Determinants of plasma levels of proglucagon and the metabolic impact of glucagon receptor signalling: a UK Biobank study.

AIMS/HYPOTHESES: Glucagon and glucagon-like peptide-1 (GLP-1) are derived from the same precursor; proglucagon, and dual agonists of their receptors are currently being explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). Elevated levels of endogenous glucagon (hyperglucagonaemia) have been linked with hyperglycaemia in individuals with type 2 diabetes but are also observed in individuals with obesity and MASLD. GLP-1 levels have been reported to be largely unaffected or even reduced in similar conditions. We investigated potential determinants of plasma proglucagon and associations of glucagon receptor signalling with metabolic diseases based on data from the UK Biobank. METHODS: We used exome sequencing data from the UK Biobank for ~410,000 white participants to identify glucagon receptor variants and grouped them based on their known or predicted signalling. Data on plasma levels of proglucagon estimated using Olink technology were available for a subset of the cohort (~40,000). We determined associations of glucagon receptor variants and proglucagon with BMI, type 2 diabetes and liver fat (quantified by liver MRI) and performed survival analyses to investigate if elevated proglucagon predicts type 2 diabetes development. RESULTS: Obesity, MASLD and type 2 diabetes were associated with elevated plasma levels of proglucagon independently of each other. Baseline proglucagon levels were associated with the risk of type 2 diabetes development over a 14 year follow-up period (HR 1.13; 95% CI 1.09, 1.17; n=1562; p=1.3×10-12). This association was of the same magnitude across strata of BMI. Carriers of glucagon receptor variants with reduced cAMP signalling had elevated levels of proglucagon (ß 0.847; 95% CI 0.04, 1.66; n=17; p=0.04), and carriers of variants with a predicted frameshift mutation had higher levels of liver fat compared with the wild-type reference group (ß 0.504; 95% CI 0.03, 0.98; n=11; p=0.04). CONCLUSIONS/INTERPRETATION: Our findings support the suggestion that glucagon receptor signalling is involved in MASLD, that plasma levels of proglucagon are linked to the risk of type 2 diabetes development, and that proglucagon levels are influenced by genetic variation in the glucagon receptor, obesity, type 2 diabetes and MASLD. Determining the molecular signalling pathways downstream of glucagon receptor activation may guide the development of biased GLP-1/glucagon co-agonist with improved metabolic benefits. DATA AVAILABILITY: All coding is available through https://github.com/nicwin98/UK-Biobank-GCG.

The role of preproglucagon peptides in regulating ß-cell morphology and responses to streptozotocin-induced diabetes.

Insulin secretion from ß-cells is tightly regulated by local signaling from preproglucagon (Gcg) products from neighboring α-cells. Physiological paracrine signaling within the microenvironment of the ß-cell is altered after metabolic stress, such as high-fat diet or the ß-cell toxin, streptozotocin (STZ). Here, we examined the role and source of Gcg peptides in ß-cell function and in response to STZ-induced hyperglycemia. We used whole body Gcg null (GcgNull) mice and mice with Gcg expression either specifically within the pancreas (GcgΔPanc) or the intestine (GcgΔIntest). With lower doses of STZ exposure, insulin levels were greater and glucose levels were lower in GcgNull mice compared with wild-type mice. When Gcg was functional only in the intestine, plasma glucagon-like peptide-1 (GLP-1) levels were fully restored but these mice did not have any additional protection from STZ-induced diabetes. Pancreatic Gcg reactivation normalized the hyperglycemic response to STZ. In animals not treated with STZ, GcgNull mice had increased pancreas mass via both α- and ß-cell hyperplasia and reactivation of Gcg in the intestine normalized ß- but not α-cell mass, whereas pancreatic reactivation normalized both ß- and α-cell mass. GcgNull and GcgΔIntest mice maintained higher ß-cell mass after treatment with STZ compared with control and GcgΔPanc mice. Although in vivo insulin response to glucose was normal, global lack of Gcg impaired glucose-stimulated insulin secretion in isolated islets. Congenital replacement of Gcg either in the pancreas or intestine normalized glucose-stimulated insulin secretion. Interestingly, mice that had intestinal Gcg reactivated in adulthood had impaired insulin response to KCl. We surmise that the expansion of ß-cell mass in the GcgNull mice compensated for decreased individual ß-cell insulin secretion, which is sufficient to normalize glucose under physiological conditions and conferred some protection after STZ-induced diabetes.NEW & NOTEWORTHY We examined the role of Gcg on ß-cell function under normal and high glucose conditions. GcgNull mice had decreased glucose-stimulated insulin secretion, increased ß-cell mass, and partial protection against STZ-induced hyperglycemia. Expression of Gcg within the pancreas normalized these endpoints. Intestinal expression of Gcg only normalized ß-cell mass and glucose-stimulated insulin secretion. Increased ß-cell mass in GcgNull mice likely compensated for decreased insulin secretion normalizing physiological glucose levels and conferring some protection after STZ-induced diabetes.

