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Objectives: We here assessed whether typical pathogens of laboratory mice affect the development of diet-induced obesity and glucose intolerance, and whether colonization affects the efficacy of the GLP-1R agonist liraglutide and of the GLP-1/GIP co-agonist MAR709 to treat obesity and diabetes. Methods: Male C57BL/6J mice were experimentally infected with Helicobacter hepaticus, Rodentibacter pneumotropicus and Staphylococcus aureus and compared to a group of uninfected specific and opportunistic pathogen free (SOPF) mice. The development of diet-induced obesity and glucose intolerance was monitored over a period of 26 weeks. To study the influence of pathogens on drug treatment, mice were then subjected for 6 days daily treatment with either the GLP-1 receptor agonist liraglutide or the GLP-1/GIP co-agonist MAR709. Results: Colonized mice did not differ from SOPF controls regarding HFD-induced body weight gain, food intake, body composition, glycemic control, or responsiveness to treatment with liraglutide or the GLP-1/GIP co-agonist MAR709. Conclusions: We conclude that the occurrence of H. hepaticus, R. pneumotropicus and S. aureus does neither affect the development of diet-induced obesity or type 2 diabetes, nor the efficacy of GLP-1-based drugs to decrease body weight and to improve glucose control 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 β-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.

Summary: Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK’s anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

Objective: Snacking, i.e., the intake of small amounts of palatable food items, is a common behavior in modern societies, promoting overeating and obesity. Shifting food intake into the daily rest phase disrupts circadian rhythms and is also known to stimulate weight gain. We therefore hypothesized that chronic snacking in the inactive phase may promote body weight gain and that this effect is based on disruption of circadian clocks. Methods: Male mice were fed a daily chocolate snack either during their rest or their active phase and body weight development and metabolic parameters were investigated. Snacking experiments were repeated in constant darkness and in clock-deficient mutant mice to examine the role of external and internal time cues in mediating the metabolic effects of snacking. Results: Chronic snacking in the rest phase increased body weight gain and disrupted metabolic circadian rhythms in energy expenditure, body temperature, and locomotor activity. Additionally, these rest phase snacking mice assimilated more energy during the inactive phase. Body weight remained increased in rest phase snacking wildtype mice in constant darkness as well as in clock-deficient mutant mice under a regular light–dark cycle compared to mice snacking in the active phase. Weight gain effects were abolished in clock-deficient mice in constant darkness. Conclusions: Our data suggest that mistimed snacking increases energy resorption and promotes body weight gain. This effect requires a functional circadian clock at least under constant darkness conditions.

Objective: The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. Methods: We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. Results: We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. Conclusions: We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

Objective: Technological advances have brought a steady increase in the availability of various types of omics data, from genomics to metabolomics. Integrating these multi-omics data is a chance and challenge for systems biology; yet, tools to fully tap their potential remain scarce. Methods: We present here a fully unsupervised and versatile correlation-based method – termed Correlation guided Network Integration (CoNI) – to integrate multi-omics data into a hypergraph structure that allows for the identification of effective modulators of metabolism. Our approach yields single transcripts of potential relevance that map to specific, densely connected, metabolic subgraphs or pathways. Results: By applying our method on transcriptomics and metabolomics data from murine livers under standard Chow or high-fat diet, we identified eleven genes with potential regulatory effects on hepatic metabolism. Five candidates, including the hepatokine INHBE, were validated in human liver biopsies to correlate with diabetes-related traits such as overweight, hepatic fat content, and insulin resistance (HOMA-IR). Conclusion: Our method's successful application to an independent omics dataset confirmed that the novel CoNI framework is a transferable, entirely data-driven, flexible, and versatile tool for multiple omics data integration and interpretation.

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.

