Male ZDF Rats Research Articles

Cardioprotective effect of cemtirestat treatment in male Zucker diabetic fatty rats: a pilot study

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Slovak Research and Development under the contract no. 21-0410 (APVV) and 20-0411 (APVV) and VEGA grant no. 2/0006/23 Introduction Type 2 Diabetes Mellitus (T2DM), the most common metabolic disorder in human population is caused by defective insulin secretion and reduced tissue responsiveness to insulin. T2DM is characterized by hyperglycaemia accompanied by oxidative stress and inflammation that results in organopathy. The Zucker diabetic fatty rats (ZDF) are established animal model for research of T2DM associated with obesity, and for efficacy of novel treatments. Myocardial electrical coupling protein, connexin43 (Cx43) is one of the key factors affecting heart function and arrhythmogenesis. We have previously shown upregulation of Cx43 in type 1 DM rats that was associated with reduced susceptibility to malignant arrhythmias. However, it is not known whether T2DM affects Cx43 expression and/or function in the heart as well as in brown adipose tissue (BAT). The purpose of our study was to examine expression of Cx43 in heart and BAT as well as the effect of treatment with highly selective and efficient aldose reductase inhibitor enriched with antioxidant activity, Cemtirestat. Methods: Forty 4-month-old male ZDF rats (fa/+ and fa/fa) were divided into 4 groups: 1) untreated control lean rats (fa/+), 2) control lean rats treated with Cemtirestat, 3) untreated diabetic fatty rats (fa/fa), 4) diabetic fatty rats treated with Cemtirestat (2,55 mg/kg/day). After 6 months of treatment, biometric parameters were registered and left ventricle (LV) + BAT were used for Western blot analysis of Cx43, PKCɛ (phosphorylates Cx43) and PKCδ (implicated in pro­apoptotic signaling). BAT and LV tissue sections were also used for immunolabeling of Cx43 to examine its topology. Results: Increase of body weight, heart weight, LV weight, adiposity, plasma triglycerides, cholesterol and blood glucose were registered in ZDF fa/fa rats. T2DM resulted in altered localisation of Cx43 that was not affected by treatment with Cemtirestat. Expression of Cx43 was significantly decreased in LV of fa/fa rats and restored with Cemtirestat. Moreover, BAT exhibited significantly lower expression of total Cx43 in fa/fa rats, that was moderately increased by Cemtirestat. The expression of PKCɛ was significantly decreased in both, LV and BAT of fa/fa rats with mild elevation of PKCɛ in LV of treated fa/fa rats. Cemtirestat slightly normalized increased expression of PKCδ in LV of fa/fa rats, but had no effect on lower PKCδ levels in BAT of fa/fa rats. Echocardiographic examination did not show significant difference between treated and untreated groups of rats and showed mild differences between diabetic and control rats. In conclusions, findings point out that T2DM downregulates Cx43 in the heart and BAT, however further studies are necessary for understanding the mechanisms implicated in every step in the development of T2DM cardiopathy and arrhythmogenesis as well as BAT alterations. Further research is required to determine factors involved in cardioprotective effects of Cemtirestat.

MP38-08 HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS AMELIORATE ERECTILE DYSFUNCTION IN TYPE I AND TYPE II DIABETES MELLITUS RATS THROUGH THE ATTENUATION OF FERROPTOSIS BY SLC7A11/GPX4/ACSL4 PATHWAY