The RNA-binding protein, HuD regulates proglucagon biosynthesis in pancreatic α cells.

Glucagon is a peptide hormone generated by pancreatic α cells. It is the counterpart of insulin and plays an essential role in the regulation of blood glucose level. Therefore, a tight regulation of glucagon levels is pivotal to maintain homeostasis of blood glucose. However, little is known about the mechanisms regulating glucagon biosynthesis. In this study, we demonstrate that the RNA-binding protein HuD regulates glucagon expression in pancreatic α cells. HuD was found in α cells from mouse pancreatic islet and mouse glucagonoma αTC1 cell line. Ribonucleoprotein immunoprecipitation analysis, followed by RT-qPCR showed the association of HuD with glucagon mRNA. Knockdown of HuD resulted in a reduction in both proglucagon expression and cellular glucagon level by decreasing its de novo synthesis. Reporter analysis using the EGFP reporter containing 3' untranslated region (3'UTR) of glucagon mRNA showed that HuD regulates proglucagon expression via its 3'UTR. In addition, the relative level of glucagon in the islets and plasma was lower in HuD knockout (KO) mice compared to age-matched control mice. Taken together, these results suggest that HuD is a novel factor regulating the biosynthesis of proglucagon in pancreatic α cells.

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon-like peptide-2 in pre-weaned lambs.

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon-like peptide -2 (GLP-2) in pre-weaned lambs using animal and cell culture experiments. In vivo, twelve 14-day-old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP-2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin-dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin-like growth factor 1 (IGF-1) in the jejunum and ileum and mRNA expression of GLP-2 receptor (GLP-2R) in the jejunum. In vitro, when jejunal cells were treated with GLP-2 for 2 hr, the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) OD, IGF-1 concentration, and the mRNA expression of IGF-1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF-1 receptor (IGF-1R) inhibitor decreased (p < .05) the mRNA expression of IGF-1, cyclin A2, cyclin D1 and CDK6 in GLP-2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP-2 in pre-weaning lambs. Furthermore, GLP-2 can indirectly promote the proliferation of jejunal cells mainly through the IGF-1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.

Targeting the gut microbiota with inulin-type fructans: preclinical demonstration of a novel approach in the management of endothelial dysfunction.

OBJECTIVE: To investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction. DESIGN: We examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe-/-) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12 weeks with or without inulin-type fructans (ITFs) supplementation for the last 15 days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed. RESULTS: ITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe-/- mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation. CONCLUSIONS: We demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.

Diversification of the functions of proglucagon and glucagon receptor genes in fish.

