dysfunction in white adipose tissue.

Mitochondrial function/dysfunction in white adipose tissue

Sihem Boudina and Timothy E. Graham

Division of Endocrinology, Metabolism and Diabetes and Program in Molecular Medicine, University of Utah School of Medicine, Salt Lake City, Utah 84112

Corresponding author: Sihem Boudina Address: Division of Endocrinology, Metabolism and Diabetes Program in Molecular Medicine 15 N 2030 E Bldg. # 533 Rm. 3410B Salt Lake City, Utah 84112 Phone: (801) 585-6833 Fax: (801) 585-0701 E-mail: [email protected]

This is an Accepted Article that has been peer-reviewed and approved for publication in the Experimental Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an Accepted Article; doi: 10.1113/expphysiol.2014.081414. This article is protected by copyright. All rights reserved. Downloaded from Exp Physiol (ep.physoc.org) at QUEEN'S UNIVERSITY on August 25, 2014

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Abstract The role of mitochondria in white adipocytes has long been neglected due in part to their lower abundance in these cells. However, accumulating evidence suggests that mitochondria are vital for maintaining metabolic homeostasis in white adipocytes because of their involvement in adipogenesis, fatty acid (FA) synthesis and esterification, branched-chain amino acid catabolism and lipolysis. Therefore, it is not surprising that white adipose tissue function can be perturbed by altering mitochondrial components or oxidative capacity. Moreover, studies in humans and animals with significantly altered fat mass, such as in obesity or lipoatrophy, indicate that impaired mitochondrial function in adipocytes may be directly linked to the development of metabolic diseases such as diabetes and insulin resistance. However, recent studies that specifically targeted mitochondrial function in adipocytes indicated a dissociation between impaired mitochondrial oxidative capacity and systemic insulin sensitivity.

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Introduction The term “mitochondria” was first introduced by Carl Brenda in 1898 (Ernster and Schatz, 1981) and originates from the Greek “mitos” (thread) and “chondros” (granule), referring to the appearance of these structures during spermogenesis. Since then, mitochondria have become an important subject of research within several disciplines of experimental biology. Indeed, early work in the 19th century focused on the development of morphometric and functional methodologies to study these organelles, followed by a detailed assessment of their role in health and disease in later years. Mitochondria are double-membrane organelles composed of five distinct parts: (1) the mitochondrial outer membrane (OMM); (2) the mitochondrial inner membrane (MIM); (3) the inter-membrane space; (4) the cristae and (5) the matrix. The OMM and MIM are composed of phospholipids and proteins, whereas the matrix contains 2/3 of mitochondrial proteins including most metabolic enzymes. In addition, mitochondrial ribosomes, tRNAs and mitochondrial DNA are contained in the matrix compartment. Mitochondria are the major source of ATP in cells using aerobic respiration, with oxygen consumption being tightly coupled to ATP production. However, in recent years, other functions have been attributed to mitochondria including the generation of several metabolites necessary for cytosolic processes, amino acid catabolism, ketogenesis, urea cycle, reactive oxygen species (ROS) formation, calcium cycling, and regulation of a variety of cell signaling pathways, including key roles as effectors of apoptosis and cell death (Starkov, 2008; Williams et al., 2013; Czabotar et al., 2014). Due to the multiplicity of mitochondrial functions, diverse regulatory processes are in place to control and protect this organelle in cells at the levels of biogenesis, dynamic networking, and turnover/degradation.


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Mitochondrial dysfunction in adipose tissue has been the subject of several recent reviews (De Pauw et al., 2009; Patti and Corvera, 2010; Kusminski and Scherer, 2012), we aim in this review to update the reader on the role of mitochondria in the homeostatic function of white adipose tissue (WAT) and to outline recent evidence that alterations in mitochondrial oxidative phosphorylation in WAT most likely is not a key determinant of systemic insulin resistance. I-

Mitochondria and the homeostatic function of white adipose tissue

White adipose tissue (WAT) had once been considered to merely be a repository for excess nutrients in the form of triglycerides. Indeed, WAT is the most plastic organ in the body capable of storing and releasing lipids in response to energy excess and need. However, in addition to being a storage depot, recent work has shown that WAT is a high active major endocrine organ impacting the function of several tissues in the body. Morphologically, brown adipose tissue (BAT) is distinguished from WAT mainly by its very high mitochondrial content and expression of uncoupling protein 1 (UCP1). Because of these differences, WAT and BAT have distinct physiological roles, with BAT being a key mediator of non-shivering thermogenesis which prevents hypothermia in small mammals and human neonates (Klingenspor, 2003). The thermogenic capacity of BAT is achieved through a UCP1-mediated mitochondrial proton leak that uncouples oxidative phosphorylation from ATP synthesis (Reed and Fain, 1968; Prusiner et al., 1968; Nedergaard et al., 1977). In contrast to brown adipocytes, white adipocytes contain fewer and smaller mitochondria, which are located in the periplasmic rim (Kopecky et al., 2001). Despite being lower in number, mitochondria in white adipocytes play a key role in coordinating energy production with nutritional cues. (1) Adipogenesis This article is protected by copyright. All rights reserved. Downloaded from Exp Physiol (ep.physoc.org) at QUEEN'S UNIVERSITY on August 25, 2014

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Electron microscopy was first used to observe changes in mitochondrial number and morphology during the course of adipogenesis both in vitro and in vivo (Sheldon et al., 1962; Williamson, 1964; Novikoff et al., 1980). These microscopic observations of mitochondrial remodeling during differentiation were further confirmed using a proteomic approach. Indeed, Wilson-Fritch et al. (Wilson-Fritch et al., 2003) reported a 20-30-fold increase in the concentration of numerous mitochondrial proteins during 3T3-L1 cells differentiation, an increase that was not entirely accounted for by enhanced transcription but also involved posttranscriptional regulation. The molecular mechanisms underlying mitochondrial biogenesis during adipogenesis are not fully characterized but appear to require main transcription factors known to regulate both processes such as the peroxisome proliferator-activated receptor gamma (PPAR) and its co-activator PGC1α (De Pauw et al., 2009). Indeed, treatment of fully differentiated 3T3-L1 adipocytes with a PPAR agonist further increased mitochondrial mass and changed mitochondrial morphology (Wilson-Fritch et al., 2003; Wilson-Fritch et al., 2004). In addition to changes in mitochondrial number and morphology during adipogenesis, higher oxidative capacity was also reported (Wilson-Fritch et al., 2003; Ducluzeau et al., 2011). Interestingly and considering the ATP needs for adipogenesis, oxygen consumption was reported to be coupled to ATP synthesis early during 3T3-L1 differentiation and then progressively became uncoupled as the cells mature to adipocytes (Ducluzeau et al., 2011). As a result of enhanced oxidative metabolism, reactive oxygen species (ROS) levels also increased during the course of differentiation and are believed to play a critical role in signaling during this process (Tormos et al., 2011). However, the amount and the duration of ROS generation is critical in the initiation of physiological versus pathological responses.


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(2) Adipokine secretion While white adipocytes have long been considered as a sink for circulating lipids, the emergence of a new role for these cells in hormone secretion has given them a new consideration. Indeed, white adipocytes secrete several cytokines known as adipokines, including leptin, adiponectin, tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), resistin and others. Adiponectin, which exerts anti-hyperglycemic and insulin-sensitizing effects, has been shown to be secreted from small adipocytes that exhibit healthy metabolic status (Yu and Zhu, 2004). Furthermore, a direct link between adiponectin secretion and mitochondrial function was provided by Koh et al. (Koh et al., 2007), who showed that manipulations that decreased or increased mitochondrial biogenesis or function directly inhibited or enhanced adiponectin synthesis and secretion. The mechanisms involved in mitochondrial dysfunctioninduced inhibition of adiponectin secretion involve enhanced endoplasmic reticulum (ER) stress-mediated activation of c-Jun NH2-terminal kinase (JNK), the induction of activating transcription factor 3 (ATF3), lack of ATP synthesis, inducible nitric oxide (iNOS) activation and enhanced oxidative stress (Jeon et al., 2012; Huh et al., 2012). (3) Lipogenesis Adipose tissue is the major site of the conversion of carbohydrates to fat or lipogenesis (Shapiro and Wertheimer, 1948), a process highly regulated especially by insulin at the level of glucose uptake (Goodman, 1963). Mitochondria are essential for the anaplerotic generation of metabolic intermediates necessary for lipogenesis (De Pauw et al., 2009). The biosynthesis of FA from glucose and pyruvate by adipose tissue involves the oxidative decarboxylation of pyruvate to acetyl-CoA within mitochondria and the synthesis of FA from acetyl-CoA outside the mitochondria (Martin and Denton, 1970). Interventions that affect mitochondrial


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coupling and ATP generation have been shown to directly impact FA synthesis in situ. Thus, addition of the uncoupler 2,4-dinitrophenol depressed lipogenesis and increased lactate production in epididymal fat pad segments (Rognstad and Katz, 1969), probably as a result of a limitation in ATP availability for pyruvate carboxylation in the mitochondria. Indeed, the activity of the key enzyme in lipogenesis pyruvate carboxylase is 3-fold higher in white adipose tissue and is regulated by the ATP/ADP ratio (Patel and Hanson, 1970). Moreover, lipogenic activity of white adipose tissue was low in adipose-specific uncoupling protein 1 (UCP1) transgenic mice, secondary to the depression in ATP/ADP ratios due to uncoupling of the electron transport chain (Kopecky et al., 2001). (4) Fatty acid (FA) esterification and glyceroneogenesis Re-esterification of free fatty acids (FFAs) in adipocyte is a futile cycle in which triglycerides (TGs) broken down during lipolysis are reutilized through their esterification with glycerol 3-phosphate, which is produced by glucose via glycolysis in the fed state. However, when the supply of glucose is limited, as in starvation or in a low-carbohydrate diet, glyceroneogenesis from pyruvate and amino acids takes place in adipocytes (Ballard et al., 1967; Reshef et al., 1969; Botion et al., 1995). One of the rate limiting steps in glyceroneogenesis is the synthesis of phosphoenolpyruvate

from

oxaloacetate

catalyzed

by

phosphoenolpyruvate

carboxykinase (PEPC-K), an enzyme that is found both in the cytosol and in the mitochondria. Indeed, adipose-specific deletion or over-expression of PEPCK-C abolished or enhanced glyceroneogenesis in WAT (Olswang et al., 2002; Franckhauser et al., 2002). The role of mitochondria in FA esterification and glyceroneogenesis is essential. As discussed below, mitochondrial ATP generation dictates the rate of lipolysis in adipocytes and lipolysis and FA esterification are


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tightly coupled. Furthermore, under conditions of glucose deprivation, the production of glycerol-3-phosphate requires not only ATP (in the conversion of pyruvate to oxaloacetate by pyruvate carboxylase) but also TCA cycle intermediates such as pyruvate and malate. Moreover, one important player in the regulation of glyceroneogenesis is mitochondrial pyruvate dehydrogenase that functions as a metabolic switch between glucose and FA utilization and its inhibition by pyruvate dehydrogenase kinase 4 (PDK4) enables pyruvate to be used for glyceroneogenesis when the glucose is low (recently reviewed (Flachs et al., 2013)). (5) Lipolysis In white adipose tissue (WAT), lipolysis generates free fatty acids (FFAs) and glycerol that are released into the blood for transport to other tissues such as cardiac muscle, skeletal muscle and liver in conditions of energy need. As such, FFAs and glycerol release during fasting is unique to WAT as other tissues usually use the products of triglyceride (TG) hydrolysis instead of releasing them on the bloodstream. However, the enzymatic control of lipolysis or TG hydrolysis are almost similar in all tissues. Because of the essential role of adipose lipolysis, numerous regulatory pathways that promote or inhibit this process exist. Indeed, WAT lipolysis is

activated

mainly

by

catecholamines

acting

through

-adrenergic

receptors/adenylate cyclase/cAMP/PKA pathway (Holm et al., 2000; Collins et al., 2004). In contrast, lipolysis is inhibited mainly by insulin and insulin-like growth factors through PI3K/Akt pathway (Langin, 2006). Lipolysis is also critical for brown adipose tissue (BAT) because of the higher rate of fat oxidation and the necessary activation of UCP1 by fatty acids during thermogenesis. Indeed, mice deficient in adipose triglyceride lipase (ATGL), a key enzyme in the first step of TG hydrolysis, exhibited whitening of BAT, increased adiposity, impaired thermogenesis but


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paradoxally maintained insulin sensitivity (Ahmadian et al., 2011). Lipolysis and mitochondrial ATP synthesis are tightly coupled and inhibitors or uncouplers of the electron transport chain (ETC) abolish lipolysis stimulated by catecholamines (Fassina et al., 1974). Furthermore, the regulation of lipolysis is sensitive to the energy status of the adipocytes and the activation of AMP-activated protein kinase (AMPK). Indeed, AMPK was recently shown to directly phosphorylate ATGL to increase its activity in adipocytes (Ahmadian et al., 2011). In addition, ATP is also required for insulin-suppression of lipolysis as it is required for insulin’s binding to its receptors (Steinfelder and Joost, 1983). (6) Branched Chain Amino Acid (BCAA) Metabolism The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine comprise 40% of essential amino acids in daily dietary intake and forms distinct class of amino acids whose degradation occurs primarily at extrahepatic sites (Ichihara and Koyama, 1966; Harper et al., 1984). Adipose tissue is one of the most important tissues capable of BCAAs catabolism (Rosenthal et al., 1974), with leucine being a significant precursor for fatty acid and sterol biosynthesis in adipocytes particularly when glucose is available (Goodman, 1963). In addition, leucine serves as a source for nitrogen for synthesis of glutamine, which is released by adipocytes (Tischler and Goldberg, 1980). The catabolism of BCAAs proceeds through transamination to the respective 2-oxo acids, followed by an intra-mitochondrial oxidative decarboxylation catalyzed by the branched-chain 2-oxo acid dehydrogenase (BCDH). Therefore, it is not surprising that during adipogenesis, when mitochondrial mass is elevated, leucine catabolism and the expression of enzymes involved in BCAA catabolism dramatically increased (Frerman et al., 1983; Kitsy et al., 2014). Furthermore, mice lacking the mitochondrial branched-chain aminotransferase enzyme (BCAT2) at the


