Evans Tokushima Fatty Rats Research Articles

Effects of CBD on the Phosphorylation of Cardiac Insulin Receptor in Rat Model of Metabolic Syndrome

Metabolic syndrome (MetS) comprises a cluster of conditions that include obesity, insulin resistance, dyslipidemia, hypertension, pro-inflammation, and pro-thrombosis. In the United States, approximately one in three adults is afflicted with MetS. If left unmanaged, it can lead to type 2 diabetes (T2D), which would increase the risk of cardiovascular diseases (CVD). The insulin signaling pathway is critical to regulate glucose homeostasis in the body. During insulin resistance, the pathway is downregulated, and glucose remains in the bloodstream leading to a hyperglycemic state. Recent studies on the benefits of cannabidiol (CBD) have shown it can mitigate CVD risk factors, but its impact on glucose metabolism within the heart remains unclear. To investigate this, the Otsuka Long Evans Tokushima Fatty (OLETF) rat, a model of MetS, and its healthy strain-control, the Long Evans Tokushima Otsuka (LETO) rat, were used. Rats were assigned to three groups (n=8/group): (1) untreated LETO, (2) untreated OLETF, and (3) OLETF + H4CBD (200mg/kg/day x 4 weeks). CBD treatment did not impact the nascent insulin receptor nor the nascent protein kinase B, but the effects of CBD on the phosphorylation and activation of these insulin signaling components have yet to be determined (analyses ongoing). Because CBD has been shown to improve glucose homeostasis previously, we anticipate it is by increasing the activation by phosphorylation of the insulin receptor. A better examination of the effects of CBD on the insulin signaling pathway in the heart will provide greater insight on its therapeutic potential during diabetic cardiomyopathy. USDA Hispanic Education Training Program, Center for Medical Cannabis Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exogenous T4 Increased Cardiac Thioredoxin Protein Expression in Insulin Resistant OLETF Rats

Thioredoxin (Trx) is a protein that regulates cellular signaling and antioxidant defense. Cellular antioxidant mechanisms are designed to protect cells from oxidative damage caused by inappropriately elevated reactive oxygen species (ROS) and other free radicals. Oxidative damage can lead to cellular damage and impaired function. Diabetic cardiomyopathy (CDM) is associated with impaired redox balance and oxidative injury. Altered fuel metabolism is a key feature of CDM characterized by an imbalance in fatty acid and glucose utilization, favoring fatty acid overutilization. Excessive fatty acid beta-oxidation in the mitochondria leads to generation of free radicals including superoxide, which can contribute to oxidative injury. Thyroid hormones regulate substrate utilization and their dysregulation may contribute to oxidative injury. Therefore, we hypothesized that exogenous thyroxine (exoT4) upregulates Trx protein expression and ameliorate oxidative injury in the hearts of insulin resistant rats. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exoT4 on heart Trx, oxidative damage, and glucose metabolism. Rats were assigned to four groups: (1) lean, control Long Evans Tokushima Otsuka (LETO; n=6), (2) LETO + T4 (8 μg/100g BM/d × 5 wks; n=7), (3) untreated, insulin resistant OLETF (n=6), and (4) OLETF + T4 (n=7). OLETF group displayed a 31% decreased in TRX compared to Leto. T4 increased Trx protein expression by 88% in OLTEF compared to untreated OLETF. This data suggests that exoT4 has the potential to upregulate cardiac Trx-dependent antioxidant mechanisms in insulin resistant OLETF rats to ameliorate oxidative injury and improve cardiac glucose metabolism. This work is supported by the American Heart Association and the Undergraduate Research Opportunity Center (UROC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exogenous Thyroxine Increases Cardiac AS160 Phosphorylation in Insulin Resistant OLETF Rats