The teleost fish-specific genome duplication gave rise to a great number of species inhabiting diverse environments with different access to nutrients and life histories. This event produced duplicated gcg genes, gcga and gcgb, for proglucagon-derived peptides, glucagon and GLP-1 and duplicated gcgr receptor genes, gcgra and gcgrb, which play key roles connecting the consumption of nutrients with glucose metabolism. We conducted a systematic survey of the genomes from 28 species of fish (24 bony (Superclass Osteichthyes), 1 lobe-finned (Class Sarcoperygii), 1 cartilaginous (Superclass Chondrichthyes), and 2 jawless (Superclass Agnatha)) and find that almost all surveyed ray-finned fish contain gcga and gcgb genes with different coding potential and duplicated gcgr genes, gcgra and gcgrb that form two separate clades in the phylogenetic tree consistent with the accepted species phylogeny. All gcgb genes encoded only glucagon and GLP-1 and gcga genes encoded glucagon, GLP-1, and GLP-2, indicating that gcga was subfunctionalized to produce GLP-2. We find a single glp2r, but no glp1r suggesting that duplicated gcgrb was neofunctionalized to bind GLP-1, as demonstrated for the zebrafish gcgrb (Oren et al., 2016). In functional experiments with zebrafish gcgrb and GLP-1 from diverse fish we find that anglerfish GLP-1a, encoded by gcga, is less biologically active than the gcgb anglerfish GLP-1b paralog. But some other fish (zebrafish, salmon, and catfish) gcga GLP-1a display similar biological activities, indicating that the regulation of glucose metabolism by GLP-1 in ray-finned fish is species-specific. Searches of genomes in cartilaginous fish identified a proglucagon gene that encodes a novel GLP-3 peptide in addition to glucagon, GLP-1, and GLP-2, as well as a single gcgr, glp2r, and a new glucagon receptor-like receptor whose identity still needs to be confirmed. The sequence of the shark GLP-1 contained an N-terminal mammalian-like extension that in mammals undergoes a proteolytic cleavage to release biologically active GLP-1. Our results indicate that early in vertebrate evolution diverse regulatory mechanisms emerged for the control of glucose metabolism by proglucagon-derived peptides and their receptors and that in ray-finned fish they included subfunctionalization and neofunctionalization of these genes.

Gcg CreERT2 knockin mice as a tool for genetic manipulation in pancreatic alpha cells.

AIMS/HYPOTHESIS: The Cre/loxP system, which enables tissue-specific manipulation of genes, is widely used in mice for diabetes research. Our aim was to develop a new Cre-driver mouse line for the specific and efficient manipulation of genes in pancreatic alpha cells. METHODS: A Gcg CreERT2 knockin mouse, which expresses a tamoxifen-inducible form of Cre from the endogenous preproglucagon (Gcg) gene locus, was generated by homologous recombination. The new Gcg CreERT2 mouse line was crossed to the Rosa26 tdTomato (R26 tdTomato ) Cre reporter mouse line in order to evaluate the tissue specificity, efficiency and tamoxifen dependency of Gcg CreERT2 -mediated recombination. Cell types of pancreatic islets were identified using immunohistochemistry. Biochemical and physiological data, including blood glucose levels, plasma glucagon and glucagon-like peptide (GLP)-1 levels, and pancreatic glucagon content, were collected and used to assess the overall effect of Gcg gene targeting on Gcg CreERT2/w heterozygous mice. RESULTS: Tamoxifen-treated Gcg CreERT2/w ;R26 tdTomato/w mice displayed Cre reporter activity, i.e. expression of tdTomato red fluorescent protein (RFP) in all known cells that produce proglucagon-derived peptides. In the adult pancreas, RFP was detected in 94-97% of alpha cells, whereas it was detected in a negligible (~ 0.2%) proportion of beta cells. While more than 98% of cells labelled with tamoxifen-induced RFP were glucagon-positive cells, 14-25% of pancreatic polypeptide (PP)-positive cells were also positive for RFP, indicating the presence of glucagon/PP bihormonal cell population. Tamoxifen-independent expression of RFP occurred in approximately 6% of alpha cells. In contrast to alpha cells and GLP-1-producing neurons, in which RFP expression persisted for at least 5 months after tamoxifen administration (presumably due to rare neogenesis in these cell types in adulthood), nearly half of RFP-positive intestinal L cells were replaced with RFP-negative L cells over the first 2 weeks after tamoxifen administration. Heterozygous Gcg CreERT2/w mice showed reduced Gcg mRNA levels in islets, but maintained normal levels of pancreatic and plasma glucagon. The mice did not exhibit any detectable baseline physiological abnormalities, at least in young adulthood. CONCLUSIONS/INTERPRETATION: The newly developed Gcg CreERT2 knockin mouse shows faithful expression of CreERT2 in pancreatic alpha cells, intestinal L cells and GLP-1-producing neurons. This mouse line will be particularly useful for manipulating genes in alpha cells, due to highly specific and efficient CreERT2-mediated recombination in this cell type in the pancreas.

