OLETF Rats Research Articles

Transcription Factor Egr1 is Involved in High Glucose-Induced Proliferation and Fibrosis in Rat Glomerular Mesangial Cells

Backgroud: Diabetic nephropathy is one of the most frequent causes of end-stage renal disease and is associated with proliferation of glomerular mesangial cells (MCs) and excessive production of the extracellular matrix (ECM). Several studies have shown that early growth response factor 1 (Egr1) plays a key role in renal fibrosis by regulating the expression of genes encoding ECM components. However, whether Egr1 also contributes to diabetic nephropathy is unclear. Methods: In the present study, we compared the expression of Egr1 in kidneys from OLETF rats with spontaneous type 2 diabetes and healthy LETO rats. We also examined whether high glucose and TGF-β1 signaling up-regulated Egr1 expression in cultured MCs, and whether Egr1 expression influenced MC proliferation and expression of ECM genes. Results: We found that higher expression of Egr1 and TGF-β1, at both the mRNA and protein levels, the kidneys from OLETF rats vs. LETO rats. High glucose or TGF-β1 signaling rapidly up-regulated expression of Egr1 mRNA and protein in cultured MCs. Overexpressing Egr1 in MCs by transfection with M61-Egr1 plasmid or treatment with high glucose up-regulated expression of fibronectin, type IV collagen and TGF-β1, and promoted MC proliferation. Conversely, siRNA-mediated silencing of Egr1 expression down-regulated these genes and inhibited MC proliferation. Chromatin immunoprecipitation (ChIP) assays revealed that Egr1 bound to the TGF-β1 promoter. Conclusion: Our results provide strong evidence that Egr1 contributes to diabetic nephropathy by enhancing MC proliferation and ECM production, in part by interacting with TGF-β1.

Ezetimibe improves hepatic steatosis in relation to autophagy in obese and diabetic rats.

To investigate whether ezetimibe ameliorates hepatic steatosis and induces autophagy in a rat model of obesity and type 2 diabetes. Male age-matched lean control LETO and obese and diabetic OLETF rats were administered either PBS or ezetimibe (10 mg/kg per day) via stomach gavage for 20 wk. Changes in weight gain and energy intake were regularly monitored. Blood and liver tissue were harvested after overnight fasting at the end of study. Histological assessment was performed in liver tissue. The concentrations of glucose, insulin, triglycerides (TG), free fatty acids (FFA), and total cholesterol (TC) in blood and TG, FFA, and TG in liver tissue were measured. mRNA and protein abundance involved in autophagy was analyzed in the liver. To investigate the effect of ezetimibe on autophagy and reduction in hepatic fat accumulation, human Huh7 hepatocytes were incubated with ezetimibe (10 μmol/L) together with or without palmitic acid (PA, 0.5 mmol/L, 24 h). Transmission electron microscopy (TEM) was employed to demonstrate effect of ezetimibe on autophagy formation. Autophagic flux was measured with bafilomycin A1, an inhibitor of autophagy and following immunoblotting for autophagy-related protein expression. In the OLETF rats that received ezetimibe (10 mg/kg per day), liver weight were significantly decreased by 20% compared to OLETF control rats without changes in food intake and body weight (P < 0.05). Lipid parameters including TG, FFA, and TC in liver tissue of ezetimibe-administrated OLETF rats were dramatically decreased at least by 30% compared to OLETF controls (P < 0.01). The serum glucose, insulin, HOMA-IR, and lipid profiles were also improved by ezetimibe (P < 0.05). In addition, autophagy-related mRNA expression including ATG5, ATG6, and ATG7 and the protein level of microtubule-associated protein light chain 3 (LC3) were significantly increased in the liver in rats that received ezetimibe (P < 0.05). Likewise, for hepatocytes cultured in vitro, ezetimibe treatment significantly decreased PA-induced fat accumulation and increased PA-reduced mRNA and protein expression involved in autophagy (P < 0.05). Ezetimibe-increased autophagosomes was observed in TEM analysis. Immunoblotting analysis of autophagy formation with an inhibitor of autophagy demonstrated that ezetimibe-increased autophagy resulted from increased autophagic flux. The present study demonstrates that ezetimibe-mediated improvement in hepatic steatosis might involve the induction of autophagy.

