Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice.

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Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Identification of glucose-regulated miRNAs from pancreatic {beta} cells reveals a role for miR-30d in insulin transcription.

MicroRNAs (miRNAs) are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. MicroRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin production as well as insulin secretion. We have recently carried out a screen in the pancreatic beta-cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of 61 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107, and miR-30d were up-regulated in the presence of high glucose. Only a few of the miRNAs, including miR-296, miR-484, and miR-690 were significantly down-regulated by high glucose treatment. Interestingly, we found that overexpression of miR-30d, one of the miRNAs up-regulated by glucose, increased insulin gene expression, while inhibition of miR-30d abolished glucose-stimulated insulin gene transcription. Overexpression or inhibition of miR-30d did not have any effect on insulin secretion. These data suggest that the putative target genes of miR-30d may be negative regulators of insulin gene expression.

miR-146a Deficiency Accelerates Hepatic Inflammation Without Influencing Diet-induced Obesity in Mice.

miR-146a, an anti-inflammatory microRNA, is shown to be a negative regulator of adipocyte inflammation. However, the functional contribution of miR-146a in the development of obesity is not defined. In order to determine whether miR-146a influences diet-induced obesity, mice that were either wild type (WT) or miR-146a deficient (KO) were fed with high (60% kcal) fat diet (HFD) for 16 weeks. Deficiency of miR-146a did not influence obesity measured as HFD-induced body weight and fat mass gain, or metabolism of glucose and insulin tolerance. In addition, adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively, were comparable between the two groups of mice. Although, miR-146a deficiency had no influence on HFD-induced hepatic lipid accumulation, interestingly, it significantly increased obesity-induced inflammatory responses in liver tissue. The present study demonstrates that miR-146a deficiency had no influence on the development of HFD-induced obesity and adipose tissue remodeling, whereas it significantly increased hepatic inflammation in obese mice. This result suggests that miR-146a regulates hepatic inflammation during development of obesity.

Induction of insulin secretion in engineered liver cells by nitric oxide.

BACKGROUND: Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs in a glucose-independent manner. In the present study we engineered liver cells to produce and secrete insulin and insulin secretion can be stimulated via the nitric oxide pathway. RESULTS: Expression of either human insulin or the beta cell specific transcription factors PDX-1, NeuroD1 and MafA in the Hepa1-6 cell line or primary liver cells via adenoviral gene transfer, results in production and secretion of insulin. Although, the secretion of insulin is not significantly increased in response to high glucose, treatment of these engineered liver cells with L-arginine stimulates insulin secretion up to three-fold. This L-arginine-mediated insulin release is dependent on the production of nitric oxide. CONCLUSION: Liver cells can be engineered to produce insulin and insulin secretion can be induced by treatment with L-arginine via the production of nitric oxide.

Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice.

Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Adenoviral gene transfer into isolated pancreatic islets.

The beta cells within the pancreatic islets are responsible for production of insulin, a peptide hormone required for maintaining normoglycemia. The establishment of efficient gene transfer into pancreatic islets is very important for studies of insulin and glucagon production and secretion, as well as for gene therapy purposes for the treatment of diabetes. We describe here in detail a protocol for adenoviral gene transfer into isolated mouse islets of the pancreas. Effective gene transfer into pancreatic islets using recombinant adenoviruses can be achieved with a multiplicity of infection (MOI) of 10. However, if the islets are not dispersed, adenoviral gene transfer is limited only to the cells on the periphery of the islets, which represent the glucagon-producing alpha cells in rodents. Dispersion of pancreatic islets with EGTA increases the efficiency of gene transfer into the cells within the core of the islets, which consist of insulin-producing beta cells.