Pancreatic alpha cell-selective deletion of Tcf7l2 impairs glucagon secretion and counter-regulatory responses to hypoglycaemia in mice.

AIMS/HYPOTHESIS: Transcription factor 7-like 2 (TCF7L2) is a high mobility group (HMG) box-containing transcription factor and downstream effector of the Wnt signalling pathway. SNPs in the TCF7L2 gene have previously been associated with an increased risk of type 2 diabetes in genome-wide association studies. In animal studies, loss of Tcf7l2 function is associated with defective islet beta cell function and survival. Here, we explore the role of TCF7L2 in the control of the counter-regulatory response to hypoglycaemia by generating mice with selective deletion of the Tcf7l2 gene in pancreatic alpha cells. METHODS: Alpha cell-selective deletion of Tcf7l2 was achieved by crossing mice with floxed Tcf7l2 alleles to mice bearing a Cre recombinase transgene driven by the preproglucagon promoter (PPGCre), resulting in Tcf7l2AKO mice. Glucose homeostasis and hormone secretion in vivo and in vitro, and islet cell mass were measured using standard techniques. RESULTS: While glucose tolerance was unaffected in Tcf7l2AKO mice, glucose infusion rates were increased (AUC for glucose during the first 60 min period of hyperinsulinaemic-hypoglycaemic clamp test was increased by 1.98 ± 0.26-fold [p < 0.05; n = 6] in Tcf7l2AKO mice vs wild-type mice) and glucagon secretion tended to be lower (plasma glucagon: 0.40 ± 0.03-fold vs wild-type littermate controls [p < 0.01; n = 6]). Tcf7l2AKO mice displayed reduced fasted plasma glucose concentration. Glucagon release at low glucose was impaired in islets isolated from Tcf7l2AKO mice (0.37 ± 0.02-fold vs islets from wild-type littermate control mice [p < 0.01; n = 6). Alpha cell mass was also reduced (72.3 ± 20.3% [p < 0.05; n = 7) in Tcf7l2AKO mice compared with wild-type mice. CONCLUSIONS/INTERPRETATION: The present findings demonstrate an alpha cell-autonomous role for Tcf7l2 in the control of pancreatic glucagon secretion and the maintenance of alpha cell mass and function.

Visualization of glucagon secretion from pancreatic α cells by bioluminescence video microscopy: Identification of secretion sites in the intercellular contact regions.

We have firstly visualized glucagon secretion using a method of video-rate bioluminescence imaging. The fusion protein of proglucagon and Gaussia luciferase (PGCG-GLase) was used as a reporter to detect glucagon secretion and was efficiently expressed in mouse pancreatic α cells (αTC1.6) using a preferred human codon-optimized gene. In the culture medium of the cells expressing PGCG-GLase, luminescence activity determined with a luminometer was increased with low glucose stimulation and KCl-induced depolarization, as observed for glucagon secretion. From immunochemical analyses, PGCG-GLase stably expressed in clonal αTC1.6 cells was correctly processed and released by secretory granules. Luminescence signals of the secreted PGCG-GLase from the stable cells were visualized by video-rate bioluminescence microscopy. The video images showed an increase in glucagon secretion from clustered cells in response to stimulation by KCl. The secretory events were observed frequently at the intercellular contact regions. Thus, the localization and frequency of glucagon secretion might be regulated by cell-cell adhesion.

Proposed mechanisms for oligonucleotide IMT504 induced diabetes reversion in a mouse model of immunodependent diabetes.