You have accessJournal of UrologyCME1 May 2022MP38-08 HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS AMELIORATE ERECTILE DYSFUNCTION IN TYPE I AND TYPE II DIABETES MELLITUS RATS THROUGH THE ATTENUATION OF FERROPTOSIS BY SLC7A11/GPX4/ACSL4 PATHWAY Huan Feng, Tao Wang, and Jihong Liu Huan FengHuan Feng More articles by this author , Tao WangTao Wang More articles by this author , and Jihong LiuJihong Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002592.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Diabetic Erectile Dysfunction (DMED) is a common complication of diabetes, which seriously impairs the life of male patients of all ages. Diabetes could destroy various cells in the corpus cavernosum, such as nerve cells, endothelial cells, smooth muscle cells. Human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) are cells with multipotential differentiation potential.The present study aims to investigate the therapeutic role and mechanism of hUCMSCs on type I and type II diabetes mellitus rats. METHODS: Fifty male Sprague-Dawley rats received intraperitoneal injection with streptozotocin for 3 days to induce type 1 diabetes. Another 50 male ZDF rats were fed with Purina 5008 to induce type 2 DMED. After 8 weeks, type 1 and type 2 DMED rats were separately selected by the apomorphine (APO) test. Both of types DMED rats were divided into normal control group (NC), diabetic group (DM), cavernous injection (1×106) group (CI), tail vein injection (1×106) group (VI). Stem cells were labeled with Edu prior to injection for subsequent observation. Primary corpus cavernous smooth muscle cells (CCSMCs) from healthy rats were cultured and treated with berberine. Mitochondria in ccSMCs were observed by transmission electron microscopy (TEM). RESULTS: None of rats received stem cell injection had obvious adverse reactions. Injection of hUCMSCs improved erectile function, reduced levels of NADPH oxidases, ROS production and tissue iron content. Among the type 1 DMED rats, the results showed that mICP/MAP of DM (0.232±0.082 mmHg) was significantly lower than NC (0.793±0.153 mmHg), CI (0.333±0.104 mmHg) and VI (0.384±0.095 mmHg) were improved compared with DM, and similar phenomenon also showed among the type 2 DMED rats. Besides, co-cultured CCSMCs with hUCMSCs exhibited a lower level of ferroptosis by SLC7A11/GPX4/ACSL4 pathway with high glucose (HG) treatment. TEM showed that the mitochondria shrunk, the cristae of mitochondria were destroyed, and exhibited high electron density in CCSMCs treated with HG, and these effects could be obviously reversed by hUCMSCs. CONCLUSIONS: Human umbilical cord mesenchymal stem cells can safely and effectively ameliorate erectile dysfunction in both type I and type II diabetes mellitus rats, and attenuate ferroptosis of CCSMCs through SLC7A11/GPX4/ACSL4 pathway. It provides preclinical evidence for application of hUCMSCs to DMED treatment and potential molecular mechanism of it. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e621 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Huan Feng More articles by this author Tao Wang More articles by this author Jihong Liu More articles by this author Expand All Advertisement PDF DownloadLoading ...

Long-Term Diabetes Improvement After Duodenal Exclusion in Zucker Diabetic Fatty Rats Is Associated with Prevention of Strain-Specific Pancreatic Remodeling and Increased Beta Cell Proliferation.

BackgroundResponse to metabolic surgery is heterogeneous and the metabolic states that underpin weight loss and metabolic improvement are still unclear. In this study, we investigate parameters of post-bariatric fasting glucoregulation and leverage artificial intelligence-assisted whole-slide image analyses to characterize associated immunohistologic features of the pancreas.Materials and MethodsWe performed either loop duodeno-jejunostomy (DJOS) with exclusion of 1/3 of total intestinal length, loop duodeno-ileostomy with exclusion of 2/3 of total intestinal length (DiOS), or a sham operation on 8-week-old male obese ZDF rats. Six months post-operative, we measured blood metabolites and hormones. Subsequently, pancreatic and intestinal tissue was removed, formalin fixed, and paraffin embedded. Immunohistologic (IHC) analyses included proliferating cell nuclear antigen (PCNA) to visualize the proliferation fraction and pancreatic and duodenal homeobox 1 (PDX 1) as a measure of pancreatic cell differentiation. For IHC quantification, all slides were digitalized and analyzed using QuPath. All analyzed slides were reviewed by two independent pathologists for correctness.ResultsDJOS and DiOS were associated with preserved fasting insulin production compared to sham. Histopathologic evaluation showed significantly higher numbers of beta cells and specifically of clustered cell organization in DJOS and DiOS compared to sham. Cell proliferation (PCNA) was significantly elevated in DJOS and DiOS compared to sham.ConclusionIn this interventional model of bariatric surgery in severe genetic diabetes, we demonstrate post-operative histologic and immunohistologic features of the pancreas associated with improved fasting glucose homeostasis.Graphical abstract

Electroacupuncture improves glucose and lipid metabolism by regulating APN/AMPK/PPARα signaling of skeletal muscle in Zucker diabetic obese rats

