Otsuka Long-Evans Tokushima Fatty Rats Research Articles

Empagliflozin, an SGLT2 Inhibitor, Reduced the Mortality Rate after Acute Myocardial Infarction with Modification of Cardiac Metabolomes and Antioxidants in Diabetic Rats

The mechanism by which SGLT2 inhibitors reduce cardiac events in diabetic patients remains unclear. Here, we examined the effects of an SGLT2 inhibitor on the acute survival rate after myocardial infarction (MI) in an animal model of type 2 diabetes mellitus (DM) and the possible involvement of modification of cardiac metabolomes and antioxidative proteins. MI was induced in DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) control rats. Treatment with empagliflozin (10 mg/kg per day, 14 days) before MI reduced blood glucose and increased blood and myocardial β-hydroxybutyrate (βOHB) levels in OLETF. Survival rate at 48 hours after MI was significantly lower in OLETF rats than in LETO rats (40% vs. 84%), and empagliflozin significantly improved the survival rate in OLETF rats to 70%, although the sizes of MI were comparable. Patterns of metabolomes and gene expression in the noninfarcted myocardium of OLETF rats were consistent with increased fatty acid oxidation and decreased glucose oxidation. The patterns were modified by empagliflozin, suggesting both increased glucose oxidation and ketone utilization in OLETF rats. Empagliflozin prevented reduction of ATP level in the noninfarcted myocardium after MI and significantly increased myocardial levels of Sirt3 and superoxide dismutase 2 in OLETF rats. Administration of βOHB partially mimicked the effects of empagliflozin in OLETF rats. The results suggest that empagliflozin prevents DM-induced increase in post-MI mortality, possibly by protective modification of cardiac energy metabolism and antioxidant proteins.

Effect of sodium-glucose cotransporter 2 inhibitor, empagliflozin, and α-glucosidase inhibitor, voglibose, on hepatic steatosis in an animal model of type 2 diabetes.

We investigated the effects of sodium-glucose cotransporter 2 inhibitor, empagliflozin, and α-glucosidase inhibitor, voglibose, on hepatic steatosis in an animal model of type 2 diabetes (T2DM). Empagliflozin (OLETF-EMPA) or voglibose (OLETF-VOG) was administered to Otsuka Long-Evans Tokushima fatty (OLETF) rats once daily for 12 weeks. Control Long-Evans Tokushima Otsuka (LETO) and OLETF (OLETF-C) rats received saline. Blood glucose levels were significantly suppressed in OLETF-EMPA and OLETF-VOG compared with the OLETF-C group. The liver fat content was significantly higher in the OLETF-C group than in the OLETF-EMPA and OLETF-VOG. Hepatic gene expressions involved in gluconeogenesis (glucose 6-phosphatase [G6Pase], fructose-1,6-bisphosphatase [FBP1], and phosphoenolpyruvate carboxykinase [PEPCK]) and lipogenesis (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and sterol regulatory element-binding transcription factor 1c [SREBP-1c]) were significantly decreased in the OLETF-EMPA group compared with other OLETF groups (OLETF-C and OLETF-VOG). Sirtuin 1 (SIRT1) expression level and SIRT1 activity were markedly reduced in OLETF-C rats; however, its expression increased in the OLETF-EMPA and OLETF-VOG. AMP-activated protein kinase (AMPK) phosphorylation level was remarkably increased by empagliflozin treatment in OLETF rats compared with other OLETF groups. Long-term empagliflozin and voglibose treatment reduced hepatic steatosis with suppression of gluconeogenesis and lipogenesis pathway in OLETF rats. We suggest that this metabolic improvement might be related to SIRT1 and AMPK pathway in T2DM. But empagliflozin is thought to have more advantage to prevent hepatic steatosis than voglibose in T2DM.

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.

Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is a chronic disease manifested by hyperglycemia. It is essential to effectively control hyperglycemia to prevent complications of T2DM. Here, we hypothesize that repression of transcriptional activity of forkhead box O1 (FoxO1) via histone deacetylase inhibitors (HDACi) ameliorates hyperglycemia in T2DM rats. Methods: Male Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in water for 20 weeks. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed for elucidation of transcriptional regulation through acetylation of FoxO1 by HDACi. Results: VPA attenuated blood glucose levels in accordance with a decrease in the expression of gluconeogenic genes in hyperglycemic OLETF rats. It has been shown that HDAC class I-specific and HDAC class IIa-specific inhibitors, as well as pan-HDAC inhibitors decrease FoxO1 enrichment at the cis-element of target gene promoters. Mutations in FoxO1 prevent its acetylation, thereby increasing its transcriptional activity. HDAC3 and HDAC4 interact with FoxO1, and knockdown of HDAC3, HDAC4, or their combination increases FoxO1 acetylation, thereby decreasing the expression of gluconeogenic genes. Conclusions: These results indicate that HDACi attenuates the transcriptional activity of FoxO1 by impeding deacetylation, thereby ameliorating hyperglycemia in T2DM rats.

Effects of activated vitamin D, alfacalcidol, and low-intensity aerobic exercise on osteopenia and muscle atrophy in type 2 diabetes mellitus model rats.

Diabetes mellitus causes secondary osteoporosis and muscle atrophy. The ability of alfacalcidol (ALF) and exercise (Exe) to inhibit osteoporosis and muscle atrophy in type 2 diabetes mellitus (T2DM) model rats was examined. Twenty-week-old Otsuka Long-Evans Tokushima Fatty rats were randomized to ALF (orally 0.1 μg/kg/day), Exe (treadmill exercise at 10 m/min, 60 min/day, 5 days/week), Comb (ALF and Exe), and Cont (T2DM control treated with vehicle and no exercise) groups (n = 8–10 per group). Sedentary Long-Evans Tokushima Otsuka rats were used as a non-hyperphagic control. After treatment for 2 or 6 weeks, blood glucose (BG) levels, cross-sectional area (CSA) of tibialis anterior muscle fibers, femoral bone mineral density (BMD), and relative quantities of muscle anabolic markers (Pax7, MyoD, and myogenin) and catabolic markers (Atrogin-1, MuRF1, and REDD1) of the soleus muscle assessed by real-time polymerase chain reaction assays were measured. Exe and Comb treatments for 6 weeks decreased BG levels compared with those of the Cont group. ALF, Exe, and Comb treatments for 2 and 6 weeks recovered the CSA compared with that of the Cont group. ALF and Comb treatments for 6 weeks increased femoral BMDs compared with those of the Cont group. After 2 weeks of treatment, Comb treatment increased MyoD expression and decreased MuRF1 expression. ALF or Exe monotherapy significantly decreased Atrogin-1 or MuRF1 expression after 2 weeks of treatment, respectively. After 6 weeks of treatment, ALF and Comb treatments decreased Atrogin-1 and REDD1. These results demonstrate that a combination of ALF and Exe improved CSA from the early phase of treatment by stimulating skeletal muscle differentiation and suppressing muscle catabolic genes. Improvements in BG, BMD, and CSA were observed as long-term effects of the combination therapy. Continued suppression of muscle catabolic genes was observed as a background to these effects.

Body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of type 2 diabetic rats.

