Otsuka Long-Evans Tokushima Fatty Research Articles

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.

Dioscorea esculenta-induced increase in muscle sex steroid hormones is associated with enhanced insulin sensitivity in a type 2 diabetes rat model.

The effects of chronic Dioscorea esculenta administration and exercise training on muscle sex steroid hormone levels and insulin resistance in type 2 diabetes rats was assessed. Twenty-week-old male Otsuka Long Evans Tokushima Fatty (OLETF) rats were assigned randomly to the control, D. esculenta treatment, D. esculenta with 5α-reductase inhibitor treatment, or the exercise training groups (running at 25 m/min for 1 h, 5 d/wk; n = 10 each group). Eight weeks of D. esculenta treatment or exercise training significantly attenuated the increase in plasma insulin and fasting glucose levels. Plasma and muscle concentrations of dehydroepiandrosterone and 5α-dihydrotestosterone (DHT) and the expression of 5α-reductase increased significantly in the D. esculenta-treated and exercise training groups, and both treatments led to the upregulation of glucose transporter-4 translocation with concomitant increases in PKB and PKC-ζ/λ phosphorylation. Furthermore, the glucose metabolic clearance rate, which represents insulin sensitivity, increased significantly in both the D. esculenta-treated and exercise training groups. These effects were suppressed by administration of the DHT synthetic inhibitor. Together, these findings suggest that the D. esculenta-induced increase in muscle sex steroid hormone levels helps decrease insulin resistance in type 2 diabetes.-Sato, K., Fujita, S., Iemitsu, M. Dioscorea esculenta-induced increase in muscle sex steroid hormones is associated with enhanced insulin sensitivity in a type 2 diabetes rat model.

Exercise Training Prevent Decrease of Muscle Strength in Otsuka Long- Evans Tokushima Fatty Rats: A Preliminary Study

Background: Decrease of muscle strength around knee and ankle are considered to contribute several movement disorders in diabetic patient such as an increased risk of falling, altered gait and balance, and increased body sway. In this study, we examine the effect of exercise training on skeletal muscle of type 2 diabetic models. Methods: Otsuka Long–Evans Tokushima fatty (OLEFT) rats, type 2 diabetic models, were divided into an exercise training group (OLETF-EX, n = 3) and a sedentary group (OLETF-SED, n = 3), while Long– Evans Tokushima Otsuka (LETO) rats were used as a control group (LETO-SED, n = 4). The OLETF-EX rats were trained on a treadmill five times a week for 15 weeks. Data were subjected to two-tailed multiple t-tests with Bonferroni correction following an ANOVA. Results: The muscle force per body weight was significantly lower tension in MG muscle of OLETFSED animals than LETO-SED animals. On the other hand, twitch tensions of OLETF-EX animals were preserved. Conclusion: These data suggest that exercise training prevent decrease of muscle strength in type 2 diabetic model.

Chronic saponin treatment attenuates damage to the pancreas in chronic alcohol-treated diabetic rats

BackgroundChronic heavy alcohol consumption may raise the risk of developing type 2 diabetes mellitus. Saponins inhibit apoptosis of pancreatic islet cells and reduce lipid parameters. The present study was designed to investigate the effect of saponin on chronic ethanol-treated diabetic rats. MethodsLong–Evans Tokushima Fatty (LETO) and Otsuka Long–Evans Tokushima Fatty (OLETF) rats were pair-fed a Lieber–DeCarli diet with and without 5% ethanol for 12 wks. Two weeks after starting the pair-feeding with the Lieber–DeCarli diet, intraperitoneal injection of saponin was performed for 10 wks. To perform the experiments, rats were divided as follows: LETO-Control (LC), LETO-Ethanol (LE), LETO-Ethanol-Saponin (LES), OLETF-Control (OC), OLETF-Ethanol (OE), and OLETF-Ethanol-Saponin (OES). ResultsThe weights of epididymal and mesenteric fat tissue in LES and OES rats were the lightest from among the LETO and OLETF groups, respectively. The secretion of alanine aminotransferase and cholesterol in OES rats decreased significantly compared to their secretion in OC and OE rats, respectively. The islets of the pancreas in LE and OE rats showed clean, unclear, and smaller morphology compared to those of LC, LES, OC, and OES rats. In addition, the expression of insulin in the islets of the pancreas in LC, LES, OC, and OES rats was higher than in LE and OE rats. ConclusionSaponin may not only be helpful in alleviating the rapid progress of diabetes due to chronic alcohol consumption in diabetic patients, but may also show potential as an antidiabetic drug candidate for diabetic patients who chronically consume alcohol.

PS 10-03 EFFECTS OF LCZ696 ON BLOOD PRESSURE AND RENAL INJURY IN TYPE 2 DIABETIC RATS WITH OVERT PROTEINURIA

Objective: Clinical studies have indicated that LCZ696, an angiotensin receptor-neprilysin inhibitor, exerts beneficial effects in patients with heart failure. Here, we aimed to examine the effect of LCZ696 on blood pressure and renal injury in type 2 diabetic Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats with overt proteinuria. Design and Method: In OLETF rats, vehicle (n = 10), valsartan (30 mg/kg/day, n = 10), LCZ696 (68 mg/kg/day, n = 9) or valsartan plus hydralazine (3 mg/kg/day, n = 10) was treated from 56 to 80 weeks of age. Results: At baseline (56-week-old), diabetic OLETF rats showed hypertension, overt proteinuria, glomerular injury (glomerular PAS positive area) and tubulointerstitial fibrosis (Sirius Red-positive area). At 80-week-old, vehicle-treated OLETF rats showed developed hypertension and proteinuria, severe glomerular injury and tubulointerstitial fibrosis, and increases in plasma BUN and creatinine levels. Treatment with LCZ696 or valsartan plus hydralazine similarly caused greater blood pressure reduction than valsartan. On the other hand, LCZ696 caused greater reductions in proteinuria, glomerular injury and tubulointerstitial fibrosis than valsartan or valsartan plus hydralazine. LCZ696 also prevented the increases in plasma BUN and creatinine levels. Conclusions: These data suggest that LCZ696 elicits a reno-protective effect in type 2 diabetes with hypertension and nephrotic-range proteinuria.

Investigation of Metabolism of Exogenous Glucose at the Early Stage and Onset of Diabetes Mellitus in Otsuka Long-Evans Tokushima Fatty Rats Using [1, 2, 3-13C]Glucose Breath Tests.

This study aimed to evaluate changes in glucose metabolism at the early stage and onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Specifically, after the oral administration of [1, 2, 3-13C]glucose, the levels of exhaled 13CO2, which most likely originated from pyruvate decarboxylation and tricarboxylic acid, were measured. Eight OLETF rats and eight control rats (Long-Evans Tokushima Otsuka [LETO]) were administered 13C-glucose. Three types of 13C-glucose breath tests were performed thrice in each period at 2-week intervals. [3-13C]glucose results in a 13C isotope at position 1 in the pyruvate molecule, which provides 13CO2. The 13C at carbons 1 and 2 of glucose is converted to 13C at carbons 2 and 1 of acetate, respectively, which produce 13CO2. Based on metabolic differences of the labeled sites, glucose metabolism was evaluated using the results of three breath tests. The increase in 13CO2 excretion in OLETF rats was delayed in all three breath tests compared to that in control rats, suggesting that OLETF rats had a lower glucose metabolism than control rats. In addition, overall glucose metabolism increased with age in both groups. The utilization of [2-13C]glucose was suppressed in OLETF rats at 6–12 weeks of age, but they showed higher [3-13C]glucose oxidation than control rats at 22–25 weeks of age. In the [1-13C]glucose breath test, no significant differences in the area under the curve until 180 minutes (AUC180) were observed between OLETF and LETO rats of any age. Glucose metabolism kinetics were different between the age groups and two groups of rats; however, these differences were not significant based on the overall AUC180 of [1-13C]glucose. We conclude that breath 13CO2 excretion is reduced in OLETF rats at the primary stage of prediabetes, indicating differences in glucose oxidation kinetics between OLETF and LETO rats.

Obesity in the Otsuka Long Evans Tokushima Fatty Rat: Mechanisms and Discoveries.

Understanding the neural systems underlying the controls of energy balance has been greatly advanced by identifying the deficits and underlying mechanisms in rodent obesity models. The current review focuses on the Otsuka Long Evans Tokushima Fatty (OLETF) rat obesity model. Since its recognition in the 1990s, significant progress has been made in identifying the causes and consequences of obesity in this model. Fundamental is a deficit in the cholecystokinin (CCK)-1 receptor gene resulting in the absence of CCK-1 receptors in both the gastrointestinal track and the brain. OLETF rats have a deficit in their ability to limit the size of meals and in contrast to CCK-1 receptor knockout mice, do not compensate for this increase in the size of their spontaneous meals, resulting in hyperphagia. Prior to becoming obese and in response to pair feeding, OLETF rats have increased expression of neuropeptide Y (NPY) in the compact region of the dorsomedial hypothalamus (DMH), and this overexpression contributes to their overall hyperphagia. Study of the OLETF rats has revealed important differences in the organization of the DMH in rats and mice and elucidated previously unappreciated roles for DMH NPY in energy balance and glucose homeostasis.

A return to ad libitum feeding following caloric restriction promotes hepatic steatosis in hyperphagic OLETF rats.

Hyperphagic Otsuka Long-Evans Tokushima fatty (OLETF) rats develop obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD), but lifestyle modifications, such as caloric restriction (CR), can prevent these conditions. We sought to determine if prior CR had protective effects on metabolic health and NAFLD development following a 4-wk return to ad libitum (AL) feeding. Four-week-old male OLETF rats (n = 8-10/group) were fed AL for 16 wk (O-AL), CR for 16 wk (O-CR; ∼70% kcal of O-AL), or CR for 12 wk followed by 4 wk of AL feeding (O-AL4wk). CR-induced benefit in prevention of NAFLD, including reduced hepatic steatosis, inflammation, and markers of Kupffer cell activation/number, was largely lost in AL4wk rats. These findings occurred in conjunction with a partial loss of CR-induced beneficial effects on obesity and serum triglycerides in O-AL4wk rats, but in the absence of changes in serum glucose or insulin. CR-induced increases in hepatic mitochondrial respiration remained significantly elevated (P < 0.01) in O-AL4wk compared with O-AL rats, while mitochondrial [1-(14)C]palmitate oxidation, citrate synthase activity, and β-hydroxyacyl-CoA dehydrogenase activity did not differ among OLETF groups. NAFLD development in O-AL4wk rats was accompanied by increases in the protein content of the de novo lipogenesis markers fatty acid synthase and stearoyl-CoA desaturase-1 and decreases in phosphorylated acetyl-CoA carboxylase (pACC)/ACC compared with O-CR rats (P < 0.05 for each). The beneficial effects of chronic CR on NAFLD development were largely lost with 4 wk of AL feeding in the hyperphagic OLETF rat, highlighting the importance of maintaining energy balance in the prevention of NAFLD.

Size effect of rebamipide ophthalmic nanodispersions on its therapeutic efficacy for corneal wound healing

In a variety of tissues including gastrointestinal mucosa, rebamipide (REB) provides cytoprotection, prevents inflammation, and promotes wound healing. Clinically, REB ophthalmic dispersions are used to treat diabetic keratopathy. In this study, we investigated the optimal particle size of REB to promote corneal wound healing using a model of diabetic keratopathy, the debrided corneal epithelium from Otsuka Long-Evans Tokushima Fatty (OLETF) rats. First, we prepared three dispersions with different REB particle sizes (REB735, REB150, REB45) by treatment with zirconia beads and Bead Smash 12 (a bead mill). The mean particle sizes of the REB735, REB150, REB45 dispersions were approximately 735 nm, 150 nm and 45 nm, respectively. Next, we measured the amounts of REB in the corneal and conjunctival tissues of rats following the instillation of the REB dispersions. The amounts of REB in the corneal and conjunctival tissues following the instillation of REB dispersions was increased by using the mill method, and the amount of REB in rats instilled with the REB150 dispersion was significantly higher than in rats instilled with the REB45 dispersion. Moreover, the corneal wound healing rate for rats instilled with the REB150 dispersion was significantly higher than for rats instilled with the REB735 or REB45 dispersions. In addition, these REB dispersions enhanced corneal epithelial cell growth, resulting an enhancement of corneal wound healing rate. Thus, we found that the ocular drug accumulation and therapeutic effect on corneal wound healing of REB dispersions is enhanced by preparing particles with a size of ca. 150 nm. These findings provide significant information that can be used to design further studies aimed at developing ophthalmic dispersions.

Aerobic exercise training in the treatment of non‐alcoholic fatty liver disease related fibrosis

Physiologically relevant rodent models of non-alcoholic steatohepatitis (NASH) that resemble the human condition are limited. Exercise training and energy restriction are first-line recommendations for the treatment of NASH. Hyperphagic Otsuka Long-Evans Tokushima fatty rats fed a western diet high in fat, sucrose and cholesterol for 24weeks developed a severe NASH with fibrosis phenotype. Moderate intensity exercise training and modest energy restriction provided some improvement in the histological features of NASH that coincided with alterations in markers of hepatic stellate cell activation and extracellular matrix remodelling. The present study highlights the importance of lifestyle modification, including exercise training and energy restriction, in the regulation of advanced liver disease. The incidence of non-alcoholic steatohepatitis (NASH) is rising but the efficacy of lifestyle modifications to improve NASH-related outcomes remain unclear. We hypothesized that a western diet (WD) would induce NASH in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat and that lifestyle modification would improve this condition. Eight-week-old Long-Evans Tokushima Otsuka (L) and OLETF (O) rats consumed a control diet (10%kcal fat, 3.5% sucrose) or a WD (45%kcal fat, 17% sucrose, 1% cholesterol) for 24weeks. At 20weeks of age, additional WD-fed OLETFs were randomized to sedentary (O-SED), food restriction (O-FR; ∼25% kcal reduction vs. O-SED) or exercise training (O-EX; treadmill running 20mmin(-1) with a 15% incline, 60minday(-1) , 5daysweek(-1) ) conditions for 12weeks. WD induced a NASH phenotype in OLETFs characterized by hepatic fibrosis (collagen 1α1 mRNA and hydroxyproline content), as well as elevated inflammation and non-alcoholic fatty liver disease activity scores, and hepatic stellate cell activation (α-smooth muscle actin) compared to Long-Evans Tokushima Otsuka rats. FR and EX modestly improved NASH-related fibrosis markers (FR: hydroxyproline content, P<0.01; EX: collagen 1α1 mRNA, P<0.05; both: fibrosis score, P<0.01) and inflammation (both: inflammation score; FR: interleukin-1β and tumor necrosis factor α) vs. O-SED. FR reduced hepatic stellate cell activation markers (transforming growth factor-β protein and α-smooth muscle actin mRNA), whereas EX increased the hepatic stellate cell senescence marker CCN1 (P<0.01vs. O-SED). Additionally, both FR and EX normalized extracellular matrix remodelling markers to levels similar to L-WD (P>0.05). Although neither EX nor FR led to complete resolution of the WD-induced NASH phenotype, both independently benefitted liver fibrosis via altered hepatic stellate cell activation and extracellular matrix remodelling.

Angiotensin Receptor Blocker ameliorates hyperlipidemia and increases lipid utilization in a rat model of metabolic syndrome

CD36 and lipoprotein lipase (LPL) are essential proteins involved in lipid mobilization and triglyceride storage in various cell types, including adipocytes. Accumulation of excess lipids in non‐adipose tissues leads to cell dysfunction, potentially cell death, and ultimately insulin resistance. Furthermore, activation of the renin‐angiotensin system (RAS) is implicated in the manifestation of metabolic syndrome, for which dyslipidemia is an associated factor. To address our hypothesis that blockade of the angiotensin II receptor (AT1) can improve lipid utilization, we measured body mass, retroperitoneal mass, adipose lipase activity, CD36 and LPL expression in five groups of rats; 1) Long‐Evans Tokushima Otsuka (LETO) normal diet (ND) (n=6), 2) Otsuka Long‐Evans Tokushima Fatty (OLETF) ND (n=8), 3) OLETF high‐fat diet (HFD, 62% fat in food) (n=8), 4) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg/d 6 wk) (n=8), and 5) OLETF HFD + ARB (n=7). Body mass increased 38% in OLETF relative to LETO; ARB decreased it by 20% relative to OLETF, and HFD+ARB decreased it by 37% relative to HFD. ARB decreased retroperitoneal fat 16% in relation to OLETF, and ARB+HFD decreased it 72% compared to HFD. CD36 decreased in response to a HFD and lipase activity increased with ARB treatment. Results suggest that AT1 activation increases lipid utilization, therefore, ameliorating the development of hyperlipidemia from a high fat diet.Support or Funding InformationUC Leadership and Excellence through Advanced Degrees (UC LEADS) United States Department of Agriculture (USDA)

Bone Geometry and Trabecular and Cortical Microarchitecture are Altered by Type 2 Diabetes, but not Insulin Resistance, in the Hyperphagic OLETF Rat

Recently, fragility fracture has been added to the list of type 2 diabetes (T2D) complications. The purpose of this study was to determine the effects of insulin resistance (IR) and T2D on bone outcomes using the hyperphagic Otsuka Long‐Evans Tokushima Fatty (OLETF) rat model of T2D. Long‐Evans Tokushima Otsuka (LETO) and calorically restricted (CR; ~30% reduction vs. OLETF) OLETF rats weight‐matched to the LETO controls were used as lean, normoglycemic controls. The skeletal effects of IR and T2D were evaluated in 20‐ and 40‐week old male OLETF rats. Femur geometry and trabecular and cortical microarchitecture were analyzed using mCT. Cortical bone strength was analyzed by torsional loading of the femur to failure. The effects of IR and T2D on bone outcomes were compared using a two‐way (group, age) ANOVA; body weight (BW) was included as a covariate for body‐size‐dependent outcomes. As expected, the hyperphagic OLETF rats had significantly greater BW than controls. Blood glucose and insulin confirmed that OLETF rats were hyperglycemic and hyperinsulinemic at 20 weeks and at 40 weeks were hypoinsulinemic due to β‐cell dysfunction. At 20 weeks, only the cortical‐to‐total area ratio (Ct.Ar/Tt.Ar) was greater in IR OLETF versus CR controls. However, at 40 weeks bone geometry, microarchitecture, and biomechanical properties differed between OLETF rats and LETO and CR controls. OLETF rats had significantly shorter femur length than LETO and CR controls. Total cross‐sectional area and cortical area were greater in T2D OLETF rats versus LETO and CR controls. Ct.Ar/Tt.Ar and polar moment of inertia were greater in OLETF compared with CR. OLETF rats showed a greater increase in trabecular spacing and greater decreases in connectivity density from 20 to 40 weeks compared with LETO and CR controls; at 40 weeks T2D OLETF rats had reduced percent bone volume. T2D OLETF rats did not show the age‐related decline in torsional stiffness (Ks) observed in controls. Relative to CR, OLETF rats had a greater decline in ultimate tensile strength (Su), and at 40 weeks OLETF rats had reduced shear modulus of elasticity (G). In summary, bone geometry, trabecular and cortical microarchitecture, and biomechanical properties are altered by T2D, but not IR, in the hyperphagic OLETF rat relative to lean controls. The results of this study suggest that early identification and treatment of T2D might reduce the skeletal complications associated with hyperglycemia and β‐cell dysfunction.Support or Funding InformationThis work was funded by the VA Career Development Award and the Department of Nutrition and Exercise Physiology at the University of Missouri.

Ipragliflozin, a sodium-glucose cotransporter 2 inhibitor, ameliorates the development of liver fibrosis in diabetic Otsuka Long-Evans Tokushima fatty rats.

It is widely understood that insulin resistance (IR) critically correlates with the development of liver fibrosis in several types of chronic liver injuries. Several experiments have proved that anti-IR treatment can alleviate liver fibrosis. Sodium-glucose cotransporter 2 (SGLT2) inhibitors comprise a new class of antidiabetic agents that inhibit glucose reabsorption in the renal proximal tubules, improving IR. The aim of this study was to elucidate the effect of an SGLT2 inhibitor on the development of liver fibrosis using obese diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and their littermate nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. Male OLETF and LETO rats were intraperitoneally injected with porcine serum twice a week for 12weeks to augment liver fibrogenesis. Different concentrations of ipragliflozin (3 and 6mg/kg) were orally administered during the experimental period. Serological and histological data were examined at the end of the experimental period. The direct effect of ipragliflozin on the proliferation of a human hepatic stellate cell (HSC) line, LX-2, was also evaluated in vitro. OLETF rats, but not LETO rats, received 12weeks of porcine serum injection to induce severe fibrosis. Treatment with ipragliflozin markedly attenuated the development of liver fibrosis and expression of hepatic fibrosis markers, such as alpha smooth muscle actin, collagen 1A1, and transforming growth factor beta (TGF-β), and improved IR in a dose-dependent manner in OLETF rats. In contrast, the proliferation of LX-2 in vitro was not affected, suggesting that ipragliflozin had no significant direct effect on the proliferation of HSCs. In conclusion, our dataset suggests that an SGLT2 inhibitor could alleviate the development of liver fibrosis by improving IR in naturally diabetic rats. This may provide the basis for creating new therapeutic strategies for chronic liver injuries with IR.

The decreased expression of electron transfer flavoprotein β is associated with tubular cell apoptosis in diabetic nephropathy.

Tubular injury is closely correlated with the development of progressive diabetic nephropathy (DN), particularly in cases of type 2 diabetes. The apoptosis of tubular cells has been recognized as a major cause of tubular atrophy, followed by tubulointerstitial fibrosis. Electron transfer flavoprotein β (ETFβ) is known as an important electron acceptor in energy metabolism, but its role in DN was not fully understood. In the present study, we examined the expression pattern of ETFβ using diabetic kidney samples and further investigated ETFβ involvement in tubular epithelial cell (TEC) apoptosis. Human renal biopsy specimens from patients with DN as well as a spontaneous rat model of diabetes using Otsuka Long-Evans Tokushima fatty (OLETF) rats, were employed in order to examine the expression of ETFβ and cell apoptosis in kidneys during the development of DN (for the rats, at 36 and 56 weeks of age respectively). Moreover, ETFβ siRNA was used to investigate the role of ETFβ in the apoptosis of renal tubular cells. Our present results showed that the expression of ETFβ in the kidneys was progressively decreased both in patients with DN and OLETF rats, which coincided with progressive renal injury and TEC apoptosis. In addition, the in vitro study demonstrated that knockdown of ETFβ caused apoptosis in tubular cells, as proven by the increased expression of pro-apoptotic proteins and TUNEL assay. Therefore, the findings of our present study suggest that ETFβ plays an important role in renal tubular cell apoptosis during the progression of DN.

Effects of candesartan cilexetil and amlodipine orotate on receptor for advanced glycation end products expression in the aortic wall of Otsuka Long-Evans Tokushima Fatty (OETFF) type 2 diabetic rats.

The receptor for advanced glycation end products (RAGE) plays a key role in the development of vascular inflammation and acceleration of atherosclerosis in type 2 diabetes. We investigated the effect of candesartan cilexetil (CDRT) and amlodipine orotate (AMDP) on the expression of RAGE in the aortic walls of Otsuka Long-Evans Tokushima Fatty (OLETF) rats and AGE-treated endothelial cells. Twenty five-week-old OLETF rats were randomized to 8week treatments consisting of CDRT (n=8), AMDP (n=8) or saline (control, n=8). Immunohistochemical and dihydroethidine staining revealed reduced RAGE and reactive oxygen species (ROS) signals in rats treated with CDRT or AMDP compared with control rats. Both CDRT and AMDP suppressed the expression of p22phox and p47phox NADPH oxidase subunits. However, only CDRT significantly reduced expression of phosphorylated extracellular signal regulated kinase (pERK)1/2 in the aortic wall of OLETF rats. In addition, both drugs reduced RAGE expression and total and mitochondrial ROS production in the AGE-treated endothelial cells. Both ARBs and CCBs reduced RAGE expression in the aortic walls of OLETF rats, which was attributed to decreased ROS production through inhibition of NADPH oxidase. In addition, only CDRT reduced aortic expression of RAGE via suppression of the ERK1/2 pathway unlike AMDP.

Diabetes and Age-Related Differences in Vascular Function of Renal Artery: Possible Involvement of Endoplasmic Reticulum Stress.

To study the time-course relationship between vascular functions and endoplasmic reticulum (ER) stress in type 2 diabetes, we investigated vascular function and associated protein expression, including cyclo-oxygenase (COX), ER stress, and apoptotic markers, in renal arteries (RA) from type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats at the young adult (4 months old) and aged (18 months old) stages. In the RA of aged OLETF (vs. young OLETF), we found: (1) Increased contractions induced by uridine adenosine tetraphosphate (Up4A) and phenylephrine, (2) decreased relaxation and increased contraction induced by acetylcholine (ACh) at lower and higher concentrations, respectively, and (3) increased expression of COX-1 and C/EBP-homologous protein (CHOP, a pro-apoptotic protein). In aged rats, the expression of COX-1, COX-2, PDI (an ER protein disulfide isomerase), Bax (a proapoptotic marker), and CHOP were increased in RA from OLETF rats (vs. age-matched control Long-Evans Tokushima Otsuka [LETO] rats). Up-regulation of PDI and Bax were seen in the RA from young OLETF (vs. young LETO) rats. No age-related alterations were apparent in the above changes in RA from LETO rats, excluding ACh-induced contraction. Short-term treatment with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 100 mg/kg per day, intraperitoneally for 1 week) to OLETF rats at the chronic stage of the disease (12 months old) could suppress renal arterial contractions induced by Up4A and ACh. These results suggest that a long-term duration of disease may be important for the development of vascular dysfunction rather than aging per se. The early regulation of ER stress may be important against the development of diabetes-associated vascular dysfunction.

Long-term effects of duodenojejunal bypass on diabetes in Otsuka Long–Evans Tokushima Fatty rats

Previous studies have shown that duodenojejunal bypass (DJB) resolves type 2 diabetes. However, this finding has been contradicted by several experimental and human trials and therefore needs to be clarified. Otsuka Long-Evans Tokushima Fatty (OLETF) rats randomly underwent a sham operation or DJB. Thereafter, we measured daily body weight, serum levels of glucose and gut hormones such as glucagon-like peptide-1, insulin, and leptin. There was no significant difference in weight loss between rats in the DJB andsham-operated groups. There were also no differences in the area under the curve of glucose tolerance between the DJB and sham-operated groups (32466±2261mg/dL·min vs. 26319±427mg/dL·min; p=0.35). Duodenojejunal bypass did not affect plasma concentrations of various gut hormones such as glucagon-like peptide-1, insulin, and leptin. We have shown that DJB alone does not improve glucose tolerance in obese, diabetic OLETF rats. Therefore, it may be that DJB alone is insufficient for diabetic control in obese diabetic rats. The addition of a restrictive component such as sleeve gastrectomy, or a new drug may be necessary for achieving diabetes reversal.

The Effects of High Fat Diet and Resveratrol on Mitochondrial Activity of Brown Adipocytes

BackgroundResveratrol (RSV) is a polyphenolic phytoalexin that has many effects on metabolic diseases such as diabetes and obesity. Given the importance of brown adipose tissue (BAT) for energy expenditure, we investigated the effects of RSV on brown adipocytes.MethodsFor the in vitro study, interscapular BAT was isolated from 7-week-old male Sprague Dawley rats. For the in vivo study, 7-week-old male Otsuka Long Evans Tokushima Fatty (OLETF) rats were divided into four groups and treated for 27 weeks with: standard diet (SD); SD+RSV (10 mg/kg body weight, daily); high fat diet (HFD); HFD+RSV. RSV was provided via oral gavage once daily during the in vivo experiments.ResultsRSV treatment of primary cultured brown preadipocytes promoted mitochondrial activity, along with over-expression of estrogen receptor α (ER-α). In OLETF rats, both HFD and RSV treatment increased the weight of BAT and the differentiation of BAT. However, only RSV increased the mitochondrial activity and ER-α expression of BAT in the HFD-fed group. Finally, RSV improved the insulin sensitivity of OLETF rats by increasing the mitochondrial activity of BAT, despite having no effects on white adipocytes and muscles in either diet group.ConclusionRSV could improve insulin resistance, which might be associated with mitochondrial activity of brown adipocyte. Further studies evaluating the activity of RSV for both the differentiation and mitochondrial activity of BAT could be helpful in investigating the effects of RSV on metabolic parameters.

Biochemical and Hormones Study on Diabetic Nephrotic Patients

Background: Diabetes mellitus is a growing epidemic and is the most common cause of chronic kidney disease (CKD) and kidney failure. Diabetic nephropathy affects approximately 20–40% of individuals who have diabetes. Diabetic nephropathy can be detected by the measurement of urine albumin or serum creatinine. Visceral adipose tissue-derived serine proteinase inhibitor (vaspin) was identified in the visceral adipose tissue of OLETF (Otsuka Long-Evans Tokushim a Fatty) rats, an animal model of obesity and type 2 diabetes mellitus (T2DM). Material and methods: In this study conducted on 70 individuals in the age group of 35-70 years, from Kirkuk General Hospital. study group consists 40 individuals with diabetic nephropathy and 30 ages and sex matched healthy individuals (control). Reasults: Serum vaspin of diabetic nephropathy patients showed a high significant relationship (p<0.0001). as compared with the controls. There is high significant decrease (p<0.001) in the serum levels of T4 and T3. There is high significant increase (p<0.001) in the serum levels of TSH as compared with the controls. There is high significant increase (p<0.001) in the serum concentration of K+ and uric acid and glucose as compared with the controls. There is high significant decrease (p<0.001) in the serum levels of Ca++ and Na+ as compared with the controls. There is high significant increase (p<0.001) in the serum levels of Cholesterol, TG, LDL and VLDL as compared with the controls. There is significant decrease (p<0.05) in the serum levels of HDL as compared with the controls. Conclusion: The hormones (vaspin, TSH, T4, T3) have higher diagnostic validity values in the current study, which may be useful as a diagnostic tool to identify recurrence of the diabetic nephropathy syndromes.

Acupuncture Alters Expression of Insulin Signaling Related Molecules and Improves Insulin Resistance in OLETF Rats

To determine effect of acupuncture on insulin resistance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to evaluate expression of insulin signaling components. Rats were divided into three groups: Sprague-Dawley (SD) rats, OLETF rats, and acupuncture+OLETF rats. Acupuncture was subcutaneously applied to Neiguan (PC6), Zusanli (ST36), and Sanyinjiao (SP6); in contrast, acupuncture to Shenshu (BL23) was administered perpendicularly. For Neiguan (PC6) and Zusanli (ST36), needles were connected to an electroacupuncture (EA) apparatus. Fasting blood glucose (FPG) was measured by glucose oxidase method. Plasma fasting insulin (FINS) and serum C peptide (C-P) were determined by ELISA. Protein and mRNA expressions of insulin signaling molecules were determined by Western blot and real-time RT-PCR, respectively. OLETF rats exhibit increased levels of FPG, FINS, C-P, and homeostasis model assessment-estimated insulin resistance (HOMA-IR), which were effectively decreased by acupuncture treatment. mRNA expressions of several insulin signaling related molecules IRS1, IRS2, Akt2, aPKCζ, and GLUT4 were decreased in OLETF rats compared to SD controls. Expression of these molecules was restored back to normal levels upon acupuncture administration. PI3K-p85α was increased in OLETF rats; this increase was also reversed by acupuncture treatment. Acupuncture improves insulin resistance in OLETF rats, possibly via regulating expression of key insulin signaling related molecules.