Preventive effects of bitter melon (Momordica charantia) against insulin resistance and diabetes are associated with the inhibition of NF-κB and JNK pathways in high-fat-fed OLETF rats

Bitter melon (BM; Momordica charantia) has been used as a treatment method for various diseases including cancer and diabetes. The objective of this study was to investigate whether BM has preventive effects against insulin resistance and diabetes and to identify the underlying mechanism by which BM ameliorates insulin resistance in obese and diabetic rats. The rats were separated into three groups as follows: (a) high-fat (HF) diet control, (b) HF diet and 1% BM and (c) HF diet and 3% BM. After 6 weeks of assigned treatments, body weight and food intake were not altered by BM administration. Bitter melon treatment significantly improved glucose tolerance and insulin sensitivity. The levels of proinflammatory cytokines were significantly down-regulated in liver, muscle and epididymal fats from BM-treated rats. The activation of nuclear factor-κB (NF-κB) in the liver and muscle was decreased by BM compared with HF controls. The 3% BM supplementation significantly increased the levels of phospho-insulin receptor substrate-1 (Tyr612) and phospho-Akt (Ser473). It also significantly decreased the levels of phospho-NF-κB (p65) (Ser536) and phospho-c-Jun N-terminal kinase (JNK) (Thr183/Tyr185) in liver, muscle and epididymal fats. The findings of this study indicate that BM exerted preventive effects against insulin resistance and diabetes through the modulation of NF-κB and JNK pathways. Therefore, BM may be useful in the prevention of insulin resistance and diabetes.

P-27 * THE EFFECTS OF CHRONIC ALCOHOL CONSUMPTION ON ADIPONECTIN AND INSULIN RESISTANCE

The purpose of this study was to understand the effects of chronic alcohol consumption on diabetes. We used 14-week-old OLETF rats which have type 2 diabetes (T2DM) and non-diabetic control LETO rats. The rats were randomized into four groups. : (1) OLETF-Control(OC, n = 10), (2) OLETF-Alcohol (OE, n = 10), (3) LETO-Control (LC, n = 10), (4) LETO-Alcohol LE, n = 10). Rats in the alcohol-fed group were fed with a liquid alcohol diet over 10 week period. After each period of 5 week and 10 week feeding, we did Intraperitoneal Glucose Tolerance Testing (IP-GTT) and measured the blood adiponectin and insulin levels. The first IP-GTT data was not shown any significant difference between groups. After 10 weeks 30 minute-glucose levels of OE group is significantly higher than OC group. And after 90 minute, the glucose level of OE group was also significantly higher than OC group. The level of insulin was increased in OE group than OC group. (p < 0.05) The level of adiponectin in LE group and OE group was significantly lower than each control group. We suggest heavy alcohol consumption reduced the level of adiponectin which regulate insulin sensitivity. Reduced adiponectin level induced the insulin resistance which is one of the main factors of worsening T2DM in diabetic animal model.

Abstract 340: Psychological Stress Impairs Both Endothelial Function and Insulin Sensitivity via Activation of Inflammation and ER Stress Pathways Mediated by Corticotropin-Releasing Hormone

Background: Psychological stress may contribute to atherogenesis, although the underlying mechanism is poorly understood yet. We studied the role of corticotropin-releasing hormone (CRH) in the pathogenesis of stress-induced endothelial dysfunction and insulin resistance with specific focus on inflammation and ER stress pathways. Methods and Results: Immobilization stress (IS) was applied (2 hr/d) for 14 d to male 8-wk OLETF rats, whereas control groups comprised of rats without IS (n≥7 for each). 14d-IS decreased acetylcholine (Ach)-induced NO production, measured by direct NO electrochemical microsensor, from the aorta along with decreased expression of eNOS mRNA compared with control group. 14d-IS did not significantly change plasma glucose, but increased plasma insulin (342.5±6.3 vs. 496.0±51.6 pg/ml, p&lt;0.05) and HOMA-IR (3.1±0.2 vs.4.7±0.5 mmol/LxμIU/mL, p&lt;0.05) compared with control group. IS increased phosphorylation of both IRS1 serine and ERK, and increased expression of ER stress marker BiP. IS also increased translocation of NF-kB subunit p65 into nucleal fraction, and increased mRNA expression of both TNF-α and MCP-1 in the liver. Real-time RT-qPCR study showed that 1d IS increased expression of CRH mRNA and urocortin 2 mRNA in the hypothalamus by 2.5 folds and expression of CRH receptor type 2 mRNA in the aorta by 7.8 folds compared with control group. In cultured monocyte U937 cells, CRH induced translocation of NF-kB subunit p65 into nuclear fraction, and increased expression of BiP, phosphorylated inositol requiring enzyme 1, and MCP-1 mRNA. In cultured HUVECs, CRH pretreatment inhibited insulin-stimulated eNOS phosphorylation at Ser 1177 . Conclusions: Psychological stress impairs both endothelial function and insulin sensitivity probably via activation of NF-kB-mediated inflammation and ER stress pathways by CRH.