This study examined the effect of changes in body temperature during exercise on signal transduction-related glucose uptake in the skeletal muscle of type 2 diabetic rats. Otsuka Long-Evans Tokushima Fatty rats (25 weeks of age), which have type 2 diabetes, were divided into the following four weight-matched groups; control (CON, n = 6), exercised under warm temperature (WEx, n = 8), exercised under cold temperature (CEx, n = 8), and heat treatment (HT, n = 6). WEx and CEx animals were subjected to running on a treadmill at 20 m/min for 30 min under warm (25°C) or cold (4°C) temperature. HT animals were exposed to single heat treatment (40–41°C for 30 min) in a heat chamber. Rectal and muscle temperatures were measured immediately after exercise and heat treatment, and the gastrocnemius muscle was sampled under anesthesia. Rectal and muscle temperatures increased significantly in rats in the WEx and HT, but not the CEx, groups. The phosphorylation levels of Akt, AS160, and TBC1D1 (Thr590) were significantly higher in the WEx and HT groups than the CON group (p < 0.05). In contrast, the phosphorylation levels of AMP-activated protein kinase, ACC, and TBC1D1 (Ser660) were significantly higher in rats in the WEx and CEx groups than the CON group (p < 0.05) but did not differ significantly between rats in the WEx and CEx groups. Body temperature elevation by heat treatment did not activate the AMPK signaling. Our data suggest that body temperature elevation during exercise is essential for activating the Akt signaling pathway in the skeletal muscle of rats with type 2 diabetic rats.

Long-term insulin treatment leads to a change in myosin heavy chain fiber distribution in OLETF rat skeletal muscle.

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 OLETF rats. Male Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats aged 24 weeks were randomly allocated to either treatment with insulin for 24 weeks or no treatment, resulting in three groups. Insulin glargine treatment in OLETF rats (OLETF-G) for 24 weeks resulted in changes 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 MHC isoforms were altered in gastrocnemius muscle of OLETF rats, and the proportions of myosin heavy chain type I and II fibers were lower and higher, respectively, in OLETF-G compared with OLETF-C rats. Activation of myokines (IL-6, IL-15, FNDC5, and myostatin) in gastrocnemius muscle was significantly inhibited in OLETF-G compared with OLETF-C rats ( P < 0.05). MyoD and myogenin levels were decreased, while IGF-I and GLUT4 levels were increased, in the skeletal muscle of OLETF-G rats ( P < 0.05). Insulin glargine treatment significantly increased the phosphorylation levels of AMPK, SIRT1, and PGC-1α. Together, our results suggested that changes in the distribution of fiber types by insulin glargine could result in downregulation of myokines and muscle regulatory proteins. The effects were likely associated with activation of the AMPK/SIRT1/PGC-1α signaling pathway. Changes in these proteins may at least partly explain the effect of insulin in skeletal muscle of diabetes mellitus.

Rosiglitazone Elicits an Adiponectin-Mediated Insulin-Sensitizing Action at the Adipose Tissue-Liver Axis in Otsuka Long-Evans Tokushima Fatty Rats.

Rosiglitazone is an agonist of peroxisome proliferator-activated receptor- (PPAR-) γ that is principally associated with insulin action. The exact mechanisms underlying its insulin-sensitizing action are still not fully elucidated. It is well known that adiponectin mostly secreted in adipose tissue is an insulin sensitizer. Here, we found that treatment of Otsuka Long-Evans Tokushima Fatty (OLETF) rats with rosiglitazone (3 mg/kg, once daily, by oral gavage for 33 weeks) attenuated the increase in fasting plasma insulin concentrations and the index of the homeostasis model assessment of insulin resistance along with the age growth and glucose concentrations during an oral glucose tolerance test. In addition, the increase in plasma alanine aminotransferase activity, concentrations of fasting plasma nonesterified fatty acids and triglyceride, and hepatic triglyceride content was also suppressed. The hepatic protein expression profile revealed that rosiglitazone increased the downregulated total protein expression of insulin receptor substrate 1 (IRS-1) and IRS-2. Furthermore, the treatment suppressed the upregulated phosphorylation of IRS-1 at Ser307 and IRS-2 at Ser731. The results indicate that rosiglitazone ameliorates hepatic and systemic insulin resistance, hepatic inflammation, and fatty liver. Mechanistically, rosiglitazone suppressed hepatic protein overexpression of both phosphorylated nuclear factor- (NF-) κBp65 and inhibitory-κB kinase-α/β, a transcription factor that primarily regulates chronic inflammatory responses and the upstream NF-κB signal transduction cascades which are necessary for activating NF-κB, respectively. More importantly, rosiglitazone attenuated the decreases in adipose adiponectin mRNA level, plasma adiponectin concentrations, and hepatic protein expression of adiponectin receptor-1 and receptor-2. Thus, we can draw the conclusion that rosiglitazone elicits an adiponectin-mediated insulin-sensitizing action at the adipose tissue-liver axis in obese rats. Our findings may provide new insights into the mechanisms of action of rosiglitazone.