To observe the effect of electroacupuncture (EA) on skeletal muscle adiponectin receptor (Adipor1) / adenylate activated protein kinase (AMPK) / peroxisome proliferator-activated receptor α (PPARα) signaling pathway and skeletal muscle morphology by the secretion of serum adiponectin in Zucker diabetic obese (ZDF) rats, so as to explore its mechanism underlying regulating glucose and lipid metabolism of type 2 diabetes mellitus (T2DM) and improving skeletal muscle insulin resistance (IR). Twelve male ZDF rats and six Zucker thin (ZL) rats were selected. The rats were fed with Purina#5008 high-fat diet for four weeks to induce T2DM model after adaptive feeding with normal diet for one week. The ZDF rats were randomly divided into model group and EA group according to blood glucose level after modeling and 6 ZL rats were used as the blank control group. Rats in the EA group were treated with "Pishu" (BL20), EA stimulation of "Yishu" (EX-B3), "Zusanli" (ST36) and "Sanyinjiao" (SP6), once a day and 6 times a week for 4 weeks. Rats of the model and blank control groups were grabbed and fixed in the same way as EA group. Fasting blood glucose (FBG) was measured before and after EA intervention. Serum levels of insulin (INS), C-peptide (C-P), adiponectin (APN) were measured by radioimmunoassay, and those of free fatty acid (FFA), low-density lipoprotein (LDL), cholesterol (TC), triglyceride (TG) content determined by enzyme colorimetry and insulin resistance index (HOMA-IR) was calculated. The protein expression levels of AdipoR1, AMPK and PPARα proteins in the quadriceps femoris tissues were detected by Western blot and histopathological changes of quadriceps femoris muscle were observed by H.E. staining. Compared with the blank control group, the levels of FBG, serum INS, C-P, FFA, LDL, TC, TG and HOMA-IR in the model group were significantly increased (P<0.01, P<0.05), while the levels of serum APN and the expressions of AdipoR1, AMPK and PPARα proteins in the skeletal muscle were significantly decreased (P<0.01, P<0.05). Compared with the model group, the levels of FBG, serum INS, C-P, FFA, LDL, TC and HOMA-IR in the EA group were significantly decreased (P<0.01), and those of serum APN and expression levels of AdipoR1, AMPK and PPARα proteins in the skeletal muscle significantly increased (P<0.01), but the serum TG level had no remarkable change in the EA group (P>0.05). In addition, H.E. staining showed disordered arrangement of skeletal muscle cells, rupture and fuzziness of muscle fibers, enlargement of the space between muscular fibers and infiltration of small number of adipose cells which were relatively milder in the EA group. EA can reduce blood glucose and lipid levels, and improve IR in ZDF rats, which may be related to its functions in up-regulating AdipoR1/AMPK/PPARα signaling and in promoting APN secretion.

A Serotonin 2A-Receptor Decoy Peptide Potently Lowers Blood Pressure in Male Zucker Diabetic, Fatty, Hypertensive Rats.

To test whether a novel 5-hydroxytryptamine 2A decoy receptor peptide, SN..8 (Sertuercept), administered via intraperitoneal injection, acutely lowers arterial blood pressure in obese, hypertensive male Zucker diabetic rats (ZDF). To examine the safety, tolerability and possible reno-protective effects following chronic alternate daily administration of Sertuercept (for 10 weeks) in the male ZDF rat. Systolic and diastolic blood pressure were determined at baseline and regular intervals for up to 48 hours after a single IP administration of either Sertuercept (2 mg/kg), vehicle (saline) or an identical concentration of a scrambled sequence of the decoy receptor peptide, LN…8, in male ZDF and Zucker lean rats using tail cuff plethysmography. Plasma autoantibodies were obtained in thirteen male ZDF rats for determination of 5-hydroxytryptamine 2A receptor-mediated neurotoxicity using an acute neurite retraction assay in mouse neuroblastoma cells. Rats were sacrificed at 25-weeks of age, the kidneys were perfused, fixed and sections were stained using Masson's trichrome for semi-quantitative determination of glomerular and interstitial fibrosis. Sertuercept (2 mg/kg IP) potently lowered systolic and diastolic blood pressure in both 11-week-old and 25-week-old male ZDF rats and in a subset of hypertensive Zucker lean rats. There was no significant blood pressure-lowering effect of vehicle (saline) or scrambled peptide sequence (LN.8). Blood pressure-lowering was rapid in onset (15-30 minutes following IP injection) and sustained for at least 24 hours. Alternate daily IP administration of 2 mg/kg dose of Sertuercept vs. scrambled peptide (for 10 weeks) was safe, well-tolerated and associated with a significant decrease in glomerulosclerosis in 25-week-old male ZDF rats. Plasma autoantibody-induced neurotoxicity correlated significantly with the global index of renal fibrosis severity in 25-week-old male ZDF rats. These data indicate potent arterial blood pressure-lowering efficacy from a decoy receptor peptide comprised of a second extracellular loop region of the human 5-hydroxytryptamine receptor. Chronic administration of the decoy receptor peptide (10 weeks) was safe, well-tolerated and protected against renal glomerulosclerosis in the male ZDF rat.

Imeglimin preserves islet β-cell mass in Type 2 diabetic ZDF rats.

