PPG neurons in the nucleus of the solitary tract modulate heart rate but do not mediate GLP-1 receptor agonist-induced tachycardia in mice.

OBJECTIVE: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are used as anti-diabetic drugs and are approved for obesity treatment. However, GLP-1RAs also affect heart rate (HR) and arterial blood pressure (ABP) in rodents and humans. Although the activation of GLP-1 receptors (GLP-1R) is known to increase HR, the circuits recruited are unclear, and in particular, it is unknown whether GLP-1RAs activate preproglucagon (PPG) neurons, the brain source of GLP-1, to elicit these effects. METHODS: We investigated the effect of GLP-1RAs on heart rate in anaesthetized adult mice. In a separate study, we manipulated the activity of nucleus tractus solitarius (NTS) PPG neurons (PPGNTS) in awake, freely behaving transgenic Glu-Cre mice implanted with biotelemetry probes and injected with AAV-DIO-hM3Dq:mCherry or AAV-mCherry-FLEX-DTA. RESULTS: Systemic administration of the GLP-1RA Ex-4 increased resting HR in anaesthetized or conscious mice, but had no effect on ABP in conscious mice. This effect was abolished by ß-adrenoceptor blockade with atenolol, but unaffected by the muscarinic antagonist atropine. Furthermore, Ex-4-induced tachycardia persisted when PPGNTS neurons were ablated, and Ex-4 did not induce expression of the neuronal activity marker cFos in PPGNTS neurons. PPGNTS ablation or acute chemogenetic inhibition of these neurons via hM4Di receptors had no effect on resting HR. In contrast, chemogenetic activation of PPGNTS neurons increased resting HR. Furthermore, the application of GLP-1 within the subarachnoid space of the middle thoracic spinal cord, a major projection target of PPG neurons, increased HR. CONCLUSIONS: These results demonstrate that both systemic application of Ex-4 or GLP-1 and chemogenetic activation of PPGNTS neurons increases HR. Ex-4 increases the activity of cardiac sympathetic preganglionic neurons of the spinal cord without recruitment of PPGNTS neurons, and thus likely recapitulates the physiological effects of PPG neuron activation. These neurons therefore do not play a significant role in controlling resting HR and ABP but are capable of inducing tachycardia and so are likely involved in cardiovascular responses to acute stress.

EJE PRIZE 2018: A gut feeling about glucagon.

Hyperglucagonaemia (in the fasting as well as in the postprandial state) is considered a core pathophysiological component of diabetes and is found to contribute substantially to the hyperglycaemic state of diabetes. Hyperglucagonaemia is usually viewed upon as a consequence of pancreatic alpha cell insensitivity to the glucagon-suppressive effects of glucose and insulin. Since we observed that the well-known hyperglucagonaemic response to oral glucose in patients with type 2 diabetes is exchanged by normal suppression of plasma glucagon levels following isoglycaemic intravenous glucose administration in these patients, we have been focusing on the gut and gut-derived factors as potential mediators of diabetic hyperglucagonaemia. In a series of clinical experiments, we have elucidated the role of gut-derived factors in diabetic hyperglucagonaemia and shown that glucose-dependent insulinotropic polypeptide promotes hyperglucagonaemia and that glucagon, hitherto considered a pancreas-specific hormone, may also be secreted from extrapancreatic tissues - most likely from proglucagon-producing enteroendocrine cells. Furthermore, our observation that fasting hyperglucagonaemia is unrelated to the diabetic state, but strongly correlates with obesity, liver fat content and circulating amino acids, has made us question the common 'pancreacentric' and 'glucocentric' understanding of hyperglucagonaemia and led to the hypothesis that steatosis-induced hepatic glucagon resistance (and reduced amino acid turnover) and compensatory glucagon secretion mediated by increased circulating amino acids constitute a complete endocrine feedback system: the liver-alpha cell axis. This article summarises the physiological regulation of glucagon secretion in humans and considers new findings suggesting that the liver and the gut play key roles in determining fasting and postabsorptive circulating glucagon levels.

Glucagon-like peptide-1 receptor activation in the ventral tegmental area attenuates cocaine seeking in rats.

Novel molecular targets are needed to develop new medications for the treatment of cocaine addiction. Here we investigated a role for glucagon-like peptide-1 (GLP-1) receptors in the reinstatement of cocaine-seeking behavior, an animal model of relapse. We showed that peripheral administration of the GLP-1 receptor agonist exendin-4 dose dependently reduced cocaine seeking in rats at doses that did not affect ad libitum food intake, meal patterns or body weight. We also demonstrated that systemic exendin-4 penetrated the brain where it putatively bound receptors on both neurons and astrocytes in the ventral tegmental area (VTA). The effects of systemic exendin-4 on cocaine reinstatement were attenuated in rats pretreated with intra-VTA infusions of the GLP-1 receptor antagonist exendin-(9-39), indicating that the suppressive effects of systemic exendin-4 on cocaine seeking were due, in part, to activation of GLP-1 receptors in the VTA. Consistent with these effects, infusions of exendin-4 directly into the VTA reduced cocaine seeking. Finally, extinction following cocaine self-administration was associated with decreased preproglucagon mRNA expression in the caudal brainstem. Thus, our study demonstrated a novel role for GLP-1 receptors in the reinstatement of cocaine-seeking behavior and identified behaviorally relevant doses of a GLP-1 receptor agonist that selectively reduced cocaine seeking and did not produce adverse effects.

Insights on glicentin, a promising peptide of the proglucagon family.

Glicentin is a proglucagon-derived peptide mainly produced in the L-intestinal cells. While the roles of other members of the proglucagon family including glucagon-like peptide 1, glucagon-like peptide 2 and oxyntomodulin has been well studied, the functions and variation of glicentin in human are not fully understood. Experimental and clinical studies have highlighted its role in both intestinal physiology and glucose metabolism, pointing to its potential interest in a wide range of pathological states including gastrointestinal and metabolic disorders. Due to its structure presenting many similarities with the other proglucagon-derived peptides, its measurement is technically challenging. The recent commercialization of specific detection methods has offered new opportunities to go further in the understanding of glicentin physiology. Here we summarize the current knowledge on glicentin biogenesis and physiological roles. In the limelight of clinical studies investigating glicentin variation in human, we discuss future directions for potential applications in clinical practice.

Proglucagons in vertebrates: Expression and processing of multiple genes in a bony fish.

In contrast to mammals, where a single proglucagon (PG) gene encodes three peptides: glucagon, glucagon-like peptide 1 and glucagon-like peptide 2 (GLP-1; GLP-2), many non-mammalian vertebrates carry multiple PG genes. Here, we investigate proglucagon mRNA sequences, their tissue expression and processing in a diploid bony fish. Copper rockfish (Sebastes caurinus) express two independent genes coding for distinct proglucagon sequences (PG I, PG II), with PG II lacking the GLP-2 sequence. These genes are differentially transcribed in the endocrine pancreas, the brain, and the gastrointestinal tract. Alternative splicing identified in rockfish is only one part of this complex regulation of the PG transcripts: the system has the potential to produce two glucagons, four GLP-1s and a single GLP-2, or any combination of these peptides. Mass spectrometric analysis of partially purified PG-derived peptides in endocrine pancreas confirms translation of both PG transcripts and differential processing of the resulting peptides. The complex differential regulation of the two PG genes and their continued presence in this extant teleostean fish strongly suggests unique and, as yet largely unidentified, roles for the peptide products encoded in each gene.

Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells.