Simultaneous GLP-1 receptor activation and angiotensin receptor blockade increase natriuresis independent of altered arterial pressure in obese OLETF rats

Obesity is associated with an inappropriately activated renin-angiotensin-aldosterone system, suppressed glucagon-like peptide-1 (GLP-1), increased renal Na+ reabsorption, and hypertension. To assess the link between GLP-1 and angiotensin receptor type 1 (AT1) signaling on obesity-associated impairment of urinary Na+ excretion (UNaV) and elevated arterial pressure, we measured mean arterial pressure (MAP) and heart rate by radiotelemetry and metabolic parameters for 40 days. We tested the hypothesis that stimulation of GLP-1 signaling provides added benefit to blockade of AT1 by increasing UNaV and further reducing arterial pressure in the following groups: (1) untreated Long-Evans Tokushima Otsuka (LETO) rats (n = 7); (2) untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats (n = 9); (3) OLETF + ARB (ARB; 10 mg olmesartan/kg/day; n = 9); (4) OLETF + GLP-1 receptor agonist (EXE; 10 µg exenatide/kg/day; n = 7); and (5) OLETF + ARB + EXE (Combo; n = 6). On day 2, UNaV was 60% and 62% reduced in the EXE and Combo groups, respectively, compared with that in the OLETF rats. On day 40, UNaV was increased 69% in the Combo group compared with that in the OLETF group. On day 40, urinary angiotensinogen was 4.5-fold greater in the OLETF than in the LETO group and was 56%, 62%, and 58% lower in the ARB, EXE, and Combo groups, respectively, than in the OLETF group. From day 2 to the end of the study, MAP was lower in the ARB and Combo groups than in the OLETF rats. These results suggest that GLP-1 receptor activation may reduce intrarenal angiotensin II activity, and that simultaneous blockade of AT1 increases UNaV in obesity; however, these beneficial effects do not translate to a further reduction in MAP.

MiR-192 prevents renal tubulointerstitial fibrosis in diabetic nephropathy by targeting Egr1.

Diabetic nephropathy (DN), as the most common and serious diabetic microvascular complication, has become the first cause of end-stage renal disease (ESRD) in many countries and regions. However, the pathogenesis of renal fibrosis during the development of DN remains unknown. The expression levels of miR-192 and early growth response factor 1 (Egr1) were determined by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western blotting in the renal tissues of Otsuka-Long-Evans-Tokushima-Fatty (OLETF) and Long-Evans-Tokushima-Otsuka (LETO) rats. The diabetic kidney environment was simulated by a high-sugar medium. The expression levels of miR-192 and Egr1 were further measured in the HK-2 cell line. Egr1 was verified as a potential target of miR-192 by using bioinformatics analysis and luciferase activity assay. The expression level of Egr1 was determined by overexpressing and knocking down the expression of miR-192. In addition, Western blotting was used to determine changes in Transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN). Compared with the kidney tissue of LETO rats, the expression of miR-192 was decreased in OLETF rats, whereas the expression of Egr1 was increased. We found the same phenomenon in the HK-2 cell line cultured in the high-glucose medium. Next, miR-192 can act on Egr1 through 3'-UTR to reduce the expression of Egr1 verified by luciferase assay. In addition, the expression levels of TGF-β1 and FN changed significantly, as the expression level of Egr1 increased or decreased. MiR-192 causes degradation of TGF-β1 and FN through targeting Egr1 and affects the progression of TIF and even DN.

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 &amp;lt; 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 &amp;lt; 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 &amp;lt; 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.

A Controlled Fermented Samjunghwan Herbal Formula Ameliorates Non-alcoholic Hepatosteatosis in HepG2 Cells and OLETF Rats.

Hepatosteatosis (HS), a clinical feature of fatty liver with the excessive intracellular accumulation of triglyceride in hepatocytes, is manifested by perturbation of the maintenance of liver lipid homeostasis. Samjunghwan (SJH) is an herbal formula used mostly in Korean traditional medicine that is effective against a number of metabolic diseases, including obesity. Herbal drugs, enriched with numerous bioactive substances, possess health-protective benefits. Meanwhile, fermented herbal products enriched with probiotics are known to improve metabolic processes. Additionally, current lines of evidence indicate that probiotics-derived metabolites, termed as postbiotics, produce the same beneficial effects as their precursors. Herein, the anti-HS effects of 5-weeks naturally fermented SJH (FSJH) was investigated with FSJH-mixed chow diet in vivo using Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats as animal models of HS and controls, respectively. In parallel, the anti-HS effects of postbiotic-metabolites of three bacterial strains [Lactobacillus brevis (LBB), Lactococcus lactis (LCL) and Lactobacillus plantarum (LBP)] isolated from FSJH were also evaluated in vitro using the FFAs-induced HepG2 cells. Feeding OLETF rats with FSJH-diet effectively reduced body, liver, and visceral adipose tissue (VAT) weights, produced marked hypolipidemic effects on serum and hepatic lipid parameters, decreased serum AST and ALT levels, and upregulated the HMGCOR, SREBP, and ACC, and downregulated the AMPK and LDLR gene expressions levels. Additionally, exposure of FFAs-induced HepG2 cells to postbiotic metabolic media (PMM) of bacterial strains also produced marked hypolipidemic effects on intracellular lipid contents and significantly unregulated the HMGCOR, SREBP, and ACC, and downregulated the AMPK and LDLR genes expressions levels. Overall, our results indicate that FSJH enriched with fermented metabolites could be an effective anti-HS formulation.

Mineralocorticoid receptor antagonism reverses diabetes-related coronary vasodilator dysfunction: A unique vascular transcriptomic signature

Coronary microvascular dysfunction predicts and may be a proximate cause of cardiac dysfunction and mortality in diabetes; however, few effective treatments exist for these conditions. We recently demonstrated that mineralocorticoid receptor (MR) antagonism reversed cardiovascular dysfunction in early-stage obesity/insulin resistance. The mechanisms underlying this benefit of MR antagonism and its relevance in the setting of long-term obesity complications like diabetes; however, remain unclear. Thus, the present study evaluated the impact of MR antagonism on diabetes-related coronary dysfunction and defines the MR-dependent vascular transcriptome in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat recapitulating later stages of human diabetes. OLETF rats were treated with spironolactone (Sp) and compared to untreated OLETF and lean Long-Evans Tokushima Otsuka rats. Sp treatment attenuated diabetes-associated adipose and cardiac inflammation/fibrosis and improved coronary endothelium-dependent vasodilation but did not alter enhanced coronary vasoconstriction, blood pressure, or metabolic parameters in OLETF rats. Further mechanistic studies using RNA deep sequencing of OLETF rat aortas revealed 157 differentially expressed genes following Sp including upregulation of genes involved in the molecular regulation of nitric oxide bioavailability (Hsp90ab1, Ahsa1, Ahsa2) as well as novel changes in α1D adrenergic receptors (Adra1d), cyclooxygenase-2 (Ptgs2), and modulatory factors of these pathways (Ackr3, Acsl4). Further, Ingenuity Pathway Analysis predicted inhibition of upstream inflammatory regulators by Sp and inhibition of ‘migration of endothelial cells’, ‘differentiation of smooth muscle’, and ‘angiogenesis’ biological functions by Sp in diabetes. Thus, this study is the first to define the MR-dependent vascular transcriptome underlying treatment of diabetes-related coronary microvascular dysfunction by Sp.

Mild hyperbaric oxygen inhibits the growth-related decline in skeletal muscle oxidative capacity and prevents hyperglycemia in rats with type 2 diabetes mellitus.

Humans and animals with type 2 diabetes mellitus (T2DM) exhibit low skeletal muscle oxidative capacity and impaired glucose metabolism. The aim of the present study was to investigate the effects of exposure to mild hyperbaric oxygen on these changes in obese rats with T2DM. Five-week-old non-diabetic Long-Evans Tokushima Otsuka (LETO) and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats were divided into normobaric (LETO-NB and OLETF-NB) and mild hyperbaric oxygen (LETO-MHO and OLETF-MHO) groups. The LETO-MHO and OLETF-MHO groups received 1266 hPa with 36% oxygen for 3 h daily for 22 weeks. Fasting and non-fasting blood glucose, HbA1c, and triglyceride levels were lower in the OLETF-MHO group than in the OLETF-NB group (P < 0.05). In the soleus muscle, peroxisome proliferator-activated receptor δ/β (Pparδ/β), Pparγ, and PPARγ coactivator-1α (Pgc-1α) mRNA levels were lower in the OLETF-NB group than in all other groups (P < 0.05), whereas myogenin (Myog) and myogenic factor 5 (Myf5) mRNA levels were higher in the OLETF-MHO group than in the LETO-NB and OLETF-NB groups (P < 0.05). The soleus muscles in the OLETF-NB group contained only low-oxidative Type I fibers, whereas those in all other groups contained high-oxidative Type IIA and Type IIC fibers in addition to Type I fibers. Exposure to mild hyperbaric oxygen inhibits the decline in skeletal muscle oxidative capacity and prevents the hyperglycemia associated with T2DM. Pgc-1α, Myog, and Myf5 mRNA levels appear to be closely associated with skeletal muscle oxidative capacity in rats with T2DM.

Changes in lipid metabolism and capillary density of the skeletal muscle following low-intensity exercise training in a rat model of obesity with hyperinsulinemia.

Although exercise is effective in improving obesity and hyperinsulinemia, the exact influence of exercise on the capillary density of skeletal muscles remains unknown. The aim of this study was to investigate the effects of low-intensity exercise training on metabolism in obesity with hyperinsulinemia, focusing specifically on the capillary density within the skeletal muscle. Otsuka Long-Evans Tokushima fatty (OLETF) rats were used as animal models of obesity with hyperinsulinemia, whereas Long-Evans Tokushima Otsuka (LETO) rats served as controls (no obesity, no hyperinsulinemia). The animals were randomly assigned to either non-exercise or exercise groups (treadmill running for 60 min/day, for 4 weeks). The exercise groups were further divided into subgroups according to training mode: single bout (60 min, daily) vs. multiple bout (three bouts of 20 min, daily). Fasting insulin levels were significantly higher in OLETF than in LETO rats. Among OLETF rats, there were no significant differences in fasting glucose levels between the exercise and the non-exercise groups, but the fasting insulin levels were significantly lower in the exercise group. Body weight and triacylglycerol levels in the liver were significantly higher in OLETF than in LETO rats; however, among OLETF rats, these levels were significantly lower in the exercise than in the non-exercise group. The capillary-to-fiber ratio of the soleus muscle was significantly higher in OLETF than in LETO rats; however, among OLETF rats, the ratio was lower in the exercise group than in the non-exercise group. No significant differences in any of the studied parameters were noted between the single-bout and multiple-bout exercise training modes among either OLETF or LETO rats. These results suggest that low-intensity exercise training improves insulin sensitivity and fatty liver. Additionally, the fact that attenuation of excessive capillarization in the skeletal muscle of OLETF rats was accompanied by improvement in increased body weight.

TREADMILL EXERCISE TRAINING PROTECTS AGAINST METABOLIC DYSFUNCTION AND DIAPHRAGM WEAKNESS IN OBESE DIABETIC RATS

Excessive weight gain is a serious public health issue, as nearly one‐third of the population worldwide is obese or overweight. Obesity is a major risk factor in the development of various metabolic disorders, including type 2 diabetes. Furthermore, obesity and diabetes results in many physical disabilities, including inspiratory muscle weakness (i.e., diaphragm dysfunction) which can lower the quality of life and increase the risk of respiratory failure. Increased physical activity (i.e., exercise training) has been shown to be a critical intervention to attenuate the metabolic derangements associated with obese diabetics. Furthermore, endurance exercise training alters the diaphragmatic phenotype to protect against various stressors; however, whether endurance exercise training can attenuate obesity/diabetes‐induced diaphragm dysfunction is unclear. Therefore, we investigated the effects of exercise training on obesity/diabetes‐induced diaphragm dysfunction. Male Otsuka Long‐Evans Tokushima Fatty (OLETF) rats, a rodent model of obesity/diabetes, were randomly assigned to either sedentary (SED: n=7) or exercise groups (Ex: n=6). Dependent measures were compared to lean long‐Evans Tokushima Otsuka rats that were sedentary (LETO: n=6). Treadmill running was conducted 5 days/week for 20 weeks from 5 weeks of age for Ex group. An intraperitoneal glucose tolerance test was performed following overnight fasting, and blood and diaphragm samples were obtained 48 h after the final exercise bout. Exercise training positively impacted whole‐body metabolic function of OLETF rats, as indicated by improved glucose tolerance, and reductions in body weight, serum triglyceride, total cholesterol, and insulin that were comparable to LETO rats. A trend (p=0.0511) for decreased blood levels of free fatty acids was also observed in exercise trained OLETF rats. Diaphragm force production in OLETF rats was lower compared to LETO controls, but was rescued by exercise training. Mechanistically, OLETF rats presented increases in myocellular markers associated with diaphragmatic weakness, including increases in angiotensin II type 1 receptor (AT1R) and tumor necrosis factor‐like weak inducer of apoptosis receptor (TWEAKR). Interestingly, in conjunction with improved diaphragm function, the rise in diaphragmatic AT1R and TWEAKR were attenuated by exercise training in OLETF rats. Additionally, increased protein acetylation was prevented in OLETF rats that were exercise trained, potentially due to restoration in the protein abundance of the deacetylase sirtuin 1 (Sirt1) by exercise training (p&lt;0.05). These findings reveal that endurance exercise training protects against metabolic dysfunction and diaphragm weakness observed with obese diabetic rats. These data suggest exercise training is a potential intervention to prevent respiratory muscle weakness associated with obese diabetic individuals.Support or Funding InformationThis work was supported by JSPS KAKENHI 15KK0131 (NIS), MEXT‐Supported Program for the Private University Research Branding Project (HN) and NIH R01, R01AR064189 (SKP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Soy Protein Isolate Suppresses Bone Resorption and Improves Trabecular Microarchitecture in Spontaneously Hyperphagic, Rapidly Growing Male OLETF Rats

BackgroundTraditionally, milk proteins have been recommended for skeletal health; recently, soy proteins have emerged as popular alternatives. Excess adiposity appears detrimental to skeletal health, as obese adolescents have increased fracture rates compared with healthy controls. However, soy protein effects on skeletal health during excess adiposity remain unknown. ObjectiveThe study objective was to examine the effects of isocaloric diets containing milk protein isolate (MPI), soy protein isolate (SPI), or a 50/50 combination (MIX) as the sole protein source on metabolic health indicators and bone outcomes in rapidly growing, hyperphagic, male Otsuka Long Evans Tokushima Fatty (OLETF) rats. MethodsOLETF rats, aged 4 wk, were randomly assigned to 3 treatment groups (MPI, SPI, or MIX, n = 20 per group) and provided with access to experimental diets ad libitum for 16 wk. ResultsBody mass did not differ between the groups, but SPI had lower percentage body fat than MPI (P = 0.026). Insulin was lower in MPI than in MIX (P = 0.033) or SPI (P = 0.044), but fasting blood glucose was not different between the groups. SPI significantly reduced serum cholesterol compared with MPI (P = 0.001) and MIX (P = 0.002). N-terminal propeptide of type I collagen (P1NP) was higher in MIX than MPI (P = 0.05); C-terminal telopeptide of type 1 collagen (CTx) was higher in MPI than SPI (P < 0.001) and MIX (P < 0.001); the P1NP to CTx ratio was significantly higher in SPI and MIX than in MPI (P < 0.001). Trabecular separation was reduced in SPI compared with MPI (P = 0.030) and MIX (P = 0.008); trabecular number was increased in SPI compared with MIX (P = 0.038). No differences were seen in cortical geometry and biomechanical properties. ConclusionsIn the context of excess adiposity, soy- and milk-based proteins have comparable effects on cortical bone geometry and biomechanical properties, whereas soy-based proteins favorably affect the trabecular microarchitecture, and the combination of both proteins may offer additional benefits to bone remodeling in rapidly growing male OLETF rats.

Effects of voluntary running exercise on bone histology in type 2 diabetic rats.

The incidence of obesity in children and adolescents, which may lead to type 2 diabetes, is increasing. Exercise is recommended to prevent and improve diabetes. However, little is known about the bone marrow environment at the onset of diabetes in the young, and it is unclear whether exercise training is useful for maintaining bone homeostasis, such as mechanical and histological properties. Thus, this study clarified the histological properties of bone and whether exercise contributes to maintaining bone homeostasis at the onset of type 2 diabetes in rats. Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF; n = 21) rats as a diabetic model and Long-Evans Tokushima Otsuka (LETO; n = 18) rats as a control were assigned randomly to four groups: the OLETF sedentary group (O-Sed; n = 11), OLETF exercise group (O-Ex; n = 10), LETO sedentary group (L-Sed; n = 9), and LETO exercise group (L-Ex; n = 9). All rats in the exercise group were allowed free access to a steel running wheel for 20 weeks (5–25 weeks of age). In the glucose tolerance test, blood glucose level was higher in the O-Sed group than that in the L-Sed and L-Ex groups, and was markedly suppressed by the voluntary running exercise of O-Ex rats. The energy to fracture and the two-dimensional bone volume at 25 weeks of age did not differ significantly among the groups, though the maximum breaking force and stiffness were lower in OLETF rats. However, bone marrow fat volume was greater in O-Sed than that in L-Sed and L-Ex rats, and was markedly suppressed by wheel running in the O-Ex rats. Our results indicate that exercise has beneficial effects not only for preventing diabetes but also on normal bone remodeling at an early age.

Ocular findings in Zucker Diabetic Fatty rats emphasize the key role of neuroglia degeneration in diabetic retinopathy pathophysiology.

Ocular findings in Zucker Diabetic Fatty rats emphasize the key role of neuroglia degeneration in diabetic retinopathy pathophysiology.

Molecular Mechanisms of Vaspin Action - From Adipose Tissue to Skin and Bone, from Blood Vessels to the Brain.

Visceral adipose tissue-derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that were specifically expressed or overexpressed in the intra-abdominal or visceral adipose tissue (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral obesity, insulin resistance, hyperinsulinemia and -glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type 2 diabetes.The follow-up study reporting the cloning, expression and functional characterization of vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.