ObjectivesType 2 diabetes (T2D) is driven by progressive dysfunction and loss of pancreatic β‐cell mass. Imeglimin is a first‐in‐class novel drug candidate that improves glycaemia and glucose‐stimulated insulin secretion in preclinical models and patients. Given evidence that imeglimin can attenuate β‐cell dysfunction and protect β cells in vitro, we postulated that imeglimin could also exert longer term effects to prevent pancreatic β‐cell death and preserve functional β‐cell mass in vivo.MethodsZucker diabetic fatty (ZDF) male rats were treated by oral gavage with imeglimin at a standard dose of 150 mg/kg or vehicle, twice daily for five weeks. At treatment completion, oral glucose tolerance tests were performed in fasted animals before a thorough histomorphometry and immunohistochemical analysis was conducted on pancreas tissue slices to assess cellular composition and disease status.ResultsImeglimin treatment significantly improved glucose‐stimulated insulin secretion (augmentation of the insulinogenic index) and improved glycaemia. Both basal insulinaemia and pancreatic insulin content were also increased by imeglimin. In ZDF control rats, islet structure was disordered with few β‐cells; after imeglimin treatment, islets appeared healthier with more normal morphology in association with a significant increase in insulin‐positive β‐cells. The increase in β‐cell mass was associated with a greater degree of β‐cell proliferation in the presence of reduced apoptosis. Unexpectedly, a decrease in as a α‐cell mass was also documented due to an apparent antiproliferative effect of imeglimin on this cell type.ConclusionIn male ZDF rats, chronic imeglimin treatment corrects a paramount component of type 2 diabetes progression: progressive loss of functional β‐cell mass. In addition, imeglimin may also moderate a‐cell turnover to further ameliorate hyperglycaemia. Cumulatively, these cellular effects suggest that imeglimin may provide for disease modifying effects to preserve functional β‐cell mass.

P734Research on the mechamism of renal sympathetic denervation in the treatment of insulin resistance

Abstract Background Insulin resistance is the main pathogenesis of type 2 diabetes. In order to better control the blood glucose of diabetic patients, the key is to improve the insulin sensitivity. With the in-depth study on the mechanism of insulin resistance, sympathetic nerve is an important part of it, but the specific mechanism is not clear. Objective This experiment aims to observe the effects of RDN on insulin resistance and skeletal muscle mitochondrial function in spontaneous diabetes ZDF rats. Methods 6 SPF healthy male ZDF (fa/+) rats were selected as control group, 12 healthy males ZDF (fa/fa) rats were randomly divided into sham group and RDN group. In the sham group and RDN group, the rats were treated with sham operation or bilateral renal sympathetic denervation respectively. Insulin sensitivity was tested in all rats with Hyperinsulinemic-euglycemic clamp test 2 weeks after RDN. The skeletal muscle ATPsyn, COXIV, UCP3 and SOD2 were tested by Western-blot method. Results The result showed that 2 weeks after RDN, the blood glucose level of the RDN group decreased significantly (P=0.000) when compared with the sham group. The glucose infusion rate (GIR) in sham and RDN group decreased significantly when compared with the control group (P=0.000). After RDN, the GIR in RDN group increased significantly. ATPsyn, COXIV which were the key enzyme of oxidative metabolism in skeletal muscle mitochondria decreased significantly in the sham and RDN group when compared with control group (P=0.001). However, parameters improved dramatically after RDN (P=0.001). Table 1. Comparision of Baseline in each group 2 weeks after RDN Control (n=6) Sham (n=6) RDN (n=6) Pa Pb Pc Weight (g) 311.1±8.5 410.8±10.7 377.9±15.3 0.000 0.001 0.002 BBG (mmol/L) 5.97±0.16 21.42±1.45 7.91±0.54 0.000 0.001 0.000 BISN (ng/mL) 100.67±4.18 162.17±3.13 134.67±2.50 0.000 0.001 0.001 TG (mmol/L) 2.71±0.28 6.90±0.24 4.88±0.23 0.000 0.001 0.002 TC (mmol/L) 2.38±0.17 6.98±0.15 5.40±0.09 0.000 0.000 0.000 HDL-C (mmol/L) 7.92±0.44 2.95±0.22 5.35±0.19 0.000 0.000 0.000 LDL-C (mmol/L) 3.72±0.37 4.12±0.17 3.98±0.17 0.001 0.000 0.000 aCompared with Control Group and Sham Group. bCompared with RDN Group and Sham Group. cCompared with Sham Group and RDN Group. Relative results Conclusion Our results showed that RDN improved the insulin sensitivity in diabetes rats. The mechanism may be related to the decrease of oxidative stress and function improvement in skeletal muscle mitochondrial by RDN.

2016-P: High Dose DACRA Treatment Improves Glucose Homeostasis, without Additional Weight Loss