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whole body level exhibited reduced adiposity and enhanced energy expenditure despite higher food intake and increased circulating levels of BCAAs (She et al., 2007). Consistent with these results, mice maintained on a leucine-deficient diet exhibited reduced fat mass, enhanced energy expenditure, increased lipolysis and reduced lipogenic activity (Cheng et al., 2010). Although adipose tissue has been shown to contribute to blood BCAA concentrations and that blood levels of BCAAs positively correlate with insulin resistance and type 2 diabetes (Herman et al., 2010; Felig et al., 1969; Adams, 2011), it is difficult to distinguish between adipose versus non-adipose contribution of altered BCAA catabolism in the pathogenesis of obesity and insulin resistance. II-

Mitochondrial dysfunction in white adipose tissue

Research in the area of obesity, Type 2 diabetes and lipoatrophies has now provided strong evidence for the importance of mitochondria in the pathogenesis of these diseases. Studies by Corvera and colleagues (Wilson-Fritch et al., 2003; WilsonFritch et al., 2004) have reported a decrease in mitochondrial mass and function in WAT of obese ob/ob mice that can be reversed by rosiglitazone treatment. Furthermore, mitochondrial DNA (mtDNA) was shown to be reduced in WAT of obese humans and animals (Rong et al., 2007; Pietilainen et al., 2008). Interestingly, alterations in mtDNA are not a consequence of obesity per se but rather caused by diabetes (Dahlman et al., 2006). In addition to mitochondrial alterations caused by obesity and diabetes in WAT, studies of human immunodeficiency virus (HIV) lipodystrophy have provided strong evidence for the importance of mitochondria in this organ, a topic that was extensively reviewed elsewhere (Villarroya et al., 2009). However, we will focus this review on the alterations in mitochondrial function in


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WAT that are associated with obesity and Type 2 diabetes as summarized in Figure 1. (1) Impaired oxidative capacity Because of the low mitochondrial yield of WAT, few studies especially using human fat tissue have been conducted to study the impact of obesity and diabetes on the oxidative capacity of mitochondria. Despite this limitation, recent studies showed that membrane potential, inorganic phosphate utilization and the activities of respiratory chain complexes I-IV were reduced in isolated mitochondria from subcutaneous depots obtained from obese patients (Chattopadhyay et al., 2011). Interestingly, the reduction in oxidative capacity was similar in obese diabetic versus obese non diabetic patients, suggesting that obesity per se impairs mitochondrial function. Similarly, adipocyte mitochondrial oxidative capacity was reduced in obese subjects independently of adipocyte hypertrophy (Yin et al., 2014). In contrast, Arner and colleagues (Dahlman et al., 2006) reported a down-regulation of several genes of the electron transport chain that was most prominent in visceral fat of Type 2 diabetic women independent of obesity. This discrepancy might be related to the use of different fat depots (subcutaneous versus visceral) and methodologies (functional versus transcriptional). Contrary to the limited data on humans, several animal studies using either genetic or dietary models of obesity have clearly showed reduced mitochondrial oxidative capacity in WAT (Wilson-Fritch et al., 2003; Choo et al., 2006). However, it remains unclear whether these changes are causal or secondary to the metabolic alterations associated with obesity such as insulin resistance and diabetes. Indeed, studies performed on high fat (HF)-fed animals revealed that mitochondrial dysfunction might result from altered systemic insulin sensitivity and glucose tolerance in mice (Sutherland et al., 2008; Wang et al., 2014).


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The mechanisms responsible for diminished mitochondrial oxidative capacity in WAT with obesity are not well characterized but may involve enhanced inflammation, higher oxidative damage, ER stress, reduced biogenesis and impaired mitochondrial dynamics (Valerio et al., 2006). Indeed, increased ROS generation in epididymal fat preceded mitochondrial impairments in HF-fed mice (Wang et al., 2014). (2) Enhanced reactive oxygen species (ROS) production The role of ROS in WAT has not always been clear since these species are both necessary and detrimental for adipose function. At low levels, ROS in the form of hydrogen peroxide (H2O2) play a key role especially in insulin signal transduction (Loh et al., 2009). Indeed, mitochondria-generated H2O2 is required for the induction of PPAR during mesenchymal stem cell differentiation to adipocytes in vitro, an effect that is dependent on the mammalian target of rapamycin complex 1 (mTORC1) (Tormos et al., 2011). However, higher levels of ROS, achieved through inhibition of the electron transport chain, have been associated with reduced proliferation and differentiation of 3T3-L1 cells, 3T3-F442A preadipocytes and human stromo-vascular cells without causing cell death, thus promoting hypertrophic growth (Carriere et al., 2004; Carriere et al., 2003). The same group demonstrated that ROS-induced impairment of adipogenesis was mediated by the induction of the adipogenic repressor CHOP-10/GADD153, an effect that could be reversed by antioxidant treatment. Similar to these in vitro findings, several studies have clearly showed that ROS and oxidative damage are elevated in WAT of obese humans and animals (Galinier et al., 2006; Frohnert et al., 2011). Moreover, in addition to mitochondrial ROS, cytosolic sources of ROS such as the NADPH oxidase have been associated with obesity in both humans and animals (Furukawa et al., 2004).


