Obesogenic diet disrupts tissue-specific mitochondrial gene signatures in the artery and capillary endothelium.

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.

Smooth muscle cell FTO regulates contractile function.

The fat mass and obesity gene (FTO) is a N6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre+ Ftofl/fl) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto-/--isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells.NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).

Endothelial-adipocyte Cx43 mediated gap junctions can regulate adiposity.

Obesity is a multifactorial metabolic disorder associated with endothelial dysfunction and increased risk of cardiovascular disease. Adipose capillary adipose endothelial cells (CaECs), plays a crucial role in lipid transport and storage. Here, we investigated the mechanisms underlying CaEC-adipocyte interaction and its impact on metabolic function. Single-cell RNA sequencing revealed an enrichment of fatty acid handling machinery in CaECs from high fat diet (HFD) mice, suggesting their specialized role in lipid metabolism. Transmission Electron microscopy (TEM) confirmed direct heterocellular contact between CaECs and adipocytes. To model this, we created an in vitro co-culture transwell system to model the heterocellular contact observed with TEM. Contact between ECs and adipocytes in vitro led to upregulation of fatty acid binding protein 4 in response to lipid stimulation, hinting intercellular signaling may be important between ECs and adipocytes. We mined our and others scRNAseq data sets to examine which connexins may be present in adipose capillaries and adipocytes and consistently identified Connexin 43 (Cx43) in mouse and humans. Genetic deletion of endothelial Cx43 resulted in increased epididymal fat pad (eWAT) adiposity and dyslipidemia in HFD mice. Consistent with this observation, phosphorylation of Cx43 at serine 368, which closes gap junctions, was increased in HFD mice and lipid treated ECs. Mice resistant to this post-translational modification, Cx43S368A, were placed on a HFD and were found to have reduced eWAT adiposity and improved lipid profiles. These findings suggest Cx43-mediated heterocellular communication as a possible regulatory mechanism of adipose tissue function.

Pannexin-3 stabilizes the transcription factor Bcl6 in a channel-independent manner to protect against vascular oxidative stress.

Targeted degradation regulates the activity of the transcriptional repressor Bcl6 and its ability to suppress oxidative stress and inflammation. Here, we report that abundance of endothelial Bcl6 is determined by its interaction with Golgi-localized pannexin 3 (Panx3) and that Bcl6 transcriptional activity protects against vascular oxidative stress. Consistent with data from obese, hypertensive humans, mice with an endothelial cell-specific deficiency in Panx3 had spontaneous systemic hypertension without obvious changes in channel function, as assessed by Ca2+ handling, ATP amounts, or Golgi luminal pH. Panx3 bound to Bcl6, and its absence reduced Bcl6 protein abundance, suggesting that the interaction with Panx3 stabilized Bcl6 by preventing its degradation. Panx3 deficiency was associated with increased expression of the gene encoding the H2O2-producing enzyme Nox4, which is normally repressed by Bcl6, resulting in H2O2-induced oxidative damage in the vasculature. Catalase rescued impaired vasodilation in mice lacking endothelial Panx3. Administration of a newly developed peptide to inhibit the Panx3-Bcl6 interaction recapitulated the increase in Nox4 expression and in blood pressure seen in mice with endothelial Panx3 deficiency. Panx3-Bcl6-Nox4 dysregulation occurred in obesity-related hypertension, but not when hypertension was induced in the absence of obesity. Our findings provide insight into a channel-independent role of Panx3 wherein its interaction with Bcl6 determines vascular oxidative state, particularly under the adverse conditions of obesity.

Physiological functions of caveolae in endothelium.

Endothelial caveolae are essential for a wide range of physiological processes and have emerged as key players in vascular biology. Our understanding of caveolar biology in endothelial cells has expanded dramatically since their discovery revealing critical roles in mechanosensation, signal transduction, eNOS regulation, lymphatic transport, and metabolic disease progression. Furthermore, caveolae are involved in the organization of membrane domains, regulation of membrane fluidity, and endocytosis which contribute to endothelial function and integrity. Additionally, recent advances highlight the impact of caveolae-mediated signaling pathways on vascular homeostasis and pathology. Together, the diverse roles of caveolae discussed here represent a breadth of cellular functions presenting caveolae as a defining feature of endothelial form and function. In light of these new insights, targeting caveolae may hold potential for the development of novel therapeutic strategies to treat a range of vascular diseases.

Extracellular vesicles and insulin-mediated vascular function in metabolic syndrome.

Metabolic Syndrome (MetS) raises cardiovascular disease risk. Extracellular vesicles (EVs) have emerged as important mediators of insulin sensitivity, although few studies on vascular function exist in humans. We determined the effect of insulin on EVs in relation to vascular function. Adults with MetS (n = 51, n = 9 M, 54.8 ± 1.0 years, 36.4 ± 0.7 kg/m2 , ATPIII: 3.5 ± 0.1 a.u., VO2 max: 22.1 ± 0.6 ml/kg/min) were enrolled in this cross-sectional study. Peripheral insulin sensitivity (M-value) was determined during a euglycemic clamp (40 mU/m2 /min, 90 mg/dl), and blood was collected for EVs (CD105+, CD45+, CD41+, TX+, and CD31+; spectral flow cytometry), inflammation, insulin, and substrates. Central hemodynamics (applanation tonometry) was determined at 0 and 120 min via aortic waveforms. Pressure myography was used to assess insulin-induced arterial vasodilation from mouse 3rd order mesenteric arteries (100-200 µm in diameter) at 0.2, 2 and 20 nM of insulin with EVs from healthy and MetS adults. Adults with MetS had low peripheral insulin sensitivity (2.6 ± 0.2 mg/kg/min) and high HOMA-IR (4.7 ± 0.4 a.u.) plus Adipose-IR (13.0 ± 1.3 a.u.). Insulin decreased total/particle counts (p < 0.001), CD45+ EVs (p = 0.002), AIx75 (p = 0.005) and Pb (p = 0.04), FFA (p < 0.001), total adiponectin (p = 0.006), ICAM (p = 0.002), and VCAM (p = 0.03). Higher M-value related to lower fasted total EVs (r = -0.40, p = 0.004) while higher Adipose-IR associated with higher fasted EVs (r = 0.42, p = 0.004) independent of VAT. Fasting CD105+ and CD45+ derived total EVs correlated with fasting AIx75 (r = 0.29, p < 0.05) and Pb (r = 0.30, p < 0.05). EVs from MetS participants blunted insulin-induced vasodilation in mesenteric arteries compared with increases from healthy controls across insulin doses (all p < 0.005). These data highlight EVs as potentially novel mediators of vascular insulin sensitivity and disease risk.

Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1.

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function.

Feeding desensitizes A1 adenosine receptors in adipose through FOXO1-mediated transcriptional regulation.

OBJECTIVE: Adipose tissue is a critical regulator of energy balance that must rapidly shift its metabolism between fasting and feeding to maintain homeostasis. Adenosine has been characterized as an important regulator of adipocyte metabolism primarily through its actions on A1 adenosine receptors (A1R). We sought to understand the role A1R plays specifically in adipocytes during fasting and feeding to regulate glucose and lipid metabolism. METHODS: We used Adora1 floxed mice with an inducible, adiponectin-Cre to generate FAdora1-/- mice, where F designates a fat-specific deletion of A1R. We used these FAdora1-/- mice along with specific agonists and antagonists of A1R to investigate changes in adenosine signaling within adipocytes between the fasted and fed state. RESULTS: We found that the adipose tissue response to adenosine is not static, but changes dynamically according to nutrient conditions through the insulin-Akt-FOXO1 axis. We show that under fasted conditions, FAdora1-/- mice had impairments in the suppression of lipolysis by insulin on normal chow and impaired glucose tolerance on high-fat diet. FAdora1-/- mice also exhibited a higher lipolytic response to isoproterenol than WT controls when fasted, however this difference was lost after a 4-hour refeeding period. We demonstrate that FOXO1 binds to the A1R promoter, and refeeding leads to a rapid downregulation of A1R transcript and desensitization of adipocytes to A1R agonism. Obesity also desensitizes adipocyte A1R, and this is accompanied by a disruption of cyclical changes in A1R transcription between fasting and refeeding. CONCLUSIONS: We propose that FOXO1 drives high A1R expression under fasted conditions to limit excess lipolysis during stress and augment insulin action upon feeding. Subsequent downregulation of A1R under fed conditions leads to desensitization of these receptors in adipose tissue. This regulation of A1R may facilitate reentrance into the catabolic state upon fasting.

RalA and PLD1 promote lipid droplet growth in response to nutrient withdrawal.

Lipid droplets (LDs) are dynamic organelles that undergo dynamic changes in response to changing cellular conditions. During nutrient depletion, LD numbers increase to protect cells against toxic fatty acids generated through autophagy and provide fuel for beta-oxidation. However, the precise mechanisms through which these changes are regulated have remained unclear. Here, we show that the small GTPase RalA acts downstream of autophagy to directly facilitate LD growth during nutrient depletion. Mechanistically, RalA performs this function through phospholipase D1 (PLD1), an enzyme that converts phosphatidylcholine (PC) to phosphatidic acid (PA) and that is recruited to lysosomes during nutrient stress in a RalA-dependent fashion. RalA inhibition prevents recruitment of the LD-associated protein perilipin 3, which is required for LD growth. Our data support a model in which RalA recruits PLD1 to lysosomes during nutrient deprivation to promote the localized production of PA and the recruitment of perilipin 3 to expanding LDs.

Cellular and Functional Effects of Insulin Based Therapies and Exercise on Endothelium.

Endothelial dysfunction is a hallmark of type 2 diabetes that can have severe consequences on vascular function, including hypertension and changes in blood flow, as well as exercise performance. Because endothelium is also the barrier for insulin movement into tissues, it acts as a gatekeeper for transport and glucose uptake. For this reason, endothelial dysfunction is a tempting area for pharmacological and/or exercise intervention with insulin-based therapies. In this review, we describe the current state of drugs that can be used to treat endothelial dysfunction in type 2 diabetes and diabetes-related diseases (e.g., obesity) at the molecular levels, and also discuss their role in exercise.