KBP is a Dual Amylin and Calcitonin Receptor Agonist (DACRA). DACRAs have shown efficacy on weight loss and glucose control in rats. However, it is unclear whether the maximum effect has been reached. High Fat Diet (HFD, 60% fat) fed male Sprague-Dawley rats entered a dose escalation regimen ending at 5 (normal dose), 50 or 500 µg/kg daily KBP (n=10/group). Male ZDF (fa/fa) rats received identical doses without dose escalation in a preventive study setup (n=9-10/group). In HFD rats, KBP treatment resulted in a transient decrease in food intake at each dose escalation step. At study end, KBP treatment resulted in a significant weight loss of 77-94 g (13-16%, P&amp;lt;0.0001) compared to vehicle. Furthermore, KBP reduced the weight of adipose tissues (P&amp;lt;0.05). Surprisingly, there was a trend of 5 µg/kg KBP causing a more pronounced adipose-specific weight loss compared to 500 µg/kg (subcutaneous 1.7 vs. 0.7 g, visceral 4.1 vs. 2.5 g adipose weight loss). During two OGTTs, KBP treatment significantly lowered blood glucose (BG) and insulin levels compared to vehicle indicating improved glucose tolerance. Lastly, 24h fecal energy loss increased by KBP treatment, though this trend was absent when accounting for food intake and total feces output. In ZDF rats, KBP treatment significantly improved fasting BG (endpoint vehicle 29.5, endpoint KBP average 18.3 mmol/L, tAUC P&amp;lt;0.0001) and HbA1c (vehicle 10.2%, KBP average 6.1%, P&amp;lt;0.0001) compared to vehicle, with no significant difference between treatment groups. Importantly, fasting and non-fasting insulin levels of KBP-treated rats were increased compared to vehicle in a dose-dependent manner during an OGTT, resulting in lowered BG levels. Increased insulin levels suggest that KBP protects against the known β-cell degradation in the ZDF model. Taken together, these data suggest that higher doses of KBP than previously investigated may preserve insulin secretion to a greater extent. With respect to weight loss, no additional benefit was observed from the high doses. Disclosure N. Sonne: None. A.T. Larsen: None. K.V. Andreassen: Employee; Self; Nordic Bioscience. M.A. Karsdal: Employee; Self; Nordic Bioscience. K. Henriksen: Employee; Self; Nordic Bioscience. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Nordic Bioscience. Other Relationship; Self; Nordic Bioscience.

Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats.

Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would bemediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P). Male ZDF rats were randomized to RYGB (n = 7) or sham surgery (n = 7) and harvested after 28days. Total tissue ceramide, ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), and S1P were quantified in the white gastrocnemius muscle using LC-ESI-MS/MS after separation with HPLC. Total SIRT1, AMPK, PGC-1α, and CS protein expression were measured by Western blot. Body weight, fasting glucose, insulin, and HOMA-IR decreased significantly after RYGB compared with sham control. These changes were paralleled by lower total ceramide (483.7 ± 32.3 vs. 280.1 ± 38.8nmol/g wwt), C18:0 ceramide subspecies (P < 0.05), higher S1P (0.83 ± 0.05 vs. 1.54 ± 0.21nmol/g wwt, P < 0.05), and a lower ceramide/S1P ratio (P < 0.05) in the RYGB versus sham group. AMPK, pAMPK, SIRT1, PGC-1α, and CS protein expression was also higher after RYGB (P < 0.05). The ceramide/S1P ratio correlated with weight loss (r = 0.48, P = 0.08), insulin resistance (r = 0.61, P = 0.02), PGC-1α (r = - 0.51, P < 0.06), CS (r = - 0.63, P = 0.01), and SIRT1 (r = - 0.54, P < 0.04). Our data demonstrate that sphingolipid balance, and increased AMPK, SIRT1, PGC-1α, and CS protein expression are part of the mechanism that contributes to the remission of diabetes after RYGB surgery.

Duodenal Exclusion but Not Sleeve Gastrectomy Preserves Insulin Secretion, Making It the More Effective Metabolic Procedure

There is an ongoing debate on which procedure provides the best treatment for type 2 diabetes. Furthermore, the pathomechanisms of diabetes improvement of partly anatomically differing operations is not fully understood. A loop duodenojejunostomy (DJOS) with exclusion of one third of intestinal length, a sleeve gastrectomy (SG), or a combination of DJOS + SG was performed in 8-week-old male ZDF rats. One, three, and six months after surgery, an oral glucose tolerance test and measurements of GLP-1, GIP, insulin, and bile acids were conducted. After an initial (4weeks) equal glucose control, DJOS and DJOS + SG showed significantly lower glucose levels than SG 3 and 6months after surgery. There was sharp decline of insulin levels in SG animals over time, whereas insulin levels in DJOS and DJOS + SG were preserved. GIP levels were significantly larger in both groups containing a sleeve at all three time points, whereas GLP-1 was equal in all groups at all time. Bile acid levels were significantly higher in the DJOS compared to the SG group at all time points. Interestingly, the additional SG in the DJOS + SG group led to lower bile acid levels 1 and 6months postoperatively. The effect of SG on glucose control was transient, whereas a duodenal exclusion was the more effective procedure in this model due to a sustained pancreatic function with a preserved insulin secretion.

Chronic hyperinsulinemia contributes to insulin resistance under dietary restriction in association with altered lipid metabolism in Zucker diabetic fatty rats.

