Peripheral cannabinoid CB1 receptor antagonists that lack central nervous system effects are emerging as promising therapies for metabolic disease, yet the role of endothelial CB1 signaling in atherosclerosis remains unclear. Here, we show that endothelial CB1 is expressed in human atherosclerotic plaques, is induced by oscillatory shear stress in atheroprone flow regions, and promotes vascular inflammation, permeability and lipid uptake. Endothelial-specific Cnr1 deletion or peripheral CB1 antagonism in mice attenuates atherosclerosis, reduces endothelial caveolae-dependent low-density lipoprotein uptake by downregulating caveolin-1 and ALK1 expression, and improves metabolic parameters in brown and white adipose tissue and the liver. The anti-atherogenic and metabolic effects are more pronounced in females, which is possibly linked to estrogen signaling. These findings identify endothelial CB1 as a proatherogenic, sex-biased regulator of vascular lipid transport and plaque development and associated metabolic dysfunction.

Intermuscular adipose tissue expansion is closely associated with cardiometabolic disease, yet its cellular organization and regulatory mechanisms remain poorly defined. Using bulk transcriptomics on human intermuscular adipose tissue, we identified a distinct gene signature and functional regulators including adipogenic transcription factor early B-cell factor 2 (EBF2). By mapping this human signature to the spatial transcriptome of intermuscular adipose tissue from mice with cardiometabolic disease, we unraveled discrete stromal niches surrounding muscle fibers, characterized by intermuscular adipose tissue expansion and the coordinated activation of adipogenic, extracellular matrix, inflammatory, and metabolic pathways. Spatial analyses showed that fibro-adipogenic progenitor abundance does not predict adipocyte formation, supporting a model of localized and context-dependent lineage transitions. Cross-species comparison revealed partial conservation of human intermuscular adipose tissue gene programs, validating the mouse model and highlighting species-specific features. Functional experiments in human primary myoblasts showed that EBF2 is sufficient to induce adipogenic reprogramming. Our findings establish intermuscular adipose tissue as an active, spatially organized remodeling niche and identify lineage plasticity as a central mechanism driving its expansion in metabolic disease.

ABSTRACTBackgroundPeripherally restricted cannabinoid CB1 receptor antagonists without central side effects hold promise for treating metabolic disorders including diabetes and obesity. In atherosclerosis, the specific effects of peripheral CB1 signaling in vascular endothelial cells (ECs) remain incompletely understood.Methods and resultsEndothelial expression of the CB1 encoding geneCNR1was detectable in human plaque single-cell RNA sequencing data.In situhybridization ofCnr1in murine aortas revealed a significantly increased endothelial expression within atheroprone compared to atheroresistant regions.In vitro,CNR1was upregulated by oscillatory shear stress (OSS) in human aortic endothelial cells (HAoECs). Endothelial CB1 deficiency (Cnr1EC-KO) in female mice on atherogenic background resulted in pronounced endothelial phenotypic changes, with reduced vascular inflammation and permeability. This resulted in attenuated plaque development with reduced lipid content in female mice, while reduced white and brown adipose tissue mass and liver steatosis were observed in both males and females.Ex vivoimaging of carotid arteries via two-photon microscopy revealed less labeled low density lipoprotein (Dil-LDL) uptake inCnr1EC-KO. This was accompanied by a significant reduction of aortic endothelial caveolin-1 (CAV1) expression, a key structural protein involved in lipid transcytosis, in femaleCnr1EC-KOmice.In vitro, pharmacological blocking with CB1 antagonist AM281 reproduced the inhibition of CAV1 expression and Dil-LDL uptake in response to atheroprone OSS in HAoECs, which was dependent on cAMP-mediated PKA activation. Conversely, the CB1 agonist ACEA increased Dil-LDL uptake and CAV1 expression in HAoECs. Finally, treatment of atherosclerotic mice with the peripheral CB1 antagonist JD-5037 reduced plaque progression, CAV1 and endothelial adhesion molecule expression in female mice.ConclusionsThese results confirm an essential role of endothelial CB1 to the pathogenesis of atherosclerosis. Peripheral CB1 antagonists may hold promise as an effective therapeutic strategy for treating atherosclerosis and related metabolic disorders.NOVELTY AND SIGNIFICANCEWhat is known?Enhanced endocannabinoid-cannabinoid CB1 receptor signaling has been implicated in metabolic disorders, atherosclerosis, and hypertension, but the cell-specific role of endothelial CB1 in atherosclerosis is not well understood.Global CB1 antagonists improve metabolic function and inhibit atherosclerotic plaque development in mouse models, but have failed in the clinic due to centrally mediated psychiatric side effects.What is new?Based on human single-cell RNA sequencing data, CB1 is expressed in human plaque ECs.Using transgenic mouse models and human primary aortic endothelial cells, we provide evidence for a key role of CB1 in endothelial shear stress response, inflammatory gene expression, and LDL uptake.The underlying signaling pathway of CB1-induced endothelial LDL uptake involves a cAMP-PKA-dependent regulation of caveolin 1 (CAV1) expression, a structural protein of the shear stress sensitive signaling domains of the plasma membrane.By limiting endothelial CAV1 and VCAM1 expression, peripherally restricted CB1 antagonists confer atheroprotection in mice.Our findings reveal that endothelial CB1 expression is induced by atheroprone shear stress responses and contributes to impaired vascular barrier function, inflammation, and lipid uptake, thereby promoting atherosclerotic lesion formation and progression. By elucidating the transcriptomic pathways regulated by endothelial CB1 and its broad influence on lipid uptake and metabolism in arteries, liver, and brown and white adipose tissue, our study provides insights into novel pathways and potential interventions for the treatment of atherosclerosis and metabolic disorders. The use of peripherally restricted CB1 antagonists that specifically target vascular inflammation and tissue lipid storage could be a complementary and safe therapeutic avenue to treat cardiovascular and metabolic disease comorbidities without altering CB1 signaling in the brain.

Background Atherosclerosis is a chronic inflammatory disease characterized by metabolic and immune dysregulation. Emerging evidence suggests that specific central nervous system (CNS) regions modulate its progression via neuroimmune cardiovascular interfaces (NICIs). While the C-X-C motif chemokine receptor 4 (CXCR4) is known to participate in atherogenesis by regulating immune cell dynamics and vascular wall responses, the role of neural CXCR4 in atherosclerotic plaque formation remains unclear. Methods We generated a Nestin-Cre-mediated conditional Cxcr4 knockout mouse model on a low-density lipoprotein receptor-deficient (Ldlr-ko) background and fed these mice a western diet (WD) for up to 12 weeks to induce atherosclerosis. To evaluate the impact of neural CXCR4 function, we quantified atherosclerotic plaque burden, systemic metabolic parameters and circulating immune cell profiles, comparing neural Cxcr4-deficient mice with corresponding genetic controls. Spatial transcriptomics and RNAscope were employed to map Cxcr4 mRNA and its non-canonical ligand macrophage migration inhibitory factor (Mif) mRNA in neuroimmune-regulatory brain regions, and to assess diet-induced expression changes in relation to neuroinflammatory responses. Results Neural conditional deletion of Cxcr4 in Ldlr-ko mice significantly reduced atherosclerotic plaque formation in the aortic arch and aorta, without affecting body weight, lipid levels, glucose tolerance, or circulating immune cells. Cxcr4 gene expression was found to be uniformly low across hypothalamic subregions implicated in neuroimmune regulation of systemic inflammation and atherogenesis. Importantly, WD feeding did not modify this consistently low expression in male Ldlr-ko mice. In contrast, Mif mRNA expression was significantly upregulated in the PVN after 5-day WD feeding, but not after 8 weeks. Exploratory spatial transcriptomic analysis of PVN-containing coronal brain sections from male Ldlr-ko mice suggested that 5-day WD exposure activated MIF-CXCR4 signaling and downstream neuroinflammatory pathways in the PVN. Conclusions This study identifies neural CXCR4 as a component of neuroimmune modulation in atherosclerosis, exerting its effect independently of systemic metabolic or inflammatory changes. Short-term WD exposure activated gene expression of the CXCR4 ligand Mif in the PVN, pointing to a neuroimmune axis that may promote vascular inflammation and atherosclerotic plaque development. These findings establish a link between CNS CXCR4 and vascular disease and suggest that MIF-CXCR4-dependent neuroimmune pathways may play a role in cardiometabolic risk. ### Competing Interest Statement T.D.M. receives research funding from Novo Nordisk, the German Research Foundation (DFG TRR296, TRR152 and GRK 2816/1) and the European Research Council ERC-CoG Trusted no. 101044445, but these funds are unrelated to the here described work. T.D.M. receives research funding by Novo Nordisk and has received speaking fees from Novo Nordisk, Eli Lilly, Boehringer Ingelheim, Merck, AstraZeneca and Mercodia. S.M.H. receives research funding from the German Research Foundation (FOR 5298) that is unrelated to the here described work. J.B. and C.W. are co-inventors of patents covering anti-MIF strategies for inflammatory and cardiovascular diseases. A.K. and J.B. are co-inventors of a patent application covering MIF-binding CXCR4 ectodomain mimics for inflammatory and cardiovascular diseases. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SFB1123-A01, SFB1123-A03, SFB1123-A04, EXC 2145 SyNergy-ID 390857198 Deutsches Zentrum für Diabetes Bavarian Ministry of Economic Affairs and Media, Energy and Technology, https://ror.org/00rspzk45, SELECKREM 41-6663a/214/12-M4-2110-0005

Adipose tissue is a central organiser of systemic lipid homeostasis and a pharmacological target in obesity, orchestrating cellular responses to environmental cues. Nutritionally regulated adipose and cardiac enriched protein (NRAC) is a small adipocyte-specific transmembrane protein with unknown function. Here, we show that Nrac directly interacts with scavenger receptor CD36 via its first transmembrane domain. Forming a complex with CD36 and caveolin-1 under low extracellular fatty acid (FA) concentrations, NRAC modulates CD36-dependent fatty acid uptake in adipocytes. Upon increase in extracellular FA levels, NRAC is ubiquitinated and internalised, leading to CD36's dissociation from caveolin-1 and clathrin-mediated endocytosis. This results in increased fatty acid uptake into fat cells, adipocyte hypertrophy, increased fat mass and elevated lipid clearance from the blood in chow-diet-fed mice. Finally, human NRAC expression and the intronic SNP rs12878589 are associated with body fat distribution and obesity. Together, these findings reveal a novel regulatory mechanism by which adipocytes sense and respond to extracellular fatty acid availability to fine-tune lipid uptake and storage at cellular and organismal level.

Die Prävalenz von Typ-2-Diabetes nimmt bei beiden Geschlechtern zu, wobei Männer meist in jüngerem Alter und mit geringerer Körperfettmasse diagnostiziert werden. Frauen weisen bei der Diagnose oft ein höheres Risikofaktorenprofil auf, insbesondere in Bezug auf Adipositas und psychosozialen Stress. Schwangerschaften und Menopause erhöhen zusätzlich das kardiometabolische Risiko. Zudem haben Frauen mit Diabetes im Vergleich zu betroffenen Männern ein höheres relatives Risiko für kardiovaskuläre Erkrankungen und Mortalität. Geschlechtsspezifische Präventionsstrategien befinden sich noch in der Entwicklung und erfordern ein besseres Verständnis der krankheitsspezifischen Unterschiede und Mechanismen. Differierende gesundheitliche Auswirkungen von Diabetes bei Männern und Frauen machen maßgeschneiderte Präventions- und Behandlungsstrategien für eine optimale Versorgung unverzichtbar.

The prevalence of type 2 diabetes is increasing in both sexes, with men usually being diagnosed at a younger age and with a lower body fat mass. Women often have a higher risk factor profile at diagnosis, particularly in relation to obesity and psychosocial stress. Pregnancy and menopause also increase the cardiometabolic risk in women. Women have a higher relative risk of cardiovascular disease and mortality compared to men. Gender-specific prevention strategies are still under development and require a better understanding of differences and mechanisms. Differing health effects of diabetes in men and women require personalized prevention and treatment strategies for optimal care.

