermore, GnRH might interact with intestinal GLP-1 and GLP-2 through the paracrine and autocrine ways and then regulate glucose metabolism and insulin secretion.

Biology of Incretins: GLP-1 and GIP

This review focuses on the mechanisms regulating the synthesis, secretion, biological actions, and therapeutic relevance of the incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). The published literature was reviewed, with emphasis on recent advances in our understanding of the biology of GIP and GLP-1. GIP and GLP-1 are both secreted within minutes of nutrient ingestion and facilitate the rapid disposal of ingested nutrients. Both peptides share common actions on islet beta-cells acting through structurally distinct yet related receptors. Incretin-receptor activation leads to glucose-dependent insulin secretion, induction of beta-cell proliferation, and enhanced resistance to apoptosis. GIP also promotes energy storage via direct actions on adipose tissue, and enhances bone formation via stimulation of osteoblast proliferation and inhibition of apoptosis. In contrast, GLP-1 exerts glucoregulatory actions via slowing of gastric emptying and glucose-dependent inhibition of glucagon secretion. GLP-1 also promotes satiety and sustained GLP-1-receptor activation is associated with weight loss in both preclinical and clinical studies. The rapid degradation of both GIP and GLP-1 by the enzyme dipeptidyl peptidase-4 has led to the development of degradation-resistant GLP-1-receptor agonists and dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes. These agents decrease hemoglobin A1c (HbA1c) safely without weight gain in subjects with type 2 diabetes. GLP-1 and GIP integrate nutrient-derived signals to control food intake, energy absorption, and assimilation. Recently approved therapeutic agents based on potentiation of incretin action provide new physiologically based approaches for the treatment of type 2 diabetes.

A human cellular model for studying the regulation of glucagon-like peptide-1 secretion.

GLP-1 (glucagon-like peptide-1) is a potent insulin secretagogue released from L cells in the intestine. The regulation of GLP-1 secretion has been described both in vivo and in vitro in several animal species, but data from human cellular models are lacking. For this purpose, factors and cell-signaling pathways regulating GLP-1 secretion were investigated in the NCI-H716 human intestinal cell line. After differentiation, these cells homogeneously produced 16.8 pmol GLP-1/mg protein with a basal release of 4.2% during a 2-h incubation period. Nutrients, such as palmitic acid, oleic acid, and meat hydrolysate, stimulated GLP-1 secretion in a dose-dependent manner, as did the cholinergic agonist carbachol and the neuromediator gastrin-releasing peptide. Along with stimulating GLP-1 release, gastrin-releasing peptide, like ionomycin, increased intracellular calcium levels. Activators of PKA and PKC were able to increase GLP-1 secretion in NCI-H716 cells. However, neither PKA activators nor meat hydrolysate increased proglucagon mRNA levels. These findings indicate that the NCI-H716 cell line constitutes a unique model to study the cellular mechanism of GLP-1 secretion in humans and suggest potential interspecies divergence in the regulation of proglucagon gene expression in enteroendocrine cells.

A Human Cellular Model for Studying the Regulation of Glucagon-Like Peptide-1 Secretion

GLP-1 (glucagon-like peptide-1) is a potent insulin secretagogue released from L cells in the intestine. The regulation of GLP-1 secretion has been described both in vivo and in vitro in several animal species, but data from human cellular models are lacking. For this purpose, factors and cell-signaling pathways regulating GLP-1 secretion were investigated in the NCI-H716 human intestinal cell line. After differentiation, these cells homogeneously produced 16.8 pmol GLP-1/mg protein with a basal release of 4.2% during a 2-h incubation period. Nutrients, such as palmitic acid, oleic acid, and meat hydrolysate, stimulated GLP-1 secretion in a dose-dependent manner, as did the cholinergic agonist carbachol and the neuromediator gastrin-releasing peptide. Along with stimulating GLP-1 release, gastrin-releasing peptide, like ionomycin, increased intracellular calcium levels. Activators of PKA and PKC were able to increase GLP-1 secretion in NCI-H716 cells. However, neither PKA activators nor meat hydrolysate increased proglucagon mRNA levels. These findings indicate that the NCI-H716 cell line constitutes a unique model to study the cellular mechanism of GLP-1 secretion in humans and suggest potential interspecies divergence in the regulation of proglucagon gene expression in enteroendocrine cells.