Hyperinsulinemia is widely thought to be a compensatory response to insulin resistance, whereas its potentially causal role in the progression of insulin resistance remains to be established. Here, we aimed to examine whether hyperinsulinemia could affect the progression of insulin resistance in Zucker fatty diabetic (ZDF) rats. Male ZDF rats at 8 wk of age were fed a diet ad libitum (AL) or dietary restriction (DR) of either 15 or 30% from AL feeding over 6 wk. Insulin sensitivity was determined by hyperinsulinemic euglycemic clamp. ZDF rats in the AL group progressively developed hyperglycemia and hyperinsulinemia by 10 wk of age, and then plasma insulin rapidly declined to nearly normal levels by 12 wk of age. Compared with AL group, DR groups showed delayed onset of hyperglycemia and persistent hyperinsulinemia, leading to weight gain and raised plasma triglycerides and free fatty acids by 14 wk of age. Notably, insulin sensitivity was significantly reduced in the DR group rather than the AL group and inversely correlated with plasma levels of insulin and triglyceride but not glucose. Moreover, enhanced lipid deposition and upregulation of genes involved in lipogenesis were detected in liver, skeletal muscle, and adipose tissues of the DR group rather than the AL group. Alternatively, continuous hyperinsulinemia induced by insulin pellet implantation produced a decrease in insulin sensitivity in ZDF rats. These results suggest that chronic hyperinsulinemia may lead to the progression of insulin resistance under DR conditions in association with altered lipid metabolism in peripheral tissues in ZDF rats.

Exercise in ZDF rats does not attenuate weight gain, but prevents hyperglycemia concurrent with modulation of amino acid metabolism and AKT/mTOR activation in skeletal muscle.

Protein metabolism is altered in obesity, accompanied by elevated plasma amino acids (AA). Previously, we showed that exercise delayed progression to type 2 diabetes in obese ZDF rats with maintenance of β cell function and reduction in hyperglucocorticoidemia. We hypothesized that exercise would correct the abnormalities we found in circulating AA and other indices of skeletal muscle protein metabolism. Male obese prediabetic ZDF rats (7-10/group) were exercised (swimming) 1 h/day, 5 days/week from ages 6-19 weeks, and compared with age-matched obese sedentary and lean ZDF rats. Food intake and weight gain were unaffected. Protein metabolism was altered in obese rats as evidenced by increased plasma concentrations of essential AA, and increased muscle phosphorylation (ph) of Akt(ser473) (187%), mTOR(ser2448) (140%), eIF4E-binding protein 1 (4E-BP1) (111%), and decreased formation of 4E-BP1*eIF4E complex (75%, 0.01 ≤ p ≤ 0.05 for all measures) in obese relative to lean rats. Exercise attenuated the increase in plasma essential AA concentrations and muscle Akt and mTOR phosphorylation. Exercise did not modify phosphorylation of S6K1, S6, and 4E-BP1, nor the formation of 4E-BP1*eIF4E complex, mRNA levels of ubiquitin or the ubiquitin ligase MAFbx. Positive correlations were observed between ph-Akt and fed circulating branched-chain AA (r = 0.56, p = 0.008), postprandial glucose (r = 0.42, p = 0.04) and glucose AUC during an IPGTT (r = 0.44, p = 0.03). Swimming exercise-induced attenuation of hyperglycemia in ZDF rats is independent of changes in body weight and could result in part from modulation of muscle AKT activation acting via alterations of systemic AA metabolism.

Roux-en Y gastric bypass is superior to duodeno-jejunal bypass in improving glycaemic control in Zucker diabetic fatty rats.

Whilst weight loss results in many beneficial metabolic consequences, the immediate improvement in glycaemia after Roux-en-Y Gastric bypass (RYGB) remains intriguing. Duodenal jejunal bypass (DJB) induces similar glycaemic effects, while not affecting calorie intake or weight loss. We studied diabetic ZDF(fa/fa) rats to compare the effects of DJB and RYGB operations on glycaemia. Male ZDF(fa/fa) rats, aged 12 weeks underwent RYGB, DJB or sham operations. Unoperated ZDF(fa/fa) and ZDF(fa/+w)ere used as controls. Body weight, food intake, fasting glucose, insulin and gut hormones were measured at baseline and on postoperative days 2, 10 and 35. An oral glucose tolerance test (OGTT) was performed on days 12 and 26. DJB had similar food intake and body weight to sham-operated and unoperated control ZDF(fa/fa) rats (p = NS), but had lower fasting glucose (p < 0.05). RYGB had lower food intake, body weight and fasting glucose compared to all groups (p < 0.001). DJB prevented the progressive decline in fasting insulin observed in the sham-operated or unoperated ZDF(fa/fa) rats, while RYGB with normalized glycaemia reduced the physiological requirement for raised fasting insulin. Bypassing the proximal small bowel with the DJB has mild to moderate body weight independent effects on glucose homeostasis and preservation of fasting insulin levels in the medium term. These effects might be further amplified by the additional anatomical and physiological changes after RYGB.

Novel effects of rosiglitazone on SMAD2 and SMAD3 signaling in white adipose tissue of diabetic rats

The effects of the proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone (ROSI) on the transforming growth factor (TGF)-β/SMAD signaling pathway in white adipose tissue (WAT) of diabetic rats were assessed. Six-week-old, male ZDF rats were fed a chow diet with (ZDF ROSI) or without (ZDF chow) ROSI (diet, 100 mg/kg) for 6 weeks. Subcutaneous (scWAT) and retroperitoneal (rpWAT) adipose tissues were excised to quantify the protein content/phosphorylation. ZDF ROSI animals showed enhanced glucose tolerance and mitochondrial protein content in both depots. The protein content of enzymes involved in fatty acid handling was increased in scWAT of ZDF ROSI animals. ZDF ROSI exhibited decreased phosphorylation of SMAD2 and SMAD3 exclusively in scWAT, along with increases in inhibitory SMAD7 and the E3 ubiquitin ligase SMURF2. In contrast, ROSI increased the protein content of SMAD4, TGF-β receptor I and II, and SMAD Anchor for Receptor Activation in scWAT. For the first time, the fact that ROSI inhibits SMAD2 and SMAD3 signaling in a depot-specific manner in diabetic rats was demonstrated. In scWAT, ROSI reduced SMAD2 and SMAD3 phosphorylation, likely through the inhibitory actions of SMAD7 and SMURF2. Induction of proximal components of the SMAD pathway may constitute a feedback mechanism to counteract ROSI-induced lipid synthesis in scWAT.

Reversal of obesity-induced hypertriglyceridemia by (R)-α-lipoic acid in ZDF (fa/fa) rats

Controlling elevated blood triacylglycerol translates into substantial health benefits. The present study aimed to evaluate the triacylglycerol-lowering properties of (R)-α-lipoic acid (LA) once circulating triacylglycerol levels have become elevated, and identify the molecular targets of LA. Nine-week old male ZDF (fa/fa) rats were fed a chow diet supplemented with 3g LA per kg diet or pair fed for two weeks (8 rats per treatment). We determined changes in blood triacylglycerol, insulin, non-esterified fatty acids, and ketone bodies concentrations. We analyzed the expression of genes and proteins involved in fatty acid and triacylglycerol metabolism in liver, epididymal fat, and skeletal muscle. Feeding LA to ZDF rats (a) corrected severe hypertriglyceridemia, (b) lowered abdominal fat mass, (c) raised circulating fibroblast growth factor-21 and Fgf21 liver gene expression, (d) repressed lipogenic gene expression of ATP-citrate synthase (Acly), acetyl-coA carboxylase 1 (Acaca), fatty acid synthase (Fasn), sn-glycerol-3-phosphate acyltransferase 1 (Gpam), adiponutrin (Pnpla3) in the liver and adipose tissue, (e) decreased hepatic protein levels of ACC1/2, FASN and 5′-AMP-activated protein kinase catalytic subunit α (AMPKα), (f) did not change phospho-AMPKα/AMPKα and phospho-ACC/ACC ratios, (g) stimulated liver gene expression of PPARα target genes carnitine O-palmitoyltransferase 1β (Cpt1b) and acyl-CoA thioesterase 1 (Acot1) but not carnitine O-palmitoyltransferase 1α (Cpt1a). This is evidence that short-term LA feeding to obese rats reverses severe hypertriglyceridemia. FGF21 may mediate the beneficial metabolic effects of LA.

Treatment with CNX-011-67, a novel GPR40 agonist, delays onset and progression of diabetes and improves beta cell preservation and function in male ZDF rats

BackgroundThe role of G protein-coupled receptor (GPR40), which is highly expressed in pancreatic beta cells, has been studied extensively in the amelioration of beta cell dysfunction in T2D using rat and mouse islets, beta cell lines and in animal models of diabetes. But its potential as a therapeutic target has not been fully explored. This aim of the study is to evaluate the therapeutic potential of CNX-011-67, a highly selective, potent and orally bioavailable GPR40 agonist, in controlling diabetes and other metabolic parameters.MethodsSeven week old male ZDF rats were treated with either vehicle or CNX-011-67, 5 mg/kg twice daily, for seven weeks. The animals were subjected to oral glucose tolerance and insulin tolerance tests. Plasma glucose, insulin, triglyceride, HbA1c, fructosamine and free fatty acids were measured at selected time points. Pancreas from control and treated animals were subjected to insulin and pancreatic and duodenal homeobox 1 (PDX1) immunohistochemistry and were also evaluated by electron microscopy. Also the potential impact of CNX-011-67 on islet insulin secretion, content, ATP levels and markers of both glucose oxidation, beta cell health in rat islets under chronic glucolipotoxic conditions was evaluated.ResultsTreatment of male ZDF rats with CNX-011-67 for 7 weeks significantly enhanced insulin secretion in response to oral glucose load, delayed the onset of fasting hyperglycemia by 3 weeks, reduced nonfasting glucose excursions, fasting free fatty acids and triglyceride levels. A significant increase in PDX1 expression and insulin content and reduction in plasma fructosamine, HOMA-IR, and beta cell apoptosis were observed. CNX-011-67 improves glucose mediated insulin secretion, insulin gene transcription and islet insulin content in cultured rat islets under chronic glucolipotoxic condition. Also enhanced glucose oxidation in the form of increased islet ATP content and overall improvement in beta cell health in the form of reduced expression of stress markers (TXNIP and CHOP mRNA) were observed.ConclusionsThese findings, suggest that long-term oral therapy with CNX-011-67 could be of clinical value to provide good glycemic control and improve islet beta cell function.