Type 1 diabetes (T1D) originates from autoimmune ß-cell destruction. IMT504 is an immunomodulatory oligonucleotide that increases mesenchymal stem cell cloning capacity and reverts toxic diabetes in rats. Here, we evaluated long-term (20 doses) and short-term (2-6 doses) effects of IMT504 (20 mg·kg(-1)·day(-1) sc) in an immunodependent diabetes model: multiple low-dose streptozotocin-injected BALB/c mice (40 mg·kg(-1)·day(-1) ip for 5 consecutive days). We determined blood glucose, glucose tolerance, serum insulin, islet morphology, islet infiltration, serum cytokines, progenitor cell markers, immunomodulatory proteins, proliferation, apoptosis, and islet gene expression. IMT504 reduced glycemia, induced ß-cell recovery, and impaired islet infiltration. IMT504 induced early blood glucose decrease and infiltration inhibition, increased ß-cell proliferation and decreased apoptosis, increased islet indoleamine 2,3-dioxygenase (IDO) expression, and increased serum tumor necrosis factor and interleukin-6 (IL-6). IMT504 affected islet gene expression; preproinsulin-2, proglucagon, somatostatin, nestin, regenerating gene-1, and C-X-C motif ligand-1 cytokine (Cxcl1) increased in islets from diabetic mice and were decreased by IMT504. IMT504 downregulated platelet endothelial cell adhesion molecule-1 (Pecam1) in islets from control and diabetic mice, whereas it increased regenerating gene-2 (Reg2) in islets of diabetic mice. The IMT504-induced increase in IL-6 and islet IDO expression and decreased islet Pecam1 and Cxcl1 mRNA expression could participate in keeping leukocyte infiltration at bay, whereas upregulation of Reg2 may mediate ß-cell regeneration. We conclude that IMT504 effectively reversed immunodependent diabetes in mice. Corroboration of these effects in a model of autoimmune diabetes more similar to human T1D could provide promising results for the treatment of this disease.

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.

Association of TCF7L2 and GCG Gene Variants with Insulin Secretion, Insulin Resistance, and Obesity in New-onset Diabetes.

This cohort study was designed to evaluate the association of transcription factor 7-like 2 (TCF7L2) and proglucagon gene (GCG) variants with disordered glucose metabolism and the incidence of type 2 diabetes mellitus (T2DM) in a rural adult Chinese population. A total of 7,751 non-T2DM participants ⋝18 years old genotyped at baseline were recruited. The same questionnaire interview and physical and blood biochemical examinations were performed at both baseline and follow-up. During a median 6 years of follow-up, T2DM developed in 227 participants. After adjustment for potential contributory factors, nominally significant associations were seen between TT genotype and the recessive model of TCF7L2 rs7903146 and increased risk of T2DM [hazard ratio (HR)=4.068, 95% confidence interval (CI): 1.270-13.026; HR=4.051, 95% CI: 1.268-12.946, respectively]. The TT genotype of rs7903146 was also significantly associated with higher fasting plasma insulin level and the homeostasis model assessment of insulin resistance in case of new-onset diabetes. In addition, the TCF7L2 rs290487 TT genotype was associated with abdominal obesity and the GCG rs12104705 CC genotype was associated with both general obesity and abdominal obesity in case of new-onset diabetes.

Two dipolar α-helices within hormone-encoding regions of proglucagon are sorting signals to the regulated secretory pathway.

Proglucagon is expressed in pancreatic α cells, intestinal L cells, and some hypothalamic and brainstem neurons. Tissue-specific processing of proglucagon yields three major peptide hormones as follows: glucagon in the α cells and glucagon-like peptides (GLP)-1 and -2 in the L cells and neurons. Efficient sorting and packaging into the secretory granules of the regulated secretory pathway in each cell type are required for nutrient-regulated secretion of these proglucagon-derived peptides. Our previous work suggested that proglucagon is directed into granules by intrinsic sorting signals after initial processing to glicentin and major proglucagon fragment (McGirr, R., Guizzetti, L., and Dhanvantari, S. (2013) J. Endocrinol. 217, 229-240), leading to the hypothesis that sorting signals may be present in multiple domains. In the present study, we show that the α-helices within glucagon and GLP-1, but not GLP-2, act as sorting signals by efficiently directing a heterologous secretory protein to the regulated secretory pathway. Biophysical characterization of these peptides revealed that glucagon and GLP-1 each encode a nonamphipathic, dipolar α-helix, whereas the helix in GLP-2 is not dipolar. Surprisingly, glicentin and major proglucagon fragment were sorted with different efficiencies, thus providing evidence that proglucagon is first sorted to granules prior to processing. In contrast to many other prohormones in which sorting is directed by ordered prodomains, the sorting determinants of proglucagon lie within the ordered hormone domains of glucagon and GLP-1, illustrating that each prohormone has its own sorting "signature."