Glucose Regulates Proinsulin and Prosomatostatin But Not Proglucagon Messenger Ribonucleic Acid Levels in Rat Pancreatic Islets1

Insulin and glucagon are the major hormones involved in the control of fuel metabolism and particularly of glucose homeostasis; in turn, nutrients tightly regulate insulin and glucagon secretion from the islets of Langerhans. Nutrients have clearly been shown to affect insulin secretion, as well as insulin biosynthesis and proinsulin gene expression; by contrast, the effects of nutrients on proglucagon gene expression have not been studied. We have investigated the effect of glucose, arginine, and palmitate on glucagon release, glucagon cell content, and proglucagon messenger RNA (mRNA) levels from isolated rat islets in 24-h incubations. We report here that concentrations of glucose that clearly regulate insulin and somatostatin release as well as proinsulin and prosomatostatin mRNA levels, do not significantly affect glucagon release, glucagon cell content or proglucagon mRNA levels. In addition, though both 10 mM arginine and 1 mM palmitate strongly stimulated glucagon release, they did not affect proglucagon mRNA levels. We conclude that, in contrast to insulin and somatostatin, glucose does not affect glucagon release and proglucagon mRNA levels, and arginine and palmitate do not coordinately regulate glucagon release and proglucagon mRNA levels.

Impaired glucose homeostasis and neonatal mortality in hepatocyte nuclear factor 3alpha-deficient mice.

Hepatocyte nuclear factors 3 (HNF-3) belong to an evolutionarily conserved family of transcription factors that are critical for diverse biological processes such as development, differentiation, and metabolism. To study the physiological role of HNF-3alpha, we generated mice that lack HNF-3alpha by homologous recombination in embryonic stem cells. Mice homozygous for a null mutation in the HNF-3alpha gene develop a complex phenotype that is characterized by abnormal feeding behavior, progressive starvation, persistent hypoglycemia, hypotriglyceridemia, wasting, and neonatal mortality between days 2 and 14. Hypoglycemia in HNF-3alpha-null mice leads to physiological counter-regulatory responses in glucocorticoid and growth hormone production and an inhibition of insulin secretion but fails to stimulate glucagon secretion. Glucagon-producing pancreatic alpha cells develop normally in HNF-3alpha-/- mice, but proglucagon mRNA levels are reduced 50%. Furthermore, the transcriptional levels of neuropeptide Y are also significantly reduced shortly after birth, implying a direct role of HNF-3alpha in the expression of these genes. In contrast, mRNA levels were increased in HNF-3 target genes phosphofructo-2-kinase/fructose-2,6-bisphophatase, insulin growth factor binding protein-1, and hexokinase I of HNF-3alpha-null mice. Mice lacking one or both HNF-3alpha alleles also show impaired insulin secretion and glucose intolerance after an intraperitoneal glucose challenge, indicating that pancreatic beta-cell function is also compromised. Our results indicate that HNF-3alpha plays a critical role in the regulation of glucose homeostasis and in pancreatic islet function.

