Intestinal epithelial adaptations to vertical sleeve gastrectomy defined at single-cell resolution.

The gut plays a key role in regulating metabolic health. Dietary factors disrupt intestinal physiology and contribute to obesity and diabetes, whereas bariatric procedures such as vertical sleeve gastrectomy (VSG) cause gut adaptations that induce robust metabolic improvements. However, our understanding of these adaptations at the cellular and molecular levels remains limited. In a validated murine model, we leverage single-cell transcriptomics to determine how VSG impacts different cell lineages of the small intestinal epithelium. We define cell type-specific genes and pathways that VSG rescues from high-fat diet perturbation and characterize additional rescue-independent changes brought about by VSG. We show that Paneth cells have increased expression of the gut peptide Reg3g after VSG. We also find that VSG restores pathways pertaining to mitochondrial respiration and cellular metabolism, especially within crypt-based cells. Overall, our study provides unprecedented molecular resolution of VSG's therapeutic effects on the gut epithelium.

Neither GLP-1 receptors nor GFRAL neurons are required for aversive or anorectic response to DON (vomitoxin).

Deoxynivalenol (DON), a type B trichothecene mycotoxin contaminating grains, promotes nausea, emesis and anorexia. With DON exposure, circulating levels of intestinally derived satiation hormones, including glucagon-like peptide 1 (GLP-1) are elevated. To directly test whether GLP-1 signaling mediates the effects of DON, we examined the response of GLP-1 or GLP-1R-deficient mice to DON injection. We found comparable anorectic and conditioned taste avoidance learning responses in GLP-1/GLP-1R deficient mice compared to control littermates, suggesting that GLP-1 is not necessary for the effects of DON on food intake and visceral illness. We then used our previously published data from translating ribosome affinity purification with RNA sequencing (TRAP-seq) analysis of area postrema neurons that express the receptor for the circulating cytokine growth differentiation factor (GDF15), growth differentiation factor a-like (GFRAL). Interestingly, this analysis showed that a cell surface receptor for DON, calcium sensing receptor (CaSR), is heavily enriched in GFRAL neurons. Given that GDF15 potently reduces food intake and can cause visceral illness by signaling through GFRAL neurons, we hypothesized that DON may also signal by activating CaSR on GFRAL neurons. Indeed, circulating GDF15 levels are elevated after DON administration but both GFRAL knockout and GFRAL neuron-ablated mice exhibited similar anorectic and conditioned taste avoidance responses compared to WT littermates. Thus, GLP-1 signaling and GFRAL signaling and neurons are not required for DON-induced visceral illness or anorexia.

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.

Glucose metabolism after bariatric surgery: implications for T2DM remission and hypoglycaemia.

Although promising therapeutics are in the pipeline, bariatric surgery (also known as metabolic surgery) remains our most effective strategy for the treatment of obesity and type 2 diabetes mellitus (T2DM). Of the many available options, Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) are currently the most widely used procedures. RYGB and VSG have very different anatomical restructuring but both surgeries are effective, to varying degrees, at inducing weight loss and T2DM remission. Both weight loss-dependent and weight loss-independent alterations in multiple tissues (such as the intestine, liver, pancreas, adipose tissue and skeletal muscle) yield net improvements in insulin resistance, insulin secretion and insulin-independent glucose metabolism. In a subset of patients, post-bariatric hypoglycaemia can develop months to years after surgery, potentially reflecting the extreme effects of potent glucose reduction after surgery. This Review addresses the effects of bariatric surgery on glucose regulation and the potential mechanisms responsible for both the resolution of T2DM and the induction of hypoglycaemia.

Differential regulation of nucleus accumbens glutamate and GABA in obesity-prone and obesity-resistant rats.

