Obese Otsuka Long-Evans Tokushima Fatty Rats Research Articles

Dietary β-Conglycinin Modulates Insulin Sensitivity, Body Fat Mass, and Lipid Metabolism in Obese Otsuka Long-Evans Tokushima Fatty (OLETF) Rats.

The physiological effects of dietary β-conglycinin (β-CON), one of the major components of soy protein (SOY), were examined in an obese animal model. Prior studies show that β-CON intake decreases plasma triglycerides and visceral adipose tissue weight, and increases plasma adiponectin in rodents. Since plasma adiponectin is known to affect both lipid and glucose metabolism, feeding a diet containing β-CON could modulate insulin sensitivity. Therefore, we examined the effects of dietary β-CON on insulin sensitivity and blood glucose levels, as well as lipid metabolism in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats (pre-symptomatic stage of type 2 diabetes mellitus). Male OLETF rats (6 weeks old) were fed diets containing 20% protein such as casein (CAS), CAS replaced with soy protein (SOY), or β-CON at a proportion of 50% for 13 weeks. Fasting blood glucose levels were measured every 3 weeks, and an insulin tolerance test (ITT; 0.75 IU/kg body weight) was conducted at week 12. During the feeding period, fasting blood glucose was comparable among the groups. Insulin sensitivity measured by the ITT revealed that the SOY and β-CON diets decreased blood glucose levels at 30 min after intraperitoneal insulin injection (vs. CAS diet). In addition, the β-CON diet increased plasma adiponectin concentrations, hepatic gene expression of insulin receptor substrate (IRS) 2, and muscle gene expression of adiponectin receptor 1 (AdipoR1) and IRS1, and with a decrease in plasma insulin concentration. Finally, the β-CON diet decreased the mesenteric adipose tissue weight and liver triglyceride concentration compared to the CAS diet. These results suggest that the metabolic effects of dietary β-CON are mediated by increasing plasma adiponectin to increase insulin sensitivity and influence the hepatic lipid metabolism in obese OLETF rats.

AT1 Receptor Blockade Improves Mitochondrial Activity in Insulin Resistant but not Type II Diabetic Hearts

Hyperglycemia increases the risk of oxidant overproduction in the heart through activation of a multitude of pathways. Oxidation of mitochondrial enzymes may impair their function resulting in accumulation of intermediates and reverse electron transfer, contributing to mitochondrial dysfunction. Furthermore, the renin‐angiotensin system (RAS) becomes inappropriately activated during metabolic syndrome, increasing oxidant production through NAPDH assemblage. To test the acute effects of an oral glucose tolerance test (oGTT) on the insulin resistant and type II diabetic hearts, 16 and 25 week old animals were given a glucose tolerance test prior to dissection. Adult male rats were randomly assigned to three groups prior to dissection (n= 5–8 animals/group/time point): 1) Long Evans Tokushima Otsuka (LETO; lean strain‐control), 2) insulin resistant, obese Otsuka Long Evans Tokushima Fatty (OLETF), and 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d x 6–8 weeks). Hearts were collected at T0, T60, and T120 minutes post‐glucose infusion and at T0, T180, and T360 in 25‐week‐old animals. Aconitase activity was not changed between LETO and OLETF rats at T0, but was increased five‐fold with ARB treatment. At T60 aconitase activity was decreased 69% in OLETF from LETO, but ARB treatment increased activity six‐fold. AT 25 weeks of age aconitase activity was two‐fold higher in OLETF compared to LETO and remained unchanged through T360. ARB treatment lowered this activity to LETO levels at T0 only. Complex I activity had no detectable difference between LETO and OLETF in 16 week old animals; however, ARB increased activity by 50%. However, in 25 week old animals complex I was lowered 32% from LETO and ARB treatment had no effect on complex I activity. At T60, complex II activity was decreased 30% from LETO in OLETF rats, and ARB treatment increased complex II activity by 79%. In 25 week old animals OLETF rats had increased complex II activity at T180 by 34%, which then was restored to baseline by T360. ARB treatment increased activity by 26%, but maintained this increase over the 6 hour span. These data demonstrate the protective effects of AT1 blockade on cardiac mitochondrial function during the manifestation of insulin resistance, but demonstrate that its protective effects diminish as animals transition into a frank diabetic stage.Support or Funding InformationM. Thorwald and R. Rodriguez were supported by NIH NCMHD9T37MD001480. R.M. Ortiz was partially supported by NIH NHLBIK02HL103787. Research was partially funded by NIH NHLBIR01HL091767.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

UDP-induced relaxation is enhanced in aorta from female obese Otsuka Long-Evans Tokushima Fatty rats.

Uridine 5'-diphosphate (UDP) plays an important role in controlling vascular tone; however, UDP-mediated response in metabolic syndromes, including obesity and type 2 diabetes in females, remains unclear. In this study, we investigated UDP-mediated response in the aorta of female obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats and control Long-Evans Tokushima Otsuka (LETO) rats. In OLETF rat aortas precontracted by phenylephrine (PE) (vs. LETO), (1) UDP-induced relaxation was increased, whereas acetylcholine (ACh)-induced relaxation was decreased; (2) no UDP- or ACh-induced relaxations were observed in endothelial denudation, whereas UDP-induced small contraction was observed; and (3) NG-nitro-L-arginine [L-NNA, a nitric oxide (NO) synthase inhibitor] eliminated UDP-induced relaxation and small contraction, whereas caused contrasting responses by ACh, including slight relaxations (LETO) and contractions (OLETF). Indomethacin, a cyclooxygenase inhibitor, eliminated the difference in UDP- and ACh-induced relaxations between the groups by increased UDP-induced relaxation in the LETO group and increased ACh-induced relaxation in the OLETF group. MRS2578, a P2Y6 receptor antagonist, eliminated the difference in UDP-induced relaxations between the groups by decreasing UDP-induced relaxation in the OLETF group. MRS2578 had no effect on UDP-induced contraction in endothelium-denuded aortas. Therefore, these findings demonstrate opposite trends of relaxations by UDP and ACh in OLETF and LETO rat aortas. These differences may be attributed to the imbalance between NO and vasoconstrictor prostanoids upon stimulations. Increased UDP-induced relaxation in OLETF rat aorta may be caused by the activation of endothelial MRS2578-sensitive P2Y6 receptor.

Soy compared with milk protein in a Western diet changes fecal microbiota and decreases hepatic steatosis in obese OLETF rats

Soy protein is effective at preventing hepatic steatosis; however, the mechanisms are poorly understood. We tested the hypothesis that soy vs. dairy protein-based diet would alter microbiota and attenuate hepatic steatosis in hyperphagic Otsuka Long-Evans Tokushima fatty (OLETF) rats. Male OLETF rats were randomized to “Western” diets containing milk protein isolate (MPI), soy protein isolate (SPI) or 50:50 MPI/SPI (MS) (n=9–10/group; 21% kcal protein) for 16 weeks. SPI attenuated (P<.05) fat mass and percent fat by ~10% compared with MS, but not compared with MPI. Serum thiobarbituric acid reactive substance and total and low-density lipoprotein cholesterol concentrations were lower (P<.05) with dietary SPI vs. MPI and MS. Histological hepatic steatosis was lower (P<.05) in SPI compared with MPI or MS. Lipidomic analyses revealed reductions (P<.05) in hepatic diacylglycerols but not triacylglycerols in SPI compared with MPI, which was associated with lower hepatic de novo lipogenesis (ACC, FAS and SCD-1 protein content, and hepatic 16:1 n-7 and 18:1 n-7 PUFA concentrations) (P<.05) compared with MPI and MS; however, MPI displayed elevated hepatic mitochondrial function compared with SPI and MS. Fecal bacterial 16S rRNA analysis revealed SPI-intake elicited increases (P<.05) in Lactobacillus and decreases (P<.05) in Blautia and Lachnospiraceae suggesting decreases in fecal secondary bile acids in SPI rats. SPI and MS exhibited greater (P<.05) hepatic Fxr, Fgfr4, Hnf4a, HmgCoA reductase and synthase mRNA expression compared with MPI. Overall, dietary SPI compared with MPI decreased hepatic steatosis and diacylglycerols, changed microbiota populations and altered bile acid signaling and cholesterol homeostasis in a rodent model of obesity.

Abstract 257: Activation of the Renin-Angiotensin II-Aldosterone-System Leads to Increase in Protein Disulfide Isomerase

Activation of the Renin-Angiotensin-Aldosterone-System (RAAS) is proposed to play a role in the development of insulin resistance and type II diabetes. Angiotensin II (AngII) is a principal effector molecule that binds AT1 receptors (AT1R) in various tissues. Recent data shows that activated endothelial cells secrete protein disulfide isomerase (PDI), a multifunctional protein that has been shown to be critical to the initiation and regulation of thrombus formation. However, the effects of RAAS on PDI regulation are unknown. We hypothesized that RAAS activation would lead to increased PDI levels thus contributing to inflammation. First, we studied the in vitro effects of AngII on EA.hy926 human endothelial cells and measured PDI activity. Our results show that AngII increased PDI activity; an event that was blocked by preincubation with 0.5 M losartan, an AT1R antagonist (ARB) (P&lt;0.05, n=6). We then studied the in vivo effects of exogenous AngII infusion in Sprague–Dawley rats under the following conditions: (1) control; (2) AngII infused; (3) AngII + ARB. In these rats, AngII infusion led to significant increases in plasma PDI levels that were partially prevented by ARB treatment (P&lt;0.05, n&gt;5). We then studied the obese Otsuka Long Evans Tokushima Fatty (OLETF) (n = 6/group) rats, a model of naturally increased AngII and RAAS mediated insulin resistance and hypertension. OLETF and their lean strain controls were randomly assigned to the following groups: (1) untreated Long Evans Tokushima Otsuka (lean, control); (2) untreated OLETF; (3) OLETF + ARB. Our results show that OLETF rats had increased insulin resistance and significantly greater circulating PDI activity than control rats (P&lt;0.05) that was likewise blocked by ARB treatment (P&lt;0.05). To assess the relevance to humans of these findings we measured circulating PDI levels in patients with type 2 diabetes. In our cohort, PDI activity was significantly greater in patients with type 2 diabetes than non diabetics (P&lt;0.001, n=134). Our data suggest that RAAS activation represents a novel mechanism for PDI secretion. Thus we posit that PDI may contribute to the deleterious effects of RAAS-mediated vascular disease.

Exercise improves femoral whole-bone and tissue-level biomechanical properties in hyperphagic OLETF rats.

An often-overlooked comorbidity of type 2 diabetes (T2D) is increased fracture risk. Since traditional T2D therapies focus solely on glucose homeostasis, there is an increased need for effective treatment strategies that improve both metabolic and bone health. The current study evaluated if previously reported protective effects of exercise (EX) on cortical bone geometry and biomechanical properties are due to the prevention of obesity compared with obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats or the unique skeletal effects of exercise. Four-week-old male OLETF rats were randomly assigned to 1 of 3 groups, each fed a standard rodent chow diet until 40 weeks of age: ad libitum-fed sedentary (O-SED), ad libitum-fed EX (O-EX), or a control group body-weight-matched to the O-EX group by caloric restriction (O-CR). Ad libitum-fed sedentary Long-Evans Tokushima Otsuka (L-SED) rats were used as a lean control. EX or CR prevented the excess body mass accumulation and elevated HbA1c observed in O-SED. Total-body bone mineral density was greater in O-EX than O-CR, but similar to L-SED and O-SED. O-EX and O-CR had lower osteocalcin and TRAP5b than O-SED and L-SED. EX or CR prevented the ∼3-fold increase in CTx in O-SED versus L-SED. EX increased femoral cortical mass accumulation and expansion at the mid-diaphysis compared with O-CR. EX or CR significantly increased tissue-level stiffness and strength compared with O-SED and L-SED, but O-EX had greater whole-bone stiffness than all other groups. In summary, EX has distinct advantages over CR for improving bone biomechanical properties in hyperphagic OLETF rats.

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.

A sodium-glucose co-transporter 2 inhibitor empagliflozin prevents abnormality of circadian rhythm of blood pressure in salt-treated obese rats.

Studies were performed to examine the effects of the selective sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin on urinary sodium excretion and circadian blood pressure in salt-treated obese Otsuka Long Evans Tokushima Fatty (OLETF) rats. Fifteen-week-old obese OLETF rats were treated with 1% NaCl (in drinking water), and vehicle (0.5% carboxymethylcellulose, n=10) or empagliflozin (10 mg kg(-1)per day, p.o., n=11) for 5 weeks. Blood pressure was continuously measured by telemetry system. Glucose metabolism and urinary sodium excretion were evaluated by oral glucose tolerance test and high salt challenge test, respectively. Vehicle-treated OLETF rats developed non-dipper type blood pressure elevation with glucose intolerance and insulin resistance. Compared with vehicle-treated animals, empagliflozin-treated OLETF rats showed an approximately 1000-fold increase in urinary glucose excretion and improved glucose metabolism and insulin resistance. Furthermore, empagliflozin prevented the development of blood pressure elevation with normalization of its circadian rhythm to a dipper profile, which was associated with increased urinary sodium excretion. These data suggest that empagliflozin elicits beneficial effects on both glucose homeostasis and hypertension in salt-replete obese states.

Chronic NOS inhibition accelerates NAFLD progression in an obese rat model.

The progression in nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis is a serious health concern, but the underlying mechanisms remain unclear. We hypothesized that chronic inhibition of nitric oxide (NO) synthase (NOS) via N(ω)-nitro-L-arginine methyl ester (L-NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and NAFLD. Obese Otsuka Long-Evans Tokushima fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats received control or L-NAME (65-70 mg·kg(-1)·day(-1))-containing drinking water for 4 wk. L-NAME treatment significantly (P < 0.05) reduced serum NO metabolites and food intake in both groups. Remarkably, despite no increase in body weight, L-NAME treatment increased hepatic triacylglycerol content (+40%, P < 0.05) vs. control OLETF rats. This increase was associated with impaired (P < 0.05) hepatic mitochondrial state 3 respiration. Interestingly, the opposite effect was found in LETO rats, where L-NAME increased (P < 0.05) hepatic mitochondrial state 3 respiration. In addition, L-NAME induced a shift toward proinflammatory M1 macrophage polarity, as indicated by elevated hepatic CD11c (P < 0.05) and IL-1β (P = 0.07) mRNA in OLETF rats and reduced expression of the anti-inflammatory M2 markers CD163 and CD206 (P < 0.05) in LETO rats. Markers of total macrophage content (CD68 and F4/80) mRNA were unaffected by L-NAME in either group. In conclusion, systemic NOS inhibition in the obese OLETF rats reduced hepatic mitochondrial respiration, increased hepatic triacylglycerol accumulation, and increased hepatic inflammation. These findings suggest an important role for proper NO metabolism in the hepatic adaptation to obesity.

Nitric oxide synthase inhibition exacerbates liver injury in an obese rat model of NAFLD (1115.3)

We tested the hypothesis that chronic disruption of constitutive nitric oxide (NO) production via the non‐selective NO synthase (NOS) inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) would intensify liver injury in a rat model of obesity, insulin resistance, and nonalcoholic fatty liver disease (NAFLD). Obese Otsuka Long‐Evans Tokushima Fatty (OLETF) and lean Long‐Evans Tokushima Otsuka (LETO) rats (16‐wks) received either control or L‐NAME (65‐70 mg/kg/d) containing drinking water (n=10/group) for 4‐wks. L‐NAME treated OLETF and LETO rats had significantly (p &lt; 0.05) reduced serum NO metabolites (NOX) and food intake. Despite reducing % body fat (‐2.5%; p &lt; 0.05) in OLETF rats, L‐NAME treatment increased hepatic triacylglycerol (TAG) content (+40%; p &lt; 0.05) compared to control OLETF rats. In addition, L‐NAME treatment caused a shift toward M1 macrophage polarity as indicated by elevated CD11c (p&lt;0.05) and IL‐1β (p=0.07) mRNA in the OLETF rats. Markers of total and resident macrophages (CD68 and CD163 mRNA, respectively), fibrosis (TGFβ mRNA), and cell adhesion (MCP‐1 and ICAM mRNA) were unaffected by L‐NAME treatment in both groups. In conclusion, chronic NOS inhibition appears to aggravate liver injury by increasing expression of macrophage activation‐related genes associated with insulin resistance and by accelerating hepatic TAG accumulation in obese, insulin resistant rats.Grant Funding Source: Supported by a Life Sciences Fellowship (RDS), NIH HL‐36088 (MHL), and VHA‐CDA2 BX001299‐02 (RSR)

Transcriptome‐wide RNA sequencing analysis of rat skeletal muscle feed arteries (1156.12)

We employed next generation RNA sequencing (RNA‐Seq) technology to determine the influence of obesity on global gene expression in skeletal muscle feed arteries. Transcriptional profiles of the gasctrocnemius and soleus muscle feed arteries (GFA and SFA, respectively) and aortic endothelial cell‐enriched samples from obese Otsuka Long Evans Tokushima Fatty (OLETF) and lean Long Evans Tokushima Otsuka (LETO) rats were examined. Obesity produced 282 upregulated and 133 downregulated genes in SFA and 163 upregulated and 77 downregulated genes in GFA (false discovery rate &lt; 10%) with an overlap of 93 genes between the arteries. Within LETO, there were 89 upregulated and 114 downregulated genes in the GFA compared to the SFA. There were 244 upregulated and 275 downregulated within OLETF (FDR &lt; 10%) in the GFA compared to the SFA, with an overlap of 76 differentially expressed genes common to both LETO and OLETF in both the GFA and SFA. A total of 396 transcripts were found to be differentially expressed between LETO and OLETF in aortic endothelial cell‐enriched samples. Overall, we found (i) the existence of heterogeneity in the transcriptional profile of the SFA and GFA within healthy LETO rats, (ii) that this between‐vessel heterogeneity was markedly exacerbated within the hyperphagic, obese OLETF rat, and (iii) a greater number of genes whose expression was altered by obesity in the SFA compared to the GFA. Also, results indicate that in OLETF rats, the GFA takes on a relatively more pro‐atherogenic phenotype compared to the SFA.Grant Funding Source: Supported by NIH RO1HL036088, T32‐AR048523, VHA‐CDA2 1299‐02

Reduced hepatic eNOS phosphorylation is associated with NAFLD and type 2 diabetes progression and is prevented by daily exercise in hyperphagic OLETF rats

We tested the hypothesis that nonalcoholic fatty liver disease (NAFLD) is associated with reduced hepatic endothelial nitric oxide synthase (eNOS) activation status via S1177 phosphorylation (p-eNOS) and is prevented by daily voluntary wheel running (VWR). Hyperphagic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an established model of obesity, type 2 diabetes (T2D) and NAFLD, and normophagic controls [Long-Evans Tokushima Otsuka (LETO)] were studied at 8, 20, and 40 wk of age. Basal hepatic eNOS phosphorylation (p-eNOS/eNOS) was similar between LETO and OLETFs with early hepatic steatosis (8 wk of age) and advanced steatosis, hyperinsulinemia, and hyperglycemia (20 wk of age). In contrast, hepatic p-eNOS/eNOS was significantly lower (P < 0.05) in OLETF rats with T2D advancement and the transition to more advanced NAFLD with inflammation and fibrosis [increased tumor necrosis factor-α (TNF-α), CD68, and CD163 mRNA expression; 40 wk of age]. Reduced hepatic eNOS activation status in 40-wk OLETF rats was significantly correlated with reduced p-Akt/Akt (r = 0.73, P < 0.05), reduced serum insulin (r = 0.59, P < 0.05), and elevated serum glucose (r = -0.78, P < 0.05), suggesting a link between impaired glycemic control and altered hepatic nitric oxide metabolism. VWR by OLETF rats, in conjunction with NAFLD and T2D prevention, normalized p-eNOS/eNOS and p-Akt/Akt to LETO levels. Basal activation of hepatic eNOS and Akt are maintained until advanced NAFLD and T2D development in obese OLETF rats. The prevention of this reduction by VWR may result from maintained insulin sensitivity and glycemic control.

Adipose tissue and vascular phenotypic modulation by voluntary physical activity and dietary restriction in obese insulin-resistant OLETF rats

Adipose tissue (AT)-derived cytokines are proposed to contribute to obesity-associated vascular insulin resistance. We tested the hypothesis that voluntary physical activity and diet restriction-induced maintenance of body weight would both result in decreased AT inflammation and concomitant improvements in insulin-stimulated vascular relaxation in the hyperphagic, obese Otsuka Long-Evans Tokushima fatty (OLETF) rat. Rats (aged 12 wk) were randomly assigned to sedentary (SED; n = 10), wheel running (WR; n = 10), or diet restriction (DR; n = 10; fed 70% of SED) for 8 wk. WR and DR rats exhibited markedly lower adiposity (7.1 ± 0.4 and 15.7 ± 1.1% body fat, respectively) relative to SED (27 ± 1.2% body fat), as well as improved blood lipid profiles and systemic markers of insulin resistance. Reduced adiposity in both WR and DR was associated with decreased AT mRNA expression of inflammatory genes (e.g., MCP-1, TNF-α, and IL-6) and markers of immune cell infiltration (e.g., CD8, CD11c, and F4/80). The extent of these effects were most pronounced in visceral AT compared with subcutaneous and periaortic AT. Markers of inflammation in brown AT were upregulated with WR but not DR. In periaortic AT, WR- and DR-induced reductions in expression and secretion of cytokines were accompanied with a more atheroprotective gene expression profile in the adjacent aortic wall. WR, but not DR, resulted in greater insulin-stimulated relaxation in the aorta; an effect that was, in part, mediated by a decrease in insulin-induced endothelin-1 activation in WR aorta. Collectively, we show in OLETF rats that lower adiposity leads to less AT and aortic inflammation, as well as an exercise-specific improvement in insulin-stimulated vasorelaxation.

Transcriptome-wide RNA sequencing analysis of rat skeletal muscle feed arteries. I. Impact of obesity.

We employed next-generation RNA sequencing (RNA-Seq) technology to determine the influence of obesity on global gene expression in skeletal muscle feed arteries. Transcriptional profiles of the gastrocnemius and soleus muscle feed arteries (GFA and SFA, respectively) and aortic endothelial cell-enriched samples from obese Otsuka Long-Evans Tokushima Fatty (OLETF) and lean Long-Evans Tokushima Otsuka (LETO) rats were examined. Obesity produced 282 upregulated and 133 downregulated genes in SFA and 163 upregulated and 77 downregulated genes in GFA [false discovery rate (FDR) < 10%] with an overlap of 93 genes between the arteries. In LETO rats, there were 89 upregulated and 114 downregulated genes in the GFA compared with the SFA. There were 244 upregulated and 275 downregulated genes in OLETF rats (FDR < 10%) in the GFA compared with the SFA, with an overlap of 76 differentially expressed genes common to both LETO and OLETF rats in both the GFA and SFA. A total of 396 transcripts were found to be differentially expressed between LETO and OLETF in aortic endothelial cell-enriched samples. Overall, we found 1) the existence of heterogeneity in the transcriptional profile of the SFA and GFA within healthy LETO rats, 2) that this between-vessel heterogeneity was markedly exacerbated in the hyperphagic, obese OLETF rat, and 3) a greater number of genes whose expression was altered by obesity in the SFA compared with the GFA. Also, results indicate that in OLETF rats the GFA takes on a relatively more proatherogenic phenotype compared with the SFA.

Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats

Here, we sought to compare the efficacy of combining exercise and metformin for the treatment of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) in hyperphagic, obese, type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (age: 20 wk, hyperglycemic and hyperinsulinemic; n = 10/group) were randomly assigned to sedentary (O-SED), SED plus metformin (O-SED + M; 300 mg·kg(-1)·day(-1)), moderate-intensity exercise training (O-EndEx; 20 m/min, 60 min/day, 5 days/wk treadmill running), or O-EndEx + M groups for 12 wk. Long-Evans Tokushima Otsuka (L-SED) rats served as nonhyperphagic controls. O-SED + M, O-EndEx, and O-EndEx + M were effective in the management of type 2 diabetes, and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, O-EndEx lowered liver triglyceride content and fasting hyperglycemia more than O-SED + M. In addition, exercise elicited greater improvements compared with metformin alone on postchallenge glycemic control, liver diacylglycerol content, hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activities and in the attenuation of markers of hepatic fatty acid uptake and de novo fatty acid synthesis. Surprisingly, combining metformin and aerobic exercise training offered little added benefit to these outcomes, and in fact, metformin actually blunted exercise-induced increases in complete mitochondrial palmitate oxidation and β-HAD activity. In conclusion, aerobic exercise training was more effective than metformin administration in the management of type 2 diabetes and NAFLD outcomes in obese hyperphagic OLETF rats. Combining therapies offered little additional benefit beyond exercise alone, and findings suggest that metformin potentially impairs exercise-induced hepatic mitochondrial adaptations.

Metformin does not Enhance Insulin‐Stimulated Vasodilation in Skeletal Muscle Resistance Arteries of the OLETF Rat

To test the hypothesis that chronic metformin treatment enhances insulin-induced vasodilation in skeletal muscle resistance arteries and arterioles. We assessed the effect of metformin treatment (from 20 to 32weeks of age) of obese Otsuka Long Evans Tokushima Fatty (OLETF) rats and lean LETO rats (300mg/kg) on insulin-stimulated vasodilation in isolated skeletal muscle feed arteries and arterioles. Metformin treatment significantly lowered food intake, body weight, percent body fat, and HbA1c in OLETF rats. Metformin resulted in a ~30% reduction in insulin-induced vasodilation of soleus feed arteries (SFA) from OLETF rats. Inhibition of endothelin-1 (ET-1) signaling produced 20% dilation and eliminated the difference between metformin-treated and untreated OLETF rats in insulin-induced dilation of SFA. In contrast to the SFA, metformin did not alter insulin-stimulated vasodilation in gastrocnemius feed arteries (GFA), or second-order arterioles in the red (G2A-R) or white (G2A-W) portions of the gastrocnemius muscle of OLETF rats. Metformin had no effects on vasomotor responses of arteries from LETO. Although metformin exerts favorable effects on body composition and HbA1c, it does not enhance the vasodilatory responses to insulin in the skeletal muscle feed arteries or arterioles of the obese OLETF rat.

Influence of regular physical activity and caloric restriction on β‐adrenergic and natriuretic peptide receptor expression in retroperitoneal adipose tissue of OLETF rats

The mechanisms underlying exercise-induced increases in adipose tissue blood flow and lipolysis involve both β-adrenergic receptor (βAR)- and natriuretic peptide receptor (NPR)-dependent processes. We hypothesized that daily wheel running (RUN) would increase the expression of NPR1, NPR2, βAR2 and βAR3 in retroperitoneal (RP) and epididymal (EPI) adipose tissues of obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Four-week-old OLETF rats were assigned to sedentary (SED, n = 6), calorie-restricted (CR, n = 8; fed 70% of SED) or RUN groups (n = 8). Rats were killed at 40 weeks of age. By design, body weight and adiposity were similar between RUN and CR animals, but each was lower than SED (P < 0.01). Compared with SED, RP depots of RUN rats exhibited 1.7- to 3.2-fold greater NPR1, NPR2, βAR2 and βAR3 mRNA levels (all P < 0.05). There were no differences between CR and SED in the expression of these genes in RP adipose tissues, and there were no differences in gene expression among groups in EPI adipose tissues. At the protein level, βAR2 and βAR3 were elevated in RUN and CR groups relative to the SED group in RP adipose tissues. In order to gain insight into the mechanisms underlying the activity-induced increases in NPR and βAR mRNAs, RP adipose tissue explants from Wistar rats were treated with atrial natriuretic peptide (ANP), adrenaline and/or S-nitroso-N-acetyl-dl-penicillamine (SNAP; a nitric oxide donor) in organ culture experiments. SNAP synergistically enhanced adrenaline- and ANP-stimulated increases in NPR2 and βAR2 mRNA levels. Our data suggest that physical activity-induced increases in nitric oxide interact with adrenaline and ANP to trigger the induction of NPR and βAR mRNAs in the RP adipose tissue depot of the OLETF rat.

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

Sterculic Oil, a natural inhibitor of SCD1, improves the metabolic state of obese OLETF rats

Abnormal lipid metabolism and excess accumulation of lipid in non-adipose tissues are defining characteristics of obesity and its comorbidities. Expression and/or activity of stearoyl-CoA desaturase-1 (SCD1), a major regulator of lipid metabolism, is increased with obesity and the reduction/ablation of this enzyme is associated with an improved metabolic profile. Sterculic oil (SO), obtained from the seeds of the Sterculia feotida tree, contains a high concentration of cyclopropenoic fatty acids which are known inhibitors of SCD1. The purpose of this study was to determine the effects of SO supplementation on the development of obesity and insulin resistance in hyperphagic, obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats received either an AIN-93G diet (control) or an AIN-93G diet containing 0.5% SO for 10 weeks. SO did not alter body weight or body composition. Importantly, the desaturase indices, a proxy for the activity of SCD1, were reduced in the liver and adipose tissue of SO supplemented animals. This reduction in SCD1 activity was associated with a reduction in fasting blood glucose concentrations and improved glucose tolerance. In addition, SO reduced intra-abdominal fat mass and adipocyte size and resulted in a ∼3-fold increase in GLUT1 gene expression in intra-abdominal fat. Liver triglyceride content and lipogenic gene expression were reduced by SO. Consistent with an improved metabolic phenotype, SO also improved plasma cholesterol, LDL-cholesterol, and triglyceride concentrations. Overall, our data demonstrate an improved metabolic phenotype with SO supplementation and suggest further studies are required to better understand the therapeutic potential of SO.

Aberrant Activation of the Intrarenal Renin-Angiotensin System in the Developing Kidneys of Type 2 Diabetic Rats

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.