Type 2 diabetes mellitus (T2DM) is the most common metabolic disease in the world. Maintenance of glucose homeostasis depends on a complex interplay between the insulin responsiveness of skeletal muscle, liver, adipose tissue and glucose-stimulated insulin secretion by pancreatic beta cells. Defects in these organs are responsible for insulin resistance and progression to hyperglycemia. Understanding the integrated pathophysiology initiating the development of insulin resistance should extend our capacity to identify novel therapeutic targets for the prevention and/or treatment of T2DM. This biology remains incompletely characterized, in part, due to the interaction of multiple organ systems. The complexity of this biology is further underscored by the progressive changes in the systemic milieu including the onset of hyperinsulinemia, elevated circulating free fatty acids and triglycerides, hyperglycemia, and the activation of systemic immune system during the development of T2DM. In skeletal muscle, loss of mitochondrial function is evident in some insulin-resistant subjects years before they develop diabetes. Mitochondria are particularly important for skeletal muscle function, given the high oxidative demands imposed on this tissue by intermittent contraction. Moreover, muscle cells must maintain metabolic flexibility, defined as the ability to rapidly modulate substrate oxidation as a function of hormonal and energetic conditions. The molecular mechanisms that control mitochondrial number and function remain poorly understood, and only a few transcription factors or coactivators (e.g., PGC-1α, NRF1, Tfam) have been associated with this process. Notably, skeletal muscle differentiation and remodelling are also controlled at the epigenetic level, via transcriptional modulation of key genes in mitochondrial biogenesis and oxidative metabolism, involving enzymes, such as members of the histone deacetylase (HDAC) family, in particular those belonging to class I and class II, which modulate post-translational modifications on target proteins. The current knowledge on HDACs is that they function in general as transcriptional repressors, however their role in vivo is likely more complex. Less is known on the effects of HDACs modulators on energy metabolism, however a recent study reported that supplementation with sodium butyrate, a dietary component active as HDAC inhibitor, promotes energy expenditure and mitochondrial function in mice fed with a high fat diet212. Given the importance of skeletal muscle metabolism in insulin resistance/diabetes, and given the role of HDACs in skeletal muscle biology, it is reasonable to speculate that modulation of these enzymes would play a role in this pathology that deserves to be investigated more deeply. Based on the evidences that mitochondrial dysfunction is often associated to whole body metabolic dysregulation, aim of this study is to better understand the role of histone deacetylases in the regulation of mitochondrial biogenesis and in the modulation of all these mechanisms underlying the pathophisiology of insulin resistance. In C2C12 myotubes treated with pan and class selective HDAC inhibitors (HDACi), such as SAHA(pan-inhibitor), MS275 (Class I HDAC inhibitor) and MC1568 (Class II HDAC inhibitor), transcriptome analysis revealed an increase of OXPHOS genes and of genes encoding fatty acid catabolic enzymes, following treatment with pan or class I HDACi. Moreover, staining of myotubes treated with SAHA and MS275 showed an increase of mitochondrial density and activity, coupled with an increase in mitochondrial DNA content. In Db/Db obese and diabetic mice we observed that treatments with SAHA and MS275 reduce glycemia, triglycerides, plasma insulin land transaminases levels and improves glucose clearance and insulin sensitivity. In vivo metabolic study revealed an increase of oxygen consumption, a net decrease of the respiratory exchange ratio and an increased heat production, implying a more oxidative metabolism, in the MS275 treated group. In order to characterize the effects of HDACi in different tissues, we performed microarray analysis in skeletal muscles, liver, brown and white adipose tissues and histological analysis. In the skeletal muscle of mice treated with MS275, and with SAHA to a lesser degree, we observed a general increase in OXPHOS genes and in genes involved in lipid and glucose metabolism. These observations were also supported by the increased oxidative capacity highlighted by SDH staining in gastrocnemius sections. In brown fat we found an increased expression of brown adipocytes markers , such as PRDM16, CIDEA, UCP1, ELOVL3 and DIO2, and an induction of mitochondrial biogenesis, after MS275 administration. In parallel, adypocyte size appeared smaller respect to the adipocytes of the control group. In white adipose tissue, we observed an increase of adipocytes markers and, even in this case, an induction of mitochondrial biogenesis; adipocytes, beyond being smaller, also showed a reduced macrophages infiltration. Gene expression also revealed an increased expression of brown adipocytes markers, such as UCP1, also confirmed by immunohistochemistry assay, ADRB3, ELOVL3 and DIO2. Nevertheless, white adipocytes remained PRDM16 negative. Collectively, our results suggest that HDACs, and in particular Class I HDACs, play an unexpected role in energy metabolism, induce mitochondrial biogenesis in different tissues and may represent key regulators in diseases based on metabolic alterations.

EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON ENERGY METABOLISM / A. Galmozzi ; Tutor: Emma De Fabiani ; Coordinatore: Francesco Bonomi. - : . Universita' degli Studi di Milano, 2010 Dec 09. ((23. ciclo, Anno Accademico 2010. [10.13130/galmozzi-andrea_phd2010-12-09].

EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON ENERGY METABOLISM

A. Galmozzi
2010-12-09

Abstract

Type 2 diabetes mellitus (T2DM) is the most common metabolic disease in the world. Maintenance of glucose homeostasis depends on a complex interplay between the insulin responsiveness of skeletal muscle, liver, adipose tissue and glucose-stimulated insulin secretion by pancreatic beta cells. Defects in these organs are responsible for insulin resistance and progression to hyperglycemia. Understanding the integrated pathophysiology initiating the development of insulin resistance should extend our capacity to identify novel therapeutic targets for the prevention and/or treatment of T2DM. This biology remains incompletely characterized, in part, due to the interaction of multiple organ systems. The complexity of this biology is further underscored by the progressive changes in the systemic milieu including the onset of hyperinsulinemia, elevated circulating free fatty acids and triglycerides, hyperglycemia, and the activation of systemic immune system during the development of T2DM. In skeletal muscle, loss of mitochondrial function is evident in some insulin-resistant subjects years before they develop diabetes. Mitochondria are particularly important for skeletal muscle function, given the high oxidative demands imposed on this tissue by intermittent contraction. Moreover, muscle cells must maintain metabolic flexibility, defined as the ability to rapidly modulate substrate oxidation as a function of hormonal and energetic conditions. The molecular mechanisms that control mitochondrial number and function remain poorly understood, and only a few transcription factors or coactivators (e.g., PGC-1α, NRF1, Tfam) have been associated with this process. Notably, skeletal muscle differentiation and remodelling are also controlled at the epigenetic level, via transcriptional modulation of key genes in mitochondrial biogenesis and oxidative metabolism, involving enzymes, such as members of the histone deacetylase (HDAC) family, in particular those belonging to class I and class II, which modulate post-translational modifications on target proteins. The current knowledge on HDACs is that they function in general as transcriptional repressors, however their role in vivo is likely more complex. Less is known on the effects of HDACs modulators on energy metabolism, however a recent study reported that supplementation with sodium butyrate, a dietary component active as HDAC inhibitor, promotes energy expenditure and mitochondrial function in mice fed with a high fat diet212. Given the importance of skeletal muscle metabolism in insulin resistance/diabetes, and given the role of HDACs in skeletal muscle biology, it is reasonable to speculate that modulation of these enzymes would play a role in this pathology that deserves to be investigated more deeply. Based on the evidences that mitochondrial dysfunction is often associated to whole body metabolic dysregulation, aim of this study is to better understand the role of histone deacetylases in the regulation of mitochondrial biogenesis and in the modulation of all these mechanisms underlying the pathophisiology of insulin resistance. In C2C12 myotubes treated with pan and class selective HDAC inhibitors (HDACi), such as SAHA(pan-inhibitor), MS275 (Class I HDAC inhibitor) and MC1568 (Class II HDAC inhibitor), transcriptome analysis revealed an increase of OXPHOS genes and of genes encoding fatty acid catabolic enzymes, following treatment with pan or class I HDACi. Moreover, staining of myotubes treated with SAHA and MS275 showed an increase of mitochondrial density and activity, coupled with an increase in mitochondrial DNA content. In Db/Db obese and diabetic mice we observed that treatments with SAHA and MS275 reduce glycemia, triglycerides, plasma insulin land transaminases levels and improves glucose clearance and insulin sensitivity. In vivo metabolic study revealed an increase of oxygen consumption, a net decrease of the respiratory exchange ratio and an increased heat production, implying a more oxidative metabolism, in the MS275 treated group. In order to characterize the effects of HDACi in different tissues, we performed microarray analysis in skeletal muscles, liver, brown and white adipose tissues and histological analysis. In the skeletal muscle of mice treated with MS275, and with SAHA to a lesser degree, we observed a general increase in OXPHOS genes and in genes involved in lipid and glucose metabolism. These observations were also supported by the increased oxidative capacity highlighted by SDH staining in gastrocnemius sections. In brown fat we found an increased expression of brown adipocytes markers , such as PRDM16, CIDEA, UCP1, ELOVL3 and DIO2, and an induction of mitochondrial biogenesis, after MS275 administration. In parallel, adypocyte size appeared smaller respect to the adipocytes of the control group. In white adipose tissue, we observed an increase of adipocytes markers and, even in this case, an induction of mitochondrial biogenesis; adipocytes, beyond being smaller, also showed a reduced macrophages infiltration. Gene expression also revealed an increased expression of brown adipocytes markers, such as UCP1, also confirmed by immunohistochemistry assay, ADRB3, ELOVL3 and DIO2. Nevertheless, white adipocytes remained PRDM16 negative. Collectively, our results suggest that HDACs, and in particular Class I HDACs, play an unexpected role in energy metabolism, induce mitochondrial biogenesis in different tissues and may represent key regulators in diseases based on metabolic alterations.
DE FABIANI, EMMA SELINA ROSA
BONOMI, FRANCESCO
energy metabolism ; histone deacetylases ; mitochondria ; type II diabetes
Settore BIO/10 - Biochimica
EFFECTS OF HISTONE DEACETYLASE INHIBITORS ON ENERGY METABOLISM / A. Galmozzi ; Tutor: Emma De Fabiani ; Coordinatore: Francesco Bonomi. - : . Universita' degli Studi di Milano, 2010 Dec 09. ((23. ciclo, Anno Accademico 2010. [10.13130/galmozzi-andrea_phd2010-12-09].
Doctoral Thesis
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