Eunhui Seo

Systemic Delivery of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Elevates Levels of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) and Prevents Type 1 Diabetes in Nonobese Diabetic Mice

Recent studies have demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is a modulator of the immune response. The relation between TRAIL and type 1 diabetes (T1D) as an autoimmune inflammatory disease in vivo is relatively unknown. To explore the potential role of TRAIL in the development of T1D, we examined its in vivo effects in nonobese diabetic (NOD) mice. NOD mice at 7 wk of age were iv injected with an adenovirus carrying either human TRAIL (Ad.hTRAIL) or β-galactosidase genes. Blood glucose was monitored weekly, and the expression of hTRAIL was evaluated in plasma and liver of mice. To investigate whether hTRAIL elicits its effect through the induction of tissue inhibitor of metalloproteinase-1 (TIMP-1), we examined the concentration of plasma TIMP-1 by ELISA and the inhibition of matrix metalloproteinase (MMP) by gelatin zymography. Here, we show that Ad.hTRAIL-transduced mice had significantly reduced blood glucose levels and markedly increased production of TIMP-1 compared with control β-galactosidase animals. Pancreatic tissue isolated from Ad.hTRAIL-treated NOD mice showed reduced MMP activities associated with significantly improved insulitis. In addition, TIMP-1 in vitro suppressed cytokine-induced apoptosis in insulin-producing INS-1 cells. These results indicate that T1D can be prevented by TRAIL overexpression through enhancement of TIMP-1 function. Elevated TIMP-1 production inhibits the activity of MMPs, which may contribute to suppress the transmigration of diabetogenic T cells into the pancreatic islets and protects pancreatic β-cells from cytokine-induced apoptosis. Therefore, TRAIL and TIMP-1 induction may be potential targets to prevent development of T1D.

The hyperleptinemia and ObRb expression in hyperphagic obese rats

Leptin resistance associated with hyperleptinemia in high-fat-diet-induced obese rats and aged obese rats is well established, but it is not clear whether hyperphagia-induced obese rats also develop leptin resistance. We investigated whether Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which are a strain of hyperphagia-induced obese rats, develop leptin resistance and whether caloric restriction reversed this leptin resistance-induced leptin receptor (ObRb) deficit. Twenty male OLETF rats, 20 male Long-Evans Tokushima Otsuka (LETO) rats, and 10 male Sprague Dawley (SD) rats were used. All rats were initially studied at 10 weeks of age and were freely fed with standard rat chow and water until they were 38 weeks of age. Daily food intake, body weight, and plasma leptin levels of OLETF rats were remarkably increased compared to LETO or SD rats from 10 to 38 weeks of age. When they were 38 weeks of age, all OLETF rats were randomly divided into two groups. One group was freely fed with standard rat chow (FD, or free diet group), and the other group (RD, or restricted diet group) was fed with only 70% of the amount consumed by the FD group. The LETO and SD rats were dismissed from further study. After 4 weeks of caloric restriction, the average body weight (636+/-33 g vs. 752+/-24 g, P<0.05) and abdominal adipose tissue weight (10.6+/-3.2g vs. 15.8+/-1.5 g, P<0.05) of the RD group were decreased compared with those of the FD group. Plasma leptin levels of the RD group were significantly decreased compared with those of the FD group (3.47+/-1.40 ng/mL vs. 11.55+/-1.16 ng/mL, P<0.05). The mRNA expression of ObRb and leptin-related suppressor of cytokine signaling 3 (SOCS3) in the hypothalamus, liver, and skeletal muscles of the RD group were significantly decreased compared with those of the FD group. Caloric restriction did not improve leptin receptor (ObRb) deficit or the downstream signaling of leptin in the liver, skeletal muscles, and hypothalamus. Thus, we demonstrated that OLETF rats, which are a strain of hyperphagia-induced obese rats, did not develop central or peripheral leptin resistance. We suggest that hyperleptinemia in OLETF rats is a compensatory mechanism to overcome obesity induced by hyperphagia.

Differential Expression of Metabolism-related Genes in Liver of Diabetic Obese Rats.

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of spontaneous type 2 diabetes (T2D), develops hyperglycemic obesity with hyperinsulinemia and insulin resistance after the age of 25 weeks, similar to patients with noninsulin-dependent diabetes mellitus (DM). In the present study, we determined whether there are differences in the pattern of gene expression related to glucose and lipid metabolism between OLETF rats and their control counterparts, Long-Evans Tokushima (LETO) rats. The experiment was done using 35-week-old OLETF and LETO rats. At week 35 male OLETF rats showed overt T2D and increases in blood glucose, plasma insulin, plasma triglycerides (TG) and plasma total cholesterol (TC). Livers of diabetic OLETF and LETO rats also showed differences in expression of mRNA for glucose and lipid metabolism related genes. Among glucose metabolism related genes, GAPDH mRNA was significantly higher and FBPase and G6Pase mRNA were significantly lower in OLETF rats. For lipid metabolism related genes, HMGCR, SCD1 and HL mRNA were substantially higher in OLETF rats. These results indicate that gluconeogenesis in OLETF rats is lower and glycolysis is higher, which means that glucose metabolism might be compensated for by a lowering of the blood glucose level. However, lipid synthesis is increased in OLETF rats so diabetes may be aggravated. These differences between OLETF and LETO rats suggest mechanisms that could be targeted during the development of therapeutic agents for diabetes.

Reduction of Food Intake by Fenofibrate is Associated with Cholecystokinin Release in Long-Evans Tokushima Rats

Fenofibrate is a selective peroxisome proliferator-activated receptor α (PPARα) activator and is prescribed to treat hyperlipidemia. The mechanism through which PPARα agonists reduce food intake, body weight, and adiposity remains unclear. One explanation for the reduction of food intake is that fenofibrate promotes fatty acid oxidation and increases the production of ketone bodies upon a standard experimental dose of the drug (100~300 mg/kg/day). We observed that low-dose treatment of fenofibrate (30 mg/kg/day), which does not cause significant changes in ketone body synthesis, reduced food intake in Long-Evans Tokushima (LETO) rats. LETO rats are the physiologically normal controls for Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which are obese and cholecystokinin (CCK)-A receptor deficient. We hypothesized that the reduced food intake by fenofibrate-treated LETO rats may be associated with CCK production. To investigate the anorexic effects of fenofibrate in vivo and to determine whether CCK production may be involved, we examined the amount of food intake and CCK production. Fenofibrate-treated OLETF rats did not significantly change their food intake while LETO rats decreased their food intake. Treatment of fenofibrate increased CCK synthesis in the duodenal epithelial cells of both LETO and OLETF rats. The absence of a change in the food intake of OLETF rats, despite the increase in CCK production, may be explained by the absence of CCK-A receptors. Contrary to the OLETF rats, LETO rats, which have normal CCK receptors, presented a decrease in food intake and an increase in CCK production. These results suggest that reduced food intake by fenofibrate treatment may be associated with CCK production.

Fenofibrate Reduces Age-related Hypercholesterolemia in Normal Rats on a Standard Diet.

Plasma cholesterol is increased in normal aging in both rodents and humans. This is associated with reduced elimination of cholesterol and decreased receptor-mediated clearance of plasma low-density lipoprotein (LDL) cholesterol. The aims of this study were: (1) to determine age-related changes in plasma lipid profiles, and (2) to determine the effect of fenofibrate, an activator of peroxisome proliferator activated receptor alpha (PPAR alpha), on plasma lipid profiles in normal rats on a standard diet. Male Sprague-Dawley (SD) rats (n=15) were fed standard chow and water from 10 to 25 weeks of age. During that period, we measured daily food intake, body weight, fasting and random blood glucose levels, plasma total cholesterol (TC), triglycerides (TG), and free fatty acid (FFA) levels. At 20 weeks of age, all rats were randomly divided into two groups: a fenofibrate group (in which rats were gavaged with 300 mg/kg/day of fenofibrate) and a control group (gavaged with water). Fenofibrate treatment lasted 5 weeks. There were no significant changes in daily food intake, blood glucose, and plasma TG level with age. Body weight, plasma TC, and FFA levels were significantly increased with age. Fenofibrate significantly decreased plasma concentrations of TC and FFA, which had been increased with age. However, fenofibrate did not influence the plasma concentration of TG, which had not increased with age. These results suggest that fenofibrate might have a novel role in preventing age-related hypercholesterolemia in SD rats on a normal diet.

Alteration of Lipid Metabolism Related Proteins in Liver of High-Fat Fed Obese Mice

Obesity and being overweight are strongly associated with the development of metabolic disease such as diabetes, hypertension, dyslipidemia. High-fat diet (HFD) is one of the most important factors which cause obesity. In this study, C57BL/6 mice were treated with a HFD for 22 weeks in order to induce obesity and hyperglycemia. Twenty-two weeks later, body weight and plasma glucose level of the HFD group were significantly increased, compared with the normal diet (ND) group. Intra-peritoneal glucose tolerance test (IPGTT) showed glucose intolerance in the HFD group compared with the ND group. These results confirmed that a HFD induced obesity and hyperglycemia in C57BL/6 mice. Plasma levels of triglyceride (TG) and total cholesterol (TC) were increased in the HFD group compared with the ND group. Hepatic levels of TG and TC were also increased by a HFD. To investigate the alteration of lipid metabolism in liver, proteins which are related to lipid metabolism were observed. Among lipid synthesis related enzymes, fatty acid synthase (FAS) and glycerol phosphate acyl transferase (GPAT) were significantly increased in the HFD group. Apolipoprotein B (apoB) and microsomal triglyceride transport protein (MTP), which are related to lipid transport, were significantly increased in the HFD group. Interestingly, protein level and phosphorylation of AMP-activated protein kinase (AMPK), which is known as a metabolic regulator, were significantly increased in the HFD group compared with the ND group. In the present study we suggest that HFD may physiologically increase the proteins which are related with lipid synthesis and lipid transport, but that HFD may paradoxically induce the activation of AMPK.

Fenofibrate antagonizes Chk2 activation by inducing Wip1 expression: implications for cell proliferation and tumorigenesis.

AIMS Fenofibrate is a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist that has been widely used to treat dyslipidemia. Previous studies have suggested that fenofibrate plays a role in cell proliferation and the development of hepatocarcinoma, but the underlying mechanism has not been fully characterized. In this report, we investigated whether fenofibrate treatment affected on the machinery of cell cycle checkpoint using nocodazole-induced cell cycle arrest. MAIN METHODS The human normal liver cell line, CCL13 cells were treated with nocodazole and fenofibrate. Flow cytometry was performed for cell cycle analysis, and checkpoint kinase 2 (Chk2) and phosphatase Wip1 were analyzed by Western blot. KEY FINDINGS Fenofibrate treatment overrode nocodazole-induced G2/M cell cycle arrest in a PPARalpha-independent manner. Mechanistically, fenofibrate treatment inhibited phosphorylation of checkpoint kinase Chk2 induced by nocodazole, and increased the expression of Wip1, a negative regulator of Chk2, suggesting that fenofibrate suppressed the nocodazole-induced G2/M cell cycle checkpoint through Wip1-mediated inhibition of Chk2 activation. SIGNIFICANCE These results reveal a novel role of fenofibrate in cell cycle checkpoint control and provide a possible mechanistic explanation for how fenofibrate promotes cell proliferation and carcinogenesis.

De novo Expression of Hepatic UCP3 Is Time-Dependently Related with Metabolic Function in Fenofibrate-Treated High Fat Diet Rats

Uncoupling protein 3 (UCP3) is a mitochondrial protein that is expressed predominantly in skeletal muscle. It may play a role in altering metabolic function. However, its major physiological roles are not fully understood. Recently de novo expression of UCP3 in rat liver by fenofibrate was reported. We also reported previously that fenofibrate-induced de novo expression of UCP3 contributes to reduction of adipose tissue in obese rats. In the present study, we investigated that fenofibrate-induced expression of UCP3 in rat liver is related with metabolic function such as body weight and hepatic lipid content by time-dependent manner in high-fat diet rats. Eight-week-old male Sprague-Dawley rats were randomly divided into two groups; the high fat diet group (HF, n=16) and fenofibrate-treated high fat diet group (HFF, n=16). The mRNA expression of hepatic UCP3 was detected as early as 1 week of fenofibrate treatment by quantitative real-time PCR and the amount of mRNA was increased time-dependently. The mean body weight of the HFF group was significantly less compared with the HF group after 6 weeks of fenofibrate treatment, even though there was no difference of food intake between the two groups. Rectal temperature was increased during 4 to 6 weeks of fenofibrate treatment and body weight was decreased after 6 weeks of treatment. These results were corresponded with the increased amount of the expression of UCP3 mRNA and protein. We suggest that de novo expression of hepatic UCP3 is increased time-dependently with fenofibrate treatment and that the amount of expression is correlated with metabolic function.