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GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL + neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL + neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL + neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL + neurons causes hypothermia and hyperglycemia., Competing Interests: Declaration of interests F.B. is a co-founder and shareholder of Roxbiosens, Inc., and Implexion Pharma AB, receives research funding from Biogaia AB, and is a member of the scientific advisory board of Bactolife A/S., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [ 13 C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [ 13 C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health., (© 2021. The Author(s).)

Mice lacking a microbiota are protected from diet-induced obesity. Previous studies have shown that feeding a Western diet causes hypothalamic inflammation, which in turn can lead to leptin resistance and weight gain. Here, we show that wild-type (WT) mice with depleted gut microbiota, i.e., germ-free (GF) and antibiotic-treated mice, have elevated levels of glucagon-like peptide-1 (GLP-1), are protected against diet-induced hypothalamic inflammation, and have enhanced leptin sensitivity when fed a Western diet. Using GLP-1 receptor (GLP-1R)-deficient mice and pharmacological inhibition of the GLP-1R in WT mice, we demonstrate that intact GLP-1R signaling is required for preventing hypothalamic inflammation and enhancing leptin sensitivity. Furthermore, we show that astrocytes express the GLP-1R, and deletion of the receptor in glial fibrillary acidic protein (GFAP)-expressing cells diminished the antibiotic-induced protection against diet-induced hypothalamic inflammation. Collectively, our results suggest that depletion of the gut microbiota attenuates diet-induced hypothalamic inflammation and enhances leptin sensitivity via GLP-1R-dependent mechanisms., Competing Interests: Declaration of interests D.J.D. has served as an advisor or consultant or speaker within the past 12 months to Forkhead Biotherapeutics, Intarcia Therapeutics, Kallyope, Eli Lilly, Merck Research Laboratories, Novo Nordisk Inc., and Pfizer Inc. Neither D.J.D. nor his family members hold stock in these companies. GLP-2 is the subject of a patent license agreement between Shire Inc. and the University of Toronto, Toronto General Hospital (UHN), and D.J.D. R.J.S. has research support from Ethicon Endo-Surgery/Johnson & Johnson, Novo Nordisk, Zafgen, Kallyope, Astra Zeneca, and Pfizer. He has been a consultant or part of a Scientific Advisory Board for Ethicon Endo-Surgery/Johnson & Johnson, Novo Nordisk, Janssen/Johnson & Johnson, Sanofi, Kallyope, Scohia, Ironwood Pharma, and GuidePoint Consultants. In addition, R.J.S. holds equity in Zafgen and Redesign Health., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Metformin is the first-line therapy for type 2 diabetes, but there are large inter-individual variations in responses to this drug. Its mechanism of action is not fully understood, but activation of AMP-activated protein kinase (AMPK) and changes in the gut microbiota appear to be important. The inhibitory role of microbial metabolites on metformin action has not previously been investigated. Here, we show that concentrations of the microbial metabolite imidazole propionate are higher in subjects with type 2 diabetes taking metformin who have high blood glucose. We also show that metformin-induced glucose lowering is not observed in mice pretreated with imidazole propionate. Furthermore, we demonstrate that imidazole propionate inhibits AMPK activity by inducing inhibitory AMPK phosphorylation, which is dependent on imidazole propionate-induced basal Akt activation. Finally, we identify imidazole propionate-activated p38γ as a novel kinase for Akt and demonstrate that p38γ kinase activity mediates the inhibitory action of imidazole propionate on metformin., Competing Interests: Declaration of Interests A.K. and F.B. are shareholders in Implexion Pharma AB., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Interactions between the gut microbiota, diet, and the host potentially contribute to the development of metabolic diseases. Here, we identify imidazole propionate as a microbially produced histidine-derived metabolite that is present at higher concentrations in subjects with versus without type 2 diabetes. We show that imidazole propionate is produced from histidine in a gut simulator at higher concentrations when using fecal microbiota from subjects with versus without type 2 diabetes and that it impairs glucose tolerance when administered to mice. We further show that imidazole propionate impairs insulin signaling at the level of insulin receptor substrate through the activation of p38γ MAPK, which promotes p62 phosphorylation and, subsequently, activation of mechanistic target of rapamycin complex 1 (mTORC1). We also demonstrate increased activation of p62 and mTORC1 in liver from subjects with type 2 diabetes. Our findings indicate that the microbial metabolite imidazole propionate may contribute to the pathogenesis of type 2 diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The enteric nervous system (ENS) is crucial for essential gastrointestinal physiologic functions such as motility, fluid secretion, and blood flow. The gut is colonized by trillions of bacteria that regulate host production of several signaling molecules including serotonin (5-HT) and other hormones and neurotransmitters. Approximately 90% of 5-HT originates from the intestine, and activation of the 5-HT 4 receptor in the ENS has been linked to adult neurogenesis and neuroprotection. Here, we tested the hypothesis that the gut microbiota could induce maturation of the adult ENS through release of 5-HT and activation of 5-HT 4 receptors. Colonization of germ-free mice with a microbiota from conventionally raised mice modified the neuroanatomy of the ENS and increased intestinal transit rates, which was associated with neuronal and mucosal 5-HT production and the proliferation of enteric neuronal progenitors in the adult intestine. Pharmacological modulation of the 5-HT 4 receptor, as well as depletion of endogenous 5-HT, identified a mechanistic link between the gut microbiota and maturation of the adult ENS through the release of 5-HT and activation of the 5-HT 4 receptor. Taken together, these findings show that the microbiota modulates the anatomy of the adult ENS in a 5-HT-dependent fashion with concomitant changes in intestinal transit., Competing Interests: Conflict of interest statement: F.B. is cofounder of and shareholder in Metabogen AB., (Copyright © 2018 the Author(s). Published by PNAS.)

Conversion of diacylglycerol to phosphatidic acid is mediated by diacylglycerol kinases (DGKs), with DGKα specifically linked to adaptive immune responses. We determined the role of DGKα in obesity and inflammatory responses to a high-fat diet (HFD). DGKα KO and WT littermates were either a ) chow-fed, b ) HFD-fed for 12 weeks (Long-Term HFD), or c ) HFD-fed for 3 days (Acute HFD). Body weight/composition, oxygen consumption, food intake, and glucose tolerance was unaltered between chow-fed DGKα KO and WT mice. Insulin concentration during the intraperitoneal glucose tolerance (IPGT) test was elevated in chow-fed DGKα KO mice, suggesting mild insulin resistance. Insulin concentration during the IPGT test was reduced in Long-Term HFD-fed DGKα KO mice, suggesting a mild enhancement in insulin sensitivity. Acute HFD increased hormone sensitive lipase protein abundance and altered expression of interleukin 1β mRNA, an inflammatory marker in perigonadal adipose tissue of DGKα KO mice. In conclusion, DGKα ablation is associated with mild alterations in insulin sensitivity. However, DGKα is dispensable for whole body insulin-mediated glucose uptake, hepatic glucose production, and energy homeostasis. Our results suggest DGKα aids in modulating the early immune response of adipose tissue following an acute exposure to HFD, possibly through modulation of acute T-cell action., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Metformin is widely used in the treatment of type 2 diabetes (T2D), but its mechanism of action is poorly defined. Recent evidence implicates the gut microbiota as a site of metformin action. In a double-blind study, we randomized individuals with treatment-naive T2D to placebo or metformin for 4 months and showed that metformin had strong effects on the gut microbiome. These results were verified in a subset of the placebo group that switched to metformin 6 months after the start of the trial. Transfer of fecal samples (obtained before and 4 months after treatment) from metformin-treated donors to germ-free mice showed that glucose tolerance was improved in mice that received metformin-altered microbiota. By directly investigating metformin-microbiota interactions in a gut simulator, we showed that metformin affected pathways with common biological functions in species from two different phyla, and many of the metformin-regulated genes in these species encoded metalloproteins or metal transporters. Our findings provide support for the notion that altered gut microbiota mediates some of metformin's antidiabetic effects.

Diacylglycerol kinases (DGKs) catalyze the phosphorylation and conversion of diacylglycerol (DAG) into phosphatidic acid. DGK isozymes have unique primary structures, expression patterns, subcellular localizations, regulatory mechanisms, and DAG preferences. DGKε has a hydrophobic segment that promotes its attachment to membranes and shows substrate specificity for DAG with an arachidonoyl acyl chain in the sn-2 position of the substrate. We determined the role of DGKε in the regulation of energy and glucose homeostasis in relation to diet-induced insulin resistance and obesity using DGKε-KO and wild-type mice. Lipidomic analysis revealed elevated unsaturated and saturated DAG species in skeletal muscle of DGKε KO mice, which was paradoxically associated with increased glucose tolerance. Although skeletal muscle insulin sensitivity was unaltered, whole-body respiratory exchange ratio was reduced, and abundance of mitochondrial markers was increased, indicating a greater reliance on fat oxidation and intracellular lipid metabolism in DGKε KO mice. Thus, the increased intracellular lipids in skeletal muscle from DGKε KO mice may undergo rapid turnover because of increased mitochondrial function and lipid oxidation, rather than storage, which in turn may preserve insulin sensitivity. In conclusion, DGKε plays a role in glucose and energy homeostasis by modulating lipid metabolism in skeletal muscle., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type littermates with a high-fat diet and showed that STK25 deficiency suppressed development of hyperglycemia and hyperinsulinemia, improved systemic glucose tolerance, reduced hepatic gluconeogenesis, and increased insulin sensitivity. Stk25(-/-) mice were protected from diet-induced liver steatosis accompanied by decreased protein levels of acetyl-CoA carboxylase, a key regulator of both lipid oxidation and synthesis. Lipid accumulation in Stk25(-/-) skeletal muscle was reduced, and expression of enzymes controlling the muscle oxidative capacity (Cpt1, Acox1, Cs, Cycs, Ucp3) and glucose metabolism (Glut1, Glut4, Hk2) was increased. These data are consistent with our previous study of STK25 knockdown in myoblasts and reciprocal to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Diacylglycerol kinase (DGK) isoforms regulate signal transduction and lipid metabolism. DGKδ deficiency leads to hyperglycemia, peripheral insulin resistance, and metabolic inflexibility. Thus, dysregulation of other DGK isoforms may play a role in metabolic dysfunction. We investigated DGK isoform mRNA expression in extensor digitorum longus (EDL) and soleus muscle, liver as well as subcutaneous and epididymal adipose tissue in C57BL/6J mice and obese and insulin-resistant ob/ob mice. All DGK isoforms, except for DGKκ, were detectable, although with varying mRNA expression. Liver DGK expression was generally lowest, with several isoforms undetectable. In soleus muscle, subcutaneous and epididymal adipose tissue, DGKδ was the most abundant isoform. In EDL muscle, DGKα and DGKζ were the most abundant isoforms. In liver, DGKζ was the most abundant isoform. Comparing obese insulin-resistant ob/ob mice to lean C57BL/6J mice, DGKβ, DGKι, and DGKθ were increased and DGKε expression was decreased in EDL muscle, while DGKβ, DGKη and DGKθ were decreased and DGKδ and DGKι were increased in soleus muscle. In liver, DGKδ and DGKζ expression was increased in ob/ob mice. DGKη was increased in subcutaneous fat, while DGKζ was increased and DGKβ, DGKδ, DGKη and DGKε were decreased in epididymal fat from ob/ob mice. In both adipose tissue depots, DGKα and DGKγ were decreased and DGKι was increased in ob/ob mice. In conclusion, DGK mRNA expression is altered in an isoform- and tissue-dependent manner in obese insulin-resistant ob/ob mice. DGK isoforms likely have divergent functional roles in distinct tissues, which may contribute to metabolic dysfunction., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Adipose tissue dysfunction may be a central factor in the pathogenesis of insulin resistance in women with polycystic ovary syndrome (PCOS). Gene expression in subcutaneous adipose tissue in PCOS and its relation to metabolic and endocrine features of the syndrome have been fragmentarily investigated. The aim was to assess in subcutaneous adipose tissue the expression of genes potentially associated with adipose tissue dysfunction and to explore their relation to features of the syndrome. Twenty-one women with PCOS (body mass index [BMI] 18.2-33.4 kg/m(2)) and 21 controls (BMI 19.2-31.7 kg/m(2)) were matched pair-wise for age, body weight, and BMI. Tissue biopsies were obtained to measure mRNA expression of 44 genes (TaqMan Low Density Array). Differential expression levels were correlated with BMI, glucose infusion rate (GIR), sex hormone binding globulin (SHBG), and sex steroids. In PCOS, expression of adiponectin receptor 2 (ADIPOR2), LPL, and twist-related protein 1 (TWIST1) was decreased, while expression of chemokine (C-C motif) ligand 2 (CCL2) and heme oxygenase (decycling 1) (HMOX1) was increased. TWIST1 and HMOX1, both novel adipokines, correlated with BMI and GIR. After BMI adjustment, LPL and ADIPOR2 expression correlated with plasma estradiol, and CCL2 expression correlated with GIR, in all women. We conclude that adipose tissue mRNA expression differed in PCOS women and controls and that two novel adipokines, TWIST1 and HMOX1, together with adiponectin, LPL, and CCL2, and their downstream pathways merit further investigation.

In rats with dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), repeated low-frequency electrical stimulation of acupuncture needles restores whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp. We hypothesized that electrical stimulation causing muscle contractions and manual stimulation causing needle sensation have different effects on insulin sensitivity and related signaling pathways in skeletal muscle and adipose tissue, with electrical stimulation being more effective in DHT-induced PCOS rats. From age 70 days, rats received manual or low-frequency electrical stimulation of needles in abdominal and hind limb muscle five times/wk for 4-5 wks; controls were handled but untreated rats. Low-frequency electrical stimulation modified gene expression (decreased Tbc1d1 in soleus, increased Nr4a3 in mesenteric fat) and protein expression (increased pAS160/AS160, Nr4a3 and decreased GLUT4) by western blot and increased GLUT4 expression by immunohistochemistry in soleus muscle; glucose clearance during oral glucose tolerance tests was unaffected. Manual stimulation led to faster glucose clearance and modified mainly gene expression in mesenteric adipose tissue (increased Nr4a3, Mapk3/Erk, Adcy3, Gsk3b), but not protein expression to the same extent; however, Nr4a3 was reduced in soleus muscle. The novel finding is that electrical and manual muscle stimulation affect glucose homeostasis in DHT-induced PCOS rats through different mechanisms. Repeated electrical stimulation regulated key functional molecular pathways important for insulin sensitivity in soleus muscle and mesenteric adipose tissue to a larger extent than manual stimulation. Manual stimulation improved whole-body glucose tolerance, an effect not observed after electrical stimulation, but did not affect molecular signaling pathways to the same extent as electrical stimulation. Although more functional signaling pathways related to insulin sensitivity were affected by electrical stimulation, our findings suggest that manual stimulation of acupuncture needles has a greater effect on glucose tolerance. The underlying mechanism of the differential effects of the intermittent manual and the continuous electrical stimulation remains to be elucidated.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that regulates lipid and glucose metabolism. We investigated the effects of Labisia pumila (LP) standardized water extract on PPARgamma transcriptional activity in adipocytes in vitro and in vivo. We used a rat model of dihydrotestosterone- (DHT-) induced polycystic ovary syndrome (PCOS), a condition characterized by insulin resistance. At 9 weeks of age, the PCOS rats were randomly subdivided into two groups: PCOS-LP (50 mg/kg/day of LP) and PCOS-control (1 mL of deionised water) for 4-5 weeks on the same schedule. Real-time RT-PCR was performed to determine the PPARgamma mRNA levels. LP upregulated PPARgamma mRNA level by 40% in the PCOS rats. Western blot analysis further demonstrated the increased PPARgamma protein levels in parallel with upregulation in mRNA. These observations were further proven by adipocytes culture. Differentiated 3T3-L1 adipocytes were treated with final concentration of 100  μ g/mL LP and compared to untreated control and 10  μ M of rosiglitazone (in type of thiazolidinediones). LP increased PPARgamma expressions at both mRNA and protein levels and enhanced the effect of glucose uptake in the insulin-resistant cells. The data suggest that LP may ameliorate insulin resistance in adipocytes via the upregulation of PPARgamma pathway.

Both low-frequency electro-acupuncture (EA) and manual acupuncture improve menstrual frequency and decrease circulating androgens in women with polycystic ovary syndrome (PCOS). We sought to determine whether low-frequency EA is more effective than manual stimulation in regulating disturbed oestrous cyclicity in rats with PCOS induced by 5α-dihydrotestosterone. To identify the central mechanisms of the effects of stimulation, we assessed hypothalamic mRNA expression of molecules that regulate reproductive and neuroendocrine function. From age 70 days, rats received 2 Hz EA or manual stimulation with the needles five times per week for 4-5 weeks; untreated rats served as control animals. Specific hypothalamic nuclei were obtained by laser microdissection, and mRNA expression was measured with TaqMan low-density arrays. Untreated rats were acyclic. During the last 2 weeks of treatment, seven of eight (88%) rats in the EA group had epithelial keratinocytes, demonstrating oestrous cycle change (P = 0.034 versus control rats). In the manual group, five of eight (62%) rats had oestrous cycle changes (n.s. versus control animals). The mRNA expression of the opioid receptors Oprk1 and Oprm1 in the hypothalamic arcuate nucleus was lower in the EA group than in untreated control rats. The mRNA expression of the steroid hormone receptors Esr2, Pgr and Kiss1r was lower in the manual group than in the control animals. In rats with 5α-dihydrotestosterone-induced PCOS, low-frequency EA restored disturbed oestrous cyclicity but did not differ from the manual stimulation group, although electrical stimulation lowered serum testosterone in responders, those with restored oestrus cyclicity, and differed from both control animals and the manual stimulation group. Thus, EA cannot in all aspects be considered superior to manual stimulation. The effects of low-frequency EA may be mediated by central opioid receptors, while manual stimulation may involve regulation of steroid hormone/peptide receptors.