Extracellular ATP and zinc are co-secreted with insulin and activate multiple P2X purinergic receptor channels expressed by islet beta-cells to potentiate insulin secretion.

It is well established that ATP is co-secreted with insulin and zinc from pancreatic beta-cells (beta-cells) in response to elevations in extracellular glucose concentration. Despite this knowledge, the physiological roles of extracellular secreted ATP and zinc are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by beta-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, we performed ELISA assays for secreted insulin at fixed time points within a "real-time" assay and confirmed that the physiological insulin secretagogue glucose stimulates secretion of ATP and zinc into the extracellular milieu along with insulin from primary rat islets. Exogenous ATP and zinc alone or together also induced insulin secretion in this model system. Most importantly, the presence of an extracellular ATP scavenger, a zinc chelator, and P2 receptor antagonists attenuated GSIS. Furthermore, mRNA and protein were expressed in immortalized beta-cells and primary islets for a unique subset of P2XR channel subtypes, P2X(2), P2X(3), P2X(4), and P2X(6), which are each gated by extracellular ATP and modulated positively by extracellular zinc. On the basis of these results, we propose that, within endocrine pancreatic islets, secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion via ATP-gated and zinc-modulated P2XR channels.

A thrombospondin-1 antagonist of transforming growth factor-beta activation blocks cardiomyopathy in rats with diabetes and elevated angiotensin II.

In diabetes and hypertension, the induction of increased transforming growth factor-beta (TGF-beta) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-beta activator thrombospondin-1 (TSP1). Because activation of latent TGF-beta is a major means of regulating TGF-beta, we addressed the role of TSP1-mediated TGF-beta activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-beta activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-beta activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-beta activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-beta activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-beta activation as a potential antifibrotic therapeutic strategy are warranted.

Thrombospondin 1 mediates angiotensin II induction of TGF-beta activation by cardiac and renal cells under both high and low glucose conditions.

Renal and cardiac fibrosis leading to organ failure are complications of both diabetes and hypertension. These disease processes, when combined, exacerbate development of fibrotic complications. Control of latent transforming growth factor (TGF)-beta activation is a potential determinant of fibrotic progression. Both glucose and angiotensin II (Ang II) upregulate thrombospondin-1 (TSP1), a major activator of latent TGF-beta, and stimulate increased TGF-beta activity. We previously showed that high glucose stimulated TSP1-dependent TGF-beta activation in rat mesangial cells (RMCs). In this paper, we examined whether Ang II similarly upregulates TSP1 production and TSP1-dependent TGF-beta activation alone or in combination with high glucose concentrations. Ang II and high glucose stimulated increases in TSP1 protein levels in the conditioned media of both rat cardiac fibroblasts (RCFs) and rat mesangial cells (RMCs). Meanwhile, Ang II stimulated increases in both TGF-beta activity and protein by RMCs, whereas, RCFs responded to both Ang II and high glucose with increased TGF-beta activity in the absence of altered TGF-beta protein levels. A combination of Ang II and high glucose induced synergistic TGF-beta activation by RCFs. Moreover, Ang II induction of TSP1 and increased TGF-beta activity were blocked by losartan, an antagonist of the Ang II type 1 (AT1) receptor. The increase in TSP1 expression leads to increased TGF-beta activity upon Ang II and/or glucose treatment, since peptide antagonists of TSP1-mediated TGF-beta activation blocked Ang II and glucose-induced TGF-beta activation. Our data support a role for TSP1 in the development and progression of renal and cardiac fibrosis in hypertension and diabetes.

Effects of early perturbation of the renin-angiotensin system on cardiovascular remodeling in spontaneously hypertensive rats.

RATIONALE: The goal of this study was to analyze cardiovascular (CV) remodeling in early, short-term CAP treated SHR and their offspring. METHODS: We treated SHR with Captopril (CAP, 100 mg/kg) from in utero to 1 month of age (OCAP). Some of these rats were mated at 3-4 months of age and we used their offspring (2nd G). Controls were untreated SHR, normotensive Wistar Kyoto rats (WKY) and SHR maintained on CAP (SCAP). At 12-14 months of age, rats were cannulated for mean arterial blood pressure (MAP) measurements. An image analysis system was used to quantitate changes in cardiac and vascular (wall-to-lumen ratios, w/l) morphology and fibrosis. RESULTS: Early, short-term CAP treatment prevented the full expression of hypertension in treated rats and their offspring. MAPs were: SHR (180+/-2.2 mm Hg); WKY 125+/-3 mm Hg); SCAP 112+/-2.5mm Hg; OCAP 138+/-2.3 mm Hg; and 2nd G (145+/-2.0 mm Hg). There were significant decreases in heart weight/body weight ratios, large and small vessel morphology, and interstitial and perivascular fibrosis in CAP-treated animals and their offspring in comparison to untreated SHR. CONCLUSIONS: The CV protective properties of early, short-term CAP treatment were not solely due to a reduction in MAP. Although MAP was higher in OCAP and 2nd G, CV structure resembled that found in WKY and SCAP. The effects of our early treatment appear to be due to chronic blockade of the renin-angiotensin system and its effects on growth of CV tissues and the development of fibrosis.

Increased PI3-kinase in presympathetic brain areas of the spontaneously hypertensive rat.

Existing evidence led us to hypothesize that increases in p85alpha, a regulatory subunit of PI3-kinase, in presympathetic brain areas contribute to hypertension. PI3-kinase p85alpha, p110alpha, and p110delta mRNA was 1.5- to 2-fold higher in the paraventricular nucleus (PVN) of spontaneously hypertensive rats (SHR) compared with their controls, Wistar Kyoto rats (WKY). The increase in p85alpha/p110delta was attenuated in SHR treated with captopril, an angiotensin (Ang)-converting enzyme inhibitor, from in utero to 6 months of age. In the rostral ventrolateral medulla (RVLM), p110delta mRNA was approximately 2-fold higher in SHR than in WKY. Moreover, the increases in mRNA were associated with higher PI3-kinase activity in both nuclei. The functional relevance was studied in neuronal cultures because SHR neurons reflect the augmented p85alpha mRNA and PI3-kinase activity. Expression of a p85 dominant-negative mutant decreased norepinephrine (NE) transporter mRNA and [3H]NE uptake by approximately 60% selectively in SHR neurons. In summary, increased p85alpha/p110delta expression in the PVN and RVLM is associated with increased PI3-kinase activity in the SHR. Furthermore, normalized PI3-kinase p85alpha/p110delta expression within the PVN might contribute to the overall effect of captopril, perhaps attributable to a consequent decrease in NE availability.