Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 µm internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions

WNT5A regulates adipose tissue angiogenesis via antiangiogenic VEGF-A165b in obese humans

Experimental studies have suggested that Wingless-related integration site 5A (WNT5A) is a proinflammatory secreted protein that is associated with metabolic dysfunction in obesity. Impaired angiogenesis in fat depots has been implicated in the development of adipose tissue capillary rarefaction, hypoxia, inflammation, and metabolic dysfunction. We have recently demonstrated that impaired adipose tissue angiogenesis is associated with overexpression of antiangiogenic factor VEGF-A165b in human fat and the systemic circulation. In the present study, we postulated that upregulation of WNT5A is associated with angiogenic dysfunction and examined its role in regulating VEGF-A165b expression in human obesity. We biopsied subcutaneous and visceral adipose tissue from 38 obese individuals (body mass index: 44 ± 7 kg/m2, age: 37 ± 11 yr) during planned bariatric surgery and characterized depot-specific protein expression of VEGF-A165b and WNT5A using Western blot analysis. In both subcutaneous and visceral fat, VEGF-A165b expression correlated strongly with WNT5A protein (r = 0.9, P \u3c 0.001). In subcutaneous adipose tissue where angiogenic capacity is greater than in the visceral depot, exogenous human recombinant WNT5A increased VEGF-A165b expression in both whole adipose tissue and isolated vascular endothelial cell fractions (P \u3c 0.01 and P \u3c 0.05, respectively). This was associated with markedly blunted angiogenic capillary sprout formation in human fat pad explants. Moreover, recombinant WNT5A increased secretion of soluble fms-like tyrosine kinase-1, a negative regulator of angiogenesis, in the sprout media (P \u3c 0.01). Both VEGF-A165b-neutralizing antibody and secreted frizzled-related protein 5, which acts as a decoy receptor for WNT5A, significantly improved capillary sprout formation and reduced soluble fms-like tyrosine kinase-1 production (P \u3c 0.05). We demonstrated a significant regulatory nexus between WNT5A and antiangiogenic VEGF-A165b in the adipose tissue of obese subjects that was linked to angiogenic dysfunction. Elevated WNT5A expression in obesity may function as a negative regulator of angiogenesis

Insulin Status and Vascular Responses to Weight Loss in Obesity

ObjectivesThe aim of this study was to determine whether the effects of weight loss on arterial function are differentially modified by insulin status.BackgroundClinical studies suggest that plasma insulin levels may predict the extent of cardiovascular benefit achieved with weight loss in obese individuals, but mechanisms are currently unknown.MethodsWe prospectively followed 208 overweight or obese patients (body mass index [BMI] ≥25 kg/m2) receiving medical/dietary (48%) or bariatric surgical (52%) weight-loss treatment during a median period of 11.7 months (interquartile range: 4.6 to 13 months). We measured plasma metabolic parameters and vascular endothelial function using ultrasound at baseline and following weight-loss intervention and stratified analyses by median plasma insulin levels.ResultsPatients age 45 ± 1 years, with BMI 45 ± 9 kg/m2, experienced 14 ± 14% weight loss during the study period. In individuals with higher baseline plasma insulin levels (above median >12 μIU/ml; n = 99), ≥10% weight loss (compared with <10%) significantly improved brachial artery macrovascular flow-mediated vasodilation and microvascular reactive hyperemia (p < 0.05 for all). By contrast, vascular function did not change significantly in the lower insulin group (≤12 μIU/ml; n = 109) despite a similar degree of weight loss. In analyses using a 5% weight loss cut point, only microvascular responses improved in the higher insulin group (p = 0.02).ConclusionsInsulin status is an important determinant of the positive effect of weight reduction on vascular function with hyperinsulinemic patients deriving the greatest benefit. Integrated improvement in both microvascular and macrovascular function was associated with ≥10% weight loss. Reversal of insulin resistance and endothelial dysfunction may represent key therapeutic targets for cardiovascular risk reduction in obesity

WNT5A-JNK regulation of vascular insulin resistance in human obesity

Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (pWNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p

Synaptic Plasticity and NO-cGMP-PKG Signaling Regulate Pre- and Postsynaptic Alterations at Rat Lateral Amygdala Synapses Following Fear Conditioning

