A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.

The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 µm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 µmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

Regulation of the Ca(2+)-independent phospholipase A2 in liver mitochondria by changes in the energetic state.

The effect of electron transport chain redox status on activity of the mitochondrial Ca(2+)-independent phospholipase A2 (iPLA2) has been examined. When oxidizing NAD-linked substrates, the enzyme is not active unless deenergization occurs. Uncoupler, rotenone, antimycin A, and cyanide are equally effective at upregulating the enzyme, while oligomycin is ineffective. Thenoyltrifluoroacetone causes deenergization and activates the enzyme, but only if succinate is the respiratory substrate. These findings show that the mitochondrial iPLA2 responds to the energetic state overall, rather than to the redox status of individual electron transport chain complexes. With NAD-linked substrates, and using rotenone to deenergize, iPLA2 activation can be reversed by adding succinate to reestablish a membrane potential. For this purpose, ascorbate plus N,N,N'N'-tetramethyl-phenylenediamine can be used instead of succinate and is equally effective. With succinate as substrate, the membrane potential can be reduced in a graded and stable fashion by adding increasing concentrations of malonate, which is a competitive inhibitor of succinate utilization. A partial and stable activation of the iPLA2 accompanies partial deenergization. These findings suggest that in addition to the several functions that have been proposed, the mitochondrial iPLA2 may help to coordinate local capillary blood flow with changing energy demands.

The mitochondrial dicarboxylate carrier mediates in vivo hepatic gluconeogenesis.

Hepatic gluconeogenesis (GNG) is essential for maintaining euglycemia during prolonged fasting. However, GNG becomes pathologically elevated and drives chronic hyperglycemia in type 2 diabetes (T2D). Lactate/pyruvate is a major GNG substrate known to be imported into mitochondria for GNG. Yet, the subsequent mitochondrial carbon export mechanisms required to supply the extra-mitochondrial canonical GNG pathway have not been genetically delineated. Here, we evaluated the role of the mitochondrial dicarboxylate carrier (DiC) in mediating GNG from lactate/pyruvate. We generated liver-specific DiC knockout (DiC LivKO) mice. During lactate/pyruvate tolerance tests, DiC LivKO decreased plasma glucose excursion and 13C-lactate/-pyruvate flux into hepatic and plasma glucose. In a Western diet (WD) feeding model of T2D, acute DiC LivKO after induction of obesity decreased lactate/pyruvate-driven GNG, hyperglycemia, and hyperinsulinemia. Our results show that mitochondrial carbon export through the DiC mediates GNG and that the DiC contributes to impaired glucose homeostasis in a mouse model of T2D.

Tubular mitochondrial pyruvate carrier disruption elicits redox adaptations that protect from acute kidney injury.

OBJECTIVE: Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Although tubular metabolism changes markedly following acute kidney injury (AKI), it remains unclear which metabolic alterations are beneficial or detrimental. By analyzing large-scale, publicly available datasets, we observed that AKI consistently leads to downregulation of the mitochondrial pyruvate carrier (MPC). This investigation aimed to understand the contribution of the tubular MPC to kidney function, metabolism, and acute injury severity. METHODS: We generated tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice and employed renal function tests, in vivo renal 13C-glucose tracing, mechanistic enzyme activity assays, and tests of injury and survival in an established rhabdomyolysis model of AKI. RESULTS: MPC TubKO mice retained normal kidney function, displayed unchanged markers of kidney injury, but exhibited coordinately increased enzyme activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, compared to WT control mice, MPC TubKO mice showed increased glycolysis, decreased kidney injury and oxidative stress markers, and strikingly increased survival. CONCLUSIONS: Our findings suggest that decreased renal tubular mitochondrial pyruvate uptake hormetically upregulates oxidant defense systems before AKI and is a beneficial adaptive response after rhabdomyolysis-induced AKI. This raises the possibility of therapeutically modulating the MPC to attenuate AKI severity.

3-hydroxykynurenine is a ROS-inducing cytotoxic tryptophan metabolite that disrupts the TCA cycle.

Tryptophan is an essential amino acid that is extensively characterized as a regulator of cellular function through its metabolism by indoleamine 2,3-deoxygenase (IDO) into the kynurenine pathway. However, despite decades of research on tryptophan metabolism, the metabolic regulatory roles of it and its metabolites are not well understood. To address this, we performed an activity metabolomics screen of tryptophan and most of its known metabolites in cell culture. We discovered that treatment of human colon cancer cells (HCT116) with 3-hydroxykynurenine (3-HK), a metabolite of kynurenine, potently disrupted TCA cycle function. Citrate and aconitate levels were increased, while isocitrate and all downstream TCA metabolites were decreased, suggesting decreased aconitase function. We hypothesized that 3HK or one of its metabolites increased reactive oxygen species (ROS) and inhibited aconitase activity. Accordingly, we observed almost complete depletion of reduced glutathione and a decrease in total glutathione levels. We observed a dose-dependent decrease in cell viability after 48 hours of 3HK treatment. These data suggest that raising the intracellular levels of 3HK could be sufficient to induce ROS-mediated apoptosis. We modulated the intracellular levels of 3HK by combined induction of IDO and knockdown of kynureninase (KYNU) in HCT116 cells. Cell viability decreased significantly after 48 hours of KYNU knockdown compared to controls, which was accompanied by increased ROS production and Annexin V staining revealing apoptosis. Finally, we identify xanthommatin production from 3-HK as a candidate radical-producing, cytotoxic mechanism. Our work indicates that KYNU may be a target for disrupting tryptophan metabolism. Interestingly, many cancers exhibit overexpression of IDO, providing a cancer-specific metabolic vulnerability that could be exploited by KYNU inhibition.

Glycerol monolaurate inhibits Francisella novicida growth and is produced intracellularly in an ISG15-dependent manner.

Glycerol Monolaurate (GML) is a naturally occurring fatty acid monoester with antimicrobial properties. Francisella tularensis is an agent of bioterrorism known for its unique lipopolysaccharide structure and low immunogenicity. Here we assessed whether exogenous GML would inhibit the growth of Francisella novicida . GML potently impeded Francisella growth and survival in vitro . To appraise the metabolic response to infection, we used GC-MS to survey the metabolome, and surprisingly, observed intracellular GML production following Francisella infection. Notably, the ubiquitin-like protein ISG15 was necessary for increased GML levels induced by bacterial infection, and enhanced ISG15 conjugation correlated with GML levels following serum starvation.

Metabolic clearance of oxaloacetate and mitochondrial complex II respiration: Divergent control in skeletal muscle and brown adipose tissue.

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.

Mitochondrial citrate metabolism and efflux regulates trophoblast differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

Deficiency of endothelial sirtuin1 in mice stimulates skeletal muscle insulin sensitivity by modifying the secretome.

Downregulation of endothelial Sirtuin1 (Sirt1) in insulin resistant states contributes to vascular dysfunction. Furthermore, Sirt1 deficiency in skeletal myocytes promotes insulin resistance. Here, we show that deletion of endothelial Sirt1, while impairing endothelial function, paradoxically improves skeletal muscle insulin sensitivity. Compared to wild-type mice, male mice lacking endothelial Sirt1 (E-Sirt1-KO) preferentially utilize glucose over fat, and have higher insulin sensitivity, glucose uptake, and Akt signaling in fast-twitch skeletal muscle. Enhanced insulin sensitivity of E-Sirt1-KO mice is transferrable to wild-type mice via the systemic circulation. Endothelial Sirt1 deficiency, by inhibiting autophagy and activating nuclear factor-kappa B signaling, augments expression and secretion of thymosin beta-4 (Tß4) that promotes insulin signaling in skeletal myotubes. Thus, unlike in skeletal myocytes, Sirt1 deficiency in the endothelium promotes glucose homeostasis by stimulating skeletal muscle insulin sensitivity through a blood-borne mechanism, and augmented secretion of Tß4 by Sirt1-deficient endothelial cells boosts insulin signaling in skeletal muscle cells.

Mitochondrial citrate metabolism and efflux regulate BeWo differentiation.

Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established BeWo cell culture model of trophoblast differentiation. Differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.

Tubular Mitochondrial Pyruvate Carrier Disruption Elicits Redox Adaptations that Protect from Acute Kidney Injury.

Energy-intensive kidney reabsorption processes essential for normal whole-body function are maintained by tubular epithelial cell metabolism. Tubular metabolism changes markedly following acute kidney injury (AKI), but which changes are adaptive versus maladaptive remain poorly understood. In publicly available data sets, we noticed a consistent downregulation of the mitochondrial pyruvate carrier (MPC) after AKI, which we experimentally confirmed. To test the functional consequences of MPC downregulation, we generated novel tubular epithelial cell-specific Mpc1 knockout (MPC TubKO) mice. 13C-glucose tracing, steady-state metabolomic profiling, and enzymatic activity assays revealed that MPC TubKO coordinately increased activities of the pentose phosphate pathway and the glutathione and thioredoxin oxidant defense systems. Following rhabdomyolysis-induced AKI, MPC TubKO decreased markers of kidney injury and oxidative damage and strikingly increased survival. Our findings suggest that decreased mitochondrial pyruvate uptake is a central adaptive response following AKI and raise the possibility of therapeutically modulating the MPC to attenuate AKI severity.

Organelle interactions compartmentalize hepatic fatty acid trafficking and metabolism.

Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity.

Membrane potential-dependent regulation of mitochondrial complex II by oxaloacetate in interscapular brown adipose tissue.

Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate-energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well-recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m-chlorophenyl hydrazone (FCCP). In succinate-energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP-treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC-MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP-altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP-like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1-dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.

Mouse tissue harvest-induced hypoxia rapidly alters the in vivo metabolome, between-genotype metabolite level differences, and 13C-tracing enrichments.

OBJECTIVE: Metabolomics as an approach to solve biological problems is exponentially increasing in use. Thus, this a pivotal time for the adoption of best practices. It is well known that disrupted tissue oxygen supply rapidly alters cellular energy charge. However, the speed and extent to which delayed mouse tissue freezing after dissection alters the broad metabolome is not well described. Furthermore, how tissue genotype may modulate such metabolomic drift and the degree to which traced 13C-isotopologue distributions may change have not been addressed. METHODS: By combined liquid chromatography (LC)- and gas chromatography (GC)-mass spectrometry (MS), we measured how levels of 255 mouse liver metabolites changed following 30-second, 1-minute, 3-minute, and 10-minute freezing delays. We then performed test-of-concept delay-to-freeze experiments evaluating broad metabolomic drift in mouse heart and skeletal muscle, differential metabolomic change between wildtype (WT) and mitochondrial pyruvate carrier (MPC) knockout mouse livers, and shifts in 13C-isotopologue abundances and enrichments traced from 13C-labled glucose into mouse liver. RESULTS: Our data demonstrate that delayed mouse tissue freezing after dissection leads to rapid hypoxia-driven remodeling of the broad metabolome, induction of both false-negative and false-positive between-genotype differences, and restructuring of 13C-isotopologue distributions. Notably, we show that increased purine nucleotide degradation products are an especially high dynamic range marker of delayed liver and heart freezing. CONCLUSIONS: Our findings provide a previously absent, systematic illustration of the extensive, multi-domain metabolomic changes occurring within the early minutes of delayed tissue freezing. They also provide a novel, detailed resource of mouse liver ex vivo, hypoxic metabolomic remodeling.

A pilot to assess target engagement of terazosin in Parkinson's disease.

BACKGROUND: Impaired brain energy metabolism is a key feature of Parkinson's disease (PD). Terazosin (TZ) binds phosphoglycerate kinase 1 and stimulates its activity, which enhances glycolysis and increases ATP levels. Preclinical and epidemiologic data suggest that TZ may be neuroprotective in PD. We aimed to assess target engagement and safety of TZ in people with PD. METHODS: We performed a 12-week pilot study in people with PD. Participants were randomized to receive 5 mg TZ or placebo. Participants and study personnel were blinded. We assessed TZ target engagement by measuring brain ATP with 31P-magnetic resonance spectroscopy (MRS) and whole blood ATP with a luminescence assay. Robust linear regression models compared changes between groups controlling for baseline brain and blood ATP levels, respectively. We also assessed clinical measures of PD and adverse events. RESULTS: Thirteen participants were randomized. Mild dizziness/lightheadedness was more common in the TZ group, and three participants taking TZ dropped out because of dizziness and/or orthostatic hypotension. Compared to the placebo group, the TZ group had a significant increase in the ratio of ßATP to inorganic phosphate in the brain. The TZ group also had a significant increase in blood ATP levels compared to the placebo group (p < 0.01). CONCLUSIONS: This pilot study suggests that TZ may engage its target and change ATP levels in the brain and blood of people with PD. Further studies may be warranted to test the disease-modifying potential of TZ.

Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival.

Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease. Although gene editing and gene transfer hold great promise as therapies when administered before the onset of severe clinical symptoms, it is unclear whether these strategies can restore muscle function and improve survival in the late stages of muscular dystrophy. Largemyd/Largemyd (myd) mice lack expression of like-acetylglucosaminyltransferase-1 (Large1) and exhibit severe muscle pathophysiology, impaired mobility, and a markedly reduced life span. Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression on dystroglycan, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and markedly extends survival. Our results in a mouse model of muscular dystrophy demonstrate that skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe muscle pathophysiology.

Distinct properties of Ca2+ efflux from brain, heart and liver mitochondria: The effects of Na+, Li+ and the mitochondrial Na+/Ca2+ exchange inhibitor CGP37157.

Mitochondrial Ca2+ transport is essential for regulating cell bioenergetics, Ca2+ signaling and cell death. Mitochondria accumulate Ca2+ via the mitochondrial Ca2+ uniporter (MCU), whereas Ca2+ is extruded by the mitochondrial Na+/Ca2+ (mtNCX) and H+/Ca2+ exchangers. The balance between these processes is essential for preventing toxic mitochondrial Ca2+ overload. Recent work demonstrated that MCU activity varies significantly among tissues, likely reflecting tissue-specific Ca2+ signaling and energy needs. It is less clear whether this diversity in MCU activity is matched by tissue-specific diversity in mitochondrial Ca2+ extrusion. Here we compared properties of mitochondrial Ca2+ extrusion in three tissues with prominent mitochondria function: brain, heart and liver. At the transcript level, expression of the Na+/Ca2+/Li+ exchanger (NCLX), which has been proposed to mediate mtNCX transport, was significantly greater in liver than in brain or heart. At the functional level, Na+ robustly activated Ca2+ efflux from brain and heart mitochondria, but not from liver mitochondria. The mtNCX inhibitor CGP37157 blocked Ca2+ efflux from brain and heart mitochondria but had no effect in liver mitochondria. Replacement of Na+ with Li+ to test the involvement of NCLX, resulted in a slowing of mitochondrial Ca2+ efflux by ∼70 %. Collectively, our findings suggest that mtNCX is responsible for Ca2+ extrusion from the mitochondria of the brain and heart, but plays only a small, if any, role in mitochondria of the liver. They also reveal that Li+ is significantly less effective than Na+ in driving mitochondrial Ca2+ efflux.

Endothelial BBSome is essential for vascular, metabolic, and retinal functions.

OBJECTIVES: Endothelial cells that line the entire vascular system play a pivotal role in the control of various physiological processes, including metabolism. Additionally, endothelial dysfunction is associated with many pathological conditions, including obesity. Here, we assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins in endothelial cells. METHODS: We studied the effects of BBSome disruption in endothelial cells on vascular function, body weight, glucose homeostasis, and the liver and retina. For this, we generated mice with selective BBSome disruption in endothelial cells through Bbs1 gene deletion. RESULTS: We found that endothelial cell-specific BBSome disruption causes endothelial dysfunction, as indicated by the impaired acetylcholine-induced vasorelaxation in both the aorta and mesenteric artery. This was associated with an increase in the contractile response to thromboxane A2 receptor agonist (U46619) in the mesenteric artery. Mechanistically, we demonstrated that mice lacking the Bbs1 gene in endothelial cells show elevated vascular angiotensinogen gene expression, implicating renin-angiotensin system activation in the vascular changes evoked by endothelial BBSome deficiency. Strikingly, our data indicate that endothelial BBSome deficiency increases body weight and fat mass and causes hepatosteatosis along with alterations in hepatic expression of lipid metabolism-related genes and metabolomics profile. In addition, electroretinogram and optical coherence tomography analyses revealed functional and structural abnormalities in the retina, evoked by absence of the endothelial BBSome. CONCLUSIONS: Our findings demonstrate that the BBSome in endothelial cells is required for the regulation of vascular function, adiposity, hepatic lipid metabolism, and retinal function.