A Physiological Level of Rhubarb Fiber Increases Proglucagon Gene Expression and Modulates Intestinal Glucose Uptake in Rats ,

Previous work demonstrated that a high fiber diet upregulates proglucagon mRNA and secretion of glucagon-like peptide-1 [GLP-1(7–37)] and insulin compared with an elemental fiber-free diet. This study examined whether similar intakes of fibers differing in physiochemical and fermentative properties alter the expression of intestinal hormones and intestinal absorptive properties. Sprague-Dawley rats were fed either a 50 g/kg cellulose or rhubarb fiber diet for 14 d. Ileal proglucagon mRNA levels were significantly higher in rats fed rhubarb fiber than in those fed cellulose fiber (9.3 ± 0.9 vs. 6.2 ± 1.0 densitometer units). Proglucagon mRNA in the colon did not differ between diet treatments. Plasma c-peptide concentrations were significantly higher 30 min after an oral glucose tolerance test in the rhubarb vs. cellulose group (1627 ± 67 vs. 1290 ± 71 pmol/L). Passive permeability, measured by the uptake of L-glucose, was significantly higher in the jejunum of rats fed cellulose compared with those fed rhubarb fiber. Adjusting total glucose uptake for passive permeability and unstirred water layer resistance resulted in a higher Km being calculated for the jejunum and ileum of the cellulose fiber group. Jejunal and ileal carrier-mediated uptakes (Vmax) were not altered by diet and reflected the lack of difference between groups in sodium-dependent glucose cotransporter (SGLT-1) and sodium-independent glucose transporter (GLUT2) mRNA levels. Replacing cellulose fiber with rhubarb fiber in a diet upregulated ileal proglucagon mRNA and resulted in a reduced passive permeability but did not affect glucose transport of the small intestine. This work establishes the importance of dietary fiber fermentability in modulating intestinal proglucagon expression and possibly glucose homeostasis.

Ileal proglucagon gene expression in the rat: Characterization in intestinal adaptation using in situ hybridization

Background: Proglucagon-derived peptides are potential mediators of the adaptive response of the terminal ileum to massive small bowel resection. Ileal proglucagon messenger RNA (mRNA) levels increase during ileal adaptation. The present study explored the cellular basis of this response. Methods: Sections of control ileum, ileum 4 days after resection, and pancreas were analyzed by in situ hybridization with 35S-labeled complementary RNA (cRNA) probes. Results: Both the proglucagon and the peptide YY cRNA probes hybridized to discrete cells in the ileal mucosa, the disposition of which corresponds to that reported for intestinal L cells. Four days after resection there was a marked increase in the intensity of the signal for both probes without an increase in cell number. Insulin and histone H3 probes were used as controls to confirm the specificity of the hybridization seen with the L-cell specific, proglucagon, and peptide YY probes. Conclusions: The increase in proglucagon mRNA levels after massive small bowel resection is caused by an increase in the cellular content. The parallel increase in PYY mRNA levels implies an L cell- rather than a proglucagon gene-specific response.

Proglucagon gene expression is regulated by a cyclic AMP-dependent pathway in rat intestine.

Expression of the gene encoding preproglucagon gives rise to different glucagon-related peptides in the pancreas and intestine. Glucagon gene expression is regulated by a protein kinase C-dependent pathway in rat islet cell lines, whereas activation of the adenylate cyclase pathway in islet cell lines is without effect. To elucidate the factors important for the control of proglucagon biosynthesis in the intestine, we have studied proglucagon gene expression and proglucagon biosynthesis in rat intestine. Analysis of intestinal cDNA clones encoding preproglucagon indicated that pancreatic and intestinal glucagon mRNA transcripts were identical. The regulation of proglucagon gene expression in rat intestine differed markedly from that previously observed in islet cell lines. Phorbol esters increased the secretion of glucagon-like immunoreactive peptides (GLI) but had no effect on proglucagon mRNA levels in rat intestinal cells. Bombesin also increased the secretion of GLI without affecting proglucagon mRNA levels or biosynthesis. In contrast, dibutyryl cyclic AMP, forskolin, and cholera toxin increased both proglucagon mRNA levels and GLI biosynthesis and secretion, suggesting that proglucagon gene expression in the intestine is regulated by a cyclic AMP-dependent pathway. These observations suggest that tissue-specific differences in both the regulation of proglucagon gene expression and the posttranslational processing of proglucagon contribute to the diversity of glucagon gene expression.