Jaume Padilla, Ph.D. - US grants

? DESCRIPTION (provided by applicant): In type 2 diabetes (T2D), insulin-stimulated blood flow to skeletal muscle is limited and this attenuates glucose uptake, thus contributing to impaired glucose homeostasis. A detailed understanding of the precipitating factors and mechanisms underlying the defects in vascular insulin action is critical for the development of therapeutic strategies aimed at improving glycemic control and protecting against cardiovascular disease. Based on our most recent preliminary data in animal models, we propose the novel hypothesis that endoplasmic reticulum (ER) stress plays an important role in mediating the impairment in insulin-stimulated blood flow in T2D patients. Specifically, we will test if chemical enhancement of ER function with oral administration of supplement tauroursodeoxycholic acid (TUDCA) increases insulin-stimulated blood flow and leg glucose uptake in T2D patients. TUDCA is a bile acid derivative that has been used to treat cholelithiasis and cholestatic liver disease in human patients. Studies in rodent models demonstrate that TUDCA can act as a chemical chaperone to protect against ER stress and improves glucose tolerance. Whether TUDCA can be used as an add-on pharmacological approach to suppress ER stress and improve vascular insulin actions in T2D patients is unknown. We will combine measures of leg blood flow via Doppler ultrasound, intra-arterial pharmacological blockade of endothelin 1 receptors, and measures of leg glucose uptake during a hyperinsulinemic-euglycemic clamp after 4 weeks of TUDCA treatment in a double-blinded, randomized, placebo controlled crossover study. In addition, continuous glucose monitoring will be used to assess the effects of TUDCA on free-living glycemic control. Collectively, this study will provide novel insight on the mechanisms by which alleviation of ER stress enhances insulin-stimulated blood flow, a regulatory site of glucose disposal and glycemic control largely underappreciated. The contribution of this work is significant as it is the first step in a continuum of research expected to lead to the development of novel therapeutic strategies targeting ER stress for prevention and treatment of vascular insulin resistance and glycemic dysregulation associated with T2D. We are poised to move diabetes research forward in an area currently receiving little attention, despite its importance and clear need for investigation.

DESCRIPTION (provided by applicant): My long-term career goal is to develop an independent, funded research program that contributes to understanding the mechanisms by which obesity and type 2 diabetes lead to impaired vascular insulin signaling and cardiovascular disease. In obesity, insulin-stimulated blood flow to skeletal muscle is limited and this attenuate glucose uptake, thus contributing to impaired glucose homeostasis. However, the mechanism by which insulin-induced vasodilation becomes impaired is largely unknown. The proposed study will test the hypothesis that endoplasmic reticulum (ER) stress mediates the impairment in insulin-stimulated vasodilation in skeletal muscle arterioles. It is reasoned that vascular ER stress and vascular insulin resistance caused by obesity is attributable to the local secretion of inflammatory cytokines by perivascular adipose tissue (PVAT). Using a well-established pig model of Western diet-induced obesity, Aim 1 will test if obesity-associated vascular ER stress underlies the imbalance between nitric oxide and endothlein-1 leading to impaired insulin- stimulated vasodilation. Aim 2 will then determine if obese skeletal muscle PVAT can cause vascular ER stress, thus contributing to impaired insulin-stimulated vasodilation. Finally, Aim 3 will examine whether in vivo genetic and chemical enhancement of ER function can restore impaired insulin-stimulated vasodilation associated with obesity. The contribution of this proposed work in pigs is significant as targeting ER stress may be a novel therapeutic strategy to correct vascular insulin resistance and ultimately prevent/treat metabolic and cardiovascular disease fueled by obesity. The University of Missouri (MU) campus has a distinguished history of research in cardiovascular science, metabolism, and exercise physiology and is the home for the Life Sciences Center, the Dalton Cardiovascular Research Center, the Health Activity Center, the Diabetes and Cardiovascular Center, the National Swine Resource & Research Center as resources for this project. These centers are filled with faculty from multiple departments and divisions that actively collaborate, providing an unparalleled research environment to pursue my independent research. Indeed, I will be interacting with a large number of senior investigators who will not only help ensure successful completion of the proposed studies, but also facilitate my career development as I progress toward becoming a successful independent investigator. James R. Sowers, MD, will be my primary mentor and Frank W. Booth, PhD will act as my co-mentor. Dr. Sowers is a clinical physician as well as a researcher with expertise in vascular insulin actions and cardiometabolic disease. Dr. Booth has expertise in adipose tissue biology and physical activity. Together, we have assembled a comprehensive research training plan and team of collaborators that will facilitate the acquisition of new molecular techniques (i.e., in vivo adenoviral transfection, proteomics) as well as techniques involving in vitro preparations of isolated intact arterioles to enhance my abilities to conduct mechanistic research. These additional skills combined with my earlier background in human vascular research will contribute to my long-term goal of establishing a research program with capabilities to conduct in vitro and in vivo mechanistic and translational research using animal models of obesity/type 2 diabetes as well as human patients. In short, the additional technical, academic, and career development afforded by my training plan will place me in an ideal position to successfully launch a productive, independent and translational research program. This NIH K01 application represents the next logical step in my career development as a young early investigator and will set the stage for my first R01 application. (End of Abstract)