Adipose Tissue and Vascular Phenotypic Modulation by Voluntary Physical Activity and Dietary Restriction in Obese Insulin Resistant OLETF Rats

Adipose Tissue and Vascular Phenotypic Modulation by Voluntary Physical Activity and Dietary Restriction in Obese Insulin Resistant OLETF Rats

The use of noun phrases in biomedical research papers written by Chinese scholars.

Grammatically, the simplest kinds of clause in English usually consist of nouns and verbs, for example: However, both in spoken and written languages, the positions before and after the verb are rarely occupied by just a single noun. More usually, they are filled by groups of two or more words. These groups of words are called noun phrases (NP), for example: NPs consist of a head noun plus one or two of the optional elements. These optional elements fit into four predetermined slots in the NP: 1= determiner and/or enumerator (e.g., the, a, first, his) 2= pre-head modifier (e.g., red, washed, painting, steel) 3= head noun 4= post-head modifier (e.g., in Leeds, which I showed you) Because of the optional nature of the slots 1, 2 and 4, NPs have a highly variable length, ranging from just single words to passages of text (1). Specifically in biomedical research papers, NPs are commonly long containing large quantities of information, which are also the common reason for long and complex sentences, a prominent linguistic feature of research papers in English. Some examplar sentences are as the following: Example 1 NP (The age-matched LETO rats that are developed from the same colony of OLETF rats but do not show DM and pancreatic fibrosis) were used as a normal control (2). The subject of this sentence is a long NP, with the head noun of rats and slots 1, 2 and 4. The slot 4, as the post-head modifier, consists of two clauses, which is the main reason for the length and complexity of the sentence. Example 2 These rats were maintained in NP (a temperature- and humidity- controlled room with a 12:12h light-dark cycle) (2). After the preposition in, there is an NP with the head noun of room. The slots 1, 2 and 4 define the number and conditions of the room. The comparatively complex NP enlarges the quantity of information contained in just one simple sentence. Such long and complex NPs are common in biomedical research papers, whose use constitutes challenge to Chinese scholars due to the differences in habitual way of thinking and language expressing between Chinese and English. Linguistically, it is known as the negative transfer of the mother tongue (3). In this paper, I categorize the inappropriate use of NP in biomedical research papers written by Chinese scholars into four types as the following.

Resistant starch improves insulin resistance and reduces adipose tissue weight and CD11c expression in the adipose tissues of OLETF rats (1030.2)

CD11s/CD18 dimers induce monocyte/macrophage infiltration into many tissues, including adipose tissues. In particular, it was reported that β2‐integrin CD11c‐positive macrophages in adipose tissues are closely associated with the development of insulin resistance. In this study, we examined whether resistant starch (RS) intake reduces macrophage accumulation in adipose tissues and inhibits the development of insulin resistance at an early stage in Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. 22‐week‐old male OLETF rats were fed a control diet (55% α‐corn starch) or an RS diet (55% RS) for 5 weeks. An oral glucose tolerance test was performed after 4 weeks of feeding, while tissues (mesenteric and epididymal adipose tissues, and liver) and tail vein blood were collected after 5 weeks of feeding the test diets. Feeding the RS diet to OLETF rats for 5 weeks improved insulin resistance, reduced the mesenteric adipose tissue weight and enhanced the number of small adipocytes. CD68 expression, a macrophage infiltration marker, was not changed by the RS diet, while the gene expression levels of integrins such as CD11c, CD11d, and CD18, but not CD11a and CD11b, were significantly reduced. CD11c protein expression was reduced by the RS diet. These findings suggest that part of the mechanism for the improved insulin resistance by the RS diet involves a reduction of CD11c expression in adipose tissues.