Nesfatin-1 stimulates glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide secretion from STC-1 cells in vitro.

Nesfatin-1 is an 82 amino acid peptide encoded in a secreted precursor, nucleobindin 2. It is an anorexigenic and insulinotropic peptide found abundantly in the hypothalamus, pancreas and gastric oxyntic mucosa. NUCB2 mRNA expression is 10 fold higher in the gastric mucosa than in brain, suggesting gastrointestinal tract as a main source of nesfatin-1. Meal responsive insulin secretion is regulated by incretins glucagon-like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP). Since both nesfatin-1 and incretins modulate insulin secretion, we hypothesized that nesfatin-1 is present in the enteroendocrine cells, and that it regulates incretin secretion. RT-PCR analysis found NUCB2 mRNA expression, and immunofluorescence microscopy determined nesfatin-1 immunoreactivity in STC-1, an enteroendocrine cell line. NUCB2/nesfatin-1 is co-localized with GLP-1 and GIP in mouse small intestinal cells. Static incubation of STC-1 cells with nesfatin-1 upregulated preproglucagon (GLP-1 precursor) mRNA (0.01, 0.1, 1 and 10 nM) and GLP-1 secretion (0.1, 1 and 10 nM). Nesfatin-1 also enhanced GIP mRNA (0.1, 1 and 10 nM) and GIP secretion (1 and 10 nM). Together, our data support the hypothesis that nesfatin-1 is present in enteroendocrine cells and that it stimulates incretin secretion. Future studies should aim for nesfatin-1 and incretin interactions in vivo.

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.

Hypoxia decreases glucagon-like peptide-1 secretion from the GLUTag cell line.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is secreted from L cells located in the intestinal epithelium. It is known that intestinal oxygen tension is decreased postprandially. In addition, we found that the expression of hypoxia-inducible factor-1α (HIF-1α), which accumulates in cells under hypoxic conditions, was significantly increased in the colons of mice with food intake, indicating that the oxygen concentration is likely reduced in the colon after eating. Therefore, we hypothesized that GLP-1 secretion is affected by oxygen tension. We found that forskolin-stimulated GLP-1 secretion from GLUTag cells, a model of intestinal L cells, is suppressed in hypoxia (1% O2). Forskolin-stimulated elevations of preproglucagon (ppGCG) and proprotein convertase 1/3 (PC1/3) mRNA expression were decreased under hypoxic conditions. The finding that H89, a protein kinase A (PKA) inhibitor, inhibited the forskolin-stimulated increase of ppGCG and PC1/3 indicated that the cAMP-PKA pathway is involved in the hypoxia-induced suppression of the genes. Hypoxia decreased hexokinase 2 mRNA and protein expression and increased lactate dehydrogenase A mRNA and protein expression. Concomitantly, lactate production was increased and ATP production was decreased. Together, the results indicate that hypoxia decreases glucose utilization for ATP production, which probably causes a decrease in cAMP production and in subsequent GLP-1 production. Our findings suggest that the postprandial decrease in oxygen tension in the intestine attenuates GLP-1 secretion.

p21-Activated protein kinases and their emerging roles in glucose homeostasis.

p21-Activated protein kinases (PAKs) are centrally involved in a plethora of cellular processes and functions. Their function as effectors of small GTPases Rac1 and Cdc42 has been extensively studied during the past two decades, particularly in the realms of cell proliferation, apoptosis, and hence tumorigenesis, as well as cytoskeletal remodeling and related cellular events in health and disease. In recent years, a large number of studies have shed light onto the fundamental role of group I PAKs, most notably PAK1, in metabolic homeostasis. In skeletal muscle, PAK1 was shown to mediate the function of insulin on stimulating GLUT4 translocation and glucose uptake, while in pancreatic ß-cells, PAK1 participates in insulin granule localization and vesicle release. Furthermore, we demonstrated that PAK1 mediates the cross talk between insulin and Wnt/ß-catenin signaling pathways and hence regulates gut proglucagon gene expression and the production of the incretin hormone glucagon-like peptide-1 (GLP-1). The utilization of chemical inhibitors of PAK and the characterization of Pak1(-/-) mice enabled us to gain mechanistic insights as well as to assess the overall contribution of PAKs in metabolic homeostasis. This review summarizes our current understanding of PAKs, with an emphasis on the emerging roles of PAK1 in glucose homeostasis.