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Further studies are needed to examine the contribution of mitochondrial ROS in the development of obesity and the systemic metabolic abnormalities associated. (3) Altered autophagy and mitochondrial turnover Autophagy is a ubiquitous intracellular trafficking system through which old or damaged cytosolic components, membrane regions, and whole organelles are engulfed in a double-membrane structure (the autophagosomes) that targets them for degradation in lysosomes. Degraded autophagic cargo can be used as components for new synthetic reactions, or as substrates to support cellular energy needs. Autophagy is highly regulated by nutrient availability, via suppression of mTOR complex 1 and/or activation of AMP-activated kinase, and is increased in many states of cellular stress, including starvation, growth factor withdrawal, protein misfolding, and inflammation. Importantly, autophagy is the sole pathway through which intact, damaged or aging mitochondria are degraded. Autophagy also plays a key role in the developmental “clearance” of mitochondria, such as in reticulocyte to erythrocyte differentiation or in crystallization of the ocular lens. Studies of fat biopsies indicated that autophagy is increased in human subjects with obesity or Type 2 diabetes, with particularly high rates of autophagy in biopsies from the omental fat depots and in individuals with a greater degree of insulin resistance (Ost et al., 2010; Kovsan et al., 2011). Adipocyte size, which is highly correlated with insulin resistance in most cohorts, was also highly correlated with expression of key autophagy regulatory genes Atg5 and Atg8 (LC3a and LC3b) (Kovsan et al., 2011). These findings are consistent with reports that Atg5 and Atg8, along with several other autophagy genes, are regulated by FoxO transcription factors whose activity is known to be unregulated in the setting of insulin resistance. In addition FoxO1 binds Atg7 and may directly regulate its activity (Zhao et al., 2010). Reduced


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phosphorylation of the ribosomal S6 kinase, suggesting reduced mTOR complex 1 activity, has been observed in adipocytes of Type 2 diabetics (Ost et al., 2010); however, this finding is consistent with an expected reduction in insulin/Akt signaling in adipocytes of these subjects, and whether mTORC1 signaling regulates adipocyte autophagy generally in fat has yet to be determined. Together these studies suggest adipocyte autophagy is increased in the setting of obesity and Type 2 diabetes. Given a multitude of observations that ER stress, oxidative stress, tissue hypoxia, inflammation, and insulin resistance are all present in adipocytes of obese and Type 2 diabetic subjects, it is possible that the observed increase in autophagy represents a normal response to the state of sustained cellular stress in adipocytes. Recent work on autophagy in adipocytes provided insight into the process of mitochondrial turnover and degradation.

Recent studies employing mice with

Cre/loxP-mediated adipocyte-specific knockout of Atg7 (adipocyte Atg7 KO mice) have suggested a critical role for autophagy in adipogenesis and obesity, which play important roles in insulin resistance and Type 2 diabetes. Two groups independently reported that mice with adipose tissue-specific genetic knockout of Atg7 via aP2promoter Cre/loxP recombination display an ultra-lean phenotype with enhanced insulin sensitivity and glucose tolerance (Zhang et al., 2009; Singh et al., 2009). These mice have normal birth weight but gain fat mass less quickly than wild-type mice after adulthood and are protected from obesity on high fat diet. WAT depots of adipocyte Atg7 knockout mice are very small and have the appearance of brown adipose tissue, including the presence of large numbers of mitochondria and multilocular small lipid droplets; in parallel, there is an expansion of the suprascapular BAT depot by brown adipocytes containing increased numbers of mitochondria (Zhang et al., 2009).


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The expanded mitochondrial populations in WAT and BAT of adipocyte Atg7 knockout mice are functional, as -oxidation of lipids is greatly increased, whole body energy expenditure is enhanced and respiratory quotient is decreased (Zhang et al., 2009; Singh et al., 2009). UCP1 and PGC1 protein expression are increased in WAT and BAT of adipocyte Atg7 knockout mice, consistent with increased mitochondrial content; however, expression of BAT lineage-specific genes such as Elovl3, Cidea, and Prdm16, was not increased. Furthermore, the number and proliferative capacity of WAT lineage-specific precursors in the adipose tissue stroma of adipocyte Atg7 knockout mice are not altered (Singh et al., 2009). One interpretation is that the absence of autophagy forces committed WAT lineage precursor cells to transdifferentiate into a BAT-like phenotype. Indeed, expansion of the mitochondrial content normally occurs during early WAT differentiation and is subsequently reduced during terminal differentiation to produce the mature white adipocyte adapted to lipid storage rather than utilization. The phenotype of Atg7 knockout adipocytes suggests that autophagy plays a crucial role in this remodeling process, such that a BAT-like phenotype results when mitochondria cannot be cleared during terminal differentiation. Furthermore, studies of Atg5 null mouse embryonic fibroblasts (MEFs) also reveals impaired in vitro adipogenesis characterized by normal early stages of adipocyte differentiation followed by failure to accumulate lipid droplets (Baerga et al., 2009). Consistent with an adipogenic impairment, Atg5 total body knockout mouse embryos exhibit reduced numbers of WAT-like adipocytes, possibly due to increased rates of apoptosis in later stages of differentiation (Baerga et al., 2009). In 3T3-L1 adipocytes, knockdown of Atg5 or Atg7, or treatment with the lysosome inhibitor chloroquine, also impairs differentiation (Baerga et al., 2009).


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There are several important caveats for interpreting the phenotype of adipocyte Atg7 KO mice vis a vis normal physiology. Because the aP2-Cre promoter used to impair autophagy in these mice is active in development as early as embryonic day 6, the mice never develop normal white adipose tissue at any point in their lives, and therefore

essentially

represent

models

of

impaired

primary

adipogenesis.

Furthermore, the enhanced insulin sensitivity and glucose tolerance observed in these mice most likely results from their extremely lean and hypercatabolic phenotype. The observed metabolic phenotypes may be ultimately or solely developmental in nature, and may not be directly relevant to the in vivo role of autophagy in mature white adipose tissue of adult rodents or in humans with obesity, insulin resistance, or Type 2 diabetes. Moreover, while the lean and insulin-sensitive phenotype of adipocyte Atg7 knockout might be viewed as “healthy”, the mice begin to die from unknown causes at 8 weeks of life and exhibit ~40% mortality by 12 weeks of age, indicating that autophagy in adipose tissue is likely necessary for life. More work in this area will be needed to understand both the physiological and pathophysiological roles of autophagy in adipocytes in obesity, insulin resistance.

III-

Adipose-specific alterations in mitochondrial function: what have we learned so far?

Several recent studies have targeted mitochondria specifically in adipose tissue or in adipocytes to enhance or reduce mitochondrial function, leading to a reconsideration of the role of mitochondria in adipose tissue. In particular, findings from these studies suggest that adipose tissue mitochondria are not just “power plants” for ATP generation, but rather important integrators of metabolic signals that can reprogram several processes to affect whole body physiology. Development of transgenic methods using the adipocyte-specific fatty acid-binding protein (aP2) promoter in the This article is protected by copyright. All rights reserved. Downloaded from Exp Physiol (ep.physoc.org) at QUEEN'S UNIVERSITY on August 25, 2014