Abstract Die geschlechtersensible Medizin (GSM+) erforscht, wie biologisches Geschlecht und soziokulturelle Geschlechterrollen Gesundheit und Krankheitsverläufe beeinflussen. Historisch standen Männer im Fokus, wodurch wichtige Unterschiede bei Frauen vernachlässigt wurden. Einbeziehung von Frauen in klinische Studien und die Berücksichtigung von Geschlecht als biologische Variable, zielen darauf ab, eine gerechtere personalisierte medizinische Versorgung für alle Geschlechter zu ermöglichen. Dies beinhaltet auch die Vielfalt von Geschlechtern anzuerkennen und Forschungsansätze anzupassen.

Die geschlechtersensible Medizin (GSM+) erforscht, wie biologisches Geschlecht und soziokulturelle Geschlechterrollen Gesundheit und Krankheitsverlaufe beeinflussen. Historisch standen Manner im Fokus, wodurch wichtige Unterschiede bei Frauen vernachlassigt wurden. Einbeziehung von Frauen in klinische Studien und die Beruck-sichtigung von Geschlecht als biologische Variable, zielen darauf ab, eine gerechtere personalisierte medizinische Versorgung fur alle Geschlechter zu erm & ouml;glichen. Dies beinhaltet auch die Vielfalt von Geschlechtern anzuerkennen und Forschungsansatze anzupassen. Sex and gender medicine investigates the impact of biological sex and sociocultural gender roles on health outcomes and the progression of diseases. Traditionally, research centered on men, leading to a significant oversight of differences in women. The incorporation of women into clinical trials and recognizing sex as a biological variable aim to enhance the accuracy of medical care for all sex and genders. This approach also involves acknowledging sex and gender diversity and refining research methodologies accordingly.

BACKGROUND:Agonism at the receptor for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular GIPR:GLP-1R co-agonists, which are among the most promising drugs in clinical development for the treatment of obesity and type 2 diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia and thus to reduce the cardiovascular disease risk independently of body weight loss has not been explored yet. METHODS:After 8 weeks on western diet, LDL receptor knockout (LDLR-/-) male mice were treated with daily subcutaneous injections of long-acting acylated GIP analog (acyl-GIP; 10nmol/kg body weight) for 28 days. Body weight, food intake, whole-body composition were monitored throughout the study. Fasting blood glucose and intraperitoneal glucose tolerance test (ipGTT) were determined on day 21 of the study. Circulating lipid levels, lipoprotein profiles and atherosclerotic lesion size was assessed at the end of the study. Acyl-GIP effects on fat depots were determined by histology and transcriptomics. RESULTS:Herein we found that treatment with acyl-GIP reduced dyslipidemia and atherogenesis in male LDLR-/- mice. Acyl-GIP administration resulted in smaller adipocytes within the inguinal fat depot and RNAseq analysis of the latter revealed that acyl-GIP may improve dyslipidemia by directly modulating lipid metabolism in this fat depot. CONCLUSIONS:This study identified an unanticipated efficacy of chronic GIPR agonism to improve dyslipidemia and cardiovascular disease independently of body weight loss, indicating that treatment with acyl-GIP may be a novel approach to alleviate cardiometabolic disease.

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.

Microcirculatory dysfunction in dermal (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans may predict cardio-metabolic disease progression. In-vivo visualization and monitoring of microvascular remodeling in these tissues remains challenging. We compared performance of multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT of diet-induced obese (DIO) mice undergoing voluntary wheel running. MSOT quantitatively visualized lipid and hemoglobin contrast in fat depots at early stages of DIO. RSOM precisely visualizes microvasculature with quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. Combination of MSOT and RSOM precisely monitor microcirculatory dysfunction and intervention response in dWAT and scWAT of DIO mice. Our findings lay out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in cardio-metabolic disease.### Competing Interest StatementV.N. is an equity owner and consultant for iThera Medical GmbH, Munich, Germany* dWAT : Dermal white adipose tissue scWAT : Subcutaneous white adipose tissue iBAT. : Interscapular brown adipose tissue MSOT : Multi-spectral optoacoustic tomography RSOM : Raster-scanning optoacoustic mesoscopy DIO : Diet-induced obesity MRI : Magnetic Resonance Imaging CT : Computed Tomography BAT : Brown adipose tissue WAT : White adipose tissue HFD : High fat diet : Chow Standard diet Hb : deoxy-haemoglobin HbO2 : oxy-haemoglobin

Microcirculatory dysfunction has been observed in the dermal white adipose tissue (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans and has been proposed as an early prediction marker for cardio-metabolic disease progression. In-vivo visualization and longitudinal monitoring of microvascular remodeling in these tissues remains challenging. We compare the performance of two optoacoustic imaging methods, i.e. multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT in a mouse model of diet-induced obesity (DIO) undergoing voluntary wheel running intervention for 32 weeks. MSOT visualized lipid and hemoglobin contrast in murine fat depots in a quantitative manner even at early stages of DIO. We show for the first time to our knowledge that RSOM allows precise visualization of the dWAT microvasculature and provides quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. The combination of MSOT and RSOM may allow precise monitoring of microcirculatory dysfunction and intervention response in dWAT and scWAT in a mouse model for DIO. Our findings have laid out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in metabolic disease.

Muscle-residing regulatory T cells (Tregs) control local tissue integrity and function. However, the molecular interface connecting Treg-based regulation with muscle function and regeneration remains largely unexplored. Here, we show that exercise fosters a stable induction of highly functional muscle -residing Tregs with increased expression of amphiregulin (Areg), EGFR, and ST2. Mechanistically, we find that mice lacking IL6Ra on T cells (TKO) harbor significant reductions in muscle Treg functionality and satellite and fibro-adipogenic progenitor cells, which are required for muscle regeneration. Using ex-ercise and sarcopenia models, IL6Ra TKO mice demonstrate deficits in Tregs, their functional matura-tion, and a more pronounced decline in muscle mass. Muscle injury models indicate that IL6Ra TKO mice have significant disabilities in muscle regeneration. Treg gain of function restores impaired muscle repair in IL6Ra TKO mice. Of note, pharmacological IL6R blockade in WT mice phenocopies deficits in muscle function identified in IL6Ra TKO mice, thereby highlighting the clinical implications of the findings.

Accumulation of excess nutrients hampers proper liver function and is linked to nonalcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post-translational modification by the small ubiquitin-like modifier (SUMO) allows for a dynamic regulation of numerous processes including transcriptional reprogramming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and refed mice, while this modification is abolished upon fasting. In the context of diet-induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte-selective knock-in of a SUMOylation-deficient Prox1 mutant into mice fed a high-fat/high-fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient-sensitive SUMO-switch on Prox1 may thus contribute to the development of "fasting-based" approaches for the preservation of metabolic health.

Chronic kidney disease (CKD) is marked by a pre-aging and dysfunctional immune system and chronic inflammation. Recent research indicates that chronic physical exercise training (ET) increases regulatory monocytes and T-cells thus modulating the inflammatory status associated with cardio-metabolic diseases. In particular, ET primes monocytes and T-cells towards a less inflammatory phenotype. We hypothesized that intradialytic ET is beneficial to patients with CKD by improving certain immune cell features and decreasing systemic inflammation. We isolated PBMCs from a subset of 18 patients with CKD (58-90 years, 38 % females) enrolled in the prospective intervention trial DiaTT (Dialysis Training Therapy; ClinicalTrials.gov: NCT03885102) at the beginning and after a 12-months intradialytic ET intervention (aerobic endurance + resistance training). Circulating immune cell populations were characterized by FACS. Ex-vivo basal and PMA-induced T-cell activation was determined by cell surface expression analysis of CD69. We found that ET reversed the shift towards intermediate monocytes, a known marker for CKD morbidity and mortality, and nonclassical monocytes in patients with CKD undergoing dialysis treatment. We observed an increase in naïve T-cells and Recent Thymic Emigrants (RTE) indicating an improvement of T-cell function. On T-cells, basal CD69 expression was significantly enhanced and PMA-induced CD69 expression was severely impaired in patients with CKD compared to healthy control subjects (n= 7). When patients with CKD underwent ET this specific T-cell function was restored to normal levels. Here, we show that even in these severely sick patients certain circulating immune cell functions can be regained potentially leading to an enhanced immunosurveillance. Our ex-vivo immune cell characterization and function analysis can be used to monitor exercise treatment effects on systemic inflammation. Disclosure S. M. Hofmann: None. S. M. Dinges: None. M. Walter: None. P. Vonkorn: None. G. D. Vongersdorff: None. K. Anding-rost: None. M. Halle: Advisory Panel; Abbott, Speaker's Bureau; Boehringer Ingelheim (Canada) Ltd., Daiichi Sankyo, Amgen Inc., BMS.

Aims/hypothesis Although insulin resistance often leads to type 2 diabetes mellitus, its early stages are often unrecognised, thus reducing the probability of successful prevention and intervention. Moreover, treatment efficacy is affected by the genetics of the individual. We used gene expression profiles from a cross-sectional study to identify potential candidate genes for the prediction of diabetes risk and intervention response. Methods Using a multivariate regression model, we linked gene expression profiles of human skeletal muscle and intermuscular adipose tissue (IMAT) to fasting glucose levels and glucose infusion rate. Based on the expression patterns of the top predictive genes, we characterised and compared individual gene expression with clinical classifications using k -nearest neighbour clustering. The predictive potential of the candidate genes identified was validated using muscle gene expression data from a longitudinal intervention study. Results We found that genes with a strong association with clinical measures clustered into three distinct expression patterns. Their predictive values for insulin resistance varied substantially between skeletal muscle and IMAT. Moreover, we discovered that individual gene expression-based classifications may differ from classifications based predominantly on clinical variables, indicating that participant stratification may be imprecise if only clinical variables are used for classification. Of the 15 top candidate genes, ST3GAL2 , AASS , ARF1 and the transcription factor SIN3A are novel candidates for predicting a refined diabetes risk and intervention response. Conclusion/interpretation Our results confirm that disease progression and successful intervention depend on individual gene expression states. We anticipate that our findings may lead to a better understanding and prediction of individual diabetes risk and may help to develop individualised intervention strategies. Graphical abstract

GIPR:GLP-1R co-agonism improves obesity and cardio-metabolic disease with superior efficacy in both male and female mice compared to mono-agonism. We detected sex-specific differences in metabolic phenotypes, which may point to differing sensitivity for GLP-1R and/or GIPR in areas of the hypothalamus regulating energy balance (ARC, VMH, DMH) and in areas of the amygdala regulating cardiovascular functions. To optimize future therapeutic benefits and minimize side effects from a perspective of sex differences, we investigated whether GLP1R and GIPR expression levels in specific cell populations of the hypothalamus and the amygdala regions are co-determined by sex and diet. Female and male C57Bl6 mice were fed normal chow or 58% high fat diet (HFD) for 21 days (n = 4, total mouse number 16). Spatial transcriptomics by in-situ hybridization method RNAscope™ was combined with immunofluorescence staining for neurons (NeuN), astrocytes (GFAP) and microglia (iba1) to identify cell type specific gene expression patterns of GLP1R and GIPR in female and male brains. In the hypothalamus, both sexes, gene expression of GLP-1R was higher compared to GIPR (ARC, VMH and DMH). In our studies GLP-1R and GIPR were mostly expressed in neurons, marginally in microglia and not observed in astrocytes in both sexes. HFD exposure reduced GLP-1R and GIPR gene expression in males only. In the Amygdala, neuronal GIPR gene expression levels were higher compared to GLP-1R in both sexes. HFD reduced GIPR and GLP-1R gene expression in males only. In females, HFD induced a slight increase of GIPR gene expression specifically in the central and basolateral amygdala. Our results reveal sex-specific dynamics in GLP-1R and GIPR expression patterns in metabolically relevant areas of the hypothalamus and the amygdala induced by HFD exposure. This may imply sex-specific dosing regimens of GIP:GLP-1R co-agonists for future treatments of cardio-metabolic disease Disclosure S. M. Hofmann: None. S. Zhang: None. R. Tom: None. T. D. Müller: Research Support; Novo Nordisk A/S, Speaker's Bureau; Eli Lilly and Company, AstraZeneca, Mercodia AB, Stock/Shareholder; Novo Nordisk A/S, Eli Lilly and Company. M. H. Tschöp: Consultant; Boehringer Ingelheim Pharma GmbH&Co. KG.

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 preclinical 1 – 3 and clinical studies 4 , 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 intake 3 , 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.