ABSTRACT
Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in murine skeletal muscle cell line C2 C12 . Myotubes as compared to myoblasts of C2 C12 cells were found to be more sensitive in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKTS473 ) was significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into myotubes of C2 C12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or myotubes of C2 C12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA in myotubes of C2 C12 . In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKTS473 and impaired glucose uptake. This observation is in contrast to published studies where NCoR has been reported to negatively regulate insulin signaling cascade. Furthermore, transient silencing of NCoR failed to improve insulin sensitivity in chronic hyperinsulinemia-induced insulin-resistant model of C2 C12 cells. Importantly, inhibition of lysosomal protein degradation pathway using ammonium chloride restored protein level of NCoR but failed to increase glucose uptake in serum-starved C2 C12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state (myoblast and myotubes) of C2 C12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C2 C12 myotubes.
Subject(s)
Co-Repressor Proteins/metabolism , Insulin/metabolism , Ammonium Chloride , Animals , Blotting, Western , Cell Differentiation/drug effects , Cell Line , Co-Repressor Proteins/antagonists & inhibitors , Co-Repressor Proteins/genetics , Glucose/metabolism , Insulin/pharmacology , Insulin Resistance , Leupeptins/pharmacology , Lysosomes/metabolism , Mice , Models, Biological , Muscle, Skeletal/cytology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Phosphorylation/drug effects , Proteolysis/drug effects , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
Amelioration of rodent type 2 diabetes by hemin has been linked to increased heme oxygenase (HO) activity, however alternative mechanisms have recently been proposed for its anti-diabetic effect. We sought to determine the anti-diabetic efficacy of heme arginate (HA), a clinically licensed preparation of heme, and whether its predominant mode of action is via increased HO activity. Intravenous administration of HA reduced hyperglycemia in diabetic (db/db) mice. Co-administration of the HO inhibitor stannous (IV) mesoporphyrin IX dichloride (SM) resulted unexpectedly in a further improvement in glycaemic control despite restoring HO activity to baseline levels. The anti-diabetic effects of HA±SM were associated with increased adiposity, increased serum adiponectin levels, reduced adipose tissue and islet inflammation and preservation of islet ß-cell function. HO activity independent effects of HA on adipogenesis and ß-cell inflammation were further confirmed in cell culture models using the 3T3-L1 pre-adipocyte and MIN6 ß-cell lines, respectively. In conclusion, our work demonstrates that the heme component of HA ameliorates experimental type 2 diabetes by promoting metabolically favourable adipogenesis and preserving islet ß-cell function, but this is not mediated via increased HO activity.
Subject(s)
Arginine/administration & dosage , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Heme Oxygenase (Decyclizing)/metabolism , Heme/administration & dosage , 3T3 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Adipogenesis/drug effects , Adiponectin/blood , Adiponectin/metabolism , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Adiposity/drug effects , Animals , Blood Glucose/drug effects , Cell Line , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/metabolism , Hyperglycemia/blood , Hyperglycemia/drug therapy , Hyperglycemia/metabolism , Inflammation/blood , Inflammation/drug therapy , Inflammation/metabolism , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Metalloporphyrins/administration & dosage , Mice , Pilot ProjectsABSTRACT
Aging is thought to be associated with a higher susceptibility to renal ischemia-reperfusion injury (IRI). To study whether defective induction of hemeoxygenase-1 (HO-1, a protective and anti-inflammatory enzyme) might contribute to this, we found that while 12-month-old mice had similar baseline renal function and HO-1 expression, the induction of HO-1 usually seen in ischemia-reperfusion was reduced. This was also associated with worsened renal function and acute tubular necrosis in the aged compared with young mice. In the older mice, heme arginate (HA) induced HO-1 in the cortex and medulla, significantly improved renal function, and reduced tissue injury. Cellular HO-1 induction in the medulla in response to injury or HA treatment was found to be interstitial rather than epithelial, as evidenced by its colocalization with macrophage markers. In vitro, HA treatment of primary macrophages resulted in marked HO-1 induction without impairment of classical activation pathways. Macrophage depletion, caused by diphtheria toxin treatment of 12-month-old CD11b-DTR transgenic animals, resulted in the loss of interstitial HO-1-positive cells and reversal of the protective phenotype of HA treatment. Thus, failure of HO-1 induction following renal IRI worsens structural and functional injury in older mice and represents a therapeutic target in the elderly. Hence, HO-1-positive renal macrophages mediate HA-induced protection in IRI.