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1990s (Ross et al., 1990), enabled assessing the roles of various direct and indirect regulators of mitochondrial function in fat in vivo, as well as their secondary effects on whole body metabolism in intact mice. This technology has recently been further improved by introduction of adiponectin promoter-based transgenic methods that considerably improve the tissue-specificity of these sorts of genetic manipulations in mice. For example, when uncoupling protein 1 (UCP1) was overexpressed in adipose tissue (aP2-UCP1 transgenic mice), mitochondrial uncoupling was dramatically increased; the mice accumulated less subcutaneous fat and resisted genetic and dietary obesity despite developing atrophy of brown adipose tissue (Kopecky et al., 1995; Kopecky et al., 1996). Resistance to adiposity in aP2-UCP1 transgenic mice is due to enhanced energy expenditure and fatty acid oxidation and to reduced lipogenesis in WAT (Rossmeisl et al., 2000). Similarly, when UCP1 was overexpressed in 3T3-L1 cells during differentiation in vitro, the cells accumulated less lipids whereas oxygen consumption and -oxidation were minimally affected, suggesting a decrease in FA synthesis (Si et al., 2007). Mechanisms for reduced FA synthesis in aP2-UCP1 transgenic mice include repression of mRNA expression of genes involved in lipogenesis, and possibly, inhibition of ATP-dependent pyruvate carboxylase, decreasing the mitochondrial pool of oxaloacetate and citrate to suppress the activity of the malate cycle that supplies the NADPH needed for lipogenesis (Kopecky et al., 1996; Si et al., 2007). Manipulation of expression of the mitochondrial biogenesis regulator PGC1 in adipocytes in vivo has revealed important information about the role of this transcriptional regulator in fat. PGC1 is strongly induced by the thiazolidinedione class of PPAR agonist insulin-sensitizer medications and is proposed to be a key regulator of mitochondrial biogenesis and function in different tissues. Using the aP2


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promoter, Pardo and colleagues (Pardo et al., 2011) demonstrated that Cre/loxPmediated genetic knockout of PGC1α in adipose tissue of mice produced a modest reduction in body weight and adipocyte size on chow diet, but had little or no impact on the expression of genes involved in oxidative phosphorylation, TCA cycle or fat oxidation. Moreover adipocyte PGC1 knockout mice showed normal mitochondrial content and a normal response to increase mitochondrial biogenesis when treated with the PPAR agonist, rosiglitazone.

In contrast, the ability of rosiglitazone to

induce brown adipocyte specific markers in WAT was severely impaired. Kleiner et al. (Kleiner et al., 2012) reported a similar phenotype of impaired UCP1 and brown adipocyte marker expression in mice with adiponectin promoter-Cre-mediated knockout of adipocyte PGC1; consistent with this, basal oxygen consumption rates of white adipocytes were normal, but there was reduction in endogenous uncoupled respiration. The adiponectin-Cre PGC1 adipocyte knockout mice were not protected from weight gain on high fat diet, and were even more susceptible to insulin resistance and glucose intolerance than their wild-type littermates. Because PGC1 is also upregulated by rosiglitazone, one possible explanation for the minimal mitochondrial phenotype of PGC1 knockout mice treated with rosiglitazone is compensation by PGC1. To test this possibility, Pardo and colleagues compared effects of siRNA knockdown of PGC1 versus PGC1 on mitochondrial gene expression in rosiglitazone-treated 3T3-L1 adipocytes in vitro. They found that only PGC1 knockdown prevented induction of mitochondrial oxidative phosphorylation enzymes, and reduced mitochondrial respiration in both the basal and uncoupled state. Subsequent characterization of mice with aP2 promoter-Cre-mediated knockout of adipocyte PGC1 confirmed that impairment of PGC1 is sufficient to prevent rosiglitazone induction of mitochondrial oxidative phosphorylation enzymes. This article is protected by copyright. All rights reserved. Downloaded from Exp Physiol (ep.physoc.org) at QUEEN'S UNIVERSITY on August 25, 2014

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Mitochondrial content, however, was not decreased, and mitochondrial biogenesis in response to rosiglitazone was normal. Importantly, adipocyte knockout of PGC1 did not alter body weight or prevent weight gain on high fat diet, and had no effect on insulin sensitivity or glucose tolerance. Whether the impaired induction of mitochondrial oxidative phosphorylation enzymes resulted in reduced respiration in adipose tissue was not tested; however maximal respiration was reduced in rosiglitazone-treated 3T3-L1 adipocytes subjected to PGC1knockdown in vitro. Reduction of mitochondrial biogenesis through deletion of mitochondrial transcription factor A (TFAM) in adipocytes altered the expression of proteins involved in the electron transport chain, decreased complex I activity, and enhanced ROS generation (Vernochet et al., 2012). However, endogenous uncoupling, -oxidation, and basal and insulin-stimulated glycolysis were increased. Despite the reduced complex I activity, these changes produced an enhanced flux through the electron transport chain that doubled mitochondrial oxygen consumption, increased total body energy expenditure, and conferred protection against HF diet-induced insulin resistance. In addition, mice with knockout of the surfeit locus protein 1 (SURF1), a key protein involved in complex IV assembly, are another example of mice with impaired mitochondrial oxidative phosphorylation in fat that exhibit protection from obesity and insulin resistance. As with the adipocyte TFAM knockout mice, SURF1 knockout mice exhibit enhanced -oxidation and increased energy expenditure. Also of interest in this regards, mice with liver- or muscle-specific knockout of apoptosis initiating factor (AIF), a mitochondrial flavoprotein that regulates the electron transport chain, also exhibit improved insulin sensitivity and protection from obesity on high fat diet. However, it should be noted that not all interventions that reduce mitochondrial electron transport chain (ETC) activity result in obesity and insulin


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resistance. Such is the case for mice overexpressing the iron regulating protein mitoNEET specifically in adipose tissue. These transgenic mice are morbidly obese, exhibited less oxygen consumption, more FA esterification, enhanced adiponectin secretion, less ROS generation and improved peripheral insulin sensitivity (Kusminski et al., 2012). A distinction should be made between reduced mitochondrial ETC activity alone (without ROS generation) and mitochondrial dysfunction (usually associated with ROS generation). Furthermore, adipose mitoNEET transgenic mice were able to maintain a higher glycolytic rate, which may have provided sufficient energy to support adiponectin production and FA esterification. Finally, these animals also exhibited an increase in adipocyte hyperplasia with the formation of smaller healthy adipocytes capable of storing lipids to minimize their spillover in the circulation and their deposition in insulin-sensitive tissues, thus maintaining insulin sensitivity. Taken together these studies indicate that interventions that modulate mitochondrial function (including oxidative phosphorylation, ATP synthesis and ROS generation) in adipose tissue or adipocytes may in turn affect the development of obesity and insulin resistance. Further studies are require to examine more specifically which components of mitochondrial dysfunction (altered ETC activity, ATP depletion or ROS generation) in adipocytes are causal for the development of these conditions.


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References Adams, S. H. (2011). Emerging perspectives on essential amino acid metabolism in obesity and the insulin-resistant state. Adv Nutr 2(6): 445-456. Ahmadian, M., Abbott, M. J., Tang, T., Hudak, C. S., Kim, Y., Bruss, M., Hellerstein, M. K., Lee, H. Y., Samuel, V. T., Shulman, G. I., Wang, Y., Duncan, R. E., Kang, C. &Sul, H. S. (2011). Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype. Cell Metab 13(6): 739-748. Baerga, R., Zhang, Y., Chen, P. H., Goldman, S. &Jin, S. (2009). Targeted deletion of autophagyrelated 5 (atg5) impairs adipogenesis in a cellular model and in mice. Autophagy 5(8): 11181130. Ballard, F. J., Hanson, R. W. &Leveille, G. A. (1967). Phosphoenolpyruvate carboxykinase and the synthesis of glyceride-glycerol from pyruvate in adipose tissue. J Biol Chem 242(11): 27462750. Botion, L. M., Kettelhut, I. C. &Migliorini, R. H. (1995). Increased adipose tissue glyceroneogenesis in rats adapted to a high protein, carbohydrate-free diet. Horm Metab Res 27(7): 310-313. Carriere, A., Carmona, M. C., Fernandez, Y., Rigoulet, M., Wenger, R. H., Penicaud, L. &Casteilla, L. (2004). Mitochondrial reactive oxygen species control the transcription factor CHOP10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect. J Biol Chem 279(39): 40462-40469. Carriere, A., Fernandez, Y., Rigoulet, M., Penicaud, L. &Casteilla, L. (2003). Inhibition of preadipocyte proliferation by mitochondrial reactive oxygen species. FEBS Lett 550(1-3): 163-167. Chattopadhyay, M., Guhathakurta, I., Behera, P., Ranjan, K. R., Khanna, M., Mukhopadhyay, S. &Chakrabarti, S. (2011). Mitochondrial bioenergetics is not impaired in nonobese subjects with type 2 diabetes mellitus. Metabolism 60(12): 1702-1710. Cheng, Y., Meng, Q., Wang, C., Li, H., Huang, Z., Chen, S., Xiao, F. &Guo, F. (2010). Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue. Diabetes 59(1): 17-25. Choo, H. J., Kim, J. H., Kwon, O. B., Lee, C. S., Mun, J. Y., Han, S. S., Yoon, Y. S., Yoon, G., Choi, K. M. &Ko, Y. G. (2006). Mitochondria are impaired in the adipocytes of type 2 diabetic mice. Diabetologia 49(4): 784-791. Collins, S., Cao, W. &Robidoux, J. (2004). Learning new tricks from old dogs: beta-adrenergic receptors teach new lessons on firing up adipose tissue metabolism. Mol Endocrinol 18(9): 2123-2131. Czabotar, P. E., Lessene, G., Strasser, A. &Adams, J. M. (2014). Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15(1): 49-63. Dahlman, I., Forsgren, M., Sjogren, A., Nordstrom, E. A., Kaaman, M., Naslund, E., Attersand, A. &Arner, P. (2006). Downregulation of electron transport chain genes in visceral adipose tissue in type 2 diabetes independent of obesity and possibly involving tumor necrosis factor-alpha. Diabetes 55(6): 1792-1799. De Pauw, A., Tejerina, S., Raes, M., Keijer, J. &Arnould, T. (2009). Mitochondrial (dys)function in adipocyte (de)differentiation and systemic metabolic alterations. Am J Pathol 175(3): 927939. Ducluzeau, P. H., Priou, M., Weitheimer, M., Flamment, M., Duluc, L., Iacobazi, F., Soleti, R., Simard, G., Durand, A., Rieusset, J., Andriantsitohaina, R. &Malthiery, Y. (2011). Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation. J Physiol Biochem 67(3): 285-296. Ernster, L. &Schatz, G. (1981). Mitochondria: a historical review. J Cell Biol 91(3 Pt 2): 227s-255s. Fassina, G., Dorigo, P. &Gaion, R. M. (1974). Equilibrium between metabolic pathways producing energy: a key factor in regulating lipolysis. Pharmacol Res Commun 6(1): 1-21. Felig, P., Marliss, E. &Cahill, G. F., Jr. (1969). Plasma amino acid levels and insulin secretion in obesity. N Engl J Med 281(15): 811-816. This article is protected by copyright. All rights reserved. Downloaded from Exp Physiol (ep.physoc.org) at QUEEN'S UNIVERSITY on August 25, 2014

21

Flachs, P., Rossmeisl, M., Kuda, O. &Kopecky, J. (2013). Stimulation of mitochondrial oxidative capacity in white fat independent of UCP1: a key to lean phenotype. Biochim Biophys Acta 1831(5): 986-1003. Franckhauser, S., Munoz, S., Pujol, A., Casellas, A., Riu, E., Otaegui, P., Su, B. &Bosch, F. (2002). Increased fatty acid re-esterification by PEPCK overexpression in adipose tissue leads to obesity without insulin resistance. Diabetes 51(3): 624-630. Frerman, F. E., Sabran, J. L., Taylor, J. L. &Grossberg, S. E. (1983). Leucine catabolism during the differentiation of 3T3-L1 cells. Expression of a mitochondrial enzyme system. J Biol Chem 258(11): 7087-7093. Frohnert, B. I., Sinaiko, A. R., Serrot, F. J., Foncea, R. E., Moran, A., Ikramuddin, S., Choudry, U. &Bernlohr, D. A. (2011). Increased adipose protein carbonylation in human obesity. Obesity (Silver Spring) 19(9): 1735-1741. Furukawa, S., Fujita, T., Shimabukuro, M., Iwaki, M., Yamada, Y., Nakajima, Y., Nakayama, O., Makishima, M., Matsuda, M. &Shimomura, I. (2004). Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 114(12): 1752-1761. Galinier, A., Carriere, A., Fernandez, Y., Carpene, C., Andre, M., Caspar-Bauguil, S., Thouvenot, J. P., Periquet, B., Penicaud, L. &Casteilla, L. (2006). Adipose tissue proadipogenic redox changes in obesity. J Biol Chem 281(18): 12682-12687. Goodman, H. M. (1963). Effects of Growth Hormone on Leucine Metabolism in Adipose Tissue in Vitro. Endocrinology 73: 421-426. Harper, A. E., Miller, R. H. &Block, K. P. (1984). Branched-chain amino acid metabolism. Annu Rev Nutr 4: 409-454. Herman, M. A., She, P., Peroni, O. D., Lynch, C. J. &Kahn, B. B. (2010). Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels. J Biol Chem 285(15): 11348-11356. Holm, C., Osterlund, T., Laurell, H. &Contreras, J. A. (2000). Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Annu Rev Nutr 20: 365-393. Huh, J. Y., Kim, Y., Jeong, J., Park, J., Kim, I., Huh, K. H., Kim, Y. S., Woo, H. A., Rhee, S. G., Lee, K. J. &Ha, H. (2012). Peroxiredoxin 3 is a key molecule regulating adipocyte oxidative stress, mitochondrial biogenesis, and adipokine expression. Antioxid Redox Signal 16(3): 229-243. Ichihara, A. &Koyama, E. (1966). Transaminase of branched chain amino acids. I. Branched chain amino acids-alpha-ketoglutarate transaminase. J Biochem 59(2): 160-169. Jeon, M. J., Leem, J., Ko, M. S., Jang, J. E., Park, H. S., Kim, H. S., Kim, M., Kim, E. H., Yoo, H. J., Lee, C. H., Park, I. S., Lee, K. U. &Koh, E. H. (2012). Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes. Exp Mol Med 44(9): 562-570. Kitsy, A., Carney, S., Vivar, J. C., Knight, M. S., Pointer, M. A., Gwathmey, J. K. &Ghosh, S. (2014). Effects of Leucine Supplementation and Serum Withdrawal on Branched-Chain Amino Acid Pathway Gene and Protein Expression in Mouse Adipocytes. PLoS One 9(7): e102615. Kleiner, S., Mepani, R. J., Laznik, D., Ye, L., Jurczak, M. J., Jornayvaz, F. R., Estall, J. L., Chatterjee Bhowmick, D., Shulman, G. I. &Spiegelman, B. M. (2012). Development of insulin resistance in mice lacking PGC-1alpha in adipose tissues. Proc Natl Acad Sci U S A 109(24): 9635-9640. Klingenspor, M. (2003). Cold-induced recruitment of brown adipose tissue thermogenesis. Exp Physiol 88(1): 141-148. Koh, E. H., Park, J. Y., Park, H. S., Jeon, M. J., Ryu, J. W., Kim, M., Kim, S. Y., Kim, M. S., Kim, S. W., Park, I. S., Youn, J. H. &Lee, K. U. (2007). Essential role of mitochondrial function in adiponectin synthesis in adipocytes. Diabetes 56(12): 2973-2981. Kopecky, J., Clarke, G., Enerback, S., Spiegelman, B. &Kozak, L. P. (1995). Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity. J Clin Invest 96(6): 2914-2923.