In vertebrate models of synaptic plasticity, signaling via the putative “retrograde messenger” nitric oxide (NO) has been hypothesized to serve as a critical link between functional and structural alterations at pre- and postsynaptic sites. In the present study, we show that auditory Pavlovian fear conditioning is associated with significant and long-lasting increases in the expression of the postsynaptically-localized protein GluR1 and the presynaptically-localized proteins synaptophysin and synapsin in the lateral amygdala (LA) within 24 hrs following training. Further, we show that rats given intra-LA infusion of either the NR2B-selective antagonist Ifenprodil, the NOS inhibitor 7-Ni, or the PKG inhibitor Rp-8-Br-PET-cGMPS exhibit significant decreases in training-induced expression of GluR1, synaptophysin, and synapsin immunoreactivity in the LA, while those rats infused with the PKG activator 8-Br-cGMP exhibit a significant increase in these proteins in the LA. In contrast, rats given intra-LA infusion of the NO scavenger c-PTIO exhibit a significant decrease in synapsin and synaptophysin expression in the LA, but no significant impairment in the expression of GluR1. Finally, we show that intra-LA infusions of the ROCK inhibitor Y-27632 or the CaMKII inhibitor KN-93 impair training-induced expression of GluR1, synapsin, and synaptophysin in the LA. These findings suggest that the NO-cGMP-PKG, Rho/ROCK, and CaMKII signaling pathways regulate fear memory consolidation, in part, by promoting both pre- and post-synaptic alterations at LA synapses. They further suggest that synaptic plasticity in the LA during auditory fear conditioning promotes alterations at presynaptic sites via NO-driven “retrograde signaling”

Effect of Bariatric Weight Loss on the Adipose Lipolytic Transcriptome in Obese Humans

Background. Dysregulated lipolysis has been implicated in mechanisms of cardiometabolic disease and inflammation in obesity. Purpose. We sought to examine the effect of bariatric weight loss on adipose tissue lipolytic gene expression and their relationship to systemic metabolic parameters in obese subjects. Methods/Results. We biopsied subcutaneous adipose tissue in 19 obese individuals (BMI 42 ± 5 kg/m2, 79% female) at baseline and after a mean period of 8 ± 5 months (range 3–15 months) following bariatric surgery. We performed adipose tissue mRNA expression of proteins involved in triglyceride hydrolysis and correlated their weight loss induced alterations with systemic parameters associated with cardiovascular disease risk. mRNA transcripts of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and lipid droplet proteins comparative gene identification 58 (CGI-58) and perilipin increased significantly after weight loss (p<0.05 for all). ATGL expression correlated inversely with plasma triglyceride (TG), hemoglobin A1C (HbA1C), and glucose, and HSL expression correlated negatively with glucose, while CGI-58 was inversely associated with HbA1C. Conclusion. We observed increased expression of adipose tissue lipolytic genes following bariatric weight loss which correlated inversely with systemic markers of lipid and glucose metabolism. Functional alterations in lipolysis in human adipose tissue may play a role in shaping cardiometabolic phenotypes in human obesity

Noncanonical Wnt Signaling Promotes Obesity-Induced Adipose Tissue Inflammation and Metabolic Dysfunction Independent of Adipose Tissue Expansion

Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH(2)-terminal kinase–dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion

The A2b Adenosine Receptor Modulates Glucose Homeostasis and Obesity

<div><h3>Background</h3><p>High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples.</p> <h3>Methodology/Principal Findings</h3><p>Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression.</p> <h3>Conclusions/Significance</h3><p>Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.</p> </div

Effect of A2bAR elimination on tissue insulin signaling post HFD.

<p>Western blot analysis of different insulin responsive tissues; one representative WT and A2bAR KO pair is shown. <b>A.</b> phospho-Thr308 Akt (60 kDa), phospho-Ser473 Akt (60 kDa), total Akt (60 kDa) and β-actin (43 kDa) in liver at base line, and in visceral fat tissue and gastrocnemius tissue 15 minutes post-insulin injection (1 U/kg Humulin R ). <b>B.</b> Tissue A2bAR mRNA levels were measured by qPCR. Twelve-week-old WT mice (C57BL/6J) were analyzed and compared to similar mice subjected to additional 16 weeks of HFD. The expression of the receptor is relative to the baseline value, measured in 12 week old mice before the 16 weeks diet (denoted as HFD), and normalized to 18 S rRNA (denoted as 18 S), p-value = 0.0087 for liver; p-value = 0.0073 for visceral fat; p-value = 0.0009 for gastrocnemious muscle.</p