Dissociable hindbrain GLP1R circuits for satiety and aversion.

The most successful obesity therapeutics, glucagon-like peptide-1 receptor (GLP1R) agonists, cause aversive responses such as nausea and vomiting1,2, effects that may contribute to their efficacy. Here, we investigated the brain circuits that link satiety to aversion, and unexpectedly discovered that the neural circuits mediating these effects are functionally separable. Systematic investigation across drug-accessible GLP1R populations revealed that only hindbrain neurons are required for the efficacy of GLP1-based obesity drugs. In vivo two-photon imaging of hindbrain GLP1R neurons demonstrated that most neurons are tuned to either nutritive or aversive stimuli, but not both. Furthermore, simultaneous imaging of hindbrain subregions indicated that area postrema (AP) GLP1R neurons are broadly responsive, whereas nucleus of the solitary tract (NTS) GLP1R neurons are biased towards nutritive stimuli. Strikingly, separate manipulation of these populations demonstrated that activation of NTSGLP1R neurons triggers satiety in the absence of aversion, whereas activation of APGLP1R neurons triggers strong aversion with food intake reduction. Anatomical and behavioural analyses revealed that NTSGLP1R and APGLP1R neurons send projections to different downstream brain regions to drive satiety and aversion, respectively. Importantly, GLP1R agonists reduce food intake even when the aversion pathway is inhibited. Overall, these findings highlight NTSGLP1R neurons as a population that could be selectively targeted to promote weight loss while avoiding the adverse side effects that limit treatment adherence.

No direct effect of SGLT2 activity on glucagon secretion.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. METHODS: We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. RESULTS: Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. CONCLUSIONS/INTERPRETATION: Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets.

AIMS/HYPOTHESIS: Intra-islet and gut-islet crosstalk are critical in orchestrating basal and postprandial metabolism. The aim of this study was to identify regulatory proteins and receptors underlying somatostatin secretion though the use of transcriptomic comparison of purified murine alpha, beta and delta cells. METHODS: Sst-Cre mice crossed with fluorescent reporters were used to identify delta cells, while Glu-Venus (with Venus reported under the control of the Glu [also known as Gcg] promoter) mice were used to identify alpha and beta cells. Alpha, beta and delta cells were purified using flow cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases. RESULTS: A database was constructed of all genes expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused pancreases, correlating with a decrease in insulin and glucagon release. The inhibition of insulin secretion by ghrelin was prevented by somatostatin receptor antagonism. CONCLUSIONS/INTERPRETATION: Our transcriptomic database of genes expressed in the principal islet cell populations will facilitate rational drug design to target specific islet cell types. The present study indicates that ghrelin acts specifically on delta cells within pancreatic islets to elicit somatostatin secretion, which in turn inhibits insulin and glucagon release. This highlights a potential role for ghrelin in the control of glucose metabolism.

Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion.

Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 (αTC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from αTC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not αTC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary ß- than in α-cells. Thus, suppressed glycerol phosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice.

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

GIPR Function in the Central Nervous System: Implications and Novel Perspectives for GIP-Based Therapies in Treating Metabolic Disorders.

During the past decade, pharmaceutical engineering of unimolecular agents has revealed the therapeutic potential of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism. From this work, one of the most intriguing findings is that engagement of GIPR enhances the weight loss profile of glucagon-like peptide 1 (GLP-1)-based therapeutics. Consequently, this pharmacological approach, in combination with novel Gipr mouse models, has provided evidence indicating that activation of GIPR in certain areas of the brain that regulate energy balance is required for the synergistic weight loss of dual GIPR and GLP-1 receptor (GLP-1R) agonism. This has led to significant interest in understanding how GIPR activity in the brain functions to reduce caloric intake, induce negative energy balance, and drive weight loss. Herein, we review key findings in this field and provide a novel perspective explaining how GIP may act in the brain to affect energy balance both alone and in concert with GLP-1R agonism.

The core clock gene, Bmal1, and its downstream target, the SNARE regulatory protein secretagogin, are necessary for circadian secretion of glucagon-like peptide-1.