Obesity is one of the leading health concerns in the United States. Studies from human and rodent models suggest that inherent differences in the function of brain motivation centers, including the nucleus accumbens (NAc), contribute to overeating and thus obesity. For example, there are basal enhancements in the excitability of NAc GABAergic medium spiny neurons (MSN) and reductions in basal expression of AMPA-type glutamate receptors in obesity-prone vs obesity-resistant rats. However, very little is known about the regulation of extracellular glutamate and GABA within the NAc of these models. Here we gave obesity-prone and obesity-resistant rats stable isotope-labeled glucose (13 C6 -glucose) and used liquid chromatography mass spectrometry (LC-MS) analysis of NAc dialysate to examine the real-time incorporation of 13 C6 -glucose into glutamate, glutamine, and GABA. This novel approach allowed us to identify differences in glucose utilization for neurotransmitter production between these selectively bred lines. We found that voluntarily ingested or gastrically infused 13 C6 -glucose rapidly enters the NAc and is incorporated into 13 C2 -glutamine, 13 C2 -glutamate, and 13 C2 -GABA in both groups within minutes. However, the magnitude of increases in NAc 13 C2 -glutamine and 13 C2 -GABA were lower in obesity-prone than in obesity-resistant rats, while basal levels of glutamate were elevated. This suggested that there may be differences in the astrocytic regulation of these analytes. Thus, we next examined NAc glutamine synthetase, GAD67, and GLT-1 protein expression. Consistent with reduced 13 C2 -glutamine and 13 C2 -GABA, NAc glutamine synthetase and GLT-1 protein expression were reduced in obesity-prone vs obesity-resistant groups. Taken together, these data show that NAc glucose utilization differs dramatically between obesity-prone and obesity-resistant rats, favoring glutamate over GABA production in obesity-prone rats and that reductions in NAc astrocytic recycling of glutamate contribute to these differences. These data are discussed in light of established differences in NAc function between these models and the role of the NAc in feeding behavior.

The gut peptide Reg3g links the small intestine microbiome to the regulation of energy balance, glucose levels, and gut function.

Changing composition of the gut microbiome is an important component of the gut adaptation to various environments, which have been implicated in various metabolic diseases including obesity and type 2 diabetes, but the mechanisms by which the microbiota influence host physiology remain contentious. Here we find that both diets high in the fermentable fiber inulin and vertical sleeve gastrectomy increase intestinal expression and circulating levels of the anti-microbial peptide Reg3g. Moreover, a number of beneficial effects of these manipulations on gut function, energy balance, and glucose regulation are absent in Reg3g knockout mice. Peripheral administration of various preparations of Reg3g improves glucose tolerance, and this effect is dependent on the putative receptor Extl3 in the pancreas. These data suggest Reg3g acts both within the lumen and as a gut hormone to link the intestinal microbiome to various aspects of host physiology that may be leveraged for novel treatment strategies.

Gut Factors Mediating the Physiological Impact of Bariatric Surgery.

Despite decades of obesity research and various public health initiatives, obesity remains a major public health concern. Our most drastic but most effective treatment of obesity is bariatric surgery with weight loss and improvements in co-morbidities, including resolution of type 2 diabetes (T2D). However, the mechanisms by which surgery elicits metabolic benefits are still not well understood. One proposed mechanism is through signals generated by the intestine (nutrients, neuronal, and/or endocrine) that communicate nutrient status to the brain. In this review, we discuss the contributions of gut-brain communication to the physiological regulation of body weight and its impact on the success of bariatric surgery. Advancing our understanding of the mechanisms that drive bariatric surgery-induced metabolic benefits will ultimately lead to the identification of novel, less invasive strategies to treat obesity.

Vertical sleeve gastrectomy induces enteroendocrine cell differentiation of intestinal stem cells through bile acid signaling.

Vertical sleeve gastrectomy (VSG) results in an increase in the number of hormone-secreting enteroendocrine cells (EECs) in the intestinal epithelium; however, the mechanism remains unclear. Notably, the beneficial effects of VSG are lost in a mouse model lacking the nuclear bile acid receptor farnesoid X receptor (FXR). FXR is a nuclear transcription factor that has been shown to regulate intestinal stem cell (ISC) function in cancer models. Therefore, we hypothesized that the VSG-induced increase in EECs is due to changes in intestinal differentiation driven by an increase in bile acid signaling through FXR. To test this, we performed VSG in mice that express EGFP in ISC/progenitor cells and performed RNA-Seq on GFP-positive cells sorted from the intestinal epithelia. We also assessed changes in EEC number (marked by glucagon-like peptide-1, GLP-1) in mouse intestinal organoids following treatment with bile acids, an FXR agonist, and an FXR antagonist. RNA-Seq of ISCs revealed that bile acid receptors are expressed in ISCs and that VSG explicitly alters expression of several genes that regulate EEC differentiation. Mouse intestinal organoids treated with bile acids and 2 different FXR agonists increased GLP-1-positive cell numbers, and administration of an FXR antagonist blocked these effects. Taken together, these data indicate that VSG drives ISC fate toward EEC differentiation through bile acid signaling.

Vertical sleeve gastrectomy increases duodenal Lactobacillus spp. richness associated with the activation of intestinal HIF2α signaling and metabolic benefits.

OBJECTIVE: Vertical Sleeve Gastrectomy (VSG) is one of the most efficacious treatments for obesity and its comorbidities. Although a range of evidence suggests that alterations of the microbiota in the distal gut following VSG are pivotal to these metabolic improvements, the effect of surgery to alter the microbiota of the proximal intestine and its effect on host physiology remain largely unknown. As the main bacteria in the upper small intestine, Lactobacillus subspecies have been appreciated as important regulators of gut function. These bacteria also regulate intestinal Hypoxia- Inducible Factor 2α (HIF2α) signaling that plays an integral role in gut physiology and iron absorption. In the present study, we sought to determine the impact of VSG on Lactobacillus spp. in the small intestine and potential downstream impacts of Lactobacillus spp. on HIF2α, specifically in the duodenum. METHODS: To determine the effects of VSG on the microbiota and HIF2α signaling in the duodenum, VSG surgeries were performed on diet-induced obese mice. To further probe the relationship between Lactobacillus spp. and HIF2α signaling in the duodenum, we applied a customized high-fat but iron-deficient diet on mice to increase duodenal HIF2α signaling and determined alterations of gut bacteria. To explore the causal role of Lactobacillus spp. in duodenal HIF2α signaling activation, we chronically administered probiotics containing Lactobacillus spp. to high-fat-fed obese mice. Lastly, we studied the effect of lactate, the major metabolite of Lactobacilli, on HIF2α in ex vivo duodenal organoids. RESULTS: There were pronounced increases in the abundance of Lactobacillus spp. in samples isolated from duodenal epithelium in VSG-operated mice as compared to sham-operated mice. This was accompanied by an increase in the expression of genes that are targets of HIF2α in the duodenum of VSG-treated mice. Activating HIF2α signaling with a high-fat but iron-deficient diet resulted in weight loss, improvements in glucose regulation, and increased Lactobacillus spp. richness in the duodenum as compared to mice on an iron-replete diet. Chronic administration of probiotics containing Lactobacillus spp. not only increased HIF2α signaling in the duodenum such as occurs after VSG but also resulted in reduced weight gain and improved glucose tolerance in high-fat-fed mice. Furthermore, lactate was able to activate HIF2α in ex vivo duodenal organoids. CONCLUSIONS: These results support a model whereby VSG increases duodenal Lactobacillus richness and potentially stimulates intestinal HIF2α signaling via increased lactate production.

Gut HIF2α signaling is increased after VSG, and gut activation of HIF2α decreases weight, improves glucose, and increases GLP-1 secretion.

Gastric bypass and vertical sleeve gastrectomy (VSG) remain the most potent and durable treatments for obesity and type 2 diabetes but are also associated with iron deficiency. The transcription factor HIF2α, which regulates iron absorption in the duodenum, increases following these surgeries. Increasing iron levels by means of dietary supplementation or hepatic hepcidin knockdown does not undermine the effects of VSG, indicating that metabolic improvements following VSG are not secondary to lower iron levels. Gut-specific deletion of Vhl results in increased constitutive duodenal HIF2α signaling and produces a profound lean, glucose-tolerant phenotype that mimics key effects of VSG. Interestingly, intestinal Vhl deletion also results in increased intestinal secretion of GLP-1, which is essential for these metabolic benefits. These data demonstrate a role for increased duodenal HIF2α signaling in regulating crosstalk between iron-regulatory systems and other aspects of systemic physiology important for metabolic regulation.

