Perspectives and Potential Applications of Mitochondria-Targeted Antioxidants in Cardiometabolic Diseases and Type 2 Diabetes

There is abundant evidence to suggest that mitochondrial dysfunction is a main cause of insulin resistance and related cardiometabolic comorbidities. On the other hand, insulin resistance is one of the main characteristics of type 2 diabetes, obesity, and metabolic syndrome. Lipid and glucose metabolism require mitochondria to generate energy, and when O2 consumption is low due to inefficient nutrient oxidation, there is an increase in reactive oxygen species, which can impair different types of molecules, including DNA, lipids, proteins, and carbohydrates, thereby inducing proinflammatory processes. Factors which contribute to mitochondrial dysfunction, such as mitochondrial biogenesis and genetics, can also lead to insulin resistance in different insulin-target tissues, and its association with mitochondrial dysfunction can culminate in the development of cardiovascular diseases. In this context, therapies that improve mitochondrial function may also improve insulin resistance. This review explains mechanisms of mitochondrial function related to the pathological effects of insulin resistance in different tissues. The pathogenesis of cardiometabolic diseases will be explained from a mitochondrial perspective and the potential beneficial effects of mitochondria-targeted antioxidants as a therapy for modulating mitochondrial function in cardiometabolic diseases, especially diabetes, will also be considered.Contract grant sponsor: PI10/1195; Contract grant sponsor: PI 12/1984; Contract grant sponsor: CIBERehd CB06/04/0071; Contract grant sponsor: PROMETEO 2010/060; Contract grant sponsor: ACOMP/2012/042; Contract grant sponsor: ACOMP/2012/045; Contract grant sponsor: ACOMP2013/061; Contract grant sponsor: European Regional Development Fund (ERDF)

Purification and characterization of an insulin-stimulated kinase

The rapid effects of insulin action appear to be mediated at least in part by the activation of a number of discreteprotein serine/threonine kinases. The cellular targets of some of these kinases include key metabolic enzymes such as acetyl-CoA carboxylase (ACC) and the ribosomal S6. While, the S6 kinases have now been purified and characterized, very little is known about the nature of the proteinkinase(s) able to phosphorylate ACC particularly on the insulin-directed site. Previous studies demonstrated that amyelin basic protein-kinase (MBP-kinase) from sea-star is able to phosphorylate the insulin-directed site on ACC. Thus, it was interesting to find out whether a mammalian homolog of this kinase existed. Rapid chromatography of supernatant fractions from rat adipose tissue by MonoQ ion exchange using fast protein liquid chromatography revealed several peaks of insulin-stimulated protein-serine/threonine kinase activity towards myelin basic protein. Progress in the purification and characterization of these kinases was initially impeded by the rapid decay in protein kinase activity of these extracts. Thus, a major concern initially was stabilization of the insulin-stimulated protein serine/threonine kinase activity which would enable subsequent purification and characterization of the kinases. Stabilization was achieved by the use of phosphatase inhibitors, particularly in combination with a rapid procedure which included ammonium sulphate precipitation.This enabled storage of the activated kinases (with apparently very little loss in activity) and further purification. After first examining the chromatographic properties using individual column techniques, the insulin-activated serine/threonine kinases were purified by a procedure which involved ammonium sulphate precipitation, and sequential chromatography on polylysine-agarose, MonoQ, heparin-agarose and a final MonoQ. The purified enzyme showed only two major silver-stained polypeptides (with subunit sizes of 200 and 44 kDa) as judged by SDS-PAGE. From analysis of Western blots performed with several different anti-kinase antibodies, the 44 kDa polypeptide was found to be immunologically related to a sea-star MBP-kinaseas well as to a family of mammalian mitogen-activated kinases designated ERKs. It is concluded that the 44 kDa MBP-kinase in rat adipose tissue is a member of the family of mitogen-activated kinases. However, the precise relationship to these kinases as well as the regulatory properties of this insulin-activated kinase await further characterization including molecular cloning of the rat adipose enzyme.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Evaluation of the roles of protein serine/threonine kinases in the metabolic actions of insulin