PROJECT SUMMARY/ABSTRACT In type 2 diabetes (T2D), insulin-stimulated blood flow to skeletal muscle is markedly blunted which significantly limits glucose uptake, thus contributing to impaired glucose homeostasis. A detailed understanding of the precipitating factors and mechanisms underlying the defects in vasodilator actions of insulin is critical for the development of therapeutic strategies aimed at improving glycemic control and protecting against cardiovascular disease. Based on our prior work and most recent preliminary data, we propose that in hyperglycemic T2D patients, protein kinase C (PKC) activation drives the upregulation of endothelin-1 (ET-1) and consequent impairment in insulin-induced dilation. Furthermore, we hypothesize that increased vascular exposure to shear stress, associated with physical activity, mitigates these toxic molecular effects of hyperglycemia on endothelial cells and lead to substantial improvements in insulin-induced dilation in T2D. Specifically, we will test the overarching hypothesis that endothelial PKC activation mediates the upregulation of ET-1 and impairment in insulin-induced dilation in patients with T2D, a defect that can be corrected with increased physical activity and shear stress. In Aims 1 and 2, ex vivo functional studies will be performed in isolated visceral resistance arteries from obese T2D and obese non-T2D patients undergoing Roux-en-Y gastric bypass surgery. Through gain- and loss-of-function experiments, we will examine the role of PKC activation in mediating impaired insulin-induced dilation in arteries from T2D patients as well as the role of hyperglycemia and shear stress in modulating insulin-induced dilation. In Aim 3, we will perform a clinical study in patients with T2D to determine the effects of increased walking and shear stress on insulin-stimulated leg blood flow. In particular, we will test the hypothesis that increased walking for 8 weeks decreases vascular PKC activation and ET-1 production, thus leading to an improvement in insulin-stimulated leg blood flow. Leg blood flow via Doppler ultrasound will be assessed during a hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies will be performed for vascular phenotypic characterization. Furthermore, we will determine if increased leg vascular shear stress using a non-exercise stimulus (i.e., leg heating intervention for 8 weeks) recapitulates the beneficial vascular effects of increased walking. Targeting PKC activation and ET-1, pharmacologically or through an increase in shear stress, may be key for correction of vascular insulin resistance and ultimately improvement of metabolic and cardiovascular outcomes in patients with T2D. Indeed, our research team is poised to move cardiovascular and diabetes research forward in an area currently receiving little attention, despite its importance and clear need for investigation.