OBJECTIVES: The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted from intestinal L-cells upon nutrient intake. While recent evidence has shown that GLP-1 is released in a circadian manner in rats, whether this occurs in mice and if this pattern is regulated by the circadian clock remain to be elucidated. Furthermore, although circadian GLP-1 secretion parallels expression of the core clock gene Bmal1, the link between the two remains largely unknown. Secretagogin (Scgn) is an exocytotic SNARE regulatory protein that demonstrates circadian expression and is essential for insulin secretion from ß-cells. The objective of the current study was to establish the necessity of the core clock gene Bmal1 and the SNARE protein SCGN as essential regulators of circadian GLP-1 secretion. METHODS: Oral glucose tolerance tests were conducted at different times of the day on 4-hour fasted C57BL/6J, Bmal1 wild-type, and Bmal1 knockout mice. Mass spectrometry, RNA-seq, qRT-PCR and/or microarray analyses, and immunostaining were conducted on murine (m) and human (h) primary L-cells and mGLUTag and hNCI-H716 L-cell lines. At peak and trough GLP-1 secretory time points, the mGLUTag cells were co-stained for SCGN and a membrane-marker, ChIP was used to analyze BMAL1 binding sites in the Scgn promoter, protein interaction with SCGN was tested by co-immunoprecipitation, and siRNA was used to knockdown Scgn for GLP-1 secretion assay. RESULTS: C57BL/6J mice displayed a circadian rhythm in GLP-1 secretion that peaked at the onset of their feeding period. Rhythmic GLP-1 release was impaired in Bmal1 knockout (KO) mice as compared to wild-type controls at the peak (p < 0.05) but not at the trough secretory time point. Microarray identified SNARE and transport vesicle pathways as highly upregulated in mGLUTag L-cells at the peak time point of GLP-1 secretion (p < 0.001). Mass spectrometry revealed that SCGN was also increased at this time (p < 0.001), while RNA-seq, qRT-PCR, and immunostaining demonstrated Scgn expression in all human and murine primary L-cells and cell lines. The mGLUTag and hNCI-H716 L-cells exhibited circadian rhythms in Scgn expression (p < 0.001). The ChIP analysis demonstrated increased binding of BMAL1 only at the peak of Scgn expression (p < 0.01). Immunocytochemistry showed the translocation of SCGN to the cell membrane after stimulation at the peak time point only (p < 0.05), while CoIP showed that SCGN was pulled down with SNAP25 and ß-actin, but only the latter interaction was time-dependent (p < 0.05). Finally, Scgn siRNA-treated cells demonstrated significantly blunted GLP-1 secretion (p < 0.01) in response to stimulation at the peak time point only. CONCLUSIONS: These data demonstrate, for the first time, that mice display a circadian pattern in GLP-1 secretion, which is impaired in Bmal1 knockout mice, and that Bmal1 regulation of Scgn expression plays an essential role in the circadian release of the incretin hormone GLP-1.

Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake.

Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.

Distribution and Stimulus Secretion Coupling of Enteroendocrine Cells along the Intestinal Tract.

The enteroendocrine system of the gut acts both locally and peripherally, regulating gastrointestinal function as well as metabolism, energy expenditure, and central appetite control through the release of a variety of hormones. The chemosensing ability of enteroendocrine cells is integral to their role in eliciting physiological changes in response to fluctuations in the composition of the intestinal lumen. Regulation of enteroendocrine cell activity is complex, and requires that these cells can integrate signals deriving from dietary sources as well as the nervous and endocrine systems. Here, we provide an overview of enteroendocrine cell form and function, with a focus on new insights into their distribution throughout the intestine and the stimulus secretion coupling mechanisms underlying the activity of these important members of the gut-brain axis. © 2018 American Physiological Society. Compr Physiol 8:1603-1638, 2018.

Identification and characterization of GLP-1 receptor-expressing cells using a new transgenic mouse model.

GLP-1 is an intestinal hormone with widespread actions on metabolism. Therapies based on GLP-1 are highly effective because they increase glucose-dependent insulin secretion in people with type 2 diabetes, but many reports suggest that GLP-1 has additional beneficial or, in some cases, potentially dangerous actions on other tissues, including the heart, vasculature, exocrine pancreas, liver, and central nervous system. Identifying which tissues express the GLP-1 receptor (GLP1R) is critical for the development of GLP-1-based therapies. Our objective was to use a method independent of GLP1R antibodies to identify and characterize the targets of GLP-1 in mice. Using newly generated glp1r-Cre mice crossed with fluorescent reporter strains, we show that major sites of glp1r expression include pancreatic ß- and δ-cells, vascular smooth muscle, cardiac atrium, gastric antrum/pylorus, enteric neurones, and vagal and dorsal root ganglia. In the central nervous system, glp1r-fluorescent cells were abundant in the area postrema, arcuate nucleus, paraventricular nucleus, and ventromedial hypothalamus. Sporadic glp1r-fluorescent cells were found in pancreatic ducts. No glp1r-fluorescence was observed in ventricular cardiomyocytes. Enteric and vagal neurons positive for glp1r were activated by GLP-1 and may contribute to intestinal and central responses to locally released GLP-1, such as regulation of intestinal secretomotor activity and appetite.

Reversible changes in pancreatic islet structure and function produced by elevated blood glucose.

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that ß-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some ß-cells begin expressing glucagon, whilst retaining many ß-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.