Protein phosphorylation plays a central role in the biological effects of insulin. A number of proteins display increases in serine/threonine (Ser/Thr) phosphorylation in response to the hormone and include acetyl-CoA carboxylase (ACC) and the ribosomal protein S6. These phosphorylations are mediated by a discrete number of Ser/Thr protein kinases which are stimulated by the hormone. While the S6 kinases have been purified and are well characterized, very little is known about the insulin stimulated ACC-kinase. This study evaluated the roles of three different classes of the mitogen-activated protein (MAP) kinase family (ERKs, JNKs and p38) and that of protein kinase B in the metabolic responses of insulin in rat white adipose tissue. Initial evidence led to the hypothesis that an insulin-stimulated myelin basic protein (MBP) kinase may be an important ACC-kinase. A MBP kinase was highly purified from rat adipose tissue and displayed a number of properties indicating it was a member of the family of MAP kinases. However, the adipose tissue MBP kinase did not phosphorylate the insulin-directed site on ACC and did not induce insulin-like activation of ACC, indicating that it was not an ACC-kinase. This result therefore, argues strongly against the initial hypothesis that MBP/MAP kinases were significant insulin-stimulated ACC-kinases. Further characterization of the fat cell MBP kinase revealed its close association with the actin cytoskeleton. It is proposed that the cytoskeleton may act as an interface to nucleate signaling elements. This would provide efficiency and fidelity in signaling. Because extracellular osmolarity (and consequent regulation of cell volume) affects ! the three classes of MAP kinases and also influences liver carbohydrate metabolism, the effects of insulin and osmolarity on fatty acid biosynthesis and the activation state of the three classes of MAP kinases were therefore, investigated in adipose tissue. Changes in extracellular osmolarity alone, over the range from hypo-osmotic (228 mOsM) to hyperosmotic (404 mOsM) did not stimulate fatty acid biosynthesis in adipose tissue. Insulin stimulated the pathway to an equivalent extent under iso-osmotic (316 mOsM), hypo- and hyper-osmotic conditions. The activation of the ERKs differed from that of fatty acid synthesis, since hypo-osmotic medium alone activated the ERKs and because the activation of the ERKs was blocked in hyper-osmotic medium. Unlike fatty acid synthesis, JNKs were activated by both hypo-osmotic and hyper-osmotic medium in the absence of insulin. The activation state of p38 judged by tyrosine phosphorylation of p38, by associated MBP kinase activity or by the activity of a downstream heat-shock protein kinase was not affected by changes in osmolarity (200-400 mOsM) or by insulin. In conclusion, within a moderate physiological range, extracellular osmolarity did not lead to changes in the rates of de novo fatty acid synthesis in rat white adipose tissue (in contrast to previous studies with isolated rat hepatocytes). Therefore, activations of ERK1 and ERK2 are neither necessary nor sufficient for the activation of fatty acid biosynthesis. The p38 pathway was insensitive to insulin and activation of JNKs did not correlate with the stimulation of fatty acid biosynthesis. A newly-discovered protein kinase B (PKB), is insulin-activated and appears to play a significant role in regulating glycogen synthase. I therefore, examined the role of PKB in ACC regulation. Insulin provoked a rapid and sustained activation of PKB in rat white adipose. The activation of PKB was associated with a retardation of the mobility of the PKB protein on SDS-PAGE gels and was especially prominent in membrane fractions of rat adipose tissue. PKB immunoprecipitates strongly phosphorylated ACC and showed a striking preference for the 265-kDa ACC isoform. The major tryptic phosphopeptide from ACC phosphorylated by PKB immunoprecipitates co-migrated on 2-dimensional thin layer and high pressure chromatography with that obtained from ACC following phosphorylation with AMP-activated protein kinase (AMP-PK). Unlike AMP-PK, however, PKB immunoprecipitates did not phosphorylate the optimum (SAMS) peptide substrate. Furthermore, unlike AMP-PK, phosphorylation of ACC with PKB immunoprecipitates did not affect the enzymatic activity of ACC. Because specific PKB inhibitors are not yet available, we examined the effects of vanadium to shed light on the potential significance of PKB activation. Vanadium activated PKB, glycogen synthase and fatty acid biosynthesis with very similar dose dependencies. The maximal effects of vanadium were similar to the maximal effects of insulin and were not additive. PKB may therefore be required in the activation of glycogen synthase and fatty acid biosynthesis but further studies will be required to confirm that (ideally with PKB antagonists). In contrast, the anti-lipolytic effects of vanadium were evident at concentrations substantially below that required for PKB activation. Therefore, not all metabolic responses of vanadium are dependent on PKB activation.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Cell volume and the metabolic actions of insulin

Insulin increases the volume of isolated hepatocytes and cells in perfused livers, but effects of the hormone on the volume of fat or muscle cells have not been demonstrated. Exogenous amino acids may stimulate swelling of liver cells and induce insulin-like effects on hepatic protein metabolism; however, swelling of liver cells can be induced by some treatment that do not induce insulin-like metabolic responses. Exogenous amino acids also influence protein metabolism of fat and muscle cells, but no relationship with cell volume has been established and no corresponding effects on metabolism of carbohydrates or lipids have been observed. Three families of mitogen-activated protein kinases are activated after changes in extracellular osmolality but they appear to play little or no role in the metabolic actions of insulin. Direct evidence against a metabolic role for the extracellular signal-regulated kinases ERK-1 and ERK-2 is discussed. The c-Jun N-terminal kinases (also called stress-activated protein kinases) and the mammalian homologs of the yeast Hog protein kinase are strongly activated by environmental stresses associated with catabolic metabolism. We conclude that cell volume and protein metabolism may be correlated in liver but there is no compelling evidence that the effects of insulin on metabolism of liver, fat, or muscle cells can be accounted for by changes in cell volume. The effects of insulin on cell volume may represent a discrete aspect of the complete physiological response rather than an obligatory intermediate step in metabolic signalling.Key words: insulin action, cell volume, osmolality, metabolic regulation, MAP kinases.</jats:p

Molecular Mechanism of Insulin-Induced Degradation of Insulin Receptor Substrate 1

Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway