OBJECTIVEAutophagy is a critical cellular system for removal of aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in the development of heart failure, the role of autophagy in the development of diabetic cardiomyopathy has not been studied. We investigated whether chronic activation of the AMP-activated protein kinase (AMPK) by metformin restores cardiac function and cardiomyocyte autophagy in OVE26 diabetic mice.RESEARCH DESIGN AND METHODSOVE26 mice and cardiac-specific AMPK dominant negative transgenic (DN)-AMPK diabetic mice were treated with metformin or vehicle for 4 months, and cardiac autophagy, cardiac functions, and cardiomyocyte apoptosis were monitored.RESULTSCompared with control mice, diabetic OVE26 mice exhibited a significant reduction of AMPK activity in parallel with reduced cardiomyocyte autophagy and cardiac dysfunction in vivo and in isolated hearts. Furthermore, diabetic OVE26 mouse hearts exhibited aggregation of chaotically distributed mitochondria between poorly organized myofibrils and increased polyubiquitinated protein and apoptosis. Inhibition of AMPK by overexpression of a cardiac-specific DN-AMPK gene reduced cardiomyocyte autophagy, exacerbated cardiac dysfunctions, and increased mortality in diabetic mice. Finally, chronic metformin therapy significantly enhanced autophagic activity and preserved cardiac functions in diabetic OVE26 mice but not in DN-AMPK diabetic mice.CONCLUSIONSDecreased AMPK activity and subsequent reduction in cardiac autophagy are important events in the development of diabetic cardiomyopathy. Chronic AMPK activation by metformin prevents cardiomyopathy by upregulating autophagy activity in diabetic OVE26 mice. Thus, stimulation of AMPK may represent a novel approach to treat diabetic cardiomyopathy.

OBJECTIVEPrevious studies showed that genetic deletion or pharmacological blockade of the receptor for advanced glycation end products (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy. To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to diabetic nephropathy in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function.RESEARCH DESIGN AND METHODSWe bred OVE26 mice with homozygous RAGE knockout (RKO) mice and examined structural changes associated with diabetic nephropathy and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Transcriptional changes in the Tgf-β1 and plasminogen activator inhibitor 1 gene products were measured to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice.RESULTSDeletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE. Increased transcription of the genes for plasminogen activator inhibitor 1, Tgf-β1, Tgf-β–induced, and α1-(IV) collagen observed in OVE26 renal cortex was significantly reduced in OVE26 RKO kidney cortex. ROCK1 activity was significantly lower in OVE26 RKO compared with OVE26 kidney cortex.CONCLUSIONSThese data provide compelling evidence for critical roles for RAGE in the pathogenesis of diabetic nephropathy and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

Coronary heart disease is a leading cause of mortality in diabetic patients. The increased susceptibility to cardiovascular diseases in type 2 diabetes is due to a constellation of risk factors, including hyperglycemia, insulin resistance, and dyslipidemia. One of the earliest phenomena for vascular injury in diabetes is endothelial dysfunction, characterized by impaired endothelium-dependent relaxation, oxidant stress, and accelerated endothelial cell apoptosis (1, 2).Statins belong to the class of lipid-lowering drugs that target and inhibit 3-hydroxy-3-methylglutaryl-CoA reductase. Several clinical trials, including the Heart Protection Study (3) and the Collaborative Atorvastatin Diabetes Study (4), have shown significant benefits with low to moderate dose statin therapy in diabetic patients with and/or without overt cardiovascular diseases. A recent study from us (5) suggests that the protection conferred by statin therapy occurs not only as a consequence of its cholesterol-lowering activity but also as a direct result of its effects imparted on endothelium function. In addition, the antithrombotic and antiinflammatory effects of statins have been shown to contribute to its overall beneficial activity (6). In humans, improved endothelium functions are one of the earliest observed clinical effects following the initiation of statin treatment (7,8). Most importantly, statin therapy improves endothelial function by virtue of its antioxidant (8, 9) and antiinflammatory (8, 9) effects as well as its ability to up-regulate endothelial nitric-oxide synthase (eNOS) 2 (10, 11). Thus, it is known that statin inhibits angiotensin II (Ang-II)-induced production of vascular reactive oxygen species (ROS) in spontaneously hypertensive and diabetic rats (12)(13)(14). However, the molecular target and biochemical mechanisms whereby statins lower ROS and exert their antiapoptotic effects remain poorly defined.The AMP-activated protein kinase (AMPK) is a serine/threonine kinase and a member of the Snf1/AMPK protein kinase family found in all eukaryotes (15,16). AMPK is considered to be a cellular energy sensor, which stimulates ATP-producing *

Diabetic nephropathy is a major complication of diabetes and a leading cause of end-stage renal diseases in the U.S. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor that has been extensively studied in diabetic retinopathy. Recently, we reported that PEDF is expressed at high levels in normal kidneys and that PEDF levels are decreased in kidneys of streptozotocin (STZ)-induced diabetic rats. In the present study, we injected STZ-diabetic rats with an adenovirus expressing PEDF (Ad-PEDF) to evaluate its effects in diabetes. The results showed that increased expression of PEDF in the kidney in response to Ad-PEDF delivery significantly alleviated microalbuminuria in early stages of diabetes. Administration of Ad-PEDF was found to prevent the overexpression of two major fibrogenic factors, transforming growth factor-␤ (TGF-␤)1 and connective tissue growth factor (CTGF), and to significantly reduce the production of an extracellular matrix (ECM) protein in the diabetic kidney. Moreover, PEDF upregulated metalloproteinase-2 expression in diabetic kidney, which is responsible for ECM degradation. In cultured human mesangial cells, PEDF significantly inhibited the overexpression of TGF-␤1 and fibronectin induced by angiotensin II. PEDF also blocked the fibronectin production induced by TGF-␤1 through inhibition of Smad3 activation. These findings suggest that PEDF functions as an endogenous anti-TGF-␤ and antifibrogenic factor in the kidney. A therapeutic potential of PEDF in diabetic nephropathy is supported by its downregulation in diabetes; its prevention of the overexpression of TGF-␤, CTGF, and ECM proteins in diabetic kidney; and its amelioration of proteinuria in diabetic rats following Ad-PEDF injection.

To define the mechanism for the hypercalciuria in spontaneously hypertensive rats (SHR), Ca clearance was evaluated in fasted 23-wk-old SHR and normotensive Wistar Kyoto (WKy) controls. There was no exaggerated calciuria before or after parathyroidectomy. Ca balance was therefore measured in the nonfasted animals, which revealed hyperabsorption in SHR of both sexes with increments 10-fold that of Ca excretion, supporting the primacy of intestinal hyperabsorption. In situ duodenal Ca uptake was also increased in the SHR. Parathyroidectomy did not affect the hyperabsorption. Hypercalcemia (total and ionized) and hypercalciuria in SHR associated with reduced adenosine 3',5'-cyclic monophosphate excretion, were abolished by fasting. Correction of hypertension for 6 mo failed to abolish the hypercalciuria. Bone Ca deposits were increased in 1-yr-old SHR. Ten-week-old SHR, in contrast, displayed mild malabsorption. Our data do not support the "renal leak" hypothesis. Instead, the adult SHR is characterized by increased Ca retention due to primary hyperabsorption, absorptive hypercalciuria, and increased bone Ca deposition. These phenomena are independent of sex, parathyroid hormone, and treatment of the established hypertension.

Previous studies indicated a salutary effect of a high-Ca diet on high blood pressure (BP). The mechanism, however, is obscure. With balance and clearance techniques, the role of parathyroid hormone (PTH), volume contraction, hypercalcemia, and PO4 deficiency was evaluated in female spontaneous hypertensive rats (SHR). The antihypertensive effects of a high (4.3%) Ca diet in intact animals (groups I and II) could be reproduced in both 9- and 22-wk-old chronic stable parathyroidectomized (PTX) rats (groups III and IV), when compared with a low (0.22%) or normal (1.2%) CA diet. In both short (7 days) and long (12 wk) term exposure to the high-Ca diet, evidence for volume contraction could not be documented despite hypercalcemia sustained through the 12th wk (10.8 vs. 9.7 mg/100 ml, group I control, P less than 0.02). When produced by ip injections, chronic hypercalcemia of similar magnitudes as oral Ca supplements failed to reduce BP in either intact (group VI) or PTX (group IV) rats. Rats in group IV fed the high-Ca diet displayed marked hypophosphatemia (3.2 vs. 6.9 mg/100 ml), hypophosphaturia (0.15 vs. 15 mg/day), hypermagnesiuria (11 vs. 7.7 mg/day), and drastically reduced net intestinal PO4 absorption (13.3 +/- 7.5 vs. 66.8 +/- 7.5 mg/day) compared with rats fed 1.2% Ca diet. To test the PO4-deficiency hypothesis, additional SHR (group V) were fed either 1.2% Ca diet and injected ip with NaCl or fed 4.3% Ca diet, with half of these animals injected with neutral NaPO4 and half with NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

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Platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD31), a 130-kD transmembrane glycoprotein that functions in adhesion and signaling, is thought to play a role in some forms of leukocyte transmigration. In the lung, PECAM-1 is highly expressed, yet there have been few studies examining its role in pulmonary pathology. We therefore examined the inflammatory response (measured by bronchoalveolar lavage cell counts and protein content) after several types of lung injury in wild-type and PECAM-1 knockout mice. Consistent with studies in other organs, instillation of an endothelial stimulant (interleukin-1) was PECAM-1-dependent. In contrast, we noted that three other forms of acute lung injury (acid aspiration, adenoviral instillation, and tumor necrosis factor instillation) were completely PECAM-1-independent. Interestingly, in situ immune complex deposition injury, another complex lung disease, was also PECAM-1-dependent. This surprising finding was investigated in more detail and found to be due to a defect in macrophage activation, and not to a blockade of leukocyte transmigration. Experiments in bone marrow chimeric mice as well as ex vivo data demonstrated that Fcgamma receptor-dependent phagocytosis and tumor necrosis factor release were significantly reduced in macrophages derived from PECAM-1 knockout mice. Although PECAM-1 may not be required for transmigration of leukocytes into the alveolar space in many forms of complex lung inflammation, it is important in the function of Fcgamma receptors on alveolar macrophages.