Benzimidazole-carboxamides as potent and bioavailable stearoyl-CoA desaturase (SCD1) inhibitors from ligand-based virtual screening and chemical optimization

The discovery of potent benzimidazole stearoyl-CoA desaturase (SCD1) inhibitors by ligand-based virtual screening is described. ROCS 3D-searching gave a favorable chemical motif that was subsequently optimized to arrive at a chemical series of potent and promising SCD1 inhibitors. In particular, compound SAR224 was selected for further pharmacological profiling based on favorable in vitro data. After oral administration to male ZDF rats, this compound significantly decreased the serum fatty acid desaturation index, thus providing conclusive evidence for SCD1 inhibition in vivo by SAR224.

AVE8134, a novel potent PPARα agonist, improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats

AVE8134 is a structurally novel potent PPARα agonist. The aim of this study is to investigate the efficacy of AVE8134 on lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats. A cell based PPAR Gal4 transactivation assay was constructed for testing the activities of AVE8134 at 3 different PPAR isoforms in vitro. Transgenic human Apo A1 (hApo A1) mice and insulin-resistant ZDF rats were used to evaluate the effects of AVE8134 in vivo. AVE8134 was a full PPARα dominated PPAR agonist (the values of EC(50) for human and rodent PPARα receptor were 0.01 and 0.3 μmol/L, respectively). AVE8134 was not active at PPARδ receptor. In female hApo A1 mice, AVE8134 (1-30 mg·kg(-1)·d(-1), po for 12 d) dose-dependently lowered the plasma triglycerides, and increased the serum HDL-cholesterol, hApo A1 and mouse Apo E levels. In female ZDF rats, AVE8134 (3-30 mg·kg(-1)·d(-1) for 2 weeks) improved insulin-sensitivity index. In pre-diabetic male ZDF rats (at the age of 7 weeks), AVE8134 (10 mg·kg(-1)·d(-1) for 8 weeks) produced an anti-diabetic action comparable to rosiglitazone, without the PPARγ mediated adverse effects on body weight and heart weight. In male ZDF rats (at the age of 6 weeks), AVE8134 (20 mg·kg(-1)·d(-1) for 12 weeks) increased mRNA levels of the target genes LPL and PDK4 about 20 fold in the liver, and there was no relevant effect with rosiglitazone. AVE8134 improves lipid profile and glucose metabolism in dyslipidemic mice and type 2 diabetic rats.

In Vivo Metabolic Phenotyping of Myocardial Substrate Metabolism in Rodents

Background— Cardiovascular disease is the leading cause of death among diabetic patients, with alteration in myocardial substrate metabolism being a likely contributor. We aimed to assess noninvasively the efficacy of metformin and rosiglitazone monotherapy in normalizing myocardial substrate metabolism in an animal model of type 2 diabetes mellitus. Methods and Results— The study used 18 male ZDF rats (fa/fa) with 6 rats in each group: an untreated group; a group treated with metformin (16.6 mg/kg/d), and a group treated with rosiglitazone (4 mg/kg). Each rat was scanned at age 14 weeks (baseline) and subsequently at 19 weeks with small-animal positron emission tomography to estimate myocardial glucose utilization (MGU) and myocardial utilization (MFAU), oxidation (MFAO), and esterification (MFAE). Treatment lasted for 5 weeks after baseline imaging. At week 19, rats were euthanized and hearts were extracted for expression analysis of select genes encoding for GLUT transporters and fatty acid transport and oxidation genes. In addition, echocardiography measurements were obtained at weeks 13 and 18 to characterize cardiac function. Metformin had no significant effect on either MGU or MFAU and MFAO. In contrast, rosiglitazone tended to enhance MGU and significantly reduced MFAU and MFAO. Rosiglitazone-induced increase in glucose uptake correlated significantly with increased expression of GLUT4, whereas diminished MFAO correlated significantly with decreased expression of FATP-1 and MCAD. Finally, changes in fractional shortening as a measure of cardiac function were unchanged throughout the study. Conclusions— Treatment with rosiglitazone enhanced glucose utilization and diminished MFAO, thus reversing the metabolic phenotype of the diabetic heart.

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N ɛ-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.