22

Kopecky, J., Rossmeisl, M., Flachs, P., Bardova, K. &Brauner, P. (2001). Mitochondrial uncoupling and lipid metabolism in adipocytes. Biochem Soc Trans 29(Pt 6): 791-797. Kopecky, J., Rossmeisl, M., Hodny, Z., Syrovy, I., Horakova, M. &Kolarova, P. (1996). Reduction of dietary obesity in aP2-Ucp transgenic mice: mechanism and adipose tissue morphology. Am J Physiol 270(5 Pt 1): E776-786. Kovsan, J., Bluher, M., Tarnovscki, T., Kloting, N., Kirshtein, B., Madar, L., Shai, I., Golan, R., HarmanBoehm, I., Schon, M. R., Greenberg, A. S., Elazar, Z., Bashan, N. &Rudich, A. (2011). Altered autophagy in human adipose tissues in obesity. J Clin Endocrinol Metab 96(2): E268-277. Kusminski, C. M., Holland, W. L., Sun, K., Park, J., Spurgin, S. B., Lin, Y., Askew, G. R., Simcox, J. A., McClain, D. A., Li, C. &Scherer, P. E. (2012). MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity. Nat Med 18(10): 1539-1549. Kusminski, C. M. &Scherer, P. E. (2012). Mitochondrial dysfunction in white adipose tissue. Trends Endocrinol Metab 23(9): 435-443. Langin, D. (2006). Adipose tissue lipolysis as a metabolic pathway to define pharmacological strategies against obesity and the metabolic syndrome. Pharmacol Res 53(6): 482-491. Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S. &Tiganis, T. (2009). Reactive oxygen species enhance insulin sensitivity. Cell Metab 10(4): 260-272. Martin, B. R. &Denton, R. M. (1970). The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. Biochem J 117(5): 861-877. Nedergaard, J., Cannon, B. &Lindberg, O. (1977). Microcalorimetry of isolated mammalian cells. Nature 267(5611): 518-520. Novikoff, A. B., Novikoff, P. M., Rosen, O. M. &Rubin, C. S. (1980). Organelle relationships in cultured 3T3-L1 preadipocytes. J Cell Biol 87(1): 180-196. Olswang, Y., Cohen, H., Papo, O., Cassuto, H., Croniger, C. M., Hakimi, P., Tilghman, S. M., Hanson, R. W. &Reshef, L. (2002). A mutation in the peroxisome proliferator-activated receptor gammabinding site in the gene for the cytosolic form of phosphoenolpyruvate carboxykinase reduces adipose tissue size and fat content in mice. Proc Natl Acad Sci U S A 99(2): 625-630. Ost, A., Svensson, K., Ruishalme, I., Brannmark, C., Franck, N., Krook, H., Sandstrom, P., Kjolhede, P. &Stralfors, P. (2010). Attenuated mTOR signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes. Mol Med 16(7-8): 235-246. Pardo, R., Enguix, N., Lasheras, J., Feliu, J. E., Kralli, A. &Villena, J. A. (2011). Rosiglitazone-induced mitochondrial biogenesis in white adipose tissue is independent of peroxisome proliferatoractivated receptor gamma coactivator-1alpha. PLoS One 6(11): e26989. Patel, M. S. &Hanson, R. W. (1970). Carboxylation of pyruvate by isolated rat adipose tissue mitochondria. J Biol Chem 245(6): 1302-1310. Patti, M. E. &Corvera, S. (2010). The role of mitochondria in the pathogenesis of type 2 diabetes. Endocr Rev 31(3): 364-395. Pietilainen, K. H., Naukkarinen, J., Rissanen, A., Saharinen, J., Ellonen, P., Keranen, H., Suomalainen, A., Gotz, A., Suortti, T., Yki-Jarvinen, H., Oresic, M., Kaprio, J. &Peltonen, L. (2008). Global transcript profiles of fat in monozygotic twins discordant for BMI: pathways behind acquired obesity. PLoS Med 5(3): e51. Prusiner, S. B., Cannon, B., Ching, T. M. &Lindberg, O. (1968). Oxidative metabolism in cells isolated from brown adipose tissue. 2. Catecholamine regulated respiratory control. Eur J Biochem 7(1): 51-57. Reed, N. &Fain, J. N. (1968). Stimulation of respiration in brown fat cells by epinephrine, dibutyryl3',5'-adenosine monophosphate, and m-chloro(carbonyl cyanide)phenylhydrazone. J Biol Chem 243(11): 2843-2848.