Thyroxine (T4) is the most abundant thyroid hormone secreted by thyroid gland and contributes to glucose metabolism via both insulin and non-insulin dependent pathways. Akt substrate 160 (AS160) is a Rab-GDP activating protein and a downstream effector of protein kinase B (Akt) required for glucose transport and upregulation of GLUT4 activating vesicle (GSV). The phosphorylation of AS160 (p-AS160) is required for the regulation of GLUT4 translocation to the membrane. It is also involved in insulin-mediated traffcking of GLUT4 in skeletal muscle and adipose tissues. The accumulation of AS160 has also been implicated in promoting cardiac hypertrophy, which is commonly associated with the metabolic syndrome (MetS). This study explores how thyroid hormones can affect the phosphorylation status of cardiac AS160 during insulin resistance. Under normal conditions, AS160 is phosphorylated by Akt to upregulate GSV, allowing glucose to cross the cell membrane through GLUT4. We hypothesize that exogenous T4 increases cardiac phosphorylation of AS160 expression during insulin resistance. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on cardiac p-AS160. Rats were assigned to four groups: (1) lean, Long Evans Tokushima Otsuka (LETO; n=6), (2) LETO + T4 (8 μg/100g BM/d × 5 wks; n=7), (3) untreated OLETF (n=6), and (4) OLETF + T4 (n=7). Exogenous T4 did not significantly alter native AS160 protein expression among all four groups. However, p-AS160 was reduced in the non-treated OLETF group by 61% (p<0.01) compared to healthy LETO control group. Exogenous T4 increased p-AS160 by 16% (p<0.05) compared to non-treated OLETF group and by 36% (p<0.05) in treated LETO compared to control LETO group. We have previously demonstrated that exogenous T4 increased the translocation of cardiac GLUT4 and the present findings suggest that this increased translocation is mediated by activation of AS160, resulting in improved glucose metabolism in the heart during insulin resistance. American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exogenous Thyroxine Normalizes Decreased Heart Rate in Insulin Resistant OLETF Rats

Diabetic cardiomyopathy (DCM) is defined as heart failure in the absence of overt clinical coronary artery disease, valvular disease, hypertension, and dyslipidemia in diabetic patients. Patients with T2DM display left ventricular dysfunction, fibrosis, and abnormal cell signaling leading to diastolic and systolic dysfunction. Increased left ventricular mass and fibrosis potentially leads to abnormal diastolic function. Substantial data exists demonstrating both insulin-dependent and non-insulin dependent diabetes impairs left ventricular function. During insulin resistance, a metabolic shift occurs favoring fatty acid metabolism at the expense of impaired glucose metabolism, leading to lipotoxicity, increased oxidative stress, and inflammation. Thyroid hormones have shown to be cardioprotective. We have previously shown exogenous thyroxine treatment increased GLUT4 translocation and nuclear factor erythroid 2-related factor 2 (Nrf2) in cardiac tissue, demonstrating the potential to improve glucose metabolism and increase antioxidant defense mechanisms. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on left ventricular cardiac function and hemodynamics to assess if the previously observed improvements in cellular metabolism translated to functional improvements. Rats were assigned to three groups: (1) lean, Long Evans Tokushima Otsuka (LETO; n=7), (2) untreated OLETF (n=6), and (3) OLETF + T4 (8 μg/100g BM/d × 6 wks, n=7). Left ventricular function was measured by the pressure-volume system. Heart mass increased in untreated OLETF (1.46 ± 0.08) and treated OLETF (1.61 ± 0.16) compared to LETO (1.19 ± 0.08). Cardiac output (CO) and stroke volume were 26% and 56% greater in untreated OLETF compared to LETO. Heart rate (HR) was 27% lesser in untreated OLETF compared to LETO. While exogenous T4 did not alter CO and SV compared to untreated OLETF, it did normalize HR to LETO levels. The differences in CO between LETO and OLETF suggest that elevated T4 alters the mechanisms (SV vs HR) by which CO is elevated during MetS. The changes may be compensatory to accommodate the cardiac hypertrophy observed in the strain. American Heart Association Award # 946746 APS Porter Physiology Development Fellow (2nd year). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exogenous thyroxine increases cardiac GLUT4 translocation in insulin resistant OLETF rats

