rottlerin and Diabetes-Mellitus--Type-2

Relaxin (Rlx) is known to antagonize diabetic cardiac fibrosis. However, its mechanism is poorly understood. Protein kinase Cδ (PKCδ) plays a crucial role in diabetic cardiomyopathy (DCM). This study explored the involvement of PKCδ in Rlx's capacity of suppressing cardiac fibrosis in a rat model of type 2 diabetes mellitus (DM). Type 2 DM of 8-week-old male Sprague-Dawley (SD) rats was induced by a high-fat diet and the injection of streptozocin (STZ, 40 mg/kg). Fourteen-week-old rats with DM and rats in control group which were pre-treated for 1 week with human recombinant relaxin (rhRlx, 30 μg/kg.d), were assessed to detect cardiac fibrosis and PKCδ expression with Western blot. Cardiac fibroblasts of neonatal rats were treated for 72 h with rhRlx (100 ng/ml) under high glucose (HG). Western blot was utilized for detecting the membranous and cytoplasmic protein expressions of PKCδ. The effects of rhRlx and PKCδ inhibitor (rottlerin) were assessed either alone or in combination on the HG-induced proliferation and differentiation of cardiac fibroblasts and the release of collagen I.Rlx treatment inhibited the differentiation of cardiac fibroblasts and the expression of collagen I. The expression of PKCδ was regulated by Rlx in diabetic rats and cardiac fibroblasts under HG condition. The effects of Rlx upon the proliferation and differentiation of cardiac fibroblasts and the excretion of collagen I under HG were blunted by rottlerin. Rlx suppressed cardiac fibrosis in type 2 diabetic rats. This beneficial effect was associated with its ability of modulating the expression of PKCδ.

Topics: Acetophenones; Animals; Animals, Newborn; Benzopyrans; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Cardiomyopathies; Disease Models, Animal; Enzyme Inhibitors; Fibroblasts; Fibrosis; Humans; Male; Myocardium; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Relaxin

Besides the crucial role of hyperinsulinemia in the development of breast cancer with Type 2 diabetes mellitus (T2DM), it has been shown that hyperglycemia could contribute to promote cancer progression. A remarkable association within hyperglycemia, PKCδ and Ubiquitin-proteasome system (UPS) has been reported, suggesting that PKCδ may mediate high glucose-induced UPS activation in breast cancer cells. Although the independent effects of PKCδ or UPS on breast cancer and T2DM are increasingly supported by experimental evidence, the complex interactional link between PKCδ and UPS is still unclear. Hence, we focus on the relationship between PKCδ and UPS in breast cancer with T2DM. We hypothesize that PKCδ may have the function to regulate the activity of UPS. Further, we speculate that PKCδ combine with proteasome α2 promoter, that indicate PKCδ regulate the function of UPS by change the composition of proteasome. Therefore, we surmise that PKCδ mediated high glucose-induced UPS activation in breast cancer cells, and specific PKCδ inhibitor rottlerin significantly suppressed elevated glucose induced the activity of UPS. We hope that our paper will stimulate further studies the relationship between PKCδ and UPS, and a new targeted therapy and early medical intervention for PKCδ could be a useful option for breast cancer cases complicated with T2DM or hyperglycemia.

Topics: Acetophenones; Benzopyrans; Breast Neoplasms; Diabetes Mellitus, Type 2; Disease-Free Survival; Enzyme Activation; Enzyme Inhibitors; Female; Glucose; Humans; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Protein Binding; Protein Kinase C-delta; Ubiquitin

Contractile responses in small intrarenal arteries are associated with diabetic nephropathy. However, the mechanisms that induce and maintain altered small vessel contraction are not clearly understood. To further understand intrarenal artery dysfunction in diabetes, phenylephrine (PE)-induced force development was assessed in the intrarenal artery [interlobar artery (ILA)] in control (lean) and type II diabetic (ob/ob) mice. PE-induced dose-dependent force development in the ILA was significantly greater in ob/ob mice than in lean mice (592.8 ± 5.2 and 770.1 ± 12.1 µ/mm tissue, respectively, following administration of 30 µM PE, n = 5). Under high-glucose conditions (twice the normal concentration of glucose), PE-induced force development in the ILA was only enhanced in ob/ob mice (946.0 ± 18.2 µN/mm tissue; n = 5). ILA dysfunction reduces blood flow to the glomerulus and may induce diabetic nephropathy. Basal overcontraction of the ILA in ob/ob mice under normal-glucose conditions was reduced by pretreatment with rottlerin, a calcium-independent protein kinase C (PKCδ) inhibitor. Total PKC activity was also reduced by rottlerin. Under high-glucose conditions, the enhanced ILA contraction in diabetic mice was suppressed by rho A and rho kinase inhibitors. Our results indicate two types of ILA dysfunction in diabetes, as follows: 1) a basal increase in PE-induced contraction under normal-glucose conditions, and 2) extracellular glucose-dependent enhancement of PE-induced contraction. We believe that these dysfunctions are mediated by the activation of the PKCδ and rho A-rho kinase pathways, respectively.

