Epigenetic Regulation Of Thermogenesis By Adipose Specific Tet1 And Role Of Dnmt3a In Endurance Exercise By Suppressing Aldh1l1-mediated Oxidative Stress

The first part of the thesis reports the role of Ten-eleven translocation methylcytosinedioxygenase 1 (TET1) in beige adipocyte thermogenesis. It has been suggested that beige fat thermogenesis is tightly controlled by epigenetic regulators that sense environmental cues such as temperature. We report that subcutaneous adipose expression of the DNA demethylase TET1 is suppressed by cold and other stimulators of beige adipocyte thermogenesis. TET1 acts as an autonomous repressor of key thermogenic genes, including Ucp1 and Ppargc1a, in beige adipocytes. Adipose-selective Tet1 knockout mice generated by using Fabp4-Cre improves cold tolerance and increases energy expenditure and protects against diet-induced obesity and insulin resistance. Moreover, the suppressive role of TET1 in the thermogenic gene regulation of beige adipocytes is largely DNA demethylase–independent. Rather, TET1 coordinates with HDAC1 to mediate the epigenetic changes to suppress thermogenic gene transcription. Taken together, TET1 is a potent beige-selective epigenetic breaker of the thermogenic gene program. Our findings from this chapter may lead to a therapeutic strategy to increase energy expenditure in obesity and related metabolic disorders. The second part of the thesis demonstrates the role of DNA (cytosine-5)-methyltransferase 3A (DNMT3A) in exercise regulation. Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress. The aim of this dissertation work was to identify and characterize the role of adipose Tet1 in the epigenetic regulation of thermogenesis and the role of skeletal muscle Dnmt3a in exercise metabolism and oxidative stress. This may lead to a therapeutic strategy in obesity, related metabolic disorders and exercise intolerance. Chapter 1 provides a detailed understanding of the mechanisms of DNMT3A and TET2, which may lead to identifying novel targets for the treatment of IR and relevant human diseases. Even though a plethora of studies have found that changes in DNA methylation are associated with metabolic dysregulation, the functional role is poorly understood. Here, I will review the currently available literature and point out the remaining questions to be answered in order to gain a better understanding of the mechanisms of DNMT3a and TET2. Chapter 2 exhibits my efforts in characterizing the epigenetic role of DNA demethylase TET1 in suppressing key thermogenic genes. TET1 suppresses the thermogenic activation of beige adipocytes. Moreover, adipose-specific TET1 loss-of function in vivo led to increased energy expenditure and protection from diet-induced obesity, insulin resistance, and glucose tolerance. TET1 coordinates with HDAC1 to suppress thermogenic gene transcription in a DNA demethylase-independent manner. These findings will open new avenues for developing therapeutic strategies to increase energy expenditure in obesity and related metabolic disorders. Chapter 3 shows the previously unknown role of DNMT3A in endurance exercise skeletal muscle mitochondrial biology. DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Mechanistically, we reveal that ALDH1L1 serves as a novel molecular link that contributes to oxidative stress and mitochondrial dysfunction following the loss of Dnmt3a in red muscle. This is of great importance from the standpoint of exercise physiology, as physical activity is strongly encouraged as a key strategy for preventing and treating a wide range of human diseases. Chapter 4 concludes my work on the role of epigenetic enzymes TET1 and DNMT3A in metabolism and related disorders. Further, it also presents future directions and additional questions to get a further understanding

TET2 facilitates PPARγ agonist–mediated gene regulation and insulin sensitization in adipocytes

Emerging evidence indicates that epigenetic mechanisms like DNA methylation directly contribute to metabolic regulation. For example, we previously demonstrated that de novo DNA methyltransferase Dnmt3a plays a causal role in the development of adipocyte insulin resistance. Recent studies suggest that DNA demethylation plays an important role in the developmental process of adipocytes. However, little is known about whether DNA demethylase ten-eleven translocation (TET) proteins regulate the metabolic functions of adipocytes.MethodsThe expression of Tet genes was assessed in the fractionated adipocytes of chow- and high fat diet-fed C57/Bl6 mice using qPCR and western blotting. The effect of Tet2 gain- or loss-of-function in fully mature 3T3-L1 adipocytes in the presence/absence of Rosiglitazone (Rosi) and TNF-α on insulin sensitivity was using the insulin-stimulated glucose uptake and insulin signaling assays. Gene expression and DNA methylation analyses of PPARγ target genes was performed in the same setting. In addition, PPARγ reporter assays, co-immunoprecipitation assays, PPARγ ChIP-PCR analyses were performed.ResultsWe found that adipose expression of TET2, alone among its family members, was significantly reduced in diet-induced insulin resistance. TET2 gain-of-function was sufficient to promote insulin sensitivity while loss-of-function was necessary to facilitate insulin sensitization in response to the PPARγ agonist Rosiglitazone (Rosi) in cultured adipocytes. Consistent with this, TET2 was required for Rosi-dependent gene activation of certain PPARγ targets accompanied by changes in DNA demethylation at the promoter regions. Furthermore, TET2 was necessary to sustain PPARγ binding to target loci upon activation with Rosi via physical interaction with PPARγ.ConclusionsOur data demonstrate that TET2 works as an epigenetic regulator of Rosi-mediated insulin sensitization and transcriptional regulation in adipocytes