GLUTag, NCI-H716, and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide-1 (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis samples; n = 9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n = 6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2, glicentin, and oxyntomodulin in a pattern (prohormone convertase (PC)1/3 dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and GLP-2, which increased after stimulation. In contrast to freshly isolated murine L-cells, all cell lines also expressed PC2 and secreted large amounts of pancreatic glucagon. Neurotensin and somatostatin storage was low and secretion was not consistently increased by stimulation. STC-1 cells released more glucose-dependent insulinotropic polypeptide than GLP-1 at baseline (P < 0.01) and KCl elevated its secretion (P < 0.05). Peptide YY, which normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of CCK, while NCI-H716 did not store this peptide and STC-1 contained low amounts. Our results show that hormone production in cell line models of the L-cell has limited similarity to the natural L-cells.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion.

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in pGIP/neo STC-1 cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3ß-O-ß-D-glycopyranoside and QA-3ß-O-ß-D-glucopyranosyl-(28→1)-ß-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

Chickens from lines artificially selected for juvenile low and high body weight differ in glucose homeostasis and pancreas physiology.

Artificial selection of White Plymouth Rock chickens for juvenile (day 56) body weight resulted in two divergent genetic lines: hypophagic low weight (LWS) chickens and hyperphagic obese high weight (HWS) chickens, with the latter more than 10-fold heavier than the former at selection age. A study was designed to investigate glucose regulation and pancreas physiology at selection age in LWS chickens and HWS chickens. Oral glucose tolerance and insulin sensitivity tests revealed differences in threshold sensitivity to insulin and glucose clearance rate between the lines. Results from real-time PCR showed greater pancreatic mRNA expression of four glucose regulatory genes (preproinsulin, PPI; preproglucagon, PPG; glucose transporter 2, GLUT2; and pancreatic duodenal homeobox 1, Pdx1) in LWS chickens, than HWS chickens. Histological analysis of the pancreas revealed that HWS chickens have larger pancreatic islets, less pancreatic islet mass, and more pancreatic inflammation than LWS chickens, all of which presumably contribute to impaired glucose metabolism.

Stimulation of proglucagon gene expression by human GPR119 in enteroendocrine L-cell line GLUTag.

GPR119 is a G protein-coupled receptor expressed on enteroendocrine L-cells that synthesize and secrete the incretin hormone glucagon-like peptide-1 (GLP-1). Although GPR119 agonists stimulate L-cell GLP-1 secretion, there is uncertainty concerning whether GLP-1 biosynthesis is under the control of GPR119. Here we report that GPR119 is functionally coupled to increased proglucagon (PG) gene expression that constitutes an essential first step in GLP-1 biosynthesis. Using a mouse L-cell line (GLUTag) that expresses endogenous GPR119, we demonstrate that PG gene promoter activity is stimulated by GPR119 agonist AS1269574. Surprisingly, transfection of GLUTag cells with recombinant human GPR119 (hGPR119) results in a constitutive and apparently ligand-independent increase of PG gene promoter activity and PG mRNA content. These constitutive actions of hGPR119 are mediated by cAMP-dependent protein kinase (PKA) but not cAMP sensor Epac2. Thus, the constitutive action of hGPR119 to stimulate PG gene promoter activity is diminished by: 1) a dominant-negative Gαs protein, 2) a dominant-negative PKA regulatory subunit, and 3) a dominant-negative A-CREB. Interestingly, PG gene promoter activity is stimulated by 6-Bn-cAMP-AM, a cAMP analog that selectively activates α and ß isoforms of type II, but not type I PKA regulatory subunits expressed in GLUTag cells. Finally, our analysis reveals that a specific inhibitor of Epac2 activation (ESI-05) fails to block the stimulatory action of 6-Bn-cAMP-AM at the PG gene promoter, nor is PG gene promoter activity stimulated by: 1) a constitutively active Epac2, or 2) cAMP analogs that selectively activate Epac proteins. Such findings are discussed within the context of ongoing controversies concerning the relative contributions of PKA and Epac2 to the control of PG gene expression.

CTCF mediates effect of insulin on glucagon expression.

Pancreatic islet α-cell development and glucagon production are mainly regulated by Pax6 in the homeobox gene families. However, the molecular mechanism fine-tuning the regulation of these events in α-cell still remains unclear. In ocular cells, Pax6 transcription is regulated by CTCF through its binding to specific sites in Pax6 promoter. In this study, CTCF-mediated regulations of islet α-cell development and glucagon production were investigated in both CTCF transgenic mice and α-TC-1-6 cells. Over-expression of CTCF in transgenic mice affected development of pancreatic islets by significantly suppressing α-cell population in both embryonic and adult pancreases. The effect of CTCF on Pax6 gene expression and subsequently, on pro-glucagon production was however, examined in pancreatic islet α-cells. Over-expression and knock-down of CTCF directly affected Pax6 expression. More importantly, the CTCF binding sites upstream from Pax6 p0 promoter were required for regulating p0 promoter activity in islet α-cells. Stimulation of α-cells with insulin resulted in a significant increase in CTCF expression and a decrease in Pax6 expression, and consequently suppressed pro-glucagon expression. In contrast, these insulin-induced effects were blocked by knockdown of CTCF mRNA with specific siRNA in α-cells. Altogether, our results demonstrated for the first time that CTCF functions as a switch-like molecule between the insulin signaling and the regulations of Pax6 and glucagon expression in pancreatic islet α-cells.

Self-inducible secretion of glucagon-like peptide-1 (GLP-1) that allows MIN6 cells to maintain insulin secretion and insure cell survival.

Based on the hypothesis that MIN6 cells could produce glucagon-like peptide-1 (GLP-1) to maintain cell survival, we analyzed the effects of GLP-1 receptor agonist, exendin-4 (Ex4), and antagonist, exendin-(9-39) (Ex9) on cell function and cell differentiation. MIN6 cells expressed proglucagon mRNAs and produced GLP-1, which was accelerated by Ex4 and suppressed by Ex9. Moreover, Ex4 further enhanced glucose-stimulated GLP-1 secretion, suggesting autocrine loop-contributed amplification of the GLP-1 signal. Ex4 up-regulated cell differentiation- and cell function-related CREBBP, Pdx-1, Pax6, proglucagon, and PC1/3 gene expressions. The confocal laser scanning images revealed that GLP-1 positive cells were dominant in the early stage of cells, but positive for insulin were more prominent in the mature stage of cells. Ex4 accelerated cell viability, while Ex9 and anti-GLP-1 receptor antibody enhanced cell apoptosis. MIN6 cells possess a mechanism of GLP-1 signal amplification in an autocrine fashion, by which the cells maintained insulin production and cell survival.

The brainstem preproglucagon system in a non-human primate (Macaca mulatta).

The nucleus of the solitary tract (NTS) contains a small population of neurons expressing preproglucagon. In these neurons preproglucagon is processed to the glucagon-like-peptides 1 and 2 (GLP-1 and GLP-2) and oxyntomodulin. Whereas the neuroanatomy of these neurons is well characterized in rodents the location and projection of preproglucagon neurons have never been described in primates. The purpose of the present study was to characterize the location of preproglucagon neurons and their projections in the non-human primate using radioactive in situ hybridization and immunohistochemistry. In situ hybridization revealed preproglucagon mRNA expressing neurons in the caudal nucleus of the solitary tract extending laterally through the intermediate reticular nucleus into the A1 area. Using an antibody raised against rat GLP-2, GLP-2-immunoreactive (-ir) cell bodies were found in the same areas as the preproglucagon mRNA. Only very few GLP-2-ir nerve fibers were observed in the caudal brainstem and mostly in the same areas as the GLP-2-ir cell bodies. The most prominent GLP-2-ir terminal fields were detected in the hypothalamus and rostrally in the bed nucleus of the stria terminalis complex. In the hypothalamus, GLP-2-ir fibers arborized extensively in the paraventricular nucleus of the hypothalamus (PVN), the dorsomedial hypothalamic nucleus (DMH) and the arcuate nucleus (Arc), the latter containing the densest fiber-plexus. The findings indicate that the brainstem preproglucagon neuronal system is highly conserved between rat and non-human primate with the exception of a much denser innervation of the mediobasal hypothalamus in the primate brain.

Pax6 controls the expression of critical genes involved in pancreatic {alpha} cell differentiation and function.

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased ß and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Huang-lian-jie-du-decoction modulates glucagon-like peptide-1 secretion in diabetic rats.

AIM OF THE STUDY: Huang-lian-jie-du-decoction (HLJDD), a well-known Chinese herbal formula, has been used for diabetic treatment. The purpose of the study was to investigate whether HLJDD affected glucagon-like peptide (GLP)-1 (7-36) amide level in diabetic rats. MATERIALS AND METHODS: Streptozotocin (STZ)-induced diabetic rats were treated with HLJDD at low dose (2 g/kg/day) or high dose (4 g/kg/day). After 5-week treatment, GLP-1 (7-36) amide level and insulin level in portal vein and tissues stimulated by oral glucose load were measured by ELISA kits. The proglucagon gene expression in intestinal tracts and the proliferation of intestinal L cell and pancreatic beta cell were measured using RT-PCR and immunohistochemistry techniques, respectively. RESULTS: It was found that 5-week HLJDD treatment attenuated alteration of glucose level and insulin level in plasma and tissues of diabetic rats induced by STZ, accompanied by improvement of diabetic syndrome. 5-week HLJDD treatment increased GLP-1 (7-36) amide level in portal vein plasma and distal ileum. Further studies showed that 5-week HLJDD treatment increased the mRNA level of proglucagon gene in distal ileum, promoted pancreatic beta cell and intestinal L cell proliferation in a dose-dependent manner. CONCLUSION: All the results indicated that HLJDD exerted its anti-diabetic effects partly via modulating GLP-1 (7-36) amide level.

Glucocorticoid regulation of preproglucagon transcription and RNA stability during stress.

Stress elicits a synchronized response of the endocrine, sympathetic, and central nervous systems to preserve homeostasis and well-being. Glucagon-like peptide-1 (GLP-1), a primary posttranslational product of the preproglucagon (PPG) gene, activates both physical and psychological stress responses. The current study examined mechanisms regulating expression of PPG gene products in the hindbrain. Our results indicate that PPG mRNA decreases rapidly after exposure to acute stressors of multiple modalities. Reduced mRNA levels are accompanied by reduced GLP-1 immunoreactivity in the paraventricular nucleus of hypothalamus, suggesting release at PPG terminals. Stress-induced decrements in PPG mRNA were attenuated in adrenalectomized-corticosterone-replaced rats, suggesting that mRNA down-regulation is due at least in part to glucocorticoid secretion. In contrast, acute stress increased levels of PPG heteronuclear RNA (hnRNA) in a glucocorticoid-dependent manner, suggesting that decreases in PPG mRNA are due to increased degradation rather than reduced transcription. Glucocorticoid administration to unstressed rats is sufficient to cause decrements in PPG mRNA and increments in PPG hnRNA. These findings suggest that glucocorticoids deplete the pool of transcribed PPG mRNA and concurrently stimulate PPG gene transcription, with the latter allowing a mechanism for replenishment of PPG mRNA after stress cessation. The combination of rapid PPG mRNA depletion and initiation of PPG transcription within 30 min is consistent with a rapid action of glucocorticoids on GLP-1 bioavailability, resulting in a transient reduction in the capacity for neuropeptidergic excitation of stress responses.

Epac is involved in cAMP-stimulated proglucagon expression and hormone production but not hormone secretion in pancreatic alpha- and intestinal L-cell lines.