23

Reshef, L., Hanson, R. W. &Ballard, F. J. (1969). Glyceride-glycerol synthesis from pyruvate. Adaptive changes in phosphoenolpyruvate carboxykinase and pyruvate carboxylase in adipose tissue and liver. J Biol Chem 244(8): 1994-2001. Rognstad, R. &Katz, J. (1969). The effect of 2,4-dinitrophenol on adipose-tissue metabolism. Biochem J 111(4): 431-444. Rong, J. X., Qiu, Y., Hansen, M. K., Zhu, L., Zhang, V., Xie, M., Okamoto, Y., Mattie, M. D., Higashiyama, H., Asano, S., Strum, J. C. &Ryan, T. E. (2007). Adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone. Diabetes 56(7): 1751-1760. Rosenthal, J., Angel, A. &Farkas, J. (1974). Metabolic fate of leucine: a significant sterol precursor in adipose tissue and muscle. Am J Physiol 226(2): 411-418. Ross, S. R., Graves, R. A., Greenstein, A., Platt, K. A., Shyu, H. L., Mellovitz, B. &Spiegelman, B. M. (1990). A fat-specific enhancer is the primary determinant of gene expression for adipocyte P2 in vivo. Proc Natl Acad Sci U S A 87(24): 9590-9594. Rossmeisl, M., Syrovy, I., Baumruk, F., Flachs, P., Janovska, P. &Kopecky, J. (2000). Decreased fatty acid synthesis due to mitochondrial uncoupling in adipose tissue. FASEB J 14(12): 1793-1800. Shapiro, B. &Wertheimer, E. (1948). The synthesis of fatty acids in adipose tissue in vitro. J Biol Chem 173(2): 725-728. She, P., Reid, T. M., Bronson, S. K., Vary, T. C., Hajnal, A., Lynch, C. J. &Hutson, S. M. (2007). Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle. Cell Metab 6(3): 181-194. Sheldon, H., Hollenberg, C. H. &Winegrad, A. I. (1962). Observations on the morphology of adipose tissue. I. The fine structure of cells from fasted and diabetic rats. Diabetes 11: 378-387. Si, Y., Palani, S., Jayaraman, A. &Lee, K. (2007). Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism. J Lipid Res 48(4): 826-836. Singh, R., Xiang, Y., Wang, Y., Baikati, K., Cuervo, A. M., Luu, Y. K., Tang, Y., Pessin, J. E., Schwartz, G. J. &Czaja, M. J. (2009). Autophagy regulates adipose mass and differentiation in mice. J Clin Invest 119(11): 3329-3339. Starkov, A. A. (2008). The role of mitochondria in reactive oxygen species metabolism and signaling. Ann N Y Acad Sci 1147: 37-52. Steinfelder, H. J. &Joost, H. G. (1983). Reversible reduction of insulin receptor affinity by ATP depletion in rat adipocytes. Biochem J 214(1): 203-207. Sutherland, L. N., Capozzi, L. C., Turchinsky, N. J., Bell, R. C. &Wright, D. C. (2008). Time course of high-fat diet-induced reductions in adipose tissue mitochondrial proteins: potential mechanisms and the relationship to glucose intolerance. Am J Physiol Endocrinol Metab 295(5): E1076-1083. Tischler, M. E. &Goldberg, A. L. (1980). Leucine degradation and release of glutamine and alanine by adipose tissue. J Biol Chem 255(17): 8074-8081. Tormos, K. V., Anso, E., Hamanaka, R. B., Eisenbart, J., Joseph, J., Kalyanaraman, B. &Chandel, N. S. (2011). Mitochondrial complex III ROS regulate adipocyte differentiation. Cell Metab 14(4): 537-544. Valerio, A., Cardile, A., Cozzi, V., Bracale, R., Tedesco, L., Pisconti, A., Palomba, L., Cantoni, O., Clementi, E., Moncada, S., Carruba, M. O. &Nisoli, E. (2006). TNF-alpha downregulates eNOS expression and mitochondrial biogenesis in fat and muscle of obese rodents. J Clin Invest 116(10): 2791-2798. Vernochet, C., Mourier, A., Bezy, O., Macotela, Y., Boucher, J., Rardin, M. J., An, D., Lee, K. Y., Ilkayeva, O. R., Zingaretti, C. M., Emanuelli, B., Smyth, G., Cinti, S., Newgard, C. B., Gibson, B. W., Larsson, N. G. &Kahn, C. R. (2012). Adipose-specific deletion of TFAM increases mitochondrial oxidation and protects mice against obesity and insulin resistance. Cell Metab 16(6): 765-776.


24

Villarroya, J., Giralt, M. &Villarroya, F. (2009). Mitochondrial DNA: an up-and-coming actor in white adipose tissue pathophysiology. Obesity (Silver Spring) 17(10): 1814-1820. Wang, P. W., Kuo, H. M., Huang, H. T., Chang, A. Y., Weng, S. W., Tai, M. H., Chuang, J. H., Chen, I. Y., Huang, S. C., Lin, T. K. &Liou, C. W. (2014). Biphasic response of mitochondrial biogenesis to oxidative stress in visceral fat of diet-induced obesity mice. Antioxid Redox Signal 20(16): 2572-2588. Williams, G. S., Boyman, L., Chikando, A. C., Khairallah, R. J. &Lederer, W. J. (2013). Mitochondrial calcium uptake. Proc Natl Acad Sci U S A 110(26): 10479-10486. Williamson, J. R. (1964). Adipose Tissue. Morphological Changes Associated with Lipid Mobilization. J Cell Biol 20: 57-74. Wilson-Fritch, L., Burkart, A., Bell, G., Mendelson, K., Leszyk, J., Nicoloro, S., Czech, M. &Corvera, S. (2003). Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone. Mol Cell Biol 23(3): 1085-1094. Wilson-Fritch, L., Nicoloro, S., Chouinard, M., Lazar, M. A., Chui, P. C., Leszyk, J., Straubhaar, J., Czech, M. P. &Corvera, S. (2004). Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone. J Clin Invest 114(9): 1281-1289. Yin, X., Lanza, I. R., Swain, J. M., Sarr, M. G., Nair, K. S. &Jensen, M. D. (2014). Adipocyte mitochondrial function is reduced in human obesity independent of fat cell size. J Clin Endocrinol Metab 99(2): E209-216. Yu, Y. H. &Zhu, H. (2004). Chronological changes in metabolism and functions of cultured adipocytes: a hypothesis for cell aging in mature adipocytes. Am J Physiol Endocrinol Metab 286(3): E402-410. Zhang, Y., Goldman, S., Baerga, R., Zhao, Y., Komatsu, M. &Jin, S. (2009). Adipose-specific deletion of autophagy-related gene 7 (atg7) in mice reveals a role in adipogenesis. Proc Natl Acad Sci U S A 106(47): 19860-19865. Zhao, Y., Yang, J., Liao, W., Liu, X., Zhang, H., Wang, S., Wang, D., Feng, J., Yu, L. &Zhu, W. G. (2010). Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nat Cell Biol 12(7): 665-675.


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New concepts in white adipose tissue physiology.

Growth hormone action predicts age-related white adipose tissue dysfunction and senescent cell burden in mice.

White adipose tissue browning and obesity.

Enzymatic intracrine regulation of white adipose tissue.

Imaging white adipose tissue with confocal microscopy.

Functional characteristics of the microcirculation in white adipose tissue.

Metabolic remodeling of white adipose tissue in obesity.

Berberine activates thermogenesis in white and brown adipose tissue.

Acute exercise regulates adipogenic gene expression in white adipose tissue.

Methods for performing lipidomics in white adipose tissue.

Mechanisms of perivascular adipose tissue dysfunction in obesity.

Pathological role of adipose tissