Background: Studies suggest that short chain fatty acids (SCFAs), which are primarily produced from fermentation of fiber, regulate insulin secretion through free fatty acid receptors 2 and 3 (FFA2 and FFA3). As these are G-protein coupled receptors (GPCRs), they have potential therapeutic value as targets for treating type 2 diabetes (T2D). The exact mechanism by which these receptors regulate insulin secretion and other aspects of pancreatic beta cell function is unclear. It has been reported that glucose-dependent release of acetate from pancreatic beta cells negatively regulates glucose stimulated insulin secretion. While these data raise the possibility of acetate's potential autocrine action on these receptors, these findings have not been independently confirmed, and multiple concerns exist with this observation, particularly the lack of specificity and precision of the acetate detection methodology used. Methods: Using Min6 cells and mouse islets, we assessed acetate and pyruvate production and secretion in response to different glucose concentrations, via liquid chromatography mass spectrometry. Results: Using Min6 cells and mouse islets, we showed that both intracellular pyruvate and acetate increased with high glucose conditions; however, intracellular acetate level increased only slightly and exclusively in Min6 cells but not in the islets. Further, extracellular acetate levels were not affected by the concentration of glucose in the incubation medium of either Min6 cells or islets. Conclusions: Our findings do not substantiate the glucose-dependent release of acetate from pancreatic beta cells, and therefore, invalidate the possibility of an autocrine inhibitory effect on glucose stimulated insulin secretion.

Short-chain fatty acids (SCFAs) are key nutrients that play a diverse set of roles in physiological function, including regulating metabolic homeostasis. Generated through the fermentation of dietary fibers in the distal colon by the gut microbiome, SCFAs and their effects are partially mediated by their cognate receptors, including free fatty acid receptor 2 (FFA2). FFA2 is highly expressed in the intestinal epithelial cells, where its putative functions are controversial, with numerous in vivo studies relying on global knockout mouse models to characterize intestine-specific roles of the receptor. Here, we used the Villin-Cre mouse line to generate a novel, intestine-specific knockout mouse model for FFA2 (Vil-FFA2) to investigate receptor function within the intestine. Because dietary changes are known to affect the composition of the gut microbiome, and can thereby alter SCFA production, we performed an obesogenic challenge on male Vil-FFA2 mice and their littermate controls (FFA2-floxed, FFA2fl/fl) to identify physiological changes on a high-fat, high-sugar 'Western diet' (WD) compared to a low-fat control diet (CD). We found that the WD-fed Vil-FFA2 mice were transiently protected from the obesogenic effects of the WD and had lower fat mass and improved glucose homeostasis compared to the WD-fed FFA2fl/fl control group during the first half of the study. Additionally, major differences in respiratory exchange ratio and energy expenditure were observed in the WD-fed Vil-FFA2 mice, and food intake was found to be significantly reduced at multiple points in the study. Taken together, this study uncovers a novel role of intestinal FFA2 in mediating the development of obesity.

Abstract Disclosure: C. Nnyamah: None. B. Wicksteed: None. B.T. Layden: None. The obesity pandemic is a major health concern driving Type 2 Diabetes (T2D) and cardiovascular disease (CVD). One novel pathogenetic factor of obesity is the gut microbiome (GM), that is the microbes that reside in the digestive tract. Changes in the GM have profound effects on obesity. These symbiotic microbes break down dietary fiber that are otherwise indigestible, releasing nutrients (in particular, short chain fatty acids, SCFAs). Free Fatty Acid Receptor 2 (FFA2) is a G-protein coupled receptor (GPCR) that senses SCFAs and has been shown to influence the function of various organs responsible for nutrient-sensing and energy balance. Among other roles, FFA2 has been suggested to modulate the secretion of the appetite-regulating proteins GLP-1 and PYY from intestinal enteroendocrine cells and secretion of insulin (the master fuel-supply hormone) from the pancreatic beta cells. However, its role in adipose tissue is less clear. While highly expressed in adipose tissue, research using global knockouts (KOs) of FFA2 in mouse models has observed conflicting phenotypic results. Here, we use novel adipose-specific FFA2 KO (AdFFA2-KO) mice to explore adipose-determined outcomes when mice are fed obesogenic challenge diets with or without fiber-induced production of SCFAs for receptor activation. After confirming that Adiponectin-Cre x FFA2 fl/fl mice have no congenital defects and are metabolically similar to FFA2 fl/fl control mice on a normal chow diet, mice were placed on either a high fat, high sugar Western Diet (WD) or on WD supplemented with 10% fructooligosaccharides – a fermentable dietary fiber. On WD, AdFFA2-KO mice had significantly decreased glucose tolerance compared to floxed controls with no other clearly observable effects on basic metabolic parameters. Interestingly, upon receptor activation with a fiber-supplemented diet, AdFFA2-KO mice were moderately protected from WD-induced weight gain. Our ongoing research aims to detangle the observed phenotypic differences and demystify the FFA2-adipose signaling cascade using a multifaceted approach including in vivo assessment of food intake and energy expenditure, ex vivo evaluation of lipolysis and fatty-acid uptake with FFA2 agonists and antagonists, as well as RTqPCR probing of adipose-derived cytokines (adipokines) with known roles in the weight gain-weight loss cycle. Presentation: Friday, June 16, 2023

Abstract Hexokinase domain containing protein-1, or HKDC1, is a widely expressed hexokinase that is genetically associated with elevated 2-hour gestational blood glucose levels during an oral glucose tolerance test, suggesting a role for HKDC1 in postprandial glucose regulation during pregnancy. Our earlier studies utilizing mice containing global HKDC1 knockdown, as well as hepatic HKDC1 overexpression and knockout, indicated that HKDC1 is important for whole-body glucose homeostasis in aging and pregnancy, through modulation of glucose tolerance, peripheral tissue glucose utilization, and hepatic energy storage. However, our knowledge of the precise role(s) of HKDC1 in regulating postprandial glucose homeostasis under normal and diabetic conditions is lacking. Since the intestine is the main entry portal for dietary glucose, here we have developed an intestine-specific HKDC1 knockout mouse model, HKDC1Int–/–, to determine the in vivo role of intestinal HKDC1 in regulating glucose homeostasis. While no overt glycemic phenotype was observed, aged HKDC1Int–/– mice fed a high-fat diet exhibited an increased glucose excursion following an oral glucose load compared with mice expressing intestinal HKDC1. This finding resulted from glucose entry via the intestinal epithelium and is not due to differences in insulin levels, enterocyte glucose utilization, or reduction in peripheral skeletal muscle glucose uptake. Assessment of intestinal glucose transporters in high-fat diet–fed HKDC1Int–/– mice suggested increased apical GLUT2 expression in the fasting state. Taken together, our results indicate that intestinal HKDC1 contributes to the modulation of postprandial dietary glucose transport across the intestinal epithelium under conditions of enhanced metabolic stress, such as high-fat diet.

Abstract The obesity pandemic is a major health concern driving Type 2 Diabetes (T2D) and cardiovascular disease (CVD). One novel pathogenetic factor of obesity is the gut microbiome (GM), that is the microbes that reside in the digestive tract. Changes in the GM have profound effects on the obesity. These symbiotic microbes break down dietary fiber that are otherwise indigestible, releasing nutrients (in particular, short chain fatty acids, SCFAs). Free Fatty Acid Receptor 2 (FFA2) is a G-protein coupled receptor (GPCR) that senses SCFAs and influences secretion of the appetite-regulating proteins GLP-1 and PYY from enteroendocrine cells. However, its role in adipose tissue is less clear. While highly expressed in adipose tissue, research using global knockouts (KOs) of FFA2 in mouse models has observed conflicting phenotypic results. Here, we use adipocytes isolated from FFA2-KO and WT mice and SCFA-supplemented media to demonstrate that acetate, a primary ligand of FFA2, leads to increased adipogenesis. This expansion of total adipocyte population rather than individual size has been shown to greatly decrease metabolic health complications in highly obese individuals. Our ongoing research aims to explore the in vitro effects of FFA2 on lipogenesis, lipolysis, and secretion of adipokines. Primarily, we are exploring these outcomes in vivo using novel adipose-specific FFA2 KO (AdFFA2-KO) mice which we show have no congenital defects and are metabolically similar to FFA2 floxxed control mice on a normal chow diet. Our ongoing research challenges the mice with obesogenic diets to observe the effects of metabolic strain on FFA2 function specifically in adipose tissue. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Background and aims Normal gestation involves reprogramming of maternal gut microbiome (GM) that may contribute to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of GM – maternal metabolism interaction. Methods The GM and plasma metabolome of CD1, NIH-Swiss and C57BL/6J mice were analyzed using 16S rRNA sequencing and untargeted LC-MS throughout gestation and postpartum. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM in the process was studied using fecal microbial transplants (FMT). Results Significant variation in gut microbial alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time-point specific, with species enriched at gestation day 15/19 (G15/19), a point of heightened IR, distinct from those enriched pre- or post- pregnancy. Untargeted and targeted metabolomics revealed elevated plasma kynurenine at G15/19 in all three mouse strains. IDO1, the rate limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine levels effects on IR derive from the GM in both mouse and human pregnancy. Conclusions GM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1.

