A Draft Pacific Ancestry Pangenome Reference.

Individuals of Pacific ancestry suffer some of the highest rates of health disparities yet remain vastly underrepresented in genomic research, including currently available linear and pangenome references. To begin addressing this, we developed the first Pacific ancestry pangenome reference using 23 individuals with diverse Pacific ancestry. We assembled 46 haploid genomes from these 23 individuals, resulting in highly accurate and contiguous genome assemblies with an average quality value of 55.0 and an average N50 of 40.7 Mb, marking the first de novo assembly of highly accurate Pacific ancestry genomes. We combined these assemblies to create a pangenome reference, which added 30.6 Mb of novel sequence missing from the Human Pangenome Reference Consortium (HPRC) reference. Mapping short reads to this pangenome reduced variant call errors and yielded more true-positive variants compared to the HPRC and T2T-CHM13 references. This Pacific ancestry pangenome reference serves as a resource to enhance genetic analyses for this underserved population.

Ceramide microdomains: the major influencers of the sphingolipid media platform.

Like 'influencers' who achieve fame and power through social media, ceramides are low abundance members of communication platforms that have a mighty impact on their surroundings. Ceramide microdomains form within sphingolipid-laden lipid rafts that confer detergent resistance to cell membranes and serve as important signaling hubs. In cells exposed to excessive amounts of saturated fatty acids (e.g. in obesity), the abundance of ceramide-rich microdomains within these rafts increases, leading to concomitant alterations in cellular metabolism and survival that contribute to cardiometabolic disease. In this mini-review, we discuss the evidence supporting the formation of these ceramide microdomains and describe the spectrum of harmful ceramide-driven metabolic actions under the context of an evolutionary theory. Moreover, we discuss the proximal 'followers' of these ceramide media stars that account for the diverse intracellular actions that allow them to influence obesity-linked disease.

Long-term ketogenic diet causes hyperlipidemia, liver dysfunction, and glucose intolerance from impaired insulin trafficking and secretion in mice.

A ketogenic diet (KD) is a very low-carbohydrate, very high-fat diet proposed to treat obesity and type 2 diabetes. While KD grows in popularity, its effects on metabolic health are understudied. Here we show that, in male and female mice, while KD protects against weight gain and induces weight loss, over long-term, mice develop hyperlipidemia, hepatic steatosis, and severe glucose intolerance. Unlike high fat diet-fed mice, KD mice are not insulin resistant and have low levels of insulin. Hyperglycemic clamp and ex vivo GSIS revealed cell-autonomous and whole-body impairments in insulin secretion. Major ER/Golgi stress and disrupted ER-Golgi protein trafficking was indicated by transcriptomic profiling of KD islets and confirmed by electron micrographs showing a dilated Golgi network likely responsible for impaired insulin granule trafficking and secretion. Overall, our results suggest long-term KD leads to multiple aberrations of metabolic parameters that caution its systematic use as a health promoting dietary intervention.

Integrating molecular and clinical variables to predict myocardial recovery.

Mechanical unloading and circulatory support with left ventricular assist devices (LVADs) mediate significant myocardial improvement in a subset of advanced heart failure (HF) patients. The clinical and biological phenomena associated with cardiac recovery are under intensive investigation. Left ventricular (LV) apical tissue, alongside clinical data, were collected from HF patients at the time of LVAD implantation (n=208). RNA was isolated and mRNA transcripts were identified through RNA sequencing and confirmed with RT-qPCR. To our knowledge this is the first study to combine transcriptomic and clinical data to derive predictors of myocardial recovery. We used a bioinformatic approach to integrate 59 clinical variables and 22,373 mRNA transcripts at the time of LVAD implantation for the prediction of post-LVAD myocardial recovery defined as LV ejection fraction (LVEF) ≥40% and LV end-diastolic diameter (LVEDD) ≤5.9cm, as well as functional and structural LV improvement independently by using LVEF and LVEDD as continuous variables, respectively. To substantiate the predicted variables, we used a multi-model approach with logistic and linear regressions. Combining RNA and clinical data resulted in a gradient boosted model with 80 features achieving an AUC of 0.731±0.15 for predicting myocardial recovery. Variables associated with myocardial recovery from a clinical standpoint included HF duration, pre-LVAD LVEF, LVEDD, and HF pharmacologic therapy, and LRRN4CL (ligand binding and programmed cell death) from a biological standpoint. Our findings could have diagnostic, prognostic, and therapeutic implications for advanced HF patients, and inform the care of the broader HF population.

Adiponectin overexpression improves metabolic abnormalities caused by acid ceramidase deficiency but does not prolong lifespan in a mouse model of Farber Disease.

Farber Disease is a debilitating and lethal childhood disease of ceramide accumulation caused by acid ceramidase deficiency. The potent induction of a ligand-gated neutral ceramidase activity promoted by adiponectin may provide sufficient lowering of ceramides to allow for the treatment of Farber Disease. In vitro, adiponectin or adiponectin receptor agonist treatments lowered total ceramide concentrations in human fibroblasts from a patient with Farber Disease. However, adiponectin overexpression in a Farber Disease mouse model did not improve lifespan or immune infiltration. Intriguingly, mice heterozygous for the Farber Disease mutation were more prone to glucose intolerance and insulin resistance when fed a high-fat diet, and adiponectin overexpression protected from these metabolic perturbations. These studies suggest that adiponectin evokes a ceramidase activity that is not reliant on the functional expression of acid ceramidase, but indicates that additional strategies are required to ameliorate outcomes of Farber Disease.

Endothelial-Specific Reduction in Arf6 Impairs Insulin-Stimulated Vasodilation and Skeletal Muscle Blood Flow Resulting in Systemic Insulin Resistance in Mice.

BACKGROUND: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as the liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance; however, the underlying mechanisms remain incompletely understood. Arf6 (ADP ribosylation factor 6) is a small GTPase that plays a critical role in endothelial cell function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. METHODS: We used mouse models of constitutive endothelial cell-specific Arf6 deletion (Arf6f/- Tie2Cre+) and tamoxifen-inducible Arf6 knockout (Arf6f/f Cdh5CreER+). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps. We used a fluorescence microsphere-based technique to measure tissue blood flow. Skeletal muscle capillary density was assessed using intravital microscopy. RESULTS: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide bioavailability but independent of altered acetylcholine-mediated or sodium nitroprusside-mediated vasodilation. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow-fed mice and glucose intolerance in high-fat diet-fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. CONCLUSIONS: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.

Ceramides are fuel gauges on the drive to cardiometabolic disease.

Ceramides are signals of fatty acid excess that accumulate when a cell's energetic needs have been met and its nutrient storage has reached capacity. As these sphingolipids accrue, they alter the metabolism and survival of cells throughout the body including in the heart, liver, blood vessels, skeletal muscle, brain, and kidney. These ceramide actions elicit the tissue dysfunction that underlies cardiometabolic diseases such as diabetes, coronary artery disease, metabolic-associated steatohepatitis, and heart failure. Here, we review the biosynthesis and degradation pathways that maintain ceramide levels in normal physiology and discuss how the loss of ceramide homeostasis drives cardiometabolic pathologies. We highlight signaling nodes that sense small changes in ceramides and in turn reprogram cellular metabolism and stimulate apoptosis. Finally, we evaluate the emerging therapeutic utility of these unique lipids as biomarkers that forecast disease risk and as targets of ceramide-lowering interventions that ameliorate disease.

Skeletal muscle-specific inducible AMPKα1/α2 knockout mice develop muscle weakness, glycogen depletion, and fibrosis that persists during disuse atrophy.

The 5' adenosine monophosphate-activated protein kinase (AMPK) is an important skeletal muscle regulator implicated as a possible therapeutic target to ameliorate the local undesired deconditioning of disuse atrophy. However, the muscle-specific role of AMPK in regulating muscle function, fibrosis, and transcriptional reprogramming during physical disuse is unknown. The purpose of this study was to determine how the absence of both catalytic subunits of AMPK in skeletal muscle influences muscle force production, collagen deposition, and the transcriptional landscape. We generated skeletal muscle-specific tamoxifen-inducible AMPKα1/α2 knockout (AMPKα-/-) mice that underwent 14 days of hindlimb unloading (HU) or remained ambulatory for 14 days (AMB). We found that AMPKα-/- during ambulatory conditions altered body weight and myofiber size, decreased muscle function, depleted glycogen stores and TBC1 domain family member 1 (TBC1D1) phosphorylation, increased collagen deposition, and altered transcriptional pathways. Primarily, pathways related to cellular senescence and mitochondrial biogenesis and function were influenced by the absence of AMPKα. The effects of AMPKα-/- persisted, but were not worsened, following hindlimb unloading. Together, we report that AMPKα is necessary to maintain skeletal muscle quality.NEW & NOTEWORTHY We determined that skeletal muscle-specific AMPKα knockout (KO) mice display functional, fibrotic, and transcriptional alterations before and during muscle disuse atrophy. We also observed that AMPKα KO drives muscle fibrosis and pathways related to cellular senescence that continues during the hindlimb unloading period.

Germline and conditional ghrelin knockout increases islet size.

Conflicting studies in recent years report that genetic or pharmacological increases or decreases in ghrelin either increase or have no effect on islet size. In this issue of the JCI, Gupta, Burstein, and colleagues applied a rigorous approach to determine the effects of reducing ghrelin on islet size in germline and conditional ghrelin-knockout mice as well as across varying ages and weight. Both germline and conditional ghrelin-knockout mice associated with increased islet size, which was further exacerbated by older age and diet-induced obesity. These findings suggest that modulation of ghrelin may open a therapeutic window to prevent or treat diabetes.

Identification of genetic drivers of plasma lipoprotein size in the Diversity Outbred mouse population.

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2-/- mice. Compared to wild-type mice, female Asah2-/- mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

Hepatic sialic acid synthesis modulates glucose homeostasis in both liver and skeletal muscle.

OBJECTIVE: Sialic acid is a terminal monosaccharide of glycans in glycoproteins and glycolipids, and its derivation from glucose is regulated by the rate-limiting enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Although the glycans on key endogenous hepatic proteins governing glucose metabolism are sialylated, how sialic acid synthesis and sialylation in the liver influence glucose homeostasis is unknown. Studies were designed to fill this knowledge gap. METHODS: To decrease the production of sialic acid and sialylation in hepatocytes, a hepatocyte-specific GNE knockdown mouse model was generated, and systemic glucose metabolism, hepatic insulin signaling and glucagon signaling were evaluated in vivo or in primary hepatocytes. Peripheral insulin sensitivity was also assessed. Furthermore, the mechanisms by which sialylation in the liver influences hepatic insulin signaling and glucagon signaling and peripheral insulin sensitivity were identified. RESULTS: Liver GNE deletion in mice caused an impairment of insulin suppression of hepatic glucose production. This was due to a decrease in the sialylation of hepatic insulin receptors (IR) and a decline in IR abundance due to exaggerated degradation through the Eph receptor B4. Hepatic GNE deficiency also caused a blunting of hepatic glucagon receptor (GCGR) function which was related to a decline in its sialylation and affinity for glucagon. An accompanying upregulation of hepatic FGF21 production caused an enhancement of skeletal muscle glucose disposal that led to an overall increase in glucose tolerance and insulin sensitivity. CONCLUSION: These collective observations reveal that hepatic sialic acid synthesis and sialylation modulate glucose homeostasis in both the liver and skeletal muscle. By interrogating how hepatic sialic acid synthesis influences glucose control mechanisms in the liver, a new metabolic cycle has been identified in which a key constituent of glycans generated from glucose modulates the systemic control of its precursor.

A Cohort Study of the Diversity in Animated Films From 1937 to 2021: In a World Less Enchanted Can We Be More Encanto?

Background Exposure to gender stereotypes in the media can develop and reinforce these attitudes in children. Individuals who are overweight, have health conditions, or are from a minority ethnic group (IMEG) are both underrepresented and poorly portrayed in the media. Role models can raise the aspirations of young children both professionally and in taking ownership of their health. We aimed to assess how the portrayal and diversity of characters in Disney, Pixar, and Dreamworks animated films have changed over time. Method A cohort study of all main characters in Disney, Pixar, and Dreamworks feature-length, theatrical, animated films from 1937 to 2021 was conducted. The portrayal of characters (R-score divided into negative, neutral, and positive -1, 0, and 1, respectively) was scored. The proportion of individuals with certain protected characteristics (sex, increased body mass index, physical or mental health conditions, being from an IMEG or part of the lesbian, gay, bisexual, transexual, and queer community) was also recorded. Results In total, 116 films and 1,275 characters were included. From the 1930s to 2020s, the proportion of women in films increased (16.7% to 47.3%, p=0.008) and their representation was more positive (mean R-score = -0.10 (SD:0.692) versus 0.49 (SD:0.837), p<0.001, respectively). The portrayal of overweight individuals has improved to a neutral position (mean R-score: -0.67 to 0.0). Both physical and mental illnesses are better portrayed (mean R-score: -0.18 to 0.34, p=0.004 and 0.5 to 1.0, p= 0.019, respectively). IMEGs introduced in 1953 now play more than just negative roles (mean R-score = -1 to 0.76, p=0.008). There is only one explicitly stated homosexual character. The most diverse film is Encanto. Conclusion This is the first study to comprehensively assess the diversity of animated film characters. We have identified an improvement in diversity and the way diverse individuals are portrayed which we hope continues.

New-Onset Diabetes after an Obesity-Related Cancer Diagnosis and Survival Outcomes in the Women's Health Initiative.

BACKGROUND: Individuals diagnosed with an obesity-related cancer (ORC survivors) are at an elevated risk of incident diabetes compared with cancer-free individuals, but whether this confers survival disadvantage is unknown. METHODS: We assessed the rate of incident diabetes in ORC survivors and evaluated the association of incident diabetes with all-cause and cancer-specific mortality among females with ORC in the Women's Health Initiative cohort (N = 14,651). Cox proportional hazards regression models stratified by exposure-risk periods (0-1, >1-3, >3-5, >5-7, and >7-10 years) from ORC diagnosis and time-varying exposure (diabetes) analyses were performed. RESULTS: Among the ORC survivors, a total of 1.3% developed diabetes within ≤1 year of follow-up and 2.5%, 2.3%, 2.3%, and 3.6% at 1-3, 3-5, 5-7, and 7-10 years of follow-up, respectively, after an ORC diagnosis. The median survival for those diagnosed with diabetes within 1-year of cancer diagnosis and those with no diabetes diagnosis in that time frame was 8.8 [95% confidence interval (CI), 7.0-14.5) years and 16.6 (95% CI, 16.1-17.0) years, respectively. New-onset compared with no diabetes as a time-varying exposure was associated with higher risk of all-cause (HR, 1.27; 95% CI, 1.16-1.40) and cancer-specific (HR, 1.17; 95% CI, 0.99-1.38) mortality. When stratified by exposure-risk periods, incident diabetes in ≤1 year of follow-up was associated with higher all-cause (HR, 1.76; 95% CI, 1.40-2.20) and cancer-specific (HR0-1, 1.82; 95% CI, 1.28-2.57) mortality, compared with no diabetes diagnosis. CONCLUSIONS: Incident diabetes was associated with worse cancer-specific and all-cause survival, particularly in the year after cancer diagnosis. IMPACT: These findings draw attention to the importance of diabetes prevention efforts among cancer survivors to improve survival outcomes.

Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Long-term follow-up in patients with coeliac disease in the pandemic-era: a view from Sheffield the NHS England national centre for adult coeliac disease.

Aim: To explore patients' follow-up preferences. Background: Optimal follow-up strategies for patients with coeliac disease remain a subject of debate. Research suggests patients' prefer review by dietitians with a doctor available as required. Methods: Patients with coeliac disease under review at our centre, completed a questionnaire assessing their views on what makes follow-up useful based on specific criteria. Bloods tests, symptoms review, dietary assessment, opportunity to ask questions and reassurance. Patients' preferences between follow-up with a hospital doctor, a hospital dietitian, a hospital dietitian with a doctor available, a general practitioner, no follow-up or access when needed were also evaluated. Results: 138 adult patients completed the questionnaire, 80% of patients reported following a strict gluten free diet (mean diagnosis was 7.2 years). Overall, 60% found their follow-up to be 'very useful' valuing their review of blood tests and symptoms (71%) reassurance (60%) and opportunity to ask questions (58%). Follow-up by a dietitian with a doctor available was the most preferred option of review (p<0.001) except when compared to hospital doctor (p=0.75). Novel modalities of follow-up such as telephone and video reviews were regarded as of equal value to face-to-face appointments (65% and 62% respectively). Digital applications were significantly less preferable (38%, p<0.001). Conclusion: Follow-up by a dietitian with a doctor available as needed was the most preferred follow-up method. However, in this study follow-up by a dietitian with doctor available and hospital doctor alone was statistically equivalent. Many patients consider telephone and video follow-up of equal value to face-to-face reviews.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

Disuse-induced muscle fibrosis, cellular senescence, and senescence-associated secretory phenotype in older adults are alleviated during re-ambulation with metformin pre-treatment.

Muscle inflammation and fibrosis underlie disuse-related complications and may contribute to impaired muscle recovery in aging. Cellular senescence is an emerging link between inflammation, extracellular matrix (ECM) remodeling and poor muscle recovery after disuse. In rodents, metformin has been shown to prevent cellular senescence/senescent associated secretory phenotype (SASP), inflammation, and fibrosis making it a potentially practical therapeutic solution. Thus, the purpose of this study was to determine in older adults if metformin monotherapy during bed rest could reduce muscle fibrosis and cellular senescence/SASP during the re-ambulation period. A two-arm controlled trial was utilized in healthy male and female older adults (n = 20; BMI: <30, age: 60 years+) randomized into either placebo or metformin treatment during a two-week run-in and 5 days of bedrest followed by metformin withdrawal during 7 days of recovery. We found that metformin-treated individuals had less type-I myofiber atrophy during disuse, reduced pro-inflammatory transcriptional profiles, and lower muscle collagen deposition during recovery. Collagen content and myofiber size corresponded to reduced whole muscle cellular senescence and SASP markers. Moreover, metformin treatment reduced primary muscle resident fibro-adipogenic progenitors (FAPs) senescent markers and promoted a shift in fibroblast fate to be less myofibroblast-like. Together, these results suggest that metformin pre-treatment improved ECM remodeling after disuse in older adults by possibly altering cellular senescence and SASP in skeletal muscle and in FAPs.

Endothelial specific reduction in Arf6 impairs insulin-stimulated vasodilation and skeletal muscle blood flow resulting in systemic insulin resistance.

Background: Much of what we know about insulin resistance is based on studies from metabolically active tissues such as liver, adipose tissue, and skeletal muscle. Emerging evidence suggests that the vascular endothelium plays a crucial role in systemic insulin resistance, however, the underlying mechanisms remain incompletely understood. ADP ribosylation factor 6 (Arf6) is a small GTPase that plays a critical role in endothelial cell (EC) function. Here, we tested the hypothesis that the deletion of endothelial Arf6 will result in systemic insulin resistance. Methods: We used mouse models of constitutive EC-specific Arf6 deletion (Arf6 f/- Tie2Cre) and tamoxifen inducible Arf6 knockout (Arf6 f/f Cdh5Cre). Endothelium-dependent vasodilation was assessed using pressure myography. Metabolic function was assessed using a battery of metabolic assessments including glucose- and insulin-tolerance tests and hyperinsulinemic-euglycemic clamps. A fluorescence microsphere-based technique was used to measure tissue blood flow. Intravital microscopy was used to assess skeletal muscle capillary density. Results: Endothelial Arf6 deletion impaired insulin-stimulated vasodilation in white adipose tissue (WAT) and skeletal muscle feed arteries. The impairment in vasodilation was primarily due to attenuated insulin-stimulated nitric oxide (NO) bioavailability but independent of altered acetylcholine- or sodium nitroprusside-mediated vasodilation. In vitro Arf6 inhibition resulted in suppressed insulin stimulated phosphorylation of Akt and endothelial NO synthase. Endothelial cell-specific deletion of Arf6 also resulted in systematic insulin resistance in normal chow fed mice and glucose intolerance in high fat diet fed obese mice. The underlying mechanisms of glucose intolerance were reductions in insulin-stimulated blood flow and glucose uptake in the skeletal muscle and were independent of changes in capillary density or vascular permeability. Conclusion: Results from this study support the conclusion that endothelial Arf6 signaling is essential for maintaining insulin sensitivity. Reduced expression of endothelial Arf6 impairs insulin-mediated vasodilation and results in systemic insulin resistance. These results have therapeutic implications for diseases that are associated with endothelial cell dysfunction and insulin resistance such as diabetes.