Both Epac and PKA are effectors of the second messenger cAMP. Utilizing an exchange protein directly activated by cAMP (Epac) pathway-specific cAMP analog (ESCA), we previously reported that Epac signaling regulates proglucagon gene (gcg) expression in the glucagon-like peptide-1 (GLP-1)-producing intestinal endocrine L-cell lines GLUTag and STC-1. We now show that Epac-2 is also expressed in glucagon-producing pancreatic alpha-cell lines, including PKA-deficient InR1-G9 cells, and that ESCA stimulates gcg promoter and mRNA expression in the InR1-G9 cells. Using a dominant-negative Epac-2 expression plasmid (Epac-2DN), we found that Epac inhibition attenuated forskolin-stimulated gcg promoter expression in the PKA-active STC-1 cell line and blocked forskolin-stimulated gcg promoter expression in the InR1-G9 cells. Consistently, ESCA was shown to stimulate glucagon and GLP-1 production in the InR1-G9 and GLUTag cell lines, respectively. Surprisingly, ESCA treatment did not show a notable stimulation of glucagon or GLP-1 secretion from these two cell lines. This is in contrast to its ability to stimulate insulin secretion from the pancreatic INS-1 beta-cell line. Our findings suggest that Epac is selectively involved in peptide hormone secretion in pancreatic and intestinal endocrine cells and that distinct signaling cascades are involved in stimulating production vs. secretion of glucagon and GLP-1 in response to cAMP elevation.

Expression of proglucagon and proglucagon-derived peptide hormone receptor genes in the chicken.

To better understand how the proglucagon system functions in birds, we utilized a molecular cloning strategy to sequence and characterize the chicken proglucagon gene that encodes glucagon, glucagon-like peptide (GLP)-1 and GLP-2. This gene has seven exons and six introns with evidence for an additional (alternate) first exon and two promoter regions. We identified two distinct classes of proglucagon mRNA transcripts (PGA and PGB) produced by alternative splicing at their 3'-ends. These were co-expressed in all tissues examined with pancreas and proventriculus showing the highest levels of each. Although both mRNA classes contained coding sequence for glucagon and GLP-1, class A mRNA lacked that portion of the coding region (CDS) containing GLP-2; whereas, class B mRNA had a larger CDS that included GLP-2. Both classes of mRNA transcripts exhibited two variants, each with a different 5'-end arising from alternate promoter and alternate first exon usage. Fasting and refeeding had no effect on proglucagon mRNA expression despite significant changes in plasma glucagon levels. To investigate potential differences in proglucagon precursor processing among tissues, mRNA expression for two prohormone convertase (PC) genes was analyzed. PC2 mRNA was predominantly expressed in pancreas and proventriculus, whereas PC1/3 mRNA was more highly expressed in duodenum and brain. We also determined mRNA expression of the specific receptor genes for glucagon, GLP-1 and GLP-2 to help define major sites of hormone action. Glucagon receptor mRNA was most highly expressed in liver and abdominal fat, whereas GLP-1 and GLP-2 receptor genes were highly expressed in the gastrointestinal tract, brain, pancreas and abdominal fat. These results offer new insights into structure and function of the chicken proglucagon gene, processing of the precursor proteins produced from it and potential activity sites for proglucagon-derived peptide hormones mediated by their cognate receptors.

Physiological effects of dietary fructans extracted from Agave tequilana Gto. and Dasylirion spp.