Topics: Acetophenones; Animals; Benzopyrans; Blood Glucose; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Male; Mice; Mice, Obese; Phenylephrine; Protein Kinase C; Protein Kinase Inhibitors; Renal Artery; Renal Circulation; Vasoconstriction; Vasoconstrictor Agents

Individuals with chronic kidney disease (CKD) and/or diabetes mellitus (DM) are at increased risk of cardiovascular events and have elevated externalization of phosphatidylserine (PS; which propagates thrombus formation) in a small subpopulation of platelets. The purpose of this study was to examine the effect of 1) removing uremic toxins by hemodialysis on PS externalization in patients with either CKD or CKD and DM and 2) ultrafiltrate (UF) from these individuals on PS externalization in healthy platelets. PS externalization was quantified by a fluorescence-activated cell sorter using annexin V in platelet-rich plasma. PS externalization was elevated threefold in CKD patients and returned to basal values during 3-h hemodialysis. In contrast, it was elevated fivefold in individuals with CKD and DM and was still threefold above control after 3-h treatment. UF significantly increased PS externalization in a small subpopulation of platelets from healthy controls. The effect of UF from individuals with CKD and DM was significantly greater than that from patients with CKD alone, and the responses were partially inhibited by the protein kinase Cdelta (PKCdelta) inhibitor rottlerin and the 5-hydroxytryptamine (5-HT)(2A/2C) receptor antagonist ritanserin. The data suggest that uremic toxins present in UF mediate PS externalization in a small subpopulation of platelets, at least in part, via the 5-HT(2A/2C) receptor and PKCdelta and demonstrate that DM further enhances platelet PS externalization in CKD patients undergoing hemodialysis. This may explain, at least in part, the additional increase in vascular damage observed in CKD patients when DM is present.

Topics: Acetophenones; Adult; Aged; Aged, 80 and over; Benzopyrans; Blood Platelets; Case-Control Studies; Chronic Disease; Diabetes Mellitus, Type 2; Enzyme Inhibitors; Female; Hemodiafiltration; Humans; Kidney Diseases; Male; Middle Aged; Phosphatidylserines; Protein Kinase C-delta; Renal Dialysis; Ritanserin; Serotonin 5-HT2 Receptor Antagonists; Serotonin Antagonists

Protein kinase C (PKC)-delta, an upstream regulator of the Akt survival pathway, contributes to cellular dysfunction in the pathogenesis of diabetes. Herein, we examined the role of PKC-delta in neuronal apoptosis through Akt in the retinas of diabetic rats.. We used retinas from 24- and 35-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) diabetic and Long-Evans Tokushima Otsuka (LETO) nondiabetic rats. To assess whether PKC-delta affects Akt signaling and cell death in OLETF rat retinas, we examined 1) PKC-delta activity and apoptosis; 2) protein levels of phosphatidylinositol 3-kinase (PI 3-kinase) p85, heat shock protein 90 (HSP90), and protein phosphatase 2A (PP2A); 3) Akt phosphorylation; and 4) Akt binding to HSP90 or PP2A in LETO and OLETF retinas in the presence or absence of rottlerin, a highly specific PKC-delta inhibitor, or small interfering RNAs (siRNAs) for PKC-delta and HSP90.. In OLETF retinas from 35-week-old rats, ganglion cell death, PKC-delta and PP2A activity, and Akt-PP2A binding were significantly increased and Akt phosphorylation and Akt-HSP90 binding were decreased compared with retinas from 24-week-old OLETF and LETO rats. Rottlerin and PKC-delta siRNA abrogated these effects in OLETF retinas from 35-week-old rats. HSP90 siRNA significantly increased ganglion cell death and Akt-PP2A complexes and markedly decreased HSP90-Akt binding and Akt phosphorylation in LETO retinas from 35-week-old rats compared with those from nontreated LETO rats.. PKC-delta activation contributes to neuro-retinal apoptosis in diabetic rats by inhibiting Akt-mediated signaling pathways.

