Loss of GIPR in LEPR cells impairs glucose control by GIP and GIP:GLP-1 co-agonism without affecting body weight and food intake in mice.

OBJECTIVE: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr). METHODS: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice. RESULTS: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the ß-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice. CONCLUSIONS: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism.

Global, neuronal or ß cell-specific deletion of inceptor improves glucose homeostasis in male mice with diet-induced obesity.

Insulin resistance is an early complication of diet-induced obesity (DIO)1, potentially leading to hyperglycaemia and hyperinsulinaemia, accompanied by adaptive ß cell hypertrophy and development of type 2 diabetes2. Insulin not only signals via the insulin receptor (INSR), but also promotes ß cell survival, growth and function via the insulin-like growth factor 1 receptor (IGF1R)3-6. We recently identified the insulin inhibitory receptor (inceptor) as the key mediator of IGF1R and INSR desensitization7. But, although ß cell-specific loss of inceptor improves ß cell function in lean mice7, it warrants clarification whether inceptor signal inhibition also improves glycaemia under conditions of obesity. We assessed the glucometabolic effects of targeted inceptor deletion in either the brain or the pancreatic ß cells under conditions of DIO in male mice. In the present study, we show that global and neuronal deletion of inceptor, as well as its adult-onset deletion in the ß cells, improves glucose homeostasis by enhancing ß cell health and function. Moreover, we demonstrate that inceptor-mediated improvement in glucose control does not depend on inceptor function in agouti-related protein-expressing or pro-opiomelanocortin neurons. Our data demonstrate that inceptor inhibition improves glucose homeostasis in mice with DIO, hence corroborating that inceptor is a crucial regulator of INSR and IGF1R signalling.

Glucose-dependent insulinotropic polypeptide regulates body weight and food intake via GABAergic neurons in mice.

The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice.

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.

Spatiotemporal GLP-1 and GIP receptor signaling and trafficking/recycling dynamics induced by selected receptor mono- and dual-agonists.

OBJECTIVE: We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. METHODS: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. RESULTS: Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. CONCLUSIONS: Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.

Gut Peptide Agonism in the Treatment of Obesity and Diabetes.

Obesity is a global healthcare challenge that gives rise to devastating diseases such as the metabolic syndrome, type-2 diabetes (T2D), and a variety of cardiovascular diseases. The escalating prevalence of obesity has led to an increased interest in pharmacological options to counteract excess weight gain. Gastrointestinal hormones such as glucagon, amylin, and glucagon-like peptide-1 (GLP-1) are well recognized for influencing food intake and satiety, but the therapeutic potential of these native peptides is overall limited by a short half-life and an often dose-dependent appearance of unwanted effects. Recent clinical success of chemically optimized GLP-1 mimetics with improved pharmacokinetics and sustained action has propelled pharmacological interest in using bioengineered gut hormones to treat obesity and diabetes. In this article, we summarize the basic biology and signaling mechanisms of selected gut peptides and discuss how they regulate systemic energy and glucose metabolism. Subsequently, we focus on the design and evaluation of unimolecular drugs that combine the beneficial effects of selected gut hormones into a single entity to optimize the beneficial impact on systems metabolism. © 2020 American Physiological Society. Compr Physiol 10:99-124, 2020.

Antioxidants protect against diabetes by improving glucose homeostasis in mouse models of inducible insulin resistance and obesity.

AIMS/HYPOTHESIS: In the context of diabetes, the health benefit of antioxidant treatment has been widely debated. In this study, we investigated the effect of antioxidant treatment during the development of insulin resistance and hyperphagia in obesity and partial lipodystrophy. METHODS: We studied the role of antioxidants in the regulation of insulin resistance using the tamoxifen-inducible fat-specific insulin receptor knockout (iFIRKO) mouse model, which allowed us to analyse the antioxidant's effect in a time-resolved manner. In addition, leptin-deficient ob/ob mice were used as a hyperphagic, chronically obese and diabetic mouse model to validate the beneficial effect of antioxidants on metabolism. RESULTS: Acute induction of insulin receptor knockout in adipocytes changed the substrate preference to fat before induction of a diabetic phenotype including hyperinsulinaemia and hyperglycaemia. In healthy chow-fed animals as well as in morbidly obese mice, this diabetic phase could be reversed within a few weeks. Furthermore, after the induction of insulin receptor knockout in mature adipocytes, iFIRKO mice were protected from subsequent obesity development through high-fat diet feeding. By genetic tracing we show that the persistent fat mass loss in mice after insulin receptor knockout in adipocytes is not caused by the depletion of adipocytes. Treatment of iFIRKO mice with antioxidants postponed and reduced hyperglycaemia by increasing insulin sensitivity. In ob/ob mice, antioxidants rescued both hyperglycaemia and hyperphagia. CONCLUSIONS/INTERPRETATION: We conclude that fat mass reduction through insulin resistance in adipocytes is not reversible. Furthermore, it seems unlikely that adipocytes undergo apoptosis during the process of extreme lipolysis, as a consequence of insulin resistance. Antioxidants have a beneficial health effect not only during the acute phase of diabetes development, but also in a temporary fashion once chronic obesity and diabetes have been established.

Publisher Correction: Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes.

In the original PDF version of this article, affiliation 1, 'Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Muenchen & German Center for Diabetes Research (DZD), Neuherberg, Germany', was incorrectly given as 'Institute of Diabetes and Regeneration Research, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health (GmbH), Neuherberg, Germany '. This has now been corrected in the PDF version of the article; the HTML version was correct at the time of publication.

Coordinated targeting of cold and nicotinic receptors synergistically improves obesity and type 2 diabetes.

Pharmacological stimulation of brown adipose tissue (BAT) thermogenesis to increase energy expenditure is progressively being pursued as a viable anti-obesity strategy. Here, we report that pharmacological activation of the cold receptor transient receptor potential cation channel subfamily M member 8 (TRPM8) with agonist icilin mimics the metabolic benefits of cold exposure. In diet-induced obese (DIO) mice, treatment with icilin enhances energy expenditure, and decreases body weight, without affecting food intake. To further potentiate the thermogenic action profile of icilin and add complementary anorexigenic mechanisms, we set out to identify pharmacological partners next to icilin. To that end, we specifically targeted nicotinic acetylcholine receptor (nAChR) subtype alpha3beta4 (α3ß4), which we had recognized as a potential regulator of energy homeostasis and glucose metabolism. Combinatorial targeting of TRPM8 and nAChR α3ß4 by icilin and dimethylphenylpiperazinium (DMPP) orchestrates synergistic anorexic and thermogenic pathways to reverse diet-induced obesity, dyslipidemia, and glucose intolerance in DIO mice.

Author Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the months on the axis labeled projected month of conception in Fig. 1a were out of order. April and March should have been the first and last months listed, respectively. The error has been corrected in the print, PDF and HTML versions of this article.

Publisher Correction: Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

In the version of this article originally published, the bars in the mean temperature graph in Fig. 1a were incorrectly aligned. The left-most bar should have been aligned with the Apr label on the projected month of conception axis. The error has been corrected in the print, PDF and HTML versions of this article.

Short-term feeding of a ketogenic diet induces more severe hepatic insulin resistance than an obesogenic high-fat diet.

KEY POINTS: A ketogenic diet is known to lead to weight loss and is considered metabolically healthy; however there are conflicting reports on its effect on hepatic insulin sensitivity. KD fed animals appear metabolically healthy in the fasted state after 3 days of dietary challenge, whereas obesogenic high-fat diet (HFD) fed animals show elevated insulin levels. A glucose challenge reveals that both KD and HFD fed animals are glucose intolerant. Glucose intolerance correlates with increased lipid oxidation and lower respiratory exchange ratio (RER); however, all animals respond to glucose injection with an increase in RER. Hyperinsulinaemic-euglycaemic clamps with double tracer show that the effect of KD is a result of hepatic insulin resistance and increased glucose output but not impaired glucose clearance or tissue glucose uptake in other tissues. ABSTRACT: Despite being a relevant healthcare issue and heavily investigated, the aetiology of type 2 diabetes (T2D) is still incompletely understood. It is well established that increased endogenous glucose production (EGP) leads to a progressive increase in glucose levels, causing insulin resistance and eventual loss of glucose homeostasis. The consumption of high carbohydrate, high-fat, western style diet (HFD) is linked to the development of T2D and obesity, whereas the consumption of a low carbohydrate, high-fat, ketogenic diet (KD) is considered healthy. However, several days of carbohydrate restriction are known to cause selective hepatic insulin resistance. In the present study, we compare the effects of short-term HFD and KD feeding on glucose homeostasis in mice. We show that, even though KD fed animals appear to be healthy in the fasted state, they exhibit decreased glucose tolerance to a greater extent than HFD fed animals. Furthermore, we show that this effect originates from blunted suppression of hepatic glucose production by insulin, rather than impaired glucose clearance and tissue glucose uptake. These data suggest that the early effects of HFD consumption on EGP may be part of a normal physiological response to increased lipid intake and oxidation, and that systemic insulin resistance results from the addition of dietary glucose to EGP-derived glucose.

Cold-induced epigenetic programming of the sperm enhances brown adipose tissue activity in the offspring.

Recent research has focused on environmental effects that control tissue functionality and systemic metabolism. However, whether such stimuli affect human thermogenesis and body mass index (BMI) has not been explored. Here we show retrospectively that the presence of brown adipose tissue (BAT) and the season of conception are linked to BMI in humans. In mice, we demonstrate that cold exposure (CE) of males, but not females, before mating results in improved systemic metabolism and protection from diet-induced obesity of the male offspring. Integrated analyses of the DNA methylome and RNA sequencing of the sperm from male mice revealed several clusters of co-regulated differentially methylated regions (DMRs) and differentially expressed genes (DEGs), suggesting that the improved metabolic health of the offspring was due to enhanced BAT formation and increased neurogenesis. The conclusions are supported by cell-autonomous studies in the offspring that demonstrate an enhanced capacity to form mature active brown adipocytes, improved neuronal density and more norepinephrine release in BAT in response to cold stimulation. Taken together, our results indicate that in humans and in mice, seasonal or experimental CE induces an epigenetic programming of the sperm such that the offspring harbor hyperactive BAT and an improved adaptation to overnutrition and hypothermia.

Hemostasis, endothelial stress, inflammation, and the metabolic syndrome.

Obesity and the metabolic syndrome (MS) are two of the pressing healthcare problems of our time. The MS is defined as increased abdominal obesity in concert with elevated fasting glucose levels, insulin resistance, elevated blood pressure, and plasma lipids. It is a key risk factor for type 2 diabetes mellitus (T2DM) and for cardiovascular complications and mortality. Here, we review work demonstrating that various aspects of coagulation and hemostasis, as well as vascular reactivity and function, become impaired progressively during chronic ingestion of a western diet, but also acutely after meals. We outline that both T2DM and cardiovascular disease should be viewed as inflammatory diseases and describe that chronic overload of free fatty acids and glucose can trigger inflammatory pathways directly or via increased production of ROS. We propose that since endothelial stress and increases in platelet activity precede inflammation and overt symptoms of the MS, they are likely the first hit. This suggests that endothelial activation and insulin resistance are probably causative in the observed chronic low-level metabolic inflammation, and thus both metabolic and cardiovascular complications linked to consumption of a western diet.

Dietary Carbohydrates Impair Healthspan and Promote Mortality.

The prospective cohort study, named PURE, found that in >135,000 participants from 18 countries, nutritive carbohydrates increase human mortality, whereas dietary fat reduces it, requesting a fundamental change of current nutritional guidelines. Experimental evidence from animal models provides synergizing mechanistic concepts as well as pharmacological options to mimic low-carb or ketogenic diets.

Adipocytes at the Core of Bone Function.

Marrow adipose tissue (MAT) is a specialized adipose tissue with endocrine function found in bone marrow. In this issue of Cell Stem Cell, Ambrosi et al. (2017) identify a stem cell population in the bone marrow that can give rise to both osteogenic and adipogenic MAT precursors and is regulated by diet and aging and affects bone functionality.

Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling.

OBJECTIVE: Adipose tissue shows a high degree of plasticity, and adipocyte hyperplasia is an important mechanism for adipose tissue expansion. Different adipose depots respond differently to an increased demand for lipid storage. Orchestrating cellular expansion in vivo requires extracellular matrix (ECM) remodeling and a high degree of interaction between cells and ECM. METHODS: We studied decellularized primary adipose stromal cell derived ECM of different adipose depots and reseeded them with primary adipose precursors. We tested ECM effect on adipocyte differentiation and analyzed ECM composition using proteomic and immunohistochemical approaches to identify factors in the ECM influencing adipogenesis. RESULTS: We show that the ECM of an adipose depot is the major determinant for the differentiation capacity of primary preadipocytes. Visceral adipose tissue stromal cells differentiate less than subcutaneous cells, which, in turn, are less adipogenic than BAT-derived cells. This effect is based on the ECM composition of the respective depot and not dependent on the precursor origin. Addition of vitamin C pronounces the pro-adipogenic effects of the ECM, indicating the importance of collagenous ECM in mediating the effect. Using a proteomic global and a targeted downstream analysis, we identify Flotillin 2 as a protein enriched in pro-adipogenic ECM, which is involved in orchestrating ECM to preadipocyte signaling. CONCLUSIONS: We show that adipose tissue SVF secretes collagenous ECM, which directly modulates terminal differentiation of adipocyte precursors in a depot specific manner. These data demonstrate the importance of the tissue microenvironment in preadipocyte differentiation.

Hairless promotes PPARγ expression and is required for white adipogenesis.

Adipose tissue is the largest compartment in the mammalian body for storing energy as fat, providing an important reservoir of fuel for maintaining whole body energy homeostasis. Herein, we identify the transcriptional cofactor hairless (HR) to be required for white adipogenesis. Moreover, forced expression of HR in non-adipogenic precursor cells induces adipogenic gene expression and enhances adipocyte formation under permissive conditions. HR exerts its proadipogenic effects by regulating the expression of PPARγ, one of the central adipogenic transcription factors. In conclusion, our data provide a new mechanism required for white adipogenesis.

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism.

BACKGROUND: FeFe-hydrogenases are the most active class of H2-producing enzymes known in nature and may have important applications in clean H2 energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O2. RESULTS: We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O2 levels eliminate growth. This pathway forms the basis for a genetic selection for O2 tolerance. Genetically selected hydrogenases did not show improved stability in O2 and in many cases had lost H2 production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer. CONCLUSIONS: Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H2 metabolism. Our results also indicate a H2-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H2-activating catalysts.