Free fatty acid receptor 3 (FFA3) is a recently-deorphanized G-protein-coupled receptor. Its ligands are short-chain fatty acids (SCFAs), which are key nutrients derived from the gut microbiome fermentation process that play diverse roles in the regulation of metabolic homeostasis and glycemic control. FFA3 is highly expressed within the intestine, where its role and its effects on physiology and metabolism are unclear. Previous in vivo studies involving this receptor have relied on global knockout mouse models, making it difficult to isolate intestine-specific roles of FFA3. To overcome this challenge, we generated an intestine-specific knockout mouse model for FFA3, Villin-Cre-FFA3 (Vil-FFA3). Model validation and general metabolic assessment of male mice fed a standard chow diet revealed no major congenital defects. Because dietary changes are known to alter gut microbial composition, and thereby SCFA production, an obesogenic challenge was performed on male Vil-FFA3 mice and their littermate controls to probe for a phenotype on a high-fat, high-sugar "Western diet" (WD) compared with a low-fat control diet (CD). Vil-FFA3 mice versus FFA3fl/fl controls on WD, but not CD, were protected from the development of diet-induced obesity and exhibited significantly less fat mass as well as smaller adipose depositions and adipocytes. Although overall glycemic control was unchanged in the WD-fed Vil-FFA3 group, fasted glucose levels trended lower. Intestinal inflammation was significantly reduced in the WD-fed Vil-FFA3 mice, supporting protection from obesogenic effects. Furthermore, we observed lower levels of gastric inhibitory protein (GIP) in the WD-fed Vil-FFA3 mice, which may contribute to phenotypic changes. Our findings suggest a novel role of intestinal FFA3 in promoting the metabolic consequences of a WD, including the development of obesity and inflammation. Moreover, these data support an intestine-specific role of FFA3 in whole body metabolic homeostasis and in the development of adiposity.NEW & NOTEWORTHY Here, we generated a novel intestine-specific knockout mouse model for FFA3 (Vil-FFA3) and performed a comprehensive metabolic characterization of mice in response to an obesogenic challenge. We found that Vil-FFA3 mice fed with a Western diet were largely protected from obesity, exhibiting significantly lower levels of fat mass, lower intestinal inflammation, and altered expression of intestinal incretin hormones. Results support an important role of intestinal FFA3 in contributing to metabolism and in the development of diet-induced obesity.

Abstract Free fatty acid receptor 2 and free fatty acid receptor 3 (FFA2/3) are two highly similar G protein-coupled receptors belonging to the free fatty acid receptor family. Their ligands are short-chain fatty acids (SCFAs), which are key nutrients that play a diverse role in physiological function, including the regulation of metabolic homeostasis and glycemic control. FFA2/3 are broadly expressed in a multitude of tissues including the intestine, pancreas, adipose and central nervous system, where they contribute to metabolic homeostasis via a summation of tissue-specific effects. Consequently, FFA2/3 have been identified as a potential drug target for metabolic diseases including obesity and type-2 diabetes. Both FFA2 and FFA3 are highly expressed within the intestinal epithelium – the major site of SCFA generation – and have been identified in hormone-secreting enteroendocrine cells as well as intestinal epithelial cells. However, due conflicting data, the respective roles of FFA2/3 within the intestine and their effects on physiology and metabolism are still largely unclear. Previous in vivo studies involving this receptor have largely relied on global knockout mouse models, making it difficult to isolate their effects in the intestine. To overcome this challenge, we generated a novel intestine-specific knockout mouse model for FFA2 and FFA3 individually, utilizing Cre-mediated recombination under the expression of the villin promoter. Here, we report the first in vivo characterization of FFA2/3 in the intestine and reveal novel insights into receptor function. Following model validation, we conducted a general metabolic assessment of male Villin-Cre-FFA2 (Vil-FFA2) and Villin-Cre-FFA3 (Vil-FFA3) mice on standard chow and observed no major congenital or time-dependent defects. Because dietary changes are known to alter gut microbial composition, and thereby SCFA production, a pilot study was performed on male Vil-FFA2 and Vil-FFA3 mice and their littermate controls to probe for a phenotype on a high-fat, high-sugar "western diet." Mice were placed on either a low-fat control diet (CD) or western diet (WD) at 10 weeks of age and metabolically profiled for 25 weeks. We found that both Vil-FFA2 and Vil-FFA3 mouse strains were largely protected from diet-induced obesity and had significantly lower fat mass as well as adipose hypertrophy. Additionally, both mouse strains had reduced intestinal inflammation and improved glucose homeostasis. These differences were driven by lower food intake in the Vil-FFA2 strain only. Our findings suggest a novel role of FFA2/3 in mediating the metabolic consequences of a western diet – a state of high inflammation, dysbiosis and metabolic stress. Moreover, these data support an intestine-specific role of FFA2/3 in whole-body metabolic homeostasis and in the development of adiposity and hyperglycemia. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

