Inhibition of Aldose Reductase Activates Hepatic Peroxisome Proliferator-Activated Receptor-α and Ameliorates Hepatosteatosis in Diabetic db/db Mice

We previously demonstrated in streptozotocin-induced diabetic mice that deficiency or inhibition of aldose reductase (AR) caused significant dephosphorylation of hepatic transcriptional factor PPARα, leading to its activation and significant reductions in serum lipid levels. Herein, we report that inhibition of AR by zopolrestat or by a short-hairpin RNA (shRNA) against AR caused a significant reduction in serum and hepatic triglycerides levels in 10-week old diabetic db/db mice. Meanwhile, hyperglycemia-induced phosphorylation of hepatic ERK1/2 and PPARα was significantly attenuated in db/db mice treated with zopolrestat or AR shRNA. Further, in comparison with the untreated db/db mice, the hepatic mRNA expression of Aco and ApoA5, two target genes for PPARα, was increased by 93% (P < 0.05) and 73% (P < 0.05) in zopolrestat-treated mice, respectively. Together, these data indicate that inhibition of AR might lead to significant amelioration in hyperglycemia-induced dyslipidemia and nonalcoholic fatty liver disease

Intraperitoneal co-administration of thymosin alpha-1 ameliorates streptozotocin-induced pancreatic lesions and diabetes in C57BL/6 mice

We investigated the effects of the in vivo administration of thymosin alpha-1 (T alpha-1) on streptozotocin (STZ)-induced pancreatic lesions and diabetes. Mice were randomly divided into four experimental groups: normo-glycemic control, STZ-treated, STZ plus 0.1 mu g/kg body weight/day T alpha-1-treated, and STZ plus 1 mu g/kg/day T alpha-1-treated. Blood glucose was assayed periodically, and serum insulin was determined at the end of the experiment using the ELISA Kit. Aldehyde fuchsin staining was used for histopathological examination of the pancreas. Parameters for oxidative stress were measured with pancreatic malondialdehyde (MDA) level, glutathione (GSH) content and enzymatic activities of superoxide dismutase and catalase. Fourteen days after the initiation of T alpha-1 treatment and up to day 35 when the treatment was stopped, both of the two STZ and T alpha-1-co-treated mouse groups had significant lower levels of blood glucose than the STZ-treated but T alpha-1-untreated mice, although both remained higher than that of the normo-glycemic controls. At the end of the T alpha-1 treatment, the serum insulin level for STZ-treated mice receiving 1 mu g/kg/day T alpha-1 for 35 days was 2-fold (P<0.001) as much as that of the T alpha-1-untreated STZ-diabetic mice, although not completely restored to the normal level. Pancreatic aldehyde fuchsin staining showed that STZ treatment caused significant pancreatitis, islet atrophy, and a significant reduction in the number of pancreatic 13 cells. These histological lesions, however, were significantly alleviated by 1 mu g/kg/day T alpha-1 treatment for 35 days. Furthermore, compared with the T alpha-1-untreated STZ-diabetic mice, the pancreatic GSH level of the 1 mu g/kg/day T alpha-1-treated STZ-induced mice was 1.92-fold that of the untreated STZ-induced mice (P<0.01), whereas the pancreatic MDA level was only 81.9% that of the untreated STZ-diabetic mice (P<0.05). Together these results demonstrate that co-administration of T alpha-1 leads to significant protection against STZ-induced pancreatic damage and diabetes, and part of the protection might be achieved through enhancing pancreatic antioxidative capability.National Science Foundation of China [30770490]; 973 Program of China [2009CB941601

Aldose Reductase Is Involved in the Development of Murine Diet-Induced Nonalcoholic Steatohepatitis

Natural Science Foundation of Fujian Province, China [2009J01180]; Science Planning Program of Fujian Province, China [2010N0023]Hepatic aldose reductase (AR) expression is known to be induced in liver diseases, including hepatitis and hepatocellular carcinoma. However, the role of AR in the development of these diseases remains unclear. We performed this current study to determine whether and how AR might be involved in the development of diet-induced nonalcoholic steatohepatitis. Our results showed that the level of AR protein expression was significantly higher in db/db mice fed the methionine-cholinedeficient (MCD) diet than in mice fed the control diet. In parallel with the elevation in AR, steatohepatitis was observed in MCD diet-fed mice, and this diet-induced steatohepatitis was significantly attenuated by lentiviral-mediated knock-down of the AR gene. This suppressive effect of AR knock-down was associated with repressed levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced mRNA and protein expression of hepatic cytochrome P450 2E1 (CYP2E1), and decreased mRNA expression of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Moreover, AR-induced elevations on the level of CYP2E1 expression, reactive oxygen species, mRNA expression of TNF-alpha and IL-6 were confirmed in AML12 hepatocytes. Further, lentiviral-mediated knock-down of AR ameliorated MCD diet-induced collagen deposition in the livers of db/db mice. With the improvement in liver fibrosis, the mRNA levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2), two genes involved in hepatic fibrogenesis, were found to be significantly suppressed, while TIMP-2 and MMP-13 were unaffected. Together these data indicate that inhibition of AR alleviates the MCD diet-induced liver inflammation and fibrosis in db/db mice, probably through dampening CYP2E1 mediated-oxidative stress and ameliorating the expression of pro-inflammatory cytokines

Inhibition of Aldose Reductase Activates Hepatic Peroxisome Proliferator-Activated Receptor-alpha and Ameliorates Hepatosteatosis in Diabetic db/db Mice

Natural Science Foundation of Fujian Province, China [2009J01180]; Science Planning Program of Fujian Province [2010J1008]; National Science Foundation of China [30970649]We previously demonstrated in streptozotocin-induced diabeticmice that deficiency or inhibition of aldose reductase (AR) caused significant dephosphorylation of hepatic transcriptional factor PPAR alpha, leading to its activation and significant reductions in serum lipid levels. Herein, we report that inhibition of AR by zopolrestat or by a short-hairpin RNA (shRNA) against AR caused a significant reduction in serum and hepatic triglycerides levels in 10-week old diabetic db/db mice. Meanwhile, hyperglycemia-induced phosphorylation of hepatic ERK1/2 and PPAR alpha was significantly attenuated in db/db mice treated with zopolrestat or AR shRNA. Further, in comparison with the untreated db/db mice, the hepatic mRNA expression of Aco and ApoA5, two target genes for PPAR alpha, was increased by 93% (P < 0.05) and 73% (P < 0.05) in zopolrestat-treated mice, respectively. Together, these data indicate that inhibition of AR might lead to significant amelioration in hyperglycemia-induced dyslipidemia and nonalcoholic fatty liver disease

Aldose Reductase Is Involved in the Development of Murine Diet-Induced Nonalcoholic Steatohepatitis

<div><p>Hepatic aldose reductase (AR) expression is known to be induced in liver diseases, including hepatitis and hepatocellular carcinoma. However, the role of AR in the development of these diseases remains unclear. We performed this current study to determine whether and how AR might be involved in the development of diet-induced nonalcoholic steatohepatitis. Our results showed that the level of AR protein expression was significantly higher in db/db mice fed the methionine-choline-deficient (MCD) diet than in mice fed the control diet. In parallel with the elevation in AR, steatohepatitis was observed in MCD diet-fed mice, and this diet-induced steatohepatitis was significantly attenuated by lentiviral-mediated knock-down of the AR gene. This suppressive effect of AR knock-down was associated with repressed levels of serum alanine aminotransferase and hepatic lipoperoxides, reduced mRNA and protein expression of hepatic cytochrome P450 2E1 (CYP2E1), and decreased mRNA expression of pro-inflammatory tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Moreover, AR-induced elevations on the level of CYP2E1 expression, reactive oxygen species, mRNA expression of TNF-α and IL-6 were confirmed in AML12 hepatocytes. Further, lentiviral-mediated knock-down of AR ameliorated MCD diet-induced collagen deposition in the livers of db/db mice. With the improvement in liver fibrosis, the mRNA levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2), two genes involved in hepatic fibrogenesis, were found to be significantly suppressed, while TIMP-2 and MMP-13 were unaffected. Together these data indicate that inhibition of AR alleviates the MCD diet-induced liver inflammation and fibrosis in db/db mice, probably through dampening CYP2E1 mediated-oxidative stress and ameliorating the expression of pro-inflammatory cytokines.</p></div

Effect of lentiviral-mediated knock-down of AR on hepatic lipoperoxide content, CYP2E1 expression in db/db mice and over-expression of AR on CYP2E1 expression and ROS levels in AML12 cells.

<p>Hepatic lipoperoxide content (A), CYP2E1 mRNA (B) and protein (C) expression were determined in mice fed the control diet, MCD diet, or MCD diet treated with lentiviruses carrying shRNA against AR for 8 weeks (<i>n = </i>4). CYP2E1 protein expression (D) and ROS levels (E) were assayed in AML12 cells transfected with pFLAG-mAR for 36 hours (<i>n = </i>3). Values are expressed as the mean ± SEM. ***, P<0.001; **, P<0.01; *, P<0.05.</p

Effect of AR on hepatic mRNA expression of proinflammatory cytokines.

<p>Total RNA was isolated and analyzed for TNF-α, IL-6 and TGF-β1 in db/db mice (A) fed the control or MCD diet and transduced with pLV-shNC or pLV-shAR for 8 weeks (<i>n = </i>4) and in AML12 cells (B) transfected with pFLAG-CMV2 or pFLAG-mAR for 36 hours (<i>n = </i>3). Values are expressed as the mean ± SEM. **, P<0.01; *, P<0.05.</p

Primer sequences used for amplification of mRNA by real-time PCR.

<p>Primer sequences used for amplification of mRNA by real-time PCR.</p