Proceedings from the Albert Charitable Trust Inaugural Workshop on 'Understanding the Acute Effects of Exercise on the Brain'.

An inaugural workshop supported by "The Leo and Anne Albert Charitable Trust," was held October 4-7, 2019 in Scottsdale, Arizona, to focus on the effects of exercise on the brain and to discuss how physical activity may prevent or delay the onset of aging-related neurodegenerative conditions. The Scientific Program Committee (led by Dr. Jeff Burns) assembled translational, clinical, and basic scientists who research various aspects of the effects of exercise on the body and brain, with the overall goal of gaining a better understanding as to how to delay or prevent neurodegenerative diseases. In particular, research topics included the links between cardiorespiratory fitness, the cerebrovasculature, energy metabolism, peripheral organs, and cognitive function, which are all highly relevant to understanding the effects of acute and chronic exercise on the brain. The Albert Trust workshop participants addressed these and related topics, as well as how other lifestyle interventions, such as diet, affect age-related cognitive decline associated with Alzheimer's and other neurodegenerative diseases. This report provides a synopsis of the presentations and discussions by the participants, and a delineation of the next steps towards advancing our understanding of the effects of exercise on the aging brain.

Intestinal-derived FGF15 protects against deleterious effects of vertical sleeve gastrectomy in mice.

Bariatric surgeries such as the Vertical Sleeve Gastrectomy (VSG) are invasive but provide the most effective improvements in obesity and Type 2 diabetes. We hypothesized a potential role for the gut hormone Fibroblast-Growth Factor 15/19 which is increased after VSG and pharmacologically can improve energy homeostasis and glucose handling. We generated intestinal-specific FGF15 knockout (FGF15INT-KO) mice which were maintained on high-fat diet. FGF15INT-KO mice lost more weight after VSG as a result of increased lean tissue loss. FGF15INT-KO mice also lost more bone density and bone marrow adipose tissue after VSG. The effect of VSG to improve glucose tolerance was also absent in FGF15INT-KO. VSG resulted in increased plasma bile acid levels but were considerably higher in VSG-FGF15INT-KO mice. These data point to an important role after VSG for intestinal FGF15 to protect the organism from deleterious effects of VSG potentially by limiting the increase in circulating bile acids.

Preproglucagon Products and Their Respective Roles Regulating Insulin Secretion.

Historically, intracellular function and metabolic adaptation within the α-cell has been understudied, with most of the attention being placed on the insulin-producing ß-cells due to their role in the pathophysiology of type 2 diabetes mellitus. However, there is a growing interest in understanding the function of other endocrine cell types within the islet and their paracrine role in regulating insulin secretion. For example, there is greater appreciation for α-cell products and their contributions to overall glucose homeostasis. Several recent studies have addressed a paracrine role for α-cell-derived glucagon-like peptide-1 (GLP-1) in regulating glucose homeostasis and responses to metabolic stress. Further, other studies have demonstrated the ability of glucagon to impact insulin secretion by acting through the GLP-1 receptor. These studies challenge the central dogma surrounding α-cell biology describing glucagon's primary role in glucose counterregulation to one where glucagon is critical in regulating both hyper- and hypoglycemic responses. Herein, this review will update the current understanding of the role of glucagon and α-cell-derived GLP-1, placing emphasis on their roles in regulating glucose homeostasis, insulin secretion, and ß-cell mass.

ARCGHR Neurons Regulate Muscle Glucose Uptake.