Liver cancer (LC) is the fourth leading cause of death from cancer malignancies. Recently, a putative fifth hexokinase, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in LC compared to healthy liver tissue. Using a combination of in vitro and in vivo tools, we examined the role of HKDC1 in LC development and progression. Importantly, HKDC1 ablation stops LC development and progression via its action at the mitochondria by promoting metabolic reprogramming and a shift of glucose flux away from the TCA cycle. HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy, which cannot be compensated by enhanced glucose uptake. Moreover, we show that the interaction of HKDC1 with the mitochondria is essential for its role in LC progression, and without this interaction, mitochondrial dysfunction occurs. As HKDC1 is highly expressed in LC cells, but only to a minimal degree in hepatocytes under normal conditions, targeting HKDC1, specifically its interaction with the mitochondria, may represent a highly selective approach to target cancer cells in LC.

Abstract Hexokinase domain containing protein-1, or HKDC1, is a widely expressed novel hexokinase that is genetically associated with elevated 2-hour gestational blood glucose levels during an oral glucose tolerance test, suggesting a role for HKDC1 in postprandial glucose regulation during pregnancy. Our earlier studies utilizing transgenic mice containing whole-body HKDC1 knockdown, or mice in which hepatic HKDC1 was overexpressed or knocked out, indicated that HKDC1 is important for whole-body glucose homeostasis in aging and pregnancy, through modulation of glucose tolerance, peripheral tissue glucose utilization, and hepatic energy storage. However, our knowledge of the precise mechanisms by which HKDC1 regulates postprandial glucose homeostasis under normal and diabetic conditions is lacking. As the intestine is the main entry portal for dietary glucose, and since HKDC1 is highly expressed within the intestine, in this study we assessed and characterized the in vivo significance of intestine-specific HKDC1 in regulating glucose homeostasis under normal and obesogenic conditions. We developed an intestine-specific HKDC1 knockout mouse model, HKDC1Int-/-, utilizing Cre-mediated recombination of HKDC1 in which Cre was expressed under the control of the villin gene promoter, leading to genetic knockout of HKDC1 solely within the intestinal epithelium. Mice were maintained until 28 weeks of age on either a normal chow diet or a high fat diet to develop obesity, hyperglycemia, and insulin resistance. While no overt glycemic phenotype was observed, 28-week-old HKDC1Int-/- mice fed a high fat diet exhibited an increased glucose excursion following an oral glucose load compared to mice expressing intestinal HKDC1. This finding was not due to differences in insulin levels, whole-body insulin tolerance, or gluconeogenesis, nor was it a result of alterations in enterocyte glucose utilization or a reduction in peripheral skeletal muscle glucose uptake. Furthermore, the enhanced glucose excursion was related to transport of glucose through the intestinal epithelium, as mice administered an intraperitoneal glucose load did not exhibit alterations in post-load glycemic excursion. Assessment of intestinal glucose transporters in high fat diet-fed HKDC1Int-/- mice indicated an increased expression of GLUT2 in the enterocyte apical membrane in the fasting state. Taken together, our results indicate that intestine-specific HKDC1 contributes to postprandial glycemic regulation by modulating dietary glucose transport across the intestinal epithelium under conditions of enhanced metabolic stress, such as obesity, hyperglycemia, and diabetes. Presentation: Saturday, June 11, 2022 1:24 p.m. - 1:29 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