PROJECT SUMMARY/ABSTRACT Endothelial dysfunction is causally implicated in the development of cardiovascular disease (CVD), the main cause of death in patients with type 2 diabetes (T2D). The endothelium regulates arterial diameter and vascular homeostasis via the production of a myriad of vasoactive substances including nitric oxide (NO). NO is a powerful vasodilator produced in response to blood flow-induced shear stress, which is detected by mechanosensitive endothelial luminal structures. The glycocalyx is such a mechanosensor. It consists of a mesh of interwoven glycoproteins and proteoglycans that, when disturbed by shear stress, converts mechanical forces into biochemical signals. The appropriate result of this process, known as mechanotransduction, is endothelium-dependent flow-mediated dilation (FMD), which is considered the gold- standard physiological measure of endothelial function. Notably, impaired FMD is highly prevalent in T2D and also represents a critical component of the mechanisms that lead to CVD. However, despite the major role that reduced FMD plays in T2D-associated CVD development, the mechanisms that lead to this abnormal response are not completely known. In addition, there are currently no specific therapeutic means to alleviate impaired FMD. A central goal of this proposal is to decipher the mechanisms underlying the impairment of FMD in T2D and discover new therapeutic targets to improve it. Based on our prior work and most recent and exciting preliminary data, we propose the novel hypothesis that increased plasma neuraminidase activity degrades glycocalyx structures via activation of ADAM17 (a disintegrin and metalloproteinase-17) and promotes endothelial dysfunction in T2D. We will test our innovative hypothesis with gain- and loss-of-function pharmacological and genetic-manipulation experiments in human cultured endothelial cells and isolated arteries, in animal models of neuraminidase ablation and T2D, and in patients with T2D. Specifically, in Aim 1, using cultured endothelial cells and isolated arteries from humans, we will determine the mechanisms by which neuraminidase activity increases endothelial ADAM17 activation and impairs FMD. Subsequently, in Aim 2, we will determine the effects of neuraminidase inhibition on endothelial function in animal models and patients with T2D. We hypothesize that neuraminidase inhibition in T2D mice or humans improves FMD and overall vascular function. Our team is poised to move cardiovascular and diabetes research forward with a project that will exert a sustained, powerful impact across a number of levels of inquiry that are novel conceptually, mechanistically, methodologically, and therapeutically. Indeed, targeting neuraminidase activity holds extraordinary promise for correcting endothelial dysfunction in T2D and ultimately preventing/treating T2D- associated CVD.

PROJECT SUMMARY/ABSTRACT Vascular insulin resistance is a hallmark of type 2 diabetes (T2D) and dampening of insulin-induced vasodilation is its primary consequence. Notably, in T2D, reduced insulin-stimulated vasodilation and blood flow to tissues such as skeletal muscle significantly limits glucose uptake and contributes to impaired glucose control. A detailed understanding of the precipitating factors and mechanisms underlying the defects in vasodilator actions of insulin is critical for the development of therapeutic strategies aimed at improving glycemic control and protecting against cardiovascular disease. Based on our prior work and most recent and exciting preliminary data, we propose the novel hypothesis that ADAM17-mediated shedding of the insulin receptor alpha (IR?) from endothelial cells impairs insulin-stimulated vasodilation in T2D. We further propose that the increased activity of endothelial ADAM17 is attributed to protein kinase-C (PKC) activation and subsequent externalization of phosphatidylserine (PS) to the outer leaflet of the cell membrane, which serves to guide ADAM17 to its targeted substrates. As exogenous PS is a competitive inhibitor of ADAM17 sheddase activity, we will also determine the efficacy of oral administration of PS for restoring vascular insulin sensitivity in T2D patients. We will test our innovative hypotheses with gain- and loss-of-function genetic-manipulation experiments in human cultured endothelial cells, in isolated resistance arteries harvested from patients undergoing abdominal surgery, and in patients with T2D. Experimental results will determine the role of PS externalization-ADAM17 activation-IR? shedding as a mechanism impairing the vasodilatory actions of insulin in T2D. Specifically, in Aim 1, we will determine the mechanism by which PKC causes the externalization of PS and whether PS externalization is needed for PKC-dependent activation of ADAM17 in endothelial cells. Next, in Aim 2, we will determine the role of ADAM17 activity in IR? shedding and subsequent impairment of insulin-stimulated vasodilation in T2D. Finally, in Aim 3, we will perform a randomized double-blind clinical trial to determine the therapeutic efficacy of oral administration of the competitive inhibitor of ADAM17 sheddase activity, PS, on insulin-stimulated leg blood flow in patients with T2D. Our team is poised to move cardiovascular and diabetes research forward with a project that will exert a sustained, powerful impact across a number of levels of inquiry that are novel conceptually, mechanistically, methodologically, and therapeutically. Indeed, this proposal represents a paradigm shift from our current mechanistic understanding of vascular insulin resistance. Targeting ADAM17 activation holds extraordinary promise for correcting vascular insulin resistance and ultimately preventing/treating T2D-associated metabolic and cardiovascular diseases.