Tokushima Fatty Rats Research Articles

Effects of long-term childhood exercise and detraining on lipid accumulation in metabolic-related organs.

The preventive effects of regular exercise on obesity-related health problems are carried over to the non-exercise detraining period, even when physical activity decreases with aging. However, it remains unknown whether regular childhood exercises can be carried over to adulthood. Therefore, this study aimed to investigate the effects of long-term childhood exercise and detraining on lipid accumulation in organs to prevent obesity in adulthood. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as obese animals. OLETF rats were allocated into sedentary and exercise groups: exercise from 4- to 12-week-old and detraining from 12- to 20-week-old. At 12-week-old immediately after the exercise period, regular exercise completely inhibited hyperphagia, obesity, enlarged pancreatic islets, lipid accumulation and lobular inflammation in the liver, hypertrophied adipocytes in the white adipose tissue (WAT), and brown adipose tissue (BAT) whitening in OLETF rats. Additionally, exercise attenuated the decrease in the ratio of muscle wet weight to body weight associated with obesity. Decreased food consumption was maintained during the detraining period, which inhibited obesity and diabetes at 20-week-old after the detraining period. Histologically, childhood exercise inhibited the enlargement of pancreatic islets after the detraining period. In addition, inhibition of lipid accumulation was completely maintained in the WAT and BAT after the detraining period. However, the effectiveness was only partially successful in lipid accumulation and inflammation in the liver. The ratio of muscle wet weight to body weight was maintained after detraining. In conclusion, early long-term regular exercise effectively prevents obesity and diabetes in childhood, and its effectiveness can be tracked later in life. The present study suggests the importance of exercise during childhood and adolescence to inhibit hyperphagia-induced lipid accumulation in metabolic-related organs in adulthood despite exercise cessation.

Mass recovery following caloric restriction reverses lipolysis and proteolysis, but not gluconeogenesis, in insulin resistant OLETF rats

Caloric restriction (CR) is one of the most important behavioral interventions to reduce excessive abdominal adiposity, which is a risk factor for the development of insulin resistance. Previous metabolomics studies have characterized substrate metabolism during healthy conditions; however, the effects of CR and subsequent mass recovery on shifts in substrate metabolism during insulin resistance (IR) have not been widely investigated. To assess the effects of acute CR and the subsequent mass recovery on shifts in substrate metabolism, a cohort of 15-week old Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were calorie restricted (CR: 50% × 10 days) with or without partial body mass recovery (PR; 73% x 7 days), along with their respective ad libitum controls. End-of-study plasma samples were analyzed for primary carbon metabolites by gas chromatography (GC) time-of-flight (TOF) mass spectrometry (MS) data acquisition. Data analysis included PCA, Pearson correlation vs previously reported variables (adipose and body masses, and insulin resistance index, IRI), and metabolomics maps (MetaMapp) generated for the most significant group comparisons. All treatments elicited a significant group differentiation in at least one principal component. CR improved TCA cycle in OLETF, and increased lipolysis and proteolysis. These changes were reversed after PR except for gluconeogenesis. Plasma lipid concentrations were inversely correlated to IRI in LETO, but not OLETF. These shifts in substrate metabolism suggest that the CR-induced decreases in adipose may not be sufficient to more permanently alter substrate metabolism to improve IR status during metabolic syndrome.

Retinal Neuropathy in IGT Stage of OLETF Rats: Another Characteristic Change of Diabetic Retinopathy.

Aims We investigated the changes of retinal structure in normal glucose tolerance (NGT), impaired glucose tolerance (IGT), diabetes mellitus (DM), and diabetic kidney disease (DKD) stages in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Methods We assigned OLETF rats to four groups based on their OGTT results and 24 h urinary microalbumin (24 h UMA) levels: NGT, IGT, DM, and DKD groups. We observed the structural and the corresponding pathological changes and quantified the expression of HIF-1α, iNOS, NF-κB, VEGF, ICAM-1, and occludin in the retina. Results Significant damage to the retinal structure, especially in retinal ganglion cells (RGCs), was observed in the IGT stage. The expression of HIF-1α, iNOS, NF-κB, VEGF, and ICAM-1 was significantly upregulated, while that of occludin was downregulated. Conclusion Significant retinal neuropathy occurs in the IGT stage. Inflammation and hypoxia may damage the blood retina barrier (BRB), leading to diabetic retinopathy.

Impact of Exercise and Detraining during Childhood on Brown Adipose Tissue Whitening in Obesity.

This study aimed to investigate the influence of childhood exercise and detraining on brown adipose tissue (BAT) whitening in obesity. Four-week-old male Long-Evans Tokushima Otsuka (LETO) rats (n = 9) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 24) were used as non-obese and obese animals, respectively. OLETF rats were divided into non-exercise sedentary (n = 9) and exercise groups. OLETF rats in the exercise group were further divided into subgroups according to the exercise period—exercise from 10- to 12-weeks-old (n = 6); and exercise from 4- to 6-weeks-old, and detraining from 6- to 12-weeks-old (n = 9). At 12-weeks-old, immediately after exercise period, BAT whitening in OLETF rats was inhibited by exercise despite the fact that hypertrophy was not caused in the plantaris muscle. However, the effectiveness was attenuated during the detraining period. Histological BAT whitening and downregulation of uncoupling protein-1 (UCP-1) were found in non-exercise sedentary OLETF rats at 12-weeks-old. The downregulation was not inhibited, even though exercise histologically inhibited BAT whitening in OLETF rats. Childhood exercise decreased BAT whitening in obesity. Detraining attenuated the inhibition of BAT whitening. These results suggest that regular exercise is needed to improve BAT whitening and downregulation of UCP-1 in obesity.

Region-specific brain area reductions and increased cholecystokinin\xa0positive neurons in diabetic OLETF rats: implication for anxiety-like behavior

Metabolic disorders can induce psychiatric comorbidities. Both brain and neuronal composition imbalances reportedly induce an anxiety-like phenotype. We hypothesized that alterations of localized brain areas and cholecystokinin (CCK) and parvalbumin (PV) expression could induce anxiety-like behavior in type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Twenty-week-old OLETF and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats were used. The areas of corticolimbic regions were smaller in OLETF rats. The densities of CCK positive neurons in the lateral and basolateral amygdala, hippocampal cornu ammonis area 2, and prelimbic cortex were higher in OLETF rats. The densities of PV positive neurons were comparable between OLETF and LETO rats. Locomotion in the center zone in the open field test was lower in OLETF rats. These results suggest that imbalances of specific brain region areas and neuronal compositions in emotion-related areas increase the prevalence of anxiety-like behaviors in OLETF rats.

Canagliflozin, a sodium-glucose cotransporter2 inhibitor, normalizes renal susceptibility to type1 cardiorenal syndrome through reduction of renal oxidative stress in diabetic rats.

Aims/IntroductionType 2 diabetes mellitus is a risk factor of acute kidney injury after myocardial infarction (MI), a form of cardiorenal syndrome. Recent clinical trials have shown that a sodium–glucose cotransporter 2 (SGLT2) inhibitor improved both cardiac and renal outcomes in patients with type 2 diabetes mellitus, but effects of an SGLT2 inhibitor on cardiorenal syndrome remain unclear.Materials and MethodsType 2 diabetes mellitus (Otsuka Long‐Evans Tokushima Fatty rats [OLETF]) and control (Long‐Evans Tokushima Otsuka rats [LETO]) were treated with canagliflozin, an SGLT2 inhibitor, for 2 weeks. Renal tissues were analyzed at 12 h after MI with or without preoperative fasting.ResultsCanagliflozin reduced blood glucose levels in OLETF, and blood β‐hydroxybutyrate levels were increased by canagliflozin only with fasting. MI increased neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 protein levels in the kidney by 3.2‐ and 1.6‐fold, respectively, in OLETF, but not in LETO. The renal messenger ribonucleic acid level of Toll‐like receptor 4 was higher in OLETF than in LETO after MI, whereas messenger ribonucleic acid levels of cytokines/chemokines were not significantly different. Levels of lipid peroxides, nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 and NOX4 proteins after MI were significantly higher in OLETF than in LETO. Canagliflozin with pre‐MI fasting suppressed MI‐induced renal expression of neutrophil gelatinase‐associated lipocalin and kidney injury molecule‐1 in OLETF, together with reductions in lipid peroxides and NOX proteins in the kidney. Blood β‐hydroxybutyrate levels before MI were inversely correlated with neutrophil gelatinase‐associated lipocalin protein levels in OLETF. Pre‐incubation with β‐hydroxybutyrate attenuated angiotensin II‐induced upregulation of NOX4 in NRK‐52E cells.ConclusionsThe findings suggest that SGLT2 inhibitor treatment with a fasting period protects kidneys from MI‐induced cardiorenal syndrome, possibly by β‐hydroxybutyrate‐mediated reduction of NOXs and oxidative stress, in type 2 diabetic rats.

Acyclic retinoid and angiotensin-II receptor blocker exert a combined protective effect against diethylnitrosamine-induced hepatocarcinogenesis in diabetic OLETF rats

BackgroundInsulin resistance (IR) is closely associated with the progression of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR) targets retinoid X receptor α and reportedly prevents HCC recurrence in clinical practice. Angiotensin-II receptor blocker (ARB) can also inhibit experimental hepatocarcinogenesis and HCC development. These are reported to suppress IR-based hepatocarcinogenesis; however, limited data are available regarding the combined effects of both these agents. This study aimed to investigate the combined chemopreventive effect of ACR and ARB on liver tumorigenesis on rats with congenital diabetes.MethodsMale diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats underwent 70% partial hepatectomy following a single intraperitoneal injection of diethylnitrosamine to induce hepatocarcinogenesis and the administration of ACR (peretinoin, 40 mg/kg/day), ARB (losartan, 30 mg/kg/day), and a combination of ACR and ARB. Six weeks thereafter, we assessed the size and number of the pre-neoplastic lesions (PNL) as well as the altered angiogenesis, oxidative stress, and chronic inflammation in the liver. Moreover, we assessed the effects exerted by ACR and ARB on in vitro cell growth in human HCC cell lines and human umbilical vascular endothelial cells (HUVECs).ResultsOLETF rats showed increase in the size and number of PNLs compared to LETO rats. ACR suppressed the augmentation in size and number of PNLs in the OLETF rats with suppression of cell growth, intrahepatic angiogenesis, lipid peroxidation, oxidative DNA damage, and proinflammatory cytokine production. Combining ACR with ARB enhanced the tumor-suppressive effect and ameliorated intrahepatic angiogenesis, lipid peroxidation, and proinflammatory status; however, cell growth and oxidative DNA damage remained unchanged. IR-mimetic condition accelerated in vitro proliferative activity in human HCC cells, while ACR inhibited this proliferation with G0/G1 arrest and apoptosis. Furthermore, ACR and ARB significantly attenuated the HUVECs proliferation and tubular formation under the IR-mimetic condition, and a combination of both agents demonstrated greater inhibitory effects on HUVEC growth than each single treatment.ConclusionsACR and ARB exert a combined inhibitory effect against IR-based hepatocarcinogenesis by the inhibition of cell growth, intrahepatic angiogenesis, and oxidative stress. Thus, this combination therapy appears to hold potential as a chemopreventive treatment therapy against HCC.

Long-Term Insulin Treatment Alters Muscle-Related Protein in OLETF Rat Skeletal Muscle

Purpose: The objective of this study was to investigate molecular and physiological changes in response to long-term insulin glargine treatment in the skeletal muscle of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Methods: Protein changes in the skeletal muscle of type 2 diabetic rats was detected by Western blot analysis, and muscle fiber type composition was assessed by real-time PCR. Results: Insulin glargine (1 U/kg) treatment in OLETF rats (OLETF-G) for 24 weeks were changed in blood glucose levels in intraperitoneal glucose tolerance tests compared with age-matched, untreated OLETF rats (OLETF-C), and the area under the curve was significantly decreased for OLETF-G rats compared with OLETF-C rats (P < 0.05). The protein levels of myosin heavy chain (MHC) isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of MHC type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (interleukin [IL]-6, IL-15, fibronectin type III domain containing 5 [FNDC5]/irisin, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats (P < 0.05). Moreover, myogenin determination gene D and myogenin levels were decreased, while insulin-like growth factor-I levels were increased, in the skeletal muscle of OLETF-G rats (P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of 5’-AMP-activated protein kinase, sirtuin 1, and peroxisome proliferator-activated receptor γ coactivator 1α. Conclusions: Our results suggest that long-term insulin glargine treatment can effectively alter protein expression in the gastrocnemius muscle of OLETF rats. Disclosure H. Kwon: None. O. Hong: None. H. Kwon: None. Y. Kang: None. S. Lee: None. S. Kim: None. S. Yoo: None.

The effects of herbal composition Gambigyeongsinhwan (4) on hepatic steatosis and inflammation in Otsuka Long‐Evans Tokushima fatty rats and HepG2 cells

Ethnopharmacological relevanceHepatic steatosis has risen rapidly in parallel with a dramatic increase in obesity. The aim of this study was to determine whether the herbal composition Gambigyeongsinhwan (4) (GGH(4)), composed of Curcuma longa L. (Zingiberaceae), Alnus japonica (Thunb.) Steud. (Betulaceae), and the fermented traditional Korean medicine Massa Medicata Fermentata, regulates hepatic steatosis and inflammation. Materials and methodsThe effects of GGH(4) on hepatic steatosis and inflammation in Otsuka Long-Evans Tokushima fatty (OLETF) rats and HepG2 cells were examined using Oil red O, hematoxylin and eosin, and toluidine blue staining, immunohistochemistry, quantitative real-time polymerase chain reaction, and peroxisome proliferator-activated receptor α (PPARα) transactivation assay. ResultsAdministration of GGH(4) to OLETF rats improved hepatic steatosis and lowered serum levels of alanine transaminase, total cholesterol, triglycerides, and free fatty acids. GGH(4) increased mRNA levels of fatty acid oxidation enzymes (ACOX, HD, CPT-1, and MCAD) and decreased mRNA levels of lipogenesis genes (FAS, ACC1, C/EBPα, and SREBP-1c) in the liver of OLETF rats. In addition, infiltration of inflammatory cells and expression of inflammatory cytokines (CD68, TNFα, and MCP-1) in liver tissue were reduced by GGH(4). Treatment of HepG2 cells with a mixture of oleic acid and palmitoleic acid induced significant lipid accumulation, but GGH(4) inhibited lipid accumulation by regulating the expression of hepatic fatty acid oxidation and lipogenic genes. GGH(4) also increased PPARα reporter gene expression. These effects of GGH(4) were similar to those of the PPARα activator fenofibrate, whereas the PPARα antagonist GW6471 reversed the inhibitory effects of GGH(4) on lipid accumulation in HepG2 cells. ConclusionsThese results suggest that GGH(4) inhibits obesity-induced hepatic steatosis and that this process may be mediated by regulation of the expression of PPARα target genes and lipogenic genes. GGH(4) also suppressed obesity-related hepatic inflammation. Thus, GGH(4) may be a promising drug for the treatment of obesity-related liver diseases.

OS 32-05 EFFECTS OF SGLT2 INHIBITORS ON CIRCADIAN RHYTHM OF BLOOD PRESSURE IN RATS

Disrupted circadian rhythm of blood pressure is associated with cardiovascular events in metabolic syndrome and obesity. Experiments were conducted to examine the effects of sodium-glucose co-transporter 2 (SGLT2) inhibitors on circadian rhythm of blood pressure in a genetic model of obese metabolic syndrome (SHR/NDmcr-cp (+/+) (SHRcp)) and salt-treated obese Otsuka Long Evans Tokushima Fatty (OLETF) rats. Luseogliflozin (10 mg/kg/day, p.o.) and empagliflozin (10 mg/kg/day, p.o.) were administered for 5 weeks in metabolic syndrome SHRcp rats and 1% NaCl (in drinking water)-treated obese OLETF rats, respectively. Blood pressure was measured continuously by telemetry system. Glucose metabolism and insulin resistance were evaluated by oral glucose tolerance test. Both SHRcp and salt-treated OLETF rats developed non-dipper type of hypertension with altered glucose metabolism and insulin resistance. Administration of luseogliflozin and empagliflozin resulted in a remarkable increase in urinary glucose excretion and improved glucose metabolism and insulin resistance in SHRcp and salt-treated OLETF rats, respectively. Furthermore, luseogliflozin and empagliflozin significantly attenuated the development of hypertension with normalization of circadian rhythm of blood pressure, which was associated with an increase in urinary sodium excretion. These data suggest that SGLT2 inhibitors elicit beneficial effects on circadian rhythm of blood pressure during the development of hypertension in subjects with metabolic syndrome and obese.

Exercise Attenuates Ischemia-reperfusion Injury of Nonalcoholic Fatty Liver in OLETF Rat

Background: Steatotic livers are increasingly used for liver transplantation and the strategy to minimize its disadvantage is required. We determined whether exercise could protect liver against ischemia-reperfusion (IR) injury in the anesthetized hyperphagic, Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of the metabolic syndrome. Methods: At 16 th week of age, male OLETF rats (n=7/group) were randomized to the groups of sedentary (OLETF-SED)-IR, exercise (OLETF-EX)-IR, and OLETF-non IR. Non-hyperphagic, control strain Long-Evans Tokushima Otsuka (LETO) rats were similarly assigned. Rats in the exercise groups were subjected to treadmill running 6 days a week for 5 weeks. Rats were anesthetized with intraperitoneal pentobarbital and subjected to partial liver ischemia (70%) for 30 min and subsequent reperfusion for 120 min. IR injury was evaluated by plasma alanine aminotransferase (ALT)concentration, bile flow rate, liver histopathology, and hepatic microcirculation of the left lateral lobe analyzed by laser Doppler flowmetry. Results: Increases in body and liver weight were attenuated in the OLETF-EX rats as compared with the OLETF-SED rats. The ALT levels after reperfusion in the OLETF-EX rats were smaller than that in the OLETF-SED rats, and were comparable to those in the LETOSED and -EX rats. Exercise attenuated the IR-induced reduction of liver blood flow and morphological hepatic damages as observed in the OLETF-SED group. In contrast, exercise provided no influences on the LETO rats. Conclusion: Exercise attenuated the liver steatosis and IRinduced disturbances of hepatic microcirculation and parenchymal injury in the hyperphagic, OLETF rats.

Use of Drosophila as an evaluation method reveals imp as a candidate gene for type 2 diabetes in rat locus Niddm22.

Type 2 diabetes (T2D) is one of the most common human diseases. QTL analysis of the diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats has identified numerous hyperglycemic loci. However, molecular characterization and/or gene identification largely remains to be elucidated due mostly to the weak genetic variances contributed by each locus. Here we utilized Drosophila melanogaster as a secondary model organism for functional evaluation of the candidate gene. We demonstrate that the tissue specific knockdown of a homologue of igf2bp2 RNA binding protein leads to increased sugar levels similar to that found in the OLETF rat. In the mutant, the expression of two of the insulin-like peptides encoded in the fly genome, dilp2 and dilp3, were found to be downregulated. Consistent with previous reports of dilp mutants, the imp mutant flies exhibited an extension of life span; in contrast, starvation tolerance was reduced. These results further reinforce the possibility that imp is involved in sugar metabolism by modulating insulin expression.

Vascular Aldosterone Production at the Pre-Diabetic Stage of Young Otsuka Long-Evans Tokushima Fatty (OLETF) Rats, Compared with Long-Evans Tokushima Otsuka (LETO) Rats

We examined the ability of aortic smooth muscle cells (AoSMC) prepared from spontaneously diabetic rats to produce aldosterone (Aldo) and the regulatory mechanism that controls their Aldo production. AoSMC of 6 week-old Long-Evans Tokushima Otsuka (LETO: the control group) and 6 week-old Otsuka Long-Evans Tokushima Fatty (OLETF: the type 2 diabetes group) rats were used in the present experiments. CYP11B2 (Aldo synthetase) mRNA expression was detected in both the LETO and OLETF AoSMC. Basal Aldo production was significantly greater (4–5 fold higher) in the OLETF AoSMC culture medium than in the LETO AoSMC culture medium. When AoSMC were co-incubated with high-density lipoproteins (HDL), supplying cholesterol as a substrate for steroidogenesis in rats, angiotensin II (AII) significantly increased greater Aldo production in the OLETF AoSMC than in the LETO AoSMC. The present data suggested that future onset of diabetic vascular dysfunction is partly caused by excess Aldo production by AoSMC in young OLETF rats. Concomitant stimulation by HDL and AII resulted in elevated Aldo production in the OLETF and the LETO AoSMC, and also demonstrated that AII-induced Aldo production is greatly enhanced by HDL in OLETF, rather than in LETO. In conclusion, our data clearly demonstrated that Aldo production in the OLETF AoSMC was significantly higher than in the LETO AoSMC, suggesting possible future onset of vascular dysfunction in diabetes, induced by local Aldo production in the AoSMC.

Inhibition of Postprandial Hyperglycemia by Either an Insulin-Dependent or -Independent Drug Reduces the Expression of Genes Related to Inflammation in Peripheral Leukocytes of OLETF Rats

Treatment with the dipeptidyl peptidase-4 inhibitor, anagliptin, or with the α-glucosidase inhibitor, miglitol, reduced the oral sucrose load-inducible expression of interleukin (IL)-1β, IL-18, tumor necrosis factor-α, S100a8, S100a9, S100a11, IL-1R2, IL-1Rn and tumor necrosis factor receptor 2 genes in peripheral leukocytes of Otsuka Long-Evans Tokushima fatty (OLETF) rats at the stage of impaired glucose tolerance. Inhibiting postprandial hyperglycemia reduced the expression of genes related to inflammation in peripheral leukocytes of OLETF rats.

Mo1046 Effects of Iron Restriction on Hepatic Mitochondrial Iron Metabolism and Oxidative Stress in Nonalcoholic Fatty Liver Disease (NAFLD) in Otsuka Long-Evans Tokushima Fatty (OLETF) Rats

Mo1046 Effects of Iron Restriction on Hepatic Mitochondrial Iron Metabolism and Oxidative Stress in Nonalcoholic Fatty Liver Disease (NAFLD) in Otsuka Long-Evans Tokushima Fatty (OLETF) Rats

Mo1050 Attenuation of Nonalcoholic Fatty Liver Disease (NAFLD) in Type 2 Diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) Rats via Regulation of Hepatic Lipogenesis and Glucose Metabolism by Administration of S-Allyl Cysteine

Mo1050 Attenuation of Nonalcoholic Fatty Liver Disease (NAFLD) in Type 2 Diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) Rats via Regulation of Hepatic Lipogenesis and Glucose Metabolism by Administration of S-Allyl Cysteine

Abstract 1279: Serum biochemistry and microarray analysis of liver and colonic mucosa in LETO and diabetic OLETF rats treated with 20% caloric restriction and high-fat diet.

Abstract We showed recently that diabetic and mildly obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats were more susceptible to azoxymethane-induced carcinogenesis than control Long-Evans Tokushima Otsuka (LETO) rats, and carcinogenesis was inhibited by 20% caloric restriction (CR) and enhanced by high-fat diet (HFD, control diet with 10% safflower oil). To elucidate the mechanism of cancer susceptibility, we performed serum biochemical analysis and microarray analysis of the liver and colonic mucosa. Male LETO and OLETF rats were given control diet, 20% CR diet or HFD for 5 weeks, and euthanized at 24 weeks of age (n=5, respectively). In both strains, blood glucose, serum triglyceride, total cholesterol, insulin and leptin levels decreased by 20% CR, and increased by HFD. Steatosis of hepatocytes in OLETF rats was ameliorated by 20% CR and enhanced by HFD. Microarray analysis of the liver showed that 20% CR up-regulated metallothionein 1A gene, and down-regulated heat shock 105kDa/110kDa protein 1 (Hsph1) and heat shock 70kDa protein 1B (Hspa1b) genes in both strains. There were no histological differences in the colonic mucosa in both strains and the effect of the dietary difference was not found. Microarray analysis of the colonic mucosa showed that 20% CR down-regulated Hsph1 and Hspa1b genes, and HFD up-regulated fatty acid binding protein 2 in both strains. These data suggest that hyperinsulinemia, hyperlipidemia and oxidative stress by overnutrition exert significant role in cancer susceptibility in OLETF rats. Citation Format: Keisuke Izumi, Chie Takasu, Tetsuyuki Takahashi, Hisanori Uehara. Serum biochemistry and microarray analysis of liver and colonic mucosa in LETO and diabetic OLETF rats treated with 20% caloric restriction and high-fat diet. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1279. doi:10.1158/1538-7445.AM2013-1279

The influence of ethanol intake on RhoA/Rho kinase signaling pathway in corpus cavernosum of OLETF rats

To investigate the effect of the ethanol on corpus cavernosum of the type 2 diabetes mellitus (type 2 DM) rat, since previous studies have reported the positive effect of moderate drinking on type 2 DM and endothelial function. Otsuka Long Evans Tokushima Fatty (OLETF, n = 28) and Long Evans Tokushima Otsuka (LETO, n = 28) male rats were assigned to four groups: control (LETO, n = 14), control/ethanol (LETO with ethanol, n = 14), fatty (OLETF, n = 14) and fatty/ethanol (OLETF with ethanol, n = 14). The control/ethanol and fatty/ethanol groups were fed Lieber-DeCarli Regular EtOH. After 10 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed. RT-PCR and Western blot analyses of the RhoA/Rho kinase in the corpus cavernosum were performed. The expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the corpus cavernosum was evaluated by immunohistochemistry. After a 10-week ethanol intake, the body weight and the IPGTT of the fatty/ethanol group were significantly lower compared with the fatty group (p < 0.05). A significantly increased mRNA and protein expression in the fatty group was observed compared with the control group by RT-PCR and Western blot analyses (p < 0.05): A significantly decreased expression was observed in the fatty/ethanol group compared with the fatty group (p < 0.05). The significantly increased expression of eNOS was observed in the fatty/ethanol group compared with the fatty group by immunohistochemistry. Long-term ethanol intake may preserve the erectile function in type 2 DM through the down-regulation of the RhoA/Rho kinase pathway.

Cerebral antioxidant enzyme increase associated with learning deficit in type 2 diabetes rats

In this study, we examined alterations in the enzymatic antioxidant defenses associated with learning deficits induced by type 2 diabetes, and studied the effects of the peroxisome proliferator-activated receptor γ agonist pioglitazone on these learning deficits. Learning ability was assessed by visual discrimination tasks in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as a model of spontaneous type 2 diabetes. Levels of the antioxidant enzymes glutathione peroxidase (GPx), Cu(2+)-Zn(2+) superoxide dismutase (CuZn-SOD) and manganese SOD were measured in the cortex, hippocampus and striatum. Half the rats received oral pioglitazone (20mg/kg/day) from the early stage of diabetes (22 weeks old) to 27 weeks old. OLETF rats showed learning deficits compared with control, Long-Evans Tokushima Otsuka (LETO) rats. GPx levels in the cortex and hippocampus were increased in OLETF rats compared with LETO rats, with an inverse correlation between GPx in the hippocampus and learning score. CuZn-SOD levels were also increased in the hippocampus in OLETF rats. Pioglitazone reduced blood glucose and increased serum adiponectin levels, but had no effect on learning tasks or antioxidant enzymes, except for CuZn-SOD. These results suggest that an oxidative imbalance reflected by increased brain antioxidant enzymes plays an important role in the development of learning deficits in type 2 diabetes. Early pioglitazone administration partly ameliorated diabetic symptoms, but was unable to completely recover cerebral oxidative imbalance and functions. These results suggest that diabetes-induced brain impairment, which results in learning deficits, may have occurred before the appearance of the symptoms of overt diabetes.

Abstract 402: Specified Proximal Tubule Segments Augmented Angiotensinogen mRNA and Protein Expressions in Diabetic Kidneys in Rats and Humans

Others and we showed the increased levels of intrarenal angiotensinogen (AGT) in diabetic humans, mice, and rats. However, the precise localization of the augmented AGT mRNA and protein in proximal tubule segments of kidneys in diabetes remains to be established. To investigate the detailed localization of AGT in 3 proximal tubule segments of the kidneys in diabetes, the Otsuka Long-Evans Tokushima fatty (OLETF) rat (15 weeks) and the age-matched genetic control, the Long-Evans Tokushima Otsuka (LETO) rat were used. Kidney tissues were also prepared from OLETF rats that were administered with angiotensin II type 1 receptor blocker, olmesartan (0.02% in chow) or a combination of vasodilator agents (0.03% hydralazine, 0.006% reserpine, and 0.012% hydrochlorothiazide in chow; HRH). Moreover, biopsied samples of human kidney cortex were obtained by fine-needle aspiration to confirm the below-mentioned results of the rat localization study. Using 3-μm sections of zinc-saturated formalin-fixed paraffin-embedded slides, we examined the co-localization of AGT mRNA or protein with segment-specific protein markers (Segment (S)1-specific anti-sodium glucose cotransporter 2 antibody, S2-specific anti-carbonic anhydrase IV antibody, and S3-specific anti-ecto-adenosine triphosphatase antibody) by double staining using fluorescence in situ hybridization and/or immunofluorescence. AGT mRNA expression was not recognized much in S1 segments of either OLETF or LETO rat kidneys. In S3 segments, the AGT mRNA expression area was augmented in the OLETF rats than in the LETO rats (2.31 ± 0.02-fold; P = 0.0029). AGT protein expression areas in S1 and S3 segments were also increased (8.50 ± 2.78-fold; P = 0.0476, and 3.71 ± 0.83-fold; P = 0.0317, respectively). Chronic treatment with olmesartan, but not with HRH, ameliorated the augmented AGT mRNA and protein expression areas. AGT mRNA and protein expression areas in S2 segments were unchanged in OLETF rats compared with LETO rats. Biopsied samples of human kidney cortex showed the same results as for rat AGT mRNA and protein expression in the S1 and S2 segments. These data suggest that the augmented AGT mRNA in S3 and AGT protein in S1 and S3 play an important role in the development and the progression of diabetic nephropathy.