Faculty Bibliography

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation and therefore contributes to the enhanced incidence of hypertension observed in diabetes. In this study, we examined the role of insulin on the association of the myosin-binding subunit of myosin phosphatase (MYPT1) to myosin phosphatase Rho-interacting protein (MRIP), a relatively novel member of the myosin phosphatase complex that directly binds RhoA in vascular smooth muscle cells (VSMCs). Through a series of molecular and cellular studies, we investigated whether insulin stimulates the binding of MRIP to MYPT1 and compared the results generated from VSMCs isolated from both Wistar-Kyoto (WKY) control and Goto-Kakizaki (GK) diabetic rats. We demonstrate for the first time that insulin stimulates the binding of MRIP to MYPT1 in a dose- and time-dependent manner, as determined by immunoprecipitation, implying a regulatory role for MRIP in insulin-induced vasodilation signaling via MYPT1 interaction. VSMCs from GK model of Type 2 diabetes had impaired insulin-induced MRIP/MYPT1 binding as well as reduced MRIP expression. Adenovirus-mediated overexpression of MRIP in GK VSMCs led to significantly improved insulin-stimulated MRIP/MYPT1 binding. Finally, insulin-stimulated MRIP translocation out of stress fibers, which was observed in control VSMCs, was impaired in GK VSMCs. We believe the impaired expression of MRIP, and therefore decreased insulin-stimulated MRIP/MYPT1 association, in the GK diabetic model may contribute to the impaired insulin-mediated vasodilation observed in the diabetic vasculature and provides a novel therapeutic strategy for the treatment of Type 2 diabetes.

Previously, we demonstrated that lipocalin-type prostaglandin D(2) synthase (L-PGDS) induces apoptosis and prevents cell cycle progression in several cell types. In this study we determined the expression of L-PGDS in a variety of human lung tumor types. While L-PGDS expression was evident in the surrounding margins, we observed significantly decreased protein and gene expression in the tumor tissue. Using RT-PCR we demonstrated that L-PGDS gene expression decreased proportionately with tumor progression. In addition, we demonstrated that exogenously added L-PGDS could suppress the hyperproliferation and PDGF-stimulated migration of A549 cells, a cultured carcinomic human alveolar basal epithelial cell line. We conclude that L-PGDS may play a key role in modulating lung cancer growth and may offer a novel diagnostic and therapeutic approach for treatment.

BACKGROUND:Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. METHODS AND RESULTS/RESULTS:EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/microL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. CONCLUSION/CONCLUSIONS:Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

Angiotensin-II (Ang-II) exerts many of its vascular effects, including the pathophysiological changes associated with type 2 diabetes, through changes in intracellular calcium concentration [Ca(2+)](i). We sought to clarify the mechanism responsible for Ang-II-induced Ca(2+) influx in cultured aortic VSMC using the Goto-Kakizaki (GK) rat model of type 2 diabetes. Ang-II-induced Ca(2+) influx was blocked by neither VDCC nor c-src inhibition but was sensitive to inositol 1,4,5-trisphosphate receptor inhibition, lanthanide and the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol. Since transient receptor potential canonical (TRPC)-3 gene expression was undetectable in both WKY and GK VSMCs and TRPC6 gene and protein expression were significantly down-regulated in GK, we believe the 1/4/5 subgroup of TRPC proteins plays a significant role. Furthermore, in GK VSMC the elevated calcium influx observed was not attributable to increased TRPC expression, but rather an alteration of TRPC activity.

Insulin resistance associated with Type 2 diabetes contributes to impaired vasorelaxation. Previously, we showed the phosphorylation of myosin-bound phosphatase substrate MYPT1, a marker of the vascular smooth muscle cell (VSMC) contraction, was negatively regulated by Akt (protein kinase B) phosphorylation in response to insulin stimulation. In this study we examined the role of Akt phosphorylation on impaired insulin-induced vasodilation in the Goto-Kakizaki (GK) rat model of Type 2 diabetes. GK VSMCs had impaired basal and insulin-induced Akt phosphorylation as well as increases in basal MYPT1 phosphorylation, inducible nitric oxide synthase (iNOS) expression, and nitrite/nitrate production compared with Wistar-Kyoto controls. Both iNOS expression and the inhibition of angiotensin (ANG) II-induced MYPT1 phosphorylation were resistant to the effects of insulin in diabetic GK VSMC. We also measured the isometric tension of intact and denuded GK aorta using a myograph and observed significantly impaired insulin-induced vasodilation. Adenovirus-mediated overexpression of constitutively active Akt in GK VSMC led to significantly improved insulin sensitivity in terms of counteracting ANG II-induced contractile signaling via MYPT1, myosin light chain dephosphorylation, and reduced iNOS expression, S-nitrosylation and survivin expression. We demonstrated for the first time the presence of Akt-independent iNOS expression in the GK diabetic model and that the defective insulin-induced vasodilation observed in the diabetic vasculature can be restored by the overexpression of active Akt, which advocates a novel therapeutic strategy for treating diabetes.