Meal feeding improves oral glucose tolerance in male rats and causes adaptations in postprandial islet hormone secretion that are independent of plasma incretins or glycemia.

Meal-fed (MF) rats with access to food for only 4 consecutive hours during the light cycle learn to eat large meals to maintain energy balance. MF animals develop behavioral and endocrine changes that permit glucose tolerance despite increased meal size. We hypothesized that enhanced activity of the enteroinsular axis mediates glucose homeostasis during MF. Cohorts of rats were allocated to MF or ad libitum (AL) regimens for 2-4 wk. Insulin secretion and glucose tolerance were determined after oral carbohydrate and intraperitoneal (ip) and intravenous (iv) glucose. MF rats ate less than AL in the first week but maintained a comparable weight trajectory thereafter. MF rats had decreased glucose excursions after a liquid mixed meal (AUC: MF 75 ± 7, AL 461 ± 28 mmol·l⁻¹·min, P < 0.001), with left-shifted insulin secretion (AUC(0-15): MF 31.0 ± 4.9, AL 9.6 ± 4.4 pM·min, P < 0.02), which peaked before a significant rise in blood glucose. Both groups had comparable fasting glucagon levels, but postprandial responses were lower with MF. However, neither intestinal expression of proGIP and proglucagon mRNA nor plasma incretin levels differed between MF and AL groups. There were no differences in the insulin response to ip or iv glucose between MF and AL rats. These findings demonstrate that MF improves oral glucose tolerance and is associated with significant changes in postprandial islet hormone secretion. Because MF enhanced ß-cell function during oral but not parenteral carbohydrate administration, and was not accounted for by changes in circulating incretins, these results support a neural mechanism of adaptive insulin secretion.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Expression of the GCG gene and secretion of active glucagon-like peptide-1 varies along the length of intestinal tract in horses.

BACKGROUND: Active glucagon-like peptide-1 (aGLP-1) has been implicated in the pathogenesis of equine insulin dysregulation (ID), but its role is unclear. Cleavage of proglucagon (coded by the GCG gene) produces aGLP-1 in enteral L cells. OBJECTIVES: The aim in vivo was to examine the sequence of the exons of GCG in horses with and without ID, where aGLP-1 was higher in the group with ID. The aims in vitro were to identify and quantify the expression of GCG in the equine intestine (as a marker of L cells) and determine intestinal secretion of aGLP-1. STUDY DESIGN: Genomic studies were case-control studies. Expression and secretion studies in vitro were cross-sectional. METHODS: The GCG gene sequence of the exons was determined using a hybridisation capture protocol. Expression and quantification of GCG in samples of stomach duodenum, jejunum, ileum, caecum and ascending and descending colon was achieved with droplet digital PCR. For secretory studies tissue explants were incubated with 12 mM glucose and aGLP-1 secretion was measured with an ELISA. RESULTS: Although the median [IQR] post-prandial aGLP-1 concentrations were higher (p = 0.03) in animals with ID (10.2 [8.79-15.5]), compared with healthy animals (8.47 [6.12-11.7]), there was 100% pairwise identity of the exons of the GCG sequence for the cohort. The mRNA concentrations of GCG and secretion of aGLP-1 differed (p < 0.001) throughout the intestine. MAIN LIMITATIONS: Only the exons of the GCG gene were sequenced and breeds were not compared. The horses used for the study in vitro were not assessed for ID and different horses were used for the small, and large, intestinal studies. CONCLUSIONS: Differences in post-prandial aGLP-1 concentration were not due to a variant in the exons of the GCG gene sequence in this cohort. Both the large and small intestine are sites of GLP-1 secretion.