The growth hormone receptor (GHR) is expressed in brain regions that are known to participate in the regulation of energy homeostasis and glucose metabolism. We generated a novel transgenic mouse line (GHRcre) to characterize GHR-expressing neurons specifically in the arcuate nucleus of the hypothalamus (ARC). Here, we demonstrate that ARCGHR+ neurons are co-localized with agouti-related peptide (AgRP), growth hormone releasing hormone (GHRH), and somatostatin neurons, which are activated by GH stimulation. Using the designer receptors exclusively activated by designer drugs (DREADD) technique to control the ARCGHR+ neuronal activity, we demonstrate that the activation of ARCGHR+ neurons elevates a respiratory exchange ratio (RER) under both fed and fasted conditions. However, while the activation of ARCGHR+ promotes feeding, under fasting conditions, the activation of ARCGHR+ neurons promotes glucose over fat utilization in the body. This effect was accompanied by significant improvements in glucose tolerance, and was specific to GHR+ versus GHRH+ neurons. The activation of ARCGHR+ neurons increased glucose turnover and whole-body glycolysis, as revealed by hyperinsulinemic-euglycemic clamp studies. Remarkably, the increased insulin sensitivity upon the activation of ARCGHR+ neurons was tissue-specific, as the insulin-stimulated glucose uptake was specifically elevated in the skeletal muscle, in parallel with the increased expression of muscle glycolytic genes. Overall, our results identify the GHR-expressing neuronal population in the ARC as a major regulator of glycolysis and muscle insulin sensitivity in vivo.

Diet-dependent sex differences in the response to vertical sleeve gastrectomy.

Nearly 80% of patients that receive bariatric surgery are women, yet mechanistic preclinical studies have focused on males. The goal of this study was to determine the metabolic impact of diet- and surgery-induced weight loss in males, females, and ovariectomized females. All mice were fed a 60% high-fat diet (HFD) before undergoing either vertical sleeve gastrectomy (VSG) or sham surgery. Mice either remained on an HFD or were switched to a standard chow diet postsurgically. When maintained on an HFD, males and females decreased fat mass and improved oral glucose tolerance after VSG. After dietary intervention, additional adiposity was lost in both surgical groups. Ovariectomized females showed a blunted decrease in fat mass on an HFD, but lost significant adiposity after dietary intervention. Energy expenditure was impacted by dietary and not surgical intervention across all groups. Males decreased hepatic triglyceride levels after VSG, which was further decreased after dietary intervention. Intact and ovariectomized females had a blunted decrease in hepatic triglycerides after VSG, but a significant decrease after dietary intervention. The more pronounced effect of VSG on hepatic lipids in males is strongly associated with changes in hepatic expression of genes and microRNAs previously linked to hepatic lipid regulation and systemic energy homeostasis. These data highlight the importance of postsurgical diet on metabolic outcomes across sexes. Furthermore, these data suggest the impact of VSG on hepatic triglycerides is diet-dependent in females and support the hypothesis that males and females achieve similar metabolic outcome, at least within the liver, via distinct mechanisms.NEW & NOTEWORTHY These data highlight the interaction of postsurgical diet after bariatric surgery on metabolic outcomes across sexes. These data suggest the impact of VSG on hepatic triglycerides is diet-dependent in females and support the hypothesis that males and females achieve similar metabolic outcome, at least within the liver, via distinct mechanisms.

The Role of Elevated Branched-Chain Amino Acids in the Effects of Vertical Sleeve Gastrectomy to Reduce Weight and Improve Glucose Regulation.

Elevated levels of branched-chain amino acids (BCAAs) and their metabolites are strongly positively associated with obesity, insulin resistance, and type 2 diabetes. Bariatric surgery is among the best treatments for weight loss and associated morbidities. Clinical studies have reported that bariatric surgery decreases the circulating levels of BCAAs. The objective of this study was to test the hypothesis that reduced BCAA levels contribute to the metabolic improvements of sustained weight loss and improved glucose tolerance after vertical sleeve gastrectomy (VSG). We find that, as in humans, circulating BCAAs are significantly lower in VSG rats and mice. To increase circulating BCAAs, we tested mice with either increased dietary intake of BCAAs or impaired BCAA catabolism by total body deletion of mitochondrial phosphatase 2C (Pp2cm). Our results show that a decrease in circulating BCAAs is not necessary for sustained body weight loss and improved glucose tolerance after VSG.