A necessary role of DNMT3A in endurance exercise by suppressing ALDH1L1-mediated oxidative stress.

Exercise can alter the skeletal muscle DNA methylome, yet little is known about the role of the DNA methylation machinery in exercise capacity. Here, we show that DNMT3A expression in oxidative red muscle increases greatly following a bout of endurance exercise. Muscle-specific Dnmt3a knockout mice have reduced tolerance to endurance exercise, accompanied by reduction in oxidative capacity and mitochondrial respiration. Moreover, Dnmt3a-deficient muscle overproduces reactive oxygen species (ROS), the major contributors to muscle dysfunction. Mechanistically, we show that DNMT3A suppresses the Aldh1l1 transcription by binding to its promoter region, altering its epigenetic profile. Forced expression of ALDH1L1 elevates NADPH levels, which results in overproduction of ROS by the action of NADPH oxidase complex, ultimately resulting in mitochondrial defects in myotubes. Thus, inhibition of ALDH1L1 pathway can rescue oxidative stress and mitochondrial dysfunction from Dnmt3a deficiency in myotubes. Finally, we show that in vivo knockdown of Aldh1l1 largely rescues exercise intolerance in Dnmt3a-deficient mice. Together, we establish that DNMT3A in skeletal muscle plays a pivotal role in endurance exercise by controlling intracellular oxidative stress

TET1 is a beige adipocyte-selective epigenetic suppressor of thermogenesis.

It has been suggested that beige fat thermogenesis is tightly controlled by epigenetic regulators that sense environmental cues such as temperature. Here, we report that subcutaneous adipose expression of the DNA demethylase TET1 is suppressed by cold and other stimulators of beige adipocyte thermogenesis. TET1 acts as an autonomous repressor of key thermogenic genes, including Ucp1 and Ppargc1a, in beige adipocytes. Adipose-selective Tet1 knockout mice generated by using Fabp4-Cre improves cold tolerance and increases energy expenditure and protects against diet-induced obesity and insulin resistance. Moreover, the suppressive role of TET1 in the thermogenic gene regulation of beige adipocytes is largely DNA demethylase-independent. Rather, TET1 coordinates with HDAC1 to mediate the epigenetic changes to suppress thermogenic gene transcription. Taken together, TET1 is a potent beige-selective epigenetic breaker of the thermogenic gene program. Our findings may lead to a therapeutic strategy to increase energy expenditure in obesity and related metabolic disorders

Determination of polycyclic aromatic hydrocarbon levels of groundwater in Ife north local government area of Osun state, Nigeria

This study determined the presence and levels of Polycyclic Aromatic Hydrocarbons (PAHs) of groundwater in Moro, Edun-Abon, Yakoyo and Ipetumodu communities in Ife-North Local Government Area of Osun State. This was with a view to create public awareness about the safety of groundwater as a source for domestic purposes (e.g., drinking, cooking etc.) in non-industrial area. Water samples were collected on seasonal basis, comprising of three months (AugustâOctober) in the wet season and three months (DecemberâFebruary) in the dry season. The PAHs in the water samples were extracted with n-hexane using liquidâliquid extraction method, while their qualitative identifications and quantitative estimations were carried out with the use of gas chromatography. Levels of PAHs detected showed predominance of light PAHs (less than four fused rings) for both wet and the dry seasons. Higher concentrations of PAHs were recorded during the wet season than the dry season. The study concluded that the groundwater in the communities was contaminated with light PAHs and the total PAHs in this area exceeded the maximum permissible limit of 10 μg Lâ1 recommended by World Health Organization (WHO) for safety of groundwater. Keywords: Polycyclic aromatic hydrocarbons, Groundwater, Water quality, Seasonal variation, Health impac