Faculty Bibliography

We evaluated ranirestat, an aldose reductase inhibitor, in diabetic cataract and neuropathy (DN) in spontaneously diabetic Torii (SDT) rats compared with epalrestat, the positive control. Animals were divided into groups and treated once daily with oral ranirestat (0.1, 1.0, 10 mg/kg) or epalrestat (100 mg/kg) for 40 weeks, normal Sprague-Dawley rats, and untreated SDT rats. Lens opacification was scored from 0 (normal) to 3 (mature cataract). The combined scores (0-6) from both lenses represented the total for each animal. DN was assessed by measuring the motor nerve conduction velocity (MNCV) in the sciatic nerve. Sorbitol and fructose levels were measured in the lens and sciatic nerve 40 weeks after diabetes onset. Cataracts developed more in untreated rats than normal rats (P < 0.01). Ranirestat significantly (P < 0.01) inhibited rapid cataract development; epalrestat did not. Ranirestat significantly reversed the MNCV decrease (40.7 +/- 0.6 m/s) in SDT rats dose-dependently (P < 0.01). Epalrestat also reversed the prevented MNCV decrease (P < 0.05). Sorbitol levels in the sciatic nerve increased significantly in SDT rats (2.05 +/- 0.10 nmol/g), which ranirestat significantly suppressed dose-dependently, (P < 0.05, <0.01, and <0.01); epalrestat did not. Ranirestat prevents DN and cataract; epalrestat prevents DN only.

Commercially available angiotensin II At(2) receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available At(2) receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and At(2) receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or At(2) receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of At(2) receptor binding or gene expression. We conclude that none of these commercially available At(2) receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study At(2) receptor expression.

Alzheimer's disease (AD) belongs to a category of adult neurodegenerative conditions, which are associated with intracellular and extracellular accumulation of neurotoxic protein aggregates. Understanding how these aggregates are formed, secreted and propagated by neurons has been the subject of intensive research, but so far no preventive or curative therapy for AD is available, and clinical trials have been largely unsuccessful. Here we show that deficiency of the lysosomal sialidase NEU1 leads to the spontaneous occurrence of an AD-like amyloidogenic process in mice. This involves two consecutive events linked to NEU1 loss-of-function--accumulation and amyloidogenic processing of an oversialylated amyloid precursor protein in lysosomes, and extracellular release of Abeta peptides by excessive lysosomal exocytosis. Furthermore, cerebral injection of NEU1 in an established AD mouse model substantially reduces beta-amyloid plaques. Our findings identify an additional pathway for the secretion of Abeta and define NEU1 as a potential therapeutic molecule for AD.

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5'-triphosphate (5'-ppp) RNA and mediate IFN production. Cytosolic 5'-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-beta independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5'-ppp dsRNA and induces IFN-beta through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-beta induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-beta production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155(-/-) mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155(-/-) livers, we identified and validated that Nr1h3 (LXRalpha) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155(-/-) mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes.

Patients with schizophrenia receive medication to alleviate various symptoms, but some efficacious second generation antipsychotics, particularly olanzapine, can cause obesity, dyslipidemia, and diabetes mellitus. It has been generally considered that olanzapine contributes to the development of diabetes by inducing obesity and subsequent insulin resistance. In this study, we examined the effect of olanzapine and risperidone, another second generation antipsychotic, on a hamster pancreatic beta cell line, and found that both evoked mild endoplasmic reticulum (ER) stress, as evidenced by mild activation of the ER stress sensor molecule PERK. Surprisingly, only olanzapine induced marked apoptosis. Phosphorylation of the alpha subunit of eukaryotic initiation factor 2, an event immediately downstream of PERK activation, was not observed in cells treated with olanzapine, protein synthesis continued despite PERK activation, and ER stress was thereby sustained. Secretion of insulin was markedly inhibited, and both proinsulin and insulin accumulated inside olanzapine-treated cells. Inhibition of protein synthesis and knockdown of insulin mRNA, which result in less unfolded protein burden, both attenuated subsequent olanzapine-induced apoptosis. Given clinical observations that some patients taking olanzapine exhibit hyperlipidemia and hyperglycemia without gaining weight, our observations suggest that damage to pancreatic beta cells may contribute to the undesirable metabolic consequences of olanzapine treatment in some cases.

Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.