Assessment of the role of FGF15 in mediating the metabolic outcomes of murine Vertical Sleeve Gastrectomy (VSG).

Vertical sleeve gastrectomy (VSG) is the best current therapy for remission of obesity and its co-morbidities. It is understood to alter the enterohepatic circulation of bile acids in vivo. Fibroblast growth factor 19 (FGF19) in human and its murine orthologue Fgf15 plays a pivotal role in this bile acid driven enterohepatic signaling. The present study evaluated the metabolic outcomes of VSG in Fgf15 deficient mice. 6-8 weeks old male wildtype mice (WT) and Fgf15 deficient mice (KO) were fed a high fat diet (HFD) for 8 weeks. At 8th week of diet, both WT and KO mice were randomly distributed to VSG or sham surgery. Post-surgery, mice were observed for 8 weeks while fed a HFD and then euthanized to collect tissues for experimental analysis. Fgf15 deficient (KO) mice lost weight post VSG, but glucose tolerance in KO mice did not improve post VSG compared to WT mice. Enteroids derived from WT and KO mice proliferated with bile acid exposure in vitro. Post VSG both WT and KO mice had similarly altered bile acid enterohepatic flux, however Fgf15 deficient mice post VSG had increased hepatic accumulation of free and esterified cholesterol leading to lipotoxicity related ER stress, inflammasome activation, and increased Fgf21 expression. Intact Fgf15 mediated enterohepatic bile acid signaling, but not changes in bile acid flux, appear to be important for the metabolic improvements post-murine bariatric surgery. These novel data introduce a potential point of distinction between bile acids acting as ligands compared to their canonical downstream signaling pathways.

Intestine-selective reduction of Gcg expression reveals the importance of the distal gut for GLP-1 secretion.

OBJECTIVE: Glucagon-like peptide-1 is a nutrient-sensitive hormone secreted from enteroendocrine L cells within the small and large bowel. Although GLP-1 levels rise rapidly in response to food ingestion, the greatest density of L cells is localized to the distal small bowel and colon. Here, we assessed the importance of the distal gut in the acute L cell response to diverse secretagogues. METHODS: Circulating levels of glucose and plasma GLP-1 were measured in response to the administration of L cell secretagogues in wild-type mice and in mice with (1) genetic reduction of Gcg expression throughout the small bowel and large bowel (GcgGut-/-) and (2) selective reduction of Gcg expression in the distal gut (GcgDistalGut-/-). RESULTS: The acute GLP-1 response to olive oil or arginine administration was markedly diminished in GcgGut-/- but preserved in GcgDistalGut-/- mice. In contrast, the increase in plasma GLP-1 levels following the administration of the GPR119 agonist AR231453, or the melanocortin-4 receptor (MC4R) agonist LY2112688, was markedly diminished in the GcgDistalGut-/- mice. The GLP-1 response to LPS was also markedly attenuated in the GcgGut-/- mice and remained submaximal in the GcgDistalGut-/- mice. Doses of metformin sufficient to lower glucose and increase GLP-1 levels in the GcgGut+/+ mice retained their glucoregulatory activity, yet they failed to increase GLP-1 levels in the GcgGut-/- mice. Surprisingly, the actions of metformin to increase plasma GLP-1 levels were substantially attenuated in the GcgDistalGut-/- mice. CONCLUSION: These findings further establish the importance of the proximal gut for the acute response to nutrient-related GLP-1 secretagogues. In contrast, we identify essential contributions of the distal gut to (i) the rapid induction of circulating GLP-1 levels in response to pharmacological selective agonism of G-protein-coupled receptors, (ii) the increased GLP-1 levels following the activation of Toll-Like Receptors with LPS, and iii) the acute GLP-1 response to metformin. Collectively, these results reveal that distal gut Gcg + endocrine cells are rapid responders to structurally and functionally diverse GLP-1 secretagogues.