During insulin resistance, the heart undergoes a metabolic shift in which fatty acids (FA) account for roughly about 99% of the ATP production. This metabolic shift is indicative of impaired glucose metabolism. A shift in FA metabolism with impaired glucose tolerance can increase reactive oxygen species (ROS), lipotoxicity, and mitochondrial dysfunction, ultimately leading to cardiomyopathy. Thyroid hormones (TH) may improve the glucose intolerance by increasing glucose reabsorption and metabolism in peripheral tissues, but little is known on its effects on cardiac tissue during insulin resistance. In the present study, insulin resistant Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on glucose metabolism in cardiac tissue. Rats were assigned to four groups: (1) lean, Long Evans Tokushima Otsuka (LETO; n=6), (2) LETO + T4 (8 μg/100 g BM/d × 5 wks; n = 7), (3) untreated OLETF (n = 6), and (4) OLETF + T4 (8 μg/100 g BM/d × 5 wks; n = 7). T4 increased GLUT4 gene expression by 85% in OLETF and increased GLUT4 protein translocation to the membrane by 294%. Additionally, T4 increased p-AS160 by 285%, phosphofructokinase-1 (PFK-1) mRNA, the rate limiting step in glycolysis, by 98% and hexokinase II by 64% in OLETF. T4 decreased both CPT2 mRNA and protein expression in OLETF. The results suggest that exogenous T4 has the potential to increase glucose uptake and metabolism while simultaneously reducing fatty acid transport in the heart of insulin resistant rats. Thus, L-thyroxine may have therapeutic value to help correct the impaired substrate metabolism associated with diabetic cardiomyopathy.

Effects of Diabetes and Voluntary Exercise on IgA Concentration and Polymeric Immunoglobulin Receptor Expression in the Submandibular Gland of Rats.

Background and Objectives: Patients with diabetes are more susceptible to upper respiratory tract infections (URTIs) because they are easily infected. Salivary IgA (sali-IgA) levels play a major role in transmitting URTIs. Sali-IgA levels are determined by salivary gland IgA production and polymeric immunoglobulin receptor (poly-IgR) expression. However, it is unknown whether salivary gland IgA production and poly-IgR expression are decreased in patients with diabetes. While exercise is reported to increase or decrease the sali-IgA levels, it is unclear how exercise affects the salivary glands of patients with diabetes. This study aimed to determine the effects of diabetes and voluntary exercise on IgA production and poly-IgR expression in the salivary glands of diabetic rats. Materials and Methods: Ten spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats (eight-week-old) were divided into two groups of five rats each: a non-exercise group (OLETF-C) and a voluntary wheel-running group (OLETF-E). Five Long-Evans Tokushima Otsuka (LETO) rats without diabetes were bred under the same conditions as the OLETF-C. Sixteen weeks after the study began, the submandibular glands (SGs) were collected and analyzed for IgA and poly-IgR expression levels. Results: IgA concentrations and poly-IgR expression levels in SGs were lower in OLETF-C and OLETF-E than in LETO (p < 0.05). These values did not differ between the OLETF-C and OLETF-E. Conclusions: Diabetes decreases IgA production and poly-IgR expression in the salivary glands of rats. Moreover, voluntary exercise increases sali-IgA levels but does not increase IgA production and poly-IgR expression in the salivary glands of diabetic rats. Increasing IgA production and poly-IgR expression in the salivary glands, which is reduced in diabetes, might require slightly higher-intensity exercise than voluntary exercise under the supervision of a doctor.