The gut microbiome has emerged as a novel determinant of type 1 diabetes (T1D), but the underlying mechanisms are unknown. In this context, major gut microbial metabolites, short-chain fatty acids (SCFAs), are considered to be an important link between the host and gut microbiome. We, along with other laboratories, have explored how SCFAs and their cognate receptors affect various metabolic conditions, including obesity, type 2 diabetes, and metabolic syndrome. Though gut microbiome and SCFA-level changes have been reported in T1D and in mouse models of the disease, the role of SCFA receptors in T1D remains under explored. In this review article, we will highlight the existing and possible roles of these receptors in T1D pathology. We conclude with a discussion of SCFA receptors as therapeutic targets for T1D, exploring an exciting new potential for novel treatments of glucometabolic disorders.

Short chain fatty acids (SCFAs), which are gut microbial fermentation byproducts with suggested positive health effects, have emerged as a therapeutic modality against metabolic diseases like obesity and type 2 diabetes. Alluringly, in vivo SCFA levels are easily modifiable by consumption of fermentable fibers (FF). Most rodent studies on dietary FF supplementation report terminal increased cecal/fecal SCFA levels but the time course of this increase remains elusive. Also, there is limited information on the effect of this sustained SCFA increase on physiology. Thus, we investigated dietary FF-dependent temporal increases in plasma SCFA levels and its metabolic effects on a western diet (WD) mouse model. C57BL/6J male mice (age 10 weeks) were fed test diets for 8 weeks. In Phase I, to establish time-course of plasma SCFA increase, mice were fed the following isocaloric diets: control (low fat + 0% FF); WD; control + 20% FF, where FF was fructooligosaccharides (FOS), inulin (In), guar gum (GG) or pectin (Pec). In Phase II mice were fed a control diet, or a WD with or without 20% FOS, Pec or GG. End points were weekly plasma SCFAs (by MS/MS), body weight, random glucose and insulin, and at the end of experimental period body fat composition and metabolic tests. Phase I. Compared to control, WD lowered while FF induced significant increases in total plasma SCFAs (FOS, Pec, GG > In) in a time dependent manner that plateaued beyond 2 weeks. All FF increased propionate and acetate but not butyrate. Phase II. WD caused metabolic dysfunctions (increased body weight and fat mass; glucose intolerance; insulin resistance; P < 0.0001 by two-way ANOVA) that were alleviated in mice fed FF enriched WD (Pec = GG > FOS). Compared to WD, food intake was similar except high in the WD-Pec group, while WD-Pec and GG showed higher energy expenditure. All 3 plasma SCFAs were significantly higher in all WD-FF groups. FF supplementation of the control diet showed no significant difference compared to control. We conclude that: 1) FF feeding induced SCFA production reaches saturation within 2 weeks, suggesting selection of specific gut bacterial features; and 2) all FFs were protective from weight gain and its metabolic consequences. 2R01 DK104927 (NIH/NIDDK) Layden.

Hepatocellular carcinoma (HCC) is a leading cause of death from cancer malignancies. Recently, hexokinase domain containing 1 (HKDC1), was shown to have significant overexpression in HCC compared to healthy tissue. Using in vitro and in vivo tools, we examined the role of HKDC1 in HCC progression. Importantly, HKDC1 ablation stops HCC progression by promoting metabolic reprogramming by shifting glucose flux away from the TCA cycle. Next, HKDC1 ablation leads to mitochondrial dysfunction resulting in less cellular energy which cannot be compensated by enhanced glucose uptake. And finally, we show that the interaction of HKDC1 with the mitochondria is essential for its role in HCC progression, and without this mitochondrial interaction mitochondrial dysfunction occurs. In sum, HKDC1 is highly expressed in HCC cells compared to normal hepatocytes, therefore targeting HKDC1, specifically its interaction with the mitochondria, reveals a highly selective approach to target cancer cells in HCC. ### Competing Interest Statement The authors have declared no competing interest.

The free fatty acid receptor 3 (FFA3) is a nutrient sensor of gut microbiota-generated nutrients, the short-chain fatty acids. Previously, we have shown that FFA3 is expressed in β-cells and inhibits islet insulin secretion ex vivo. Here, we determined the physiological relevance of the above observation by challenging wild-type (WT) and FFA3 knockout (KO) male mice with 1) hyperglycemia and monitoring insulin response via highly sensitive hyperglycemic clamps, 2) dietary high fat (HF), and 3) chemical-induced diabetes. As expected, FFA3 KO mice exhibited significantly higher insulin secretion and glucose infusion rate in hyperglycemic clamps. Predictably, under metabolic stress induced by HF-diet feeding, FFA3 KO mice exhibited less glucose intolerance compared with the WT mice. Moreover, similar islet architecture and β-cell area in HF diet-fed FFA3 KO and WT mice was observed. Upon challenge with streptozotocin (STZ), FFA3 KO mice initially exhibited a tendency for an accelerated incidence of diabetes compared with the WT mice. However, this difference was not maintained. Similar glycemia and β-cell mass loss was observed in both genotypes 10 days post-STZ challenge. Higher resistance to STZ-induced diabetes in WT mice could be due to higher basal islet autophagy. However, this difference was not protective because in response to STZ, similar autophagy induction was observed in both WT and FFA3 KO islets. These data demonstrate that FFA3 plays a role in modulating insulin secretion and β-cell response to stressors. The β-cell FFA3 and autophagy link warrant further research.

Results from epidemiological and prospective studies indicate a close association between periodontitis and diabetes. However the mechanisms by which periodontal pathogens influence the development of prediabetes/diabetes are not clear. We previously reported that oral administration of a periodontal pathogen, Porphyromonas gingivalis (Pg) to WT mice results in insulin resistance, hyperinsulinemia, and glucose intolerance and that Pg translocates to the pancreas. In the current study, we determined the specific localization of Pg in relation to mouse and human pancreatic α- and β-cells using 3-D confocal and immunofluorescence microscopy and orthogonal analyses. Pg/gingipain is intra- or peri-nuclearly localized primarily in β-cells in experimental mice and also in human post-mortem pancreatic samples. We also identified bihormonal cells in experimental mice as well as human pancreatic samples. A low percentage of bihormonal cells has intracellular Pg in both humans and experimental mice. Our data show that the number of Pg translocated to the pancreas correlates with the number of bihormonal cells in both mice and humans. Our findings suggest that Pg/gingipain translocates to pancreas, particularly β-cells in both humans and mice, and this is strongly associated with emergence of bihormonal cells.

Brown adipose tissue is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. β-Adrenergic stimulation of brown adipocytes leads to an increase in oxygen consumption and induction of a thermogenic gene program that includes uncoupling protein 1 ( Ucp1 ) and fibroblast growth factor 21 ( Fgf21 ). In kinase inhibitor screens, we have identified glycogen synthase kinase 3 (GSK3) as a negative regulator of basal and β-adrenergically stimulated Fgf21 expression in cultured brown adipocytes. In addition, inhibition of GSK3 also caused increased Ucp1 expression and oxygen consumption. β-Adrenergic stimulation triggered an inhibitory phosphorylation of GSK3 in a protein kinase A (PKA)-dependent manner. Mechanistically, inhibition of GSK3 activated the mitogen activated protein kinase (MAPK) kinase 3/6-p38 MAPK-activating transcription factor 2 signaling module. In summary, our data describe GSK3 as a novel negative regulator of β-adrenergic signaling in brown adipocytes.

Somatic gene therapy is a promising approach for treating otherwise terminal or debilitating diseases. The human skin is a promising conduit for genetic engineering, as it is the largest and most accessible organ, epidermal autografts and tissue-engineered skin equivalents have been successfully deployed in clinical applications, and skin epidermal stem/progenitor cells for generating such grafts are easy to obtain and expand in vitro. Here, we develop skin grafts from mouse and human epidermal progenitors that were engineered by CRISPR-mediated genome editing to controllably release GLP-1 (glucagon-like peptide 1), a critical incretin that regulates blood glucose homeostasis. GLP-1 induction from engineered mouse cells grafted onto immunocompetent hosts increased insulin secretion and reversed high-fat-diet-induced weight gain and insulin resistance. Taken together, these results highlight the clinical potential of developing long-lasting, safe, and versatile gene therapy approaches based on engineering epidermal progenitor cells.