Recent data reported that inulin-type fructans extracted from chicory roots regulate appetite and lipid/glucose metabolism, namely, by promoting glucagon-like peptide-1 (GLP-1) production in the colon. The Agave genus growing in different regions of Mexico also contains important amounts of original fructans, with interesting nutritional and technological properties, but only few data report their physiological effect when added in the diet. Therefore, we decided to evaluate in parallel the effect of supplementation with 10 % agave or chicory fructans on glucose and lipid metabolism in mice. Male C57Bl/6J mice were fed a standard (STD) diet or diet supplemented with Raftilose P95 (RAF), fructans from Agave tequilana Gto. (TEQ) or fructans from Dasylirion spp. (DAS) for 5 weeks. The body weight gain and food intake in mice fed fructans-containing diets were significantly lower than the ones of mice fed the STD diet, TEQ leading to the lowest value. Serum glucose and cholesterol were similarly lower in all fructans-fed groups than in the STD group and correlated to body weight gain. Only RAF led to a significant decrease in serum TAG. As previously shown for RAF, the supplementation with agave fructans (TEQ and DAS) induced a higher concentration of GLP-1 and its precursor, proglucagon mRNA, in the different colonic segments, thus suggesting that fermentable fructans from different botanical origin and chemical structure are able to promote the production of satietogenic/incretin peptides in the lower part of the gut, with promising effects on glucose metabolism, body weight and fat mass development.

Upregulation of the brainstem preproglucagon system in the obese Zucker rat.

A group of neurons in the caudal nucleus of the solitary tract (NTS) processes preproglucagon to glucagon-like peptide 1 (GLP-1), GLP-2 and oxyntomodulin. Whereas the anorectic capacity of all three neuropeptides has been demonstrated, only relatively little is known of preproglucagon mRNA regulation in the brain stem. Using in situ hybridization and fluorescence immunohistochemistry, we examined hindbrain preproglucagon expression in lean and obese Zucker rats under different metabolic perturbations. First, the effect of an acute 48-h fast was examined in male Sprague-Dawley as well as in lean and obese Zucker rats. Whereas fasting had no effect on preproglucagon expression in either genotype, mRNA levels were strongly up regulated in obese Zucker rats. Using a direct immunostaining procedure and a monoclonal GLP-2 antibody, we found a doubling of the immunofluorescence signal emanating from the preproglucagon neurons in caudal brainstem suggesting that indeed the high mRNA levels observed using in situ hybridization histochemistry also reflect a higher translational activity. To investigate the effects of long-term body weight perturbations, lean and obese Zucker rats were either free-fed, voluntarily overfed (chocolate spread enriched chow) or food restricted for 35 days. Preproglucagon levels remained high in the obese Zucker rats irrespective of diet. Finally, in order to functionally validate the apparent hyperactivity in the preproglucagon system in the Zucker rat, we examined the effect of central GLP-1 receptor blockade. ICV administration of 20 microg of the GLP-1 receptor antagonist Des-His-Exendin-9-39 in the morning increased 4-h food intake in obese but not in lean Zucker rats, pointing to an increased activity in central preproglucagon containing pathways in leptin receptor deficient rats. Our data suggest that the preproglucagon neurons in the brainstem are influenced by leptin signaling and point to a role of preproglucagon neurons in the integration of metabolic signals that occurs in the nucleus of the solitary tract.

Glucagon-like peptide-1 receptor and proglucagon expression in mouse skin.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone expressed by alternative post-translational processing of proglucagon in the intestines, endocrine pancreas, and brain. The multiple antidiabetogenic actions of GLP-1 include stimulation of the proliferation and differentiation of the insulin-producing beta cells in the pancreas. The GLP-1 receptor is widely distributed and has been identified in the endocrine pancreas, intestinal tract, brain, lung, kidney, and heart. Here we report the expression of the GLP-1 receptor and proglucagon in the skin of newborn mice located predominantly in the hair follicles, as well as in cultures of skin-derived cells that also express nestin, a marker of cultured cells that have dedifferentiated by epithelial to mesenchymal transition. In cultured skin cells, GLP-1 activates the MAPK/ERK signal transduction pathway, associated with cellular proliferation, differentiation, and cytoprotection. No evidence was found for the activation of cAMP or Ca2+ signaling pathways. Further, redifferentiation of cultured skin-derived cells by incubation in differentiation medium containing GLP-1 induced expression of the proinsulin-derived peptide, C-peptide. These findings suggest a possible paracrine/autocrine role for GLP-1 and its receptor in skin development and possibly also in folliculogenesis.