Objective: The contribution of brown adipose tissue (BAT) to adult human metabolic control is a topic of ongoing investigation. In context, understanding the cellular events leading to BAT uncoupling, heat production, and energy expenditure is anticipated to produce significant insight into this endeavor. The phosphoinositide interacting regulator of transient receptor potentials (Pirt) was recently put forward as a key protein regulating cold sensing downstream of the transient receptor potential melastatin 8 (TRPM8). Notably, TRPM8 has been identified as a non-canonical regulator of BAT thermogenesis. The aim of this investigation was to delineate the role of Pirt in energy homeostasis and glucose metabolism - and the possible involvement of Pirt in TRPM8-elicited energy expenditure. Methods: To this end, we metabolically phenotyped male and female Pirt deficient (Pirt−/−) mice exposed to a low-fat chow diet or to a high-fat, high-sugar (HFHS) diet. Results: We identified that chow-fed female Pirt−/− mice have an increased susceptibility to develop obesity and glucose intolerance. This effect is abrogated when the mice are exposed to a HFHS diet. Conversely, Pirt−/− male mice display no metabolic phenotype on either diet relative to wild-type (WT) control mice. Finally, we observed that Pirt is dispensable for TRPM8-evoked energy expenditure. Conclusion: We here report subtle metabolic abnormalities in female, but not male, Pirt−/− mice. Future studies are required to tease out if metabolic stressors beyond dietary interventions, e.g. temperature fluctuations, are interacting with Pirt-signaling and metabolic control in a sex-specific fashion. Keywords: Signaling molecule, Sex differences, Body weight, Energy metabolism, TRPM8, Brown adipose tissue

Objective: Reorganization of the extracellular matrix is a prerequisite for healthy adipose tissue expansion, whereas fibrosis is a key feature of adipose dysfunction and inflammation. However, very little is known about the direct effects of impaired cell–matrix interaction in adipocyte function and insulin sensitivity. The objective of this study was to determine whether integrin activity can regulate insulin sensitivity in adipocytes and thereby systemic metabolism. Methods: We characterized integrin activity in adipose tissue and its consequences on whole-body metabolism using adipose-selective deletion of β1 integrin (Itgb1adipo-cre) and Kindlin-2 (Kind2adipo-cre) in mice. Results: We demonstrate that integrin signaling regulates white adipocyte insulin action and systemic metabolism. Consequently, loss of adipose integrin activity, similar to loss of adipose insulin receptors, results in a lipodystrophy-like phenotype and systemic insulin resistance. However, brown adipose tissue of Kind2adipo-cre and Itgb1adipo-cre mice is chronically hyperactivated and has increased substrate delivery, reduced endothelial basement membrane thickness, and increased endothelial vesicular transport. Conclusions: Thus, we establish integrin-extracellular matrix interactions as key regulators of white and brown adipose tissue function and whole-body metabolism.

Objective: The worldwide prevalence of obesity has increased to 10% in men and 15% in women and is associated with severe comorbidities such as diabetes, cancer, and cardiovascular disease. Animal models of obesity are central to experimental studies of disease mechanisms and therapeutic strategies. Diet-induced obesity (DIO) models in rodents have provided important insights into the pathophysiology of obesity and, in most instances, are the first in line for exploratory pharmacology studies. To deepen the relevance towards translation to human patients, we established a corresponding DIO model in Göttingen minipigs (GM). Methods: Young adult female ovariectomized GM were fed a high-fat/high-energy diet for a period of 70 weeks. The ration was calculated to meet the requirements and maintain body weight (BW) of lean adult minipigs (L-GM group) or increased stepwise to achieve an obese state (DIO-GM group). Body composition, blood parameters and intravenous glucose tolerance were determined at regular intervals. A pilot chronic treatment trial with a GLP1 receptor agonist was conducted in DIO-GM. At the end of the study, the animals were necropsied and a biobank of selected tissues was established. Results: DIO-GM developed severe subcutaneous and visceral adiposity (body fat >50% of body mass vs. 22% in L-GM), increased plasma cholesterol, triglyceride, and free fatty acid levels, insulin resistance (HOMA-IR >5 vs. 2 in L-GM), impaired glucose tolerance and increased heart rate when resting and active. However, fasting glucose concentrations stayed within normal range throughout the study. Treatment with a long-acting GLP1 receptor agonist revealed substantial reduction of food intake and body weight within four weeks, with increased drug sensitivity relative to observations in other DIO animal models. Extensive adipose tissue inflammation and adipocyte necrosis was observed in visceral, but not subcutaneous, adipose tissue of DIO-GM. Conclusions: The Munich DIO-GM model resembles hallmarks of the human metabolic syndrome with extensive adipose tissue inflammation and adipocyte necrosis reported for the first time. DIO-GM may be used for evaluating novel treatments of obesity and associated comorbidities. They may help to identify triggers and mechanisms of fat tissue inflammation and mechanisms preventing complete metabolic decompensation despite morbid obesity. Keywords: Pig, High fat diet, Obesity, Diabetes, Metabolic syndrome, Adipose tissue inflammation