PS-BPC06-5: EXOGENOUS THYROXINE INCREASES GLUT4 TRANSLOCATION IN MEMBRANE AND IMPROVES CARDIAC METABOLISM INDEPENDENT OF REDUCED SBP IN INSULIN RESISTANT OLETF RATS

Objective: During insulin resistance the heart undergoes a metabolic shift where fatty acids (FA) account for about 99% of the ATP production, indicative of impaired glucose metabolism. This results in increased reactive oxygen species and lipotoxicity leading to impaired mitochondrial function. Thyroid disorders such as hyperthyroidism and hypothyroidism both have the potential to lead to hypertension. Thyroid hormones (TH) may improve glucose intolerance by increasing glucose reabsorption and metabolism and therefore improve cardiac function and lower the risk of developing hypertension. However, incongruent data exists showing that TH promote oxidative stress and cardiac hypertrophy. Thus, the beneficial effects of TH in the heart during insulin resistance remains controversial. The glucose transport protein, GLUT4, is a downstream target of the TH response element indicating a possible mechanism by which TH regulate glucose metabolism. Design: Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on glucose metabolism in cardiac tissue. Rats were assigned to four groups: 1) lean, Long Evans Tokushima Otsuka (LETO; n = 6), 2) LETO + T4 (8 μg/100 g BM/d × 5 wks; n = 7), 3) untreated OLETF (n = 6), and 4) OLETF + T4 (n = 7). T4 was administered via osmotic minipumps implanted subcutaneously. Systolic blood pressure (SBP) was measured weekly in conscious rats by tail-cuff plethysmography. Animals were provided water and food ad libitum. Results: SBP increased by 15% in OLETF compared to lean LETO (138 ± 2 vs. 117 ± 1 mm Hg), but T4 did not alter SBP is OLETF strain. T4 increased GLUT4 gene expression by 85% and GLUT4 protein translocation to the membrane by 328% in OLETF. Additionally, T4 increased phosphofructokinase-1, the rate limiting step in glycolysis, by 98% and decreased CD36 gene expression for FA transport by 20% in OLETF. We expect our recent metabolomics and lipidomics data to help better explain these molecular changes. Conclusions: Five-week dose of exogenous T4 did not reduce SBP is OLETF. The results suggest that increased T4 has the potential to increase glucose reabsorption and metabolism in the heart of insulin resistant rats and restore impaired substrate metabolism, but these improvements in substrate metabolism do not translate to sustained reduction in arterial pressure.

Simultaneous SGLT2 inhibition and caloric restriction improves insulin resistance and kidney function in OLETF rats

SGLT2 inhibitors (SGLT2i) are emerging as a novel therapy for type 2 diabetes due to their effective hypoglycemic and potential cardio- and nephroprotective effects, while caloric restriction (CR) is a common behavioral modification to improve adiposity and insulin resistance. Therefore, both interventions simultaneously may potentially further improve metabolic syndrome by enhancing carbohydrate metabolism. To test this hypothesis, cohorts of 10-week old, male Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were treated with SGLT2i (10 mg luseoglifozin/kg/day x 4 wks) (OLETF only) and/or 30% CR (2 wks at 12 weeks of age). CR maintained body mass in both strains while SGLT2i alone did not have any effect on body mass. Simultaneous treatments decreased SBP in OLETF vs SGLT2i alone, decreased insulin resistance index (IRI), and increased creatinine clearance vs OLETF ad lib. Conversely, CR decreased albuminuria independent of SGLT2i. In conclusion, SGLT2i treatment by itself did not elicit significant improvements in insulin resistance, kidney function or blood pressure. However, when combined with CR, these changes where more profound than with CR alone without inducing chronic hypoglycemia.

Exogenous thyroxine increases GLUT4 translocation to the membrane in cardiomyocytes in insulin resistant OLETF rats Dora A Mendez1, Jose G. Soñanez‐Organis2, Guillermo Vasquez‐Anaya1, Daisuke Nakano3, Akira Nishiyama3, Rudy M Ortiz11Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA, USA 2Division of Science and Engineering, Department of Chemical Biological and Agropecuary Sciences, University of Sonora,

During insulin resistance the heart undergoes a metabolic shift in which fatty acids (FA) accounts for about 99% of the ATP production. This metabolic shift is indicative of impaired glucose metabolism. A shift in FA metabolism with impaired glucose tolerance can cause an increase in reactive oxygen species (ROS) and lipotoxicity ultimately leading to impaired mitochondrial function. Thyroid hormones (TH) may improve glucose intolerance by increasing glucose reabsorption and metabolism in peripheral tissues but its effects on cardiac tissue during insulin resistance is not well known. The glucose transport protein, GLUT4, is a downstream target of the TH response element (TRE) indicating a possible mechanism by which THs regulate glucose metabolism. Non‐classical TH signaling may also phosphorylate protein kinase B (Akt) and increase activity of phosphoinositide‐3‐kinase (PI3K), which are two key regulators in insulin‐mediated glucose uptake. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on glucose metabolism in cardiac tissue. Rats were assigned to four groups: 1) lean, Long Evans Tokushima Otsuka (LETO; n=6), 2) LETO + T4 (8 μg/100g BM/d × 5 wks; n=7), 3) untreated OLETF (n=6), and 4) OLETF + T4 (n=7). T4 increased GLUT4 gene expression by 85% (p&lt;0.05) in OLETF and increased GLUT4 protein translocation to the membrane by 328% (p&lt;0.05). Additionally, T4 increased phosphofructokinase‐1 (PFK‐1) gene expression, the rate limiting step in glycolysis, by 98% (p&lt;0.05) in OLETF. T4 increased AKT and p‐AKT by 177% and 134% (p&lt;0.05), respectively, in LETO, but not in OLETF. The results suggest that increased T4 has the potential to increase glucose reabsorption and metabolism in the heart of insulin resistant rats to help restore impaired substrate metabolism.

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

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

The Effect of Resistance Exercise on Myocardial Inflammatory Cytokines and ERK1/2 Expression in Type 2 Diabetic Rats

PURPOSE: The purpose of this study was to investigate the effect of resistance ladder exercise on myocardial inflammatory cytokines and extracellular signal regulated kinase (ERK)1/2 expression in type 2 diabetic rats.METHODS: A total of 21 8-week-old male Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were divided into three groups: LETO group (CON, n=7), OLETF group (DM, n=7), and resistance exercise group (DM+EXE, n=7). Resistance exercise referred to a climbing exercise on a slope. The rats performed this exercise 3 full d per week, for 8 week. The expression of myocardial inflammatory cytokines and ERK1/2 was analyzed via western blotting after 8 week of exercise intervention.RESULTS: After 8 weeks of resistance exercise for the management of type 2 diabetes, the production of anti-inflammatory factor Interleukin (IL)-10 in the myocardium increased. This inhibited the production of pro-inflammatory factors IL-6 and tumor necrosis factor-alpha. However, resistance exercise did not affect the expression of ERK1 and ERK2, which are apoptosis regulatory proteins.CONCLUSIONS: In type 2 diabetes, resistance exercise is thought to be effective in reducing inflammatory factors by mediating cytokines. Although the effects of resistance ladder exercise were confirmed, further studies on a variety of exercise types and intensities should be conducted to prevent and improve the conditions of those with diabetes.

In Situ Gel Incorporating Disulfiram Nanoparticles Rescues the Retinal Dysfunction via ATP Collapse in Otsuka Long-Evans Tokushima Fatty Rats.

We attempted to design an ophthalmic in situ gel formulation incorporating disulfiram (DIS) nanoparticles (Dis-NPs/ISG) and demonstrated the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in 15-month-old Otsuka Long–Evans Tokushima Fatty (OLETF) rats, a rat model of diabetes. The DIS particles were crushed using a bead mill to prepare the nanoparticles, and the Dis-NPs/ISG was prepared using a combination of the DIS nanoparticles and an in situ gelling system based on methylcellulose (MC). The particle size of the Dis-NPs/ISG was 80–250 nm, and there was no detectable precipitation or aggregation for 1 month. Moreover, the Dis-NPs/ISG was gelled at 37 °C, and the drug was delivered into the retina by instillation. Only diethyldithiocarbamate (DDC) was detected in the retina (DIS was not detected) when the Dis-NPs/ISG was instilled in the right eye, and the DDC levels in the right retina were significantly higher than those in the left retina. In addition, the retinal residence time of the drug was prolonged by the application of the in situ gelling system, since the DDC levels in the retinas of rats instilled with Dis-NPs/ISG were higher than those in DIS nanoparticles without MC. Furthermore, repetitive instillation of the Dis-NPs/ISG attenuated the deterioration of electroretinograms (ERGs) in 15-month-old OLETF rats by preventing the collapse of ATP production via excessive nitric oxide and recovered the decrease in retinal function. These findings provide important information for the development of novel therapeutic approaches to diabetic retinopathy.

Partial Body Mass Recovery After Caloric Restriction Abolishes Improved Glucose Tolerance in Obese, Insulin Resistant Rats.

Caloric restriction, among other behavioral interventions, has demonstrated benefits on improving glycemic control in obesity-associated diabetic subjects. However, an acute and severe intervention without proper maintenance could reverse the initial benefits, with additional metabolic derangements. To assess the effects of an acute caloric restriction in a metabolic syndrome model, a cohort of 15-week old Long Evans Tokushima Otsuka (LETO) and Otsuka Long Evans Tokushima Fatty (OLETF) rats were calorie restricted (CR: 50% × 10 days) with or without a 10-day body mass (BM) recovery period, along with their respective ad libitum controls. An oral glucose tolerance test (oGTT) was performed after CR and BM recovery. Both strains had higher rates of mass gain during recovery vs. ad lib controls; however, the regain was partial (ca. 50% of ad lib controls) over the measurement period. Retroperitoneal and epididymal adipose masses decreased 30% (8.8 g, P < 0.001) in OLETF; however, this loss only accounted for 11.5% of the total BM loss. CR decreased blood glucose AUC 16% in LETO and 19% in OLETF, without significant decreases in insulin. Following CR, hepatic expression of the gluconeogenic enzyme, PEPCK, was reduced 55% in OLETF compared to LETO, and plasma triglycerides (TG) decreased 86%. Acute CR induced improvements in glucose tolerance and TG suggestive of improvements in metabolism; however, partial recovery of BM following CR abolished the improvement in glucose tolerance. The present study highlights the importance of proper maintenance of BM after CR as only partial recovery of the lost BM reversed benefits of the initial mass loss.

Inhibition of Ceramide Accumulation in Podocytes by Myriocin Prevents Diabetic Nephropathy.

BackgroundCeramides are associated with metabolic complications including diabetic nephropathy in patients with diabetes. Recent studies have reported that podocytes play a pivotal role in the progression of diabetic nephropathy. Also, mitochondrial dysfunction is known to be an early event in podocyte injury. Thus, we tested the hypothesis that ceramide accumulation in podocytes induces mitochondrial damage through reactive oxygen species (ROS) production in patients with diabetic nephropathy.MethodsWe used Otsuka Long Evans Tokushima Fatty (OLETF) rats and high-fat diet (HFD)-fed mice. We fed the animals either a control- or a myriocin-containing diet to evaluate the effects of the ceramide. Also, we assessed the effects of ceramide on intracellular ROS generation and on podocyte autophagy in cultured podocytes.ResultsOLETF rats and HFD-fed mice showed albuminuria, histologic features of diabetic nephropathy, and podocyte injury, whereas myriocin treatment effectively treated these abnormalities. Cultured podocytes exposed to agents predicted to be risk factors (high glucose, high free fatty acid, and angiotensin II in combination [GFA]) showed an increase in ceramide accumulation and ROS generation in podocyte mitochondria. Pretreatment with myriocin reversed GFA-induced mitochondrial ROS generation and prevented cell death. Myriocin-pretreated cells were protected from GFA-induced disruption of mitochondrial integrity.ConclusionWe showed that mitochondrial ceramide accumulation may result in podocyte damage through ROS production. Therefore, this signaling pathway could become a pharmacological target to abate the development of diabetic kidney disease.

제 2형 당뇨 쥐에서 저항성 운동과 커큐민 섭취가 대장 조직 내 비만 및 염증 관련 인자의 mRNA 발현에 미치는 영향

The purpose of this study was to investigate the effects of resistance exercise and intake of curcumin on mRNA expression of obesity and inflammation-related factors in large intestine tissue of rats with type 2 diabetes mellitus. Thirty-two otsuka long evans tokushima fatty (OLETF) rats were randomly allocated to the control group (CON, n=8), the diabetes group (D, n=8), the exercise+diabetes group (Ex+D, n=8), and the exercise+diabetes+curcumin group (Ex+D+C, n=8). Resistance exercise involved climbing a ladder for 30 minutes per session, 3 times a day for 8 weeks. Curcumin (100 mg/kg/day) was consumed 30 minutes before the exercise. The results indicated that the degree of mRNA expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) and leptin, which are obesity-related factors in large intestine tissue, significantly increased in the D group compared to those in the CON group. However, the Ex+D and Ex+D+C groups showed a significant decrease in those values in comparison to the D group. In addition, the inflammation-related factors, such as interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and chemokine ligand 20 (CCL20) also showed substantial decreases in their mRNA expression in the Ex+D and Ex+D+C groups compared to those in the D group. The level of mRNA expression of chemokine ligand 19 (CCL19) was significantly low in the Ex+D+C group when compared to that in the D group. The results of this study suggest that resistance exercise and concurrent intake of curcumin have a positive influence on reduction of the level of obesity and inflammation-related factors in large intestine tissue of rats with type 2 diabetes mellitus.

OR04-5 Stimulation of Protein Disulfide Isomerase Activity by Activation of The Renin-Angiotensin System

Understanding the molecular and cellular mechanisms that are affected by activation of the renin-angiotensin system (RAS) are important for development of novel therapeutic strategies to more specifically treat hypertension and reduce cardiovascular risk. RAS regulates blood pressure (BP) in part via its effector molecule, angiotensin II (AngII), a potent vasopressor that stimulates the production of reactive oxygen species (ROS). Protein disulfide isomerase (PDI) is a multifunctional oxidoreductase that catalyzes thiol/disulfide interchange reactions within the endoplasmic reticulum and regulates ROS production. Also, PDI is present in circulation and at the cell surface of platelets, lymphocytes, erythrocytes and endothelial cells and plays a critical role in initiating thrombus formation. However, the interaction of RAS and PDI remain unclear. We hypothesized that RAS activation would lead to increased PDI levels thus contributing to oxidative stress. First, we studied the in vitro effects of AngII on EA.hy926, a human endothelial cell line, and measured PDI activity. Our results show that AngII dose and time dependently increased extracellular PDI activity (p<0.05, n=6); an event that was reduced to baseline levels by preincubation with 0.5 uM losartan, an AngII type 1 receptor antagonist (ARB). Consistent with these results, AngII stimulated: 1) a 3-fold rise of extracellular PDI levels as determined by ELISA (p<0.05, n=4); 2) a 2.5-fold increase in PDI mRNA levels by RT-PCR (p<0.05, n=4); and 3) increased PDI protein levels by western blot analyses that were blocked by losartan. We also studied the effect of AngII on extracellular PDI activity in differentiated HL-60 cells, a human polymorphonuclear neutrophil cell line, and observed significantly increased extracellular PDI activity following incubation with AngII (p<0.05, n=4). We then characterized the in vivo effects of exogenous AngII infusion on PDI in Sprague-Dawley rats, a model of increased BP, inflammation and target organ damage. We studied three conditions: (1) control; (2) AngII infused (80 ng/min x 28 days); (3) AngII + ARB (10 mg losartan/kg/day x 21 days). AngII infusion led to significant increases in plasma PDI levels that were reduced by 20% following ARB treatment (p<0.05, n=5/group). We then studied a model of naturally increased AngII and hypertension, the Otsuka Long Evans Tokushima Fatty rats (OLETF) and their strain controls that were randomly assigned to the following groups: (1) untreated strain controls; (2) untreated OLETF; (3) OLETF + ARB. OLETF rats had increased BP and significantly greater circulating PDI activity than control rats that was likewise blocked by ARB treatment (p<0.05; n=6/group). Thus, RAS activation represents a novel mechanism for increased PDI levels and activity that may contribute to the deleterious effects of disordered RAS in cardiovascular disease.

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.

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.

Houttuynia cordata Facilitates Metformin on Ameliorating Insulin Resistance Associated with Gut Microbiota Alteration in OLETF Rats.

Metformin and Houttuynia cordata are representative anti-diabetic therapeutics in western and oriental medicine, respectively. The current study examined the synergistic anti-diabetic effect of Houttuynia cordata extraction (HCE) and metformin combination in Otsuka Long–Evans Tokushima Fatty (OLETF) rats. Fecal microbiota were analyzed by denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Combining HCE + metformin resulted in significantly ameliorated glucose tolerance (oral glucose tolerance test (OGTT))—the same as metformin alone. Particularly, results of the insulin tolerance test (ITT) showed that combining HCE + metformin dramatically improved insulin sensitivity as compared to metformin treatment alone. Both fecal and serum endotoxin, as well as cytokines (tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6)) were significantly ameliorated by HCE + metformin compared to metformin alone. Meanwhile, the activation of AMPK (adenosine monophosphate-activated protein kinase) by metformin was distinctly enhanced by HCE. Both of HCE and metformin evidently changed the gut microbiota composition, causing the alteration of bacterial metabolite, like short-chain fatty acids. H. cordata, together with metformin, exerts intensive sensibilization to insulin; the corresponding mechanisms are associated with alleviation of endotoxemia via regulation of gut microbiota, particularly Roseburia, Akkermansia, and Gram-negative bacterium.

Obesity and type 2 diabetes, not a diet high in fat, sucrose, and cholesterol, negatively impacts bone outcomes in the hyperphagic Otsuka Long Evans Tokushima Fatty rat

BackgroundObesity and type 2 diabetes (T2D) increase fracture risk; however, the association between obesity/T2D may be confounded by consumption of a diet high in fat, sucrose, and cholesterol (HFSC). ObjectiveThe study objective was to determine the main and interactive effects of obesity/T2D and a HFSC diet on bone outcomes using hyperphagic Otuska Long Evans Tokushima Fatty (OLETF) rats and normophagic Long Evans Tokushima Otsuka (LETO) controls. MethodsAt 8weeks of age, male OLETF and LETO rats were randomized to either a control (CON, 10 en% from fat as soybean oil) or HFSC (45 en% from fat as soybean oil/lard, 17 en% sucrose, and 1wt%) diet, resulting in four treatment groups. At 32weeks, total body bone mineral content (BMC) and density (BMD) and body composition were measured by dual-energy X-ray absorptiometry, followed by euthanasia and collection of blood and tibiae. Bone turnover markers and sclerostin were measured using ELISA. Trabecular microarchitecture of the proximal tibia and geometry of the tibia mid-diaphysis were measured using microcomputed tomography; whole-bone and tissue-level biomechanical properties were evaluated using torsional loading of the tibia. Two-factor ANOVA was used to determine main and interactive effects of diet (CON vs. HFSC) and obesity/T2D (OLETF vs. LETO) on bone outcomes. ResultsHyperphagic OLEFT rats had greater final body mass, body fat, and fasting glucose than normophagic LETO, with no effect of diet. Total body BMC and serum markers of bone formation were decreased, and bone resorption and sclerostin were increased in obese/T2D OLETF rats. Trabecular bone volume and microarchitecture were adversely affected by obesity/T2D, but not diet. Whole-bone and tissue-level biomechanical properties of the tibia were not affected by obesity/T2D; the HFSC diet improved biomechanical properties only in LETO rats. ConclusionsObesity/T2D, regardless of diet, negatively impacted the balance between bone formation and resorption and trabecular bone volume and microarchitecture in OLETF rats.