Topics: Acetophenones; Animals; Apoptosis; Benzopyrans; Body Weight; Diabetes Mellitus, Type 2; HSP90 Heat-Shock Proteins; Immunoblotting; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Male; Neurons; Pentobarbital; Phosphatidylinositol 3-Kinases; Propoxycaine; Protein Binding; Protein Kinase C-delta; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Rats; Rats, Inbred OLETF; Retina; Retinal Ganglion Cells; RNA, Small Interfering; Signal Transduction

Advanced glycation end products (AGE) are substantially elevated in individuals with diabetes and/or chronic kidney disease (CKD). These patients are at greatly increased risk of cardiovascular events. The purpose of this study was to investigate the novel hypothesis that AGE elicit externalization of the platelet membrane phospholipid phosphatidylserine (PS). This contributes to hemostasis through propagation of the coagulation cascade leading to thrombus formation. Platelet-rich plasma (PRP) was prepared by differential centrifugation, and PS externalization was quantified by a fluorescence-activated cell sorter using annexin V-FITC. Human serum albumin (HSA)-AGE was generated by incubating HSA with glucose for 2, 4, or 6 wk, and total HSA-AGE was assessed by fluorescence intensity. The 2-wk HSA-AGE preparation (0-2 mg/ml) stimulated a concentration-dependent increase in PS externalization in a subpopulation of platelets that was threefold at 2 mg/ml. In contrast, the 4- and 6-wk preparations were maximal at 0.5 mg/ml and fivefold in magnitude. These effects mirrored the change in total HSA-AGE content of the preparations. The PS response was maximal at 10 min and inhibited by the PKC-delta inhibitor rottlerin and the serotonin [5-hydroxytryptamine (5-HT)](2A/2C) receptor antagonist ritanserin in a dose-dependent manner. Moreover, the 5-HT(2A/2C) receptor agonist 1,2,5-dimethoxy-4-iodophenyl-2-aminopropane mimicked the effect of HSA-AGE on PS externalization. These data demonstrate, for the first time, that HSA-AGE stimulates PS externalization in a subpopulation of platelets via the 5-HT(2A/2C) receptor. This may have important consequences for platelet involvement in inflammatory responses and the increased cardiovascular risk observed in individuals with diabetes and/or CKD.

Topics: Acetophenones; Amphetamines; Benzopyrans; Blood Coagulation; Blood Platelets; Case-Control Studies; Cell Membrane; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Glycation End Products, Advanced; Humans; Phosphatidylserines; Platelet Activation; Protein Kinase C-delta; Protein Kinase Inhibitors; Receptor, Serotonin, 5-HT2A; Receptor, Serotonin, 5-HT2C; Renal Insufficiency, Chronic; Ritanserin; Serotonin Antagonists; Serotonin Receptor Agonists; Serum Albumin; Serum Albumin, Human; Time Factors

We investigated the relationship between phosphorylation of alphaB-crystallin (alphaBC) and retinal apoptosis in type 2 diabetes. The retinas of male Otsuka Long-Evans Tokushima fatty (OLETF) rats at 24 and 35 weeks were used as an animal model for type 2 diabetes and sex- and age-matched Long-Evans Tokushima Otsuka (LETO) rats were used as controls. In the retinas of 35-week OLETF rats, the interaction between alphaBC and protein kinase C delta (PKC delta) among the PKC isozymes, alphaBC phosphorylation at Ser45 (S45p-alphaBC), TUNEL-positive apoptotic ganglion cells, several apoptotic signs, and co-localization of S45p-alphaBC and TUNEL significantly increased as compared with other groups while the alphaBC-Bax interaction greatly decreased. These changes were abolished by rottlerin treatment, a highly specific PKC delta inhibitor. These results suggest that PKC delta is involved in regulation of anti-apoptotic function of alphaBC in the retina of type 2 diabetes.

Topics: Acetophenones; Age Factors; alpha-Crystallin B Chain; Animals; Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Blood Glucose; Body Weight; Diabetes Mellitus, Type 2; Disease Models, Animal; Enzyme Inhibitors; Female; Gene Expression Regulation; Immunoprecipitation; In Situ Nick-End Labeling; Male; Protein Kinase C-delta; Rats; Rats, Inbred OLETF; Rats, Long-Evans; Retina; Time Factors