Transcriptome‐wide RNA sequencing analysis of rat skeletal muscle feed arteries: impact of exercise training in obesity (1106.23)

We employed next generation RNA sequencing (RNA‐Seq) technology to determine the extent to which exercise alters global gene expression in skeletal muscle feed arteries and aortic endothelial cells (AEC) of obese OLETF rats. Transcriptional profiles of the soleus and gasctrocnemius muscle feed arteries (SFA and GFA, respectively) and AEC‐enriched samples from rats that underwent an endurance exercise training program(EndEx; n=12), interval sprint training program (IST; n=12), or remained sedentary (Sed; n=12) were examined. In response to EndEx, there were 39 upregulated and 20 downregulated genes in SFA and 1 upregulated and 1 downregulated gene in GFA (FDR &lt; 10%). In response to IST, there were 305 upregulated and 324 downregulated genes in SFA and 101 upregulated and 66 downregulated genes in GFA, with an overlap of 32 genes between arteries. Furthermore, in AEC, there were 183 up‐regulated and 141 downregulated genes with EndEx and 71 upregulated and 69 downregulated genes with IST, with an overlap of 35 between exercise programs. Only expression of two genes (i.e., Tubb2b and Slc9a3r2) was altered (i.e., increased) by exercise in all three arteries. The finding that both EndEx and IST produced greater transcriptional changes in the SFA compared to the GFA is intriguing when considering the fact that treadmill exercise is associated with greater relative increases in blood flow to the gastrocnemius muscle compared to the soleus muscle.Grant Funding Source: NIH RO1HL036088

Lowered extracellular pH is involved in the pathogenesis of skeletal muscle insulin resistance

Insulin resistance in the skeletal muscle is manifested by diminished insulin-stimulated glucose uptake and is a core factor in the pathogenesis of type 2 diabetes mellitus (DM), but the mechanism causing insulin resistance is still unknown. Our recent study has shown that pH of interstitial fluids was lowered in early developmental stage of insulin resistance in OLETF rats, a model of type 2 DM. Therefore, in the present study, we confirmed effects of the extracellular pH on the insulin signaling pathway in a rat skeletal muscle-derived cell line, L6 cell. The phosphorylation level (activation) of the insulin receptor was significantly diminished in low pH media. The phosphorylation level of Akt, which is a downstream target of the insulin signaling pathway, also decreased in low pH media. Moreover, the insulin binding to its receptor was reduced by lowering extracellular pH, while the expression of insulin receptors on the plasma membrane was not affected by the extracellular pH. Finally, insulin-stimulated 2-deoxyglucose uptake in L6 cells was diminished in low pH media. Our present study suggests that lowered extracellular pH conditions may produce the pathogenesis of insulin resistance in skeletal muscle cells.

Type 2 diabetes induces subendocardium-predominant reduction in transient outward K+ current with downregulation of Kv4.2 and KChIP2

In the present study, we examined if and how cardiac ion channels are modified by type 2 diabetes mellitus (T2DM). Subendocardial (Endo) myocytes and subepicardial (Epi) myocytes were isolated from left ventricles of Otsuka-Long-Evans-Tokushima Fatty rats (OLETF) rats, a rat model of T2DM, and Otsuka-Long-Evans-Tokushima (LETO) rats (nondiabetic control rats). Endo and Epi myocytes were used for whole cell patch-clamp recordings and for protein and mRNA analyses. Action potential durations in Endo and Epi myocytes were longer in OLETF rats than in LETO rats, and the difference was larger in Endo myocytes. Steady-state transient outward K+ current (Ito) density was reduced in Endo but not Epi myocytes of OLETF rats compared with LETO rats, although the contribution of the fast component of Ito recovery from inactivation was smaller in both Endo and Epi myocytes of OLETF rats than in LETO rats. Kv4.2 protein was reduced only in Endo myocytes in OLETF rats, although voltage-gated K+ channel-interacting protein 2 (KChIP2) protein levels in both Endo and Epi myocytes were lower in OLETF rats than in LETO rats. Corresponding regional differences in mRNA levels of KChIP2 and Kv4.2 were observed between OLETF and LETO rats. mRNA levels of Iroquois homeobox 5 in Endo myocytes were 53% higher in OLETF rats than in LETO rats. Densities of inward rectifier K+ current and L-type Ca2+ current and mRNA levels of Kv4.3 and Kv1.4 were similar in OLETF and LETO rats. In conclusion, T2DM induces Endo-predominant prolongation of the action potential duration via a reduction of the fast component of Ito recovery from inactivation and reduced steady-state Ito, in which downregulation of Kv4.2 and KChIP2 may be involved. Increased Iroquois homeobox 5 expression may underlie Kv4.2 downregulation in T2DM.

LBP-4a improves insulin resistance via translocation and activation of GLUT4 in OLETF rats

Lycium barbarum polysaccharide (LBP) has been shown to ameliorate insulin resistance, but the identification of compounds from LBP and the mechanisms have not been clarified. In this study, LBP-4a was purified from Lycium barbarum by DEAE cellulose and Sephadex G-100 column chromatography, and the effects of LBP-4a on insulin resistance were investigated. The results indicated that LBP-4a caused translocation of the glucose transporter isoform 4 (GLUT4) to the cell surface, which in turn stimulated glucose uptake, and the effect was sensitive to wortmannin, an inhibitor of phosphoinositol 3-kinase (PI3-K), and SB203580, an inhibitor of p38 mitogen activated protein kinase (p38 MAPK (α, β)). Furthermore, the effects of LBP-4a on p38 MAPK activities were abrogated by pretreatment of rat adipocytes using SB203580. In summary, LBP-4a improved insulin resistance via translocation and activation of GLUT4 in OLETF rats, and the activation of PI3-K and p38 MAPK contributed to these effects.

Taurine Alleviates the Progression of Diabetic Nephropathy in Type 2 Diabetic Rat Model

The overexpression of vascular endothelial growth factor (VEGF) is known to be involved in the pathogenesis of diabetic nephropathy. In this study, the protective effects of taurine on diabetic nephropathy along with its underlying mechanism were investigated. Experimental animals were divided into three groups: LETO rats as normal group (n = 10), OLETF rats as diabetic control group (n = 10), and OLETF rats treated with taurine group (n = 10). We treated taurine (200 mg/kg/day) for 20 weeks and treated high glucose (HG, 30 mM) with or without taurine (30 mM) in mouse cultured podocyte. After taurine treatment, blood glucose level was decreased and insulin secretion was increased. Taurine significantly reduced albuminuria and ACR. Also it decreased glomerular volume, GBM thickness and increased open slit pore density through decreased VEGF and increased nephrin mRNA expressions in renal cortex. The antioxidant effects of taurine were confirmed by the reduction of urine MDA in taurine treated diabetic group. Also reactive oxygen species (ROS) levels were decreased in HG condition with taurine treated podocytes compared to without taurine. These results indicate that taurine lowers glucose level via increased insulin secretion and ameliorates the progression of diabetic nephropathy through antifibrotic and antioxidant effects in type 2 diabetes rat model.

Epigallocatechin gallate attenuates ET-1-induced contraction in carotid artery from type 2 diabetic OLETF rat at chronic stage of disease

AimsThere is a growing body of evidence suggesting that epigallocatechin gallate (EGCG), a major catechin isolated from green tea, has several beneficial effects, such as anti-oxidant and anti-inflammatory activities. However, whether treatment with EGCG can suppress the endothelin-1 (ET-1)-induced contraction in carotid arteries from type 2 diabetic rats is unknown, especially at the chronic stage of the disease. We hypothesized that long-term treatment with EGCG would attenuate ET-1-induced contractions in type 2 diabetic arteries. Main methodsOtsuka Long–Evans Tokushima fatty (OLETF) rats (43weeks old) were treated with EGCG (200mg/kg/day for 2months, p.o.), and the responsiveness to ET-1, phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP) was measured in common carotid artery (CA) from EGCG-treated and -untreated OLETF rats and control Long–Evans Tokushima Otsuka (LETO) rats. Key findingsIn OLETF rats, EGCG attenuated responsiveness to ET-1 in CA compared to untreated groups. However, EGCG did not alter PE-induced contractions in CA from OLETF rats. In endothelium-denuded arteries, EGCG did not affect ET-1-induced contractions in either the OLETF or LETO group. Acetylcholine-induced relaxation was increased by EGCG treatment in CA from the OLETF group. The expressions of ET receptors, endothelial nitric oxide synthase, superoxide dismutases, and gp91phox [an NAD(P)H oxidase component] in CA were not altered by EGCG treatment in either group. SignificanceOur data suggest that, within the timescale investigated here, EGCG attenuates ET-1-induced contractions in CA from type 2 diabetic rats, and one of the mechanisms may involve normalizing endothelial function.

Nicotinamide improves glucose metabolism and affects the hepatic NAD-sirtuin pathway in a rodent model of obesity and type 2 diabetes

Nicotinic acid (NA) and nicotinamide (NAM) are major forms of niacin and exert their physiological functions as precursors of nicotinamide adenine dinucleotide (NAD). Sirtuins, which are NAD-dependent deacetylases, regulate glucose and lipid metabolism and are implicated in the pathophysiology of aging, diabetes, and hepatic steatosis. The aim of this study was to investigate the effects of two NAD donors, NA and NAM, on glucose metabolism and the hepatic NAD-sirtuin pathway. The effects were investigated in OLETF rats, a rodent model of obesity and type 2 diabetes. OLETF rats were divided into five groups: (1) high fat (HF) diet, (2) HF diet and 10 mg NA/kg body weight (BW)/day (NA 10), (3) HF diet and 100 mg NA/kg BW/day (NA 100), (4) HF diet and 10 mg NAM/kg BW/day (NAM 10), and (5) HF diet and 100 mg NAM/kg BW/day (NAM 100). NA and NAM were delivered via drinking water for four weeks. NAM 100 treatment affected glucose control significantly, as shown by lower levels of accumulative area under the curve during oral glucose tolerance test, serum fasting glucose, serum fasting insulin, and homeostasis model assessment of insulin resistance, and higher levels of serum adiponectin. With regard to NAD-sirtuin pathway, intracellular nicotinamide phosphoribosyltransferase, NAD, the NAD/NADH ratio, Sirt1, 2, 3, and 6 mRNA expressions, and Sirt1 activity all increased in livers of NAM 100-treated rats. These alterations were accompanied by the increased levels of proliferator-activated receptor gamma, coactivator 1 alpha and mitochondrial DNA. The effect of NA treatment was less evident than that of NAM 100. These results demonstrate that NAM is more effective than NA on the regulation of glucose metabolism and the NAD-sirtuin pathway, which may relate to the altered mitochondrial biogenesis.

Abstract 637: Angiotensin Receptor Blockade Ameliorates the Hypertension Associated with Acute Glucagon-like Peptide-1 Receptor Activation

Insulin resistance (IR) is associated with elevated renin-angiotensin system (RAS), impaired urinary Na + excretion (U Na V), and high blood pressure. RAS blockade lowers blood pressure, decreases IR and fasting plasma glucose (FPG). Glucagon-like peptide 1 (GLP-1), has been reported to have natriuretic effects as well as blood glucose lowering effects, however, these findings are still controversial. To assess the contributions of GLP-1 receptor and angiotensin receptor type 1(AT1) activation on renal Na + handling, systolic blood pressure (SBP) and FPG, metabolic measurements were taken at days 0, 2, 7, and every week thereafter for six weeks, SBP was measured daily using radio telemetry in five groups of rats: 1) untreated, lean LETO (n=7), 2) untreated, obese OLETF (n=5), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan /kg/d; n=4), 4) OLETF + GLP-1 mimetic (Ex; 10 ug exenatide/kg/d; n=7), and 5) OLETF + ARB + Exenatide (combo; n=6). FPG increased (101 ± 4 vs 131 ± 3 mg/dL) in OLETF compared to LETO, and ARB treatment decreased it (120 ± 2 mg/dL); however, the changes in Ex and combo groups were not significant. OLETF rats exhibited elevated SBP at the onset of the study compared to LETO (119 ± 1 vs. 130 ± 1 mmHg). U Na V decreased (1.07 ± 0.07 vs 0.43 ± 0.14 &amp; 0.31 ± 0.11 mmol/day) in Ex and combo groups on day 2 compared to OLETF, and the decrease in U Na V in Ex was associated with increased (132 ± 1 vs 142 ± 1 mmHg) SBP. However, reduced U Na V in combo group was associated with reduced (114 ± 3 mmHg) SBP suggesting that activation of AT1 has a greater contribution to the insulin resistance-associated increase in SBP than the impaired U Na V. After this initial increase in SBP in Ex, it decreased to a nadir (124 ± 2 mmHg) at day 11 of treatment before increasing to 130 for the duration of the study. After six weeks of treatment U Na V increased (1.02 ± 0.05 vs 1.35 ± 0.09 mmol/day) only in combo group compared to OLETF. The results suggest that chronic GLP-1 receptor activation acutely increases SBP by decreasing U Na V, but co-treatment with an ARB abolishes this effect. These results also suggest that despite the lack of improved FBG, chronic GLP-1 receptor activation may protect against the development of high blood pressure associated with insulin resistance independent of increased U Na V.

제 2형 당뇨모델 OLETF 랫드에서 동충하초의 항당뇨 효과

본 연구는 동충하초(Isaria tenuipes)를 유전적 당뇨 질환 동물모델인 OLETF 랫드에서 4주간 경구 투여하여 항당뇨 효과에 대한 연구결과로써, OLETF 랫드 동충하초(Isaria tenuipes) 투여군은 당뇨대조군과 비교하여 투여 4주에는 사료 섭취량과 체중이 증가하였다. 이는 동충하초(Isaria tenuipes)를 섭취한 OLETF 랫드는 고지방식 사료만 섭취한 OLETF 랫드에 비해 당뇨 유발이 개선된 결과로 판단된다. 혈중 포도당 농도, 인슐린 농도 및 인슐린 저항성 지표인 HOMA-IR 수치가 용량 의존적으로 감소하였다. 또한 GLUT4 유전자 발현을 측정한 결과 대조군에 비해 동충하초(Isaria tenuipes) 투여군의 mRNA 및 단백질이 용량 의존적으로 증가하는 경향을 보였다. 따라서, 동충하초(Isaria tenuipes)의 섭취가 제2형 당뇨에서 인슐린의 감수성이나 저항성에 영향을 미치고, 혈당을 강화시키는 것으로 사료되므로 당뇨병환자의 혈당저하에 기여할 것이라 기대한다. 이에 본 연구는 천연물질을 이용한 당뇨병의 예방 및 치료에 동충하초(Isaria tenuipes)를 사용할 수 있는 이론적 토대를 제공할 것으로 생각된다. We evaluated the anti-diabetic effects of Isaria tenuipes in diabetes mellitus type 2. For the experiments, the diabetic animal model OLETF rats were divided to 4 groups: Isaria tenuipes was administered mixed with the high fat diet 45% at dose levels of 0.0%, 0.1%, 1.0%, and 5.0% for 4 weeks. All animals have free access to water and high fat diet 45%. The diabetic clinical markers, including clinical signs, body weight and food intake, organ weights, blood glucose level, insulin level and HOMA-IR index, oral glucose tolerance test, GLUT4 mRNA and protein were measured at a time. After administration for 4 weeks, the blood glucose levels, insulin levels and HOMA-IR index of test groups were decreased compared with control group in dose-dependent manner. The body weight and diet consumptions were reduced in control group at 4 weeks. The treatments of Isaria tenuipes also showed high expression of GLUT4 mRNA and protein in the muscle of OLETF rats. The results suggest that Isaria tenuipes has anti-hyperglycemic effect attenuating blood glucose in the animal model of type 2 diabetes and might be useful as a functional diet for human diabetic diseases.

Retinal Neurodegeneration in Type II Diabetic Otsuka Long-Evans Tokushima Fatty Rats

We evaluated whether diabetes causes longitudinal thinning of retinal layers as a sign of retinal neurodegeneration in a type II diabetic animal model, the Otuska Long-Evans Tokushima fatty rats (OLETF), using spectral domain-optical coherence tomography (SD-OCT) and investigated the corresponding histology. Retinal thickness in OLETF and Long-Evans Tokushima Otsuka rats (LETO) was measured at 1300 μm from the center of the optic nerve head (ONH) at 12, 20, 28, and 36 weeks using SD-OCT. Total retinal thickness (TRT) was measured automatically by built-in software. The retinal nerve fiber layer (RNFL) and other layers' thicknesses were measured manually. At 36 weeks, eyes were processed for morphometric analysis and detection of apoptosis based on active caspase-3(+) and TUNEL(+) analysis. TRT was significantly thinner in OLETF at 28 and 36 weeks (236.26 ± 5.64 and 235.98 ± 5.42 μm, respectively) than in LETO (243.82 ± 7.36 and 239.58 ± 6.99 μm, respectively; P = 0.042 and P = 0.01). At 28 weeks, RNFL thickness was significantly lower in OLETF (21.52 ± 1.91 μm) than in LETO (24.75 ± 2.10 μm, P = 0.042). The change of TRT correlated significantly with change of RNFL thickness. At 36 weeks, OLETF (24.0 ± 3.1/cross-section) had significantly fewer ganglion cells than LETO (28.4 ± 6.7/cross-section, P = 0.028). Active caspase-3(+) and TUNEL(+) cells in RNFL were observed significantly more frequently in OLETF (6.8 ± 5.2/cross-section and 3.75 ± 0.96/cross-section, respectively) than in LETO (1.5 ± 2.3/cross-section, P = 0.008 and 1.0 ± 0.8/cross-section, P = 0.029, respectively). OLETF exhibited a significantly reduced TRT, especially RNFL thickness, based on SD-OCT. Further, histology revealed increased apoptosis and a decrease in the number of ganglion cells. These results suggest that retinal neurodegeneration occurs in type II diabetic OLETF.

Abstract 188: Immobilization Stress Exacerbates Insulin Resistance and Endothelial Dysfunction via Inhibition of Insulin-Stimulated eNOS Activation and via Enhancement of NF-kB Activation by Corticotropin-Releasing Hormone

Objectives Psychological stress may contribute to the development of atherosclerosis and insulin resistance. However, its mechanism is poorly understood. We studied if stress induces insulin resistance and endothelial dysfunction, and assessed the role of stress-associated corticotropin-releasing hormone (CRH) in inflammation and insulin signaling pathways. Methods and Results Immobilization stress (IS) was applied (2 hr/d) for 14 d to male 8-wk (YI2O: before DM phenotype) and 21-wk (EI2O: DM phenotype) OLETF rats, whereas control groups comprised of rats without IS with same age (YCO and ECO, respectively; n=7~10 for each group). Systolic blood pressure was significantly increased after 14d-IS in YI2O and EI2O, whereas it was not changed in control groups. Acetylcholine-induced, but not nitroprusside-induced, vasorelaxation, measured in superior mesenteric artery, was significantly decreased by 14d IS compared with each control groups, suggesting that IS induced endothelial dysfunction. 14d-IS increased plasma insulin (342.5±6.3 vs 496±51.6 pg/ml) and HOMA-IR (3.1±0.2 vs 4.7±0.5 mmol/LxμIU/mL) in 8-wk OLETF rats (control vs stress, all p &lt;0.05). Western blot analysis using thoracic aorta showed 14d-IS increased expression of Rho-associated kinase-1 and p22 phox subunit of NAD(P)H oxidase compared with control groups. eNOS phosphorylation at Ser 1177 was increased by treatment with insulin (2nM) in cultured HUVEC cells, but this was blocked by pretreatment with CRH (100 nM). On the contrary, an increase in ERK phosphorylation by insulin treatment was further enhanced by CRH pretreatment in HUVEC cells. CRH also induced translocation of NF-kB subunit p65 into nuclear fraction in monocyte cell line U937 cells. Conclusions Immobilization stress exacerbates insulin resistance and endothelial dysfunction in a prediabetic animal model at least partly via inhibition of insulin-mediated eNOS activation and via enhancement of inflammation through ERK pathway and NF-kB activation by CRH.

Voluntary exercise at young stage prevents glucose intolerance in type 2 diabetic rats

PurposeTo investigate the effects of voluntary exercise at young stage on whole‐body metabolism and glucose tolerance in type 2 diabetic rats.MethodsOtsuka Long‐Evans Tokushima Fatty (OLETF) rats as an animal model of type 2 diabetes and their counterparts (Long‐Evans Tokushima Otsuka; LETO) were used. Twenty male OLETF and LETO rats were divided into three groups, respectively: Sedentary (SED, n=8), Wheel Running (WR, n=6), or Wheel Running‐Sedentary (WS, n=6) group. WR and WS groups were allowed to access a running wheel freely from 5 to 25 week‐old. In WS group, however, a running wheel was locked from 15 to 25 week‐old and rats could not run in the wheel. At 25 week‐old, resting oxygen consumption (VO2 rest) of each rat was measured in a metabolic chamber. Subsequently an intraperitoneal glucose tolerance test (IPGTT) was performed.ResultsThere were no significant differences in the areas under the curve of IPGTT (AUC) and VO2 rest (ml/kg/min) among three LETO groups. In OLETF rats, AUC in WR and WS groups were significantly lower than SED group, with AUC being lower for WR group. Moreover, VO2 rest of OLETF in WR and WS groups were significantly higher than SED group.ConclusionOur data suggest that voluntary exercise at young stage improves resting metabolism and prevents glucose intolerance in grown‐up type 2 diabetic rats, and these effects remained following exercise cessation.