Meta-inflammation of hypothalamic areas governing energy homeostasis has recently emerged as a process of potential pathophysiological relevance for the development of obesity and its metabolic sequelae. The current model suggests that diet-induced neuronal injury triggers microgliosis and astrocytosis, conditions which ultimately may induce functional impairment of hypothalamic circuits governing feeding behavior, systemic metabolism, and body weight. Epidemiological data indicate that low circulating HDL levels, besides conveying cardiovascular risk, also correlate strongly with obesity. We simulated that condition by using a genetic loss of function mouse model (apoA-I−/−) with markedly reduced HDL levels to investigate whether HDL may directly modulate hypothalamic inflammation. Astrogliosis was significantly enhanced in the hypothalami of apoA-I−/− compared with apoA-I+/+ mice and was associated with compromised mitochondrial function. apoA-I−/− mice exhibited key components of metabolic disease, like increased fat mass, fasting glucose levels, hepatic triglyceride content, and hepatic glucose output compared with apoA-I+/+ controls. Administration of reconstituted HDL (CSL-111) normalized hypothalamic inflammation and mitochondrial function markers in apoA-I−/− mice. Treatment of primary astrocytes with apoA-I resulted in enhanced mitochondrial activity, implying that circulating HDL levels are likely important for astrocyte function. HDL-based therapies may consequently avert reactive gliosis in hypothalamic astrocytes by improving mitochondrial bioenergetics and thereby offering potential treatment and prevention for obesity and metabolic disease.

Objective: Obesity is a major health threat that affects men and women equally. Despite this fact, weight-loss potential of pharmacotherapies is typically first evaluated in male mouse models of diet-induced obesity (DIO). To address this disparity we herein determined whether a monomeric peptide with agonism at the receptors for glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon is equally efficient in correcting DIO, dyslipidemia, and glucose metabolism in DIO female mice as it has been previously established for DIO male mice. Methods: Female C57BL/6J mice and a cohort of fatmass-matched C57BL/6J male mice were treated for 27 days via subcutaneous injections with either the GLP-1/GIP/glucagon triagonist or PBS. A second cohort of C57BL/6J male mice was included to match the females in the duration of the high-fat, high-sugar diet (HFD) exposure. Results: Our results show that GLP-1/GIP/glucagon triple agonism inhibits food intake and decreases body weight and body fat mass with comparable potency in male and female mice that have been matched for body fat mass. Treatment improved dyslipidemia in both sexes and reversed diet-induced steatohepatitis to a larger extent in female mice compared to male mice. Conclusions: We herein show that a recently developed unimolecular peptide triagonist is equally efficient in both sexes, suggesting that this polypharmaceutical strategy might be a relevant alternative to bariatric surgery for the treatment of obesity and related metabolic disorders. Keywords: Obesity, Sex differences, Diabetes, Glucose homeostasis, Dyslipidemia, Pharmacotherapy

Objective: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice. Methods: To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity. Results: TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. Conclusions: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes. Keywords: FAP, FGF21, DPP4, Metabolic regulation, Obesity, Diabetes