Previously, we demonstrated that lipocalin-type prostaglandin D(2) synthase (L-PGDS) knockout mice become glucose intolerant and display signs of diabetic nephropathy and accelerated atherosclerosis. In the current study we sought to explain the link between L-PGDS and glucose tolerance. Using the insulin-sensitive rat skeletal muscle cell line, L6, we showed that L-PGDS could stimulate glucose transport approximately 2-fold as well as enhance insulin-stimulated glucose transport, as measured by 2-deoxy-[(3)H]-glucose uptake. The increased glucose transport was not attributed to increased GLUT4 production but rather the stimulation of GLUT4 translocation to the plasma membrane, a phenomenon that was lost when cells were cultured under hyperglycemic (20 mM) conditions or pretreated with wortmannin. There was however, an increase in GLUT1 expression as well as a 3-fold increase in hexokinase III expression, which was increased to nearly 5-fold in the presence of insulin, in response to L-PGDS at 20 mM glucose. In addition, adipocytes isolated from L-PGDS knockout mice were significantly less sensitive to insulin-stimulated glucose transport than wild-type. We conclude that L-PGDS, via production of prostaglandin D(2), is an important mediator of muscle and adipose glucose transport which is modulated by glycemic conditions and plays a significant role in the glucose intolerance associated with type 2 diabetes.

Diabetes and insulin resistance are associated with an increased risk of hypertension and cardiovascular disease. Recent evidence demonstrates that AT2 receptors (AT2R) play an important role in the hemodynamic control of hypertension by vasodilation. The quantitative significance of AT2R in the establishment of diabetic vascular dysfunction, however, is not well defined and needs further investigation. Goto-Kakizaki (GK) rats, a polygenic model of spontaneous normotensive type 2 diabetes, were used to examine any abnormalities in cardiovascular function associated with AT2R at the early stage of the disease without endothelium influence. Using a myograph to measure the isometric force, we observed that ANG II-induced contraction was impaired in denuded GK aorta compared with control Wistar-Kyoto (WKY) aorta and exhibited a retarded AT1R antagonist response and enhanced Rho kinase signaling. When AT1R were blocked, ANG II induced a significant vasodilation of precontracted GK aorta via AT2R. The protein and mRNA of AT2R were increased in diabetic GK denuded aorta. Blocking AT2R restored the ANG II-induced contraction in the GK vasculature to control levels, demonstrating a counteractive role for AT2R in AT1R-induced contraction. Inhibition of inducible nitric oxide synthase (iNOS) by NG-monomethyl-L-arginine mimicked AT2R inhibition in denuded GK aorta, suggesting that AT2R-induced vasodilation was dependent on iNOS/NO generation. The protein and mRNA of iNOS were also increased in GK aorta. In conclusion, these results clearly demonstrate that enhanced AT2R and iNOS-induced, NO-mediated vasodilation impair ANG II-induced contraction in an endothelium-independent manner at the early stage of type 2 diabetes.

Lipocalin-type prostaglandin D(2) synthase (L-PGDS) is a highly glycosylated protein found in several body fluids. Elevated L-PGDS levels have been observed in the serum of patients with renal impairment, diabetes mellitus, and hypertension. Recently, we demonstrated the ability of L-PGDS to induce apoptosis in a variety of cell types including epithelial cells, neuronal cells, and vascular smooth muscle cells (VSMCs). The aim of this study was to investigate the effect several site-directed mutations had on L-PGDS-induced apoptosis in order to identify potential sites of regulation. Point mutations created in a glycosylation site (Asn51), a protein kinase C phosphorylation site (Ser106), and the enzymatic active site (Cys65) all inhibited L-PGDS-induced apoptosis as determined by both terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and caspase3 activity. We also compared the L-PGDS isoforms present in GK rat serum to WKY control serum using two-dimensional gel electrophoresis and observed distinct differences which vanished after PNGase F glycolytic digestion. We conclude that post-translational modification of L-PGDS, by either glycosylation or phosphorylation, enhances its apoptotic activity and inhibits VSMC hyperproliferation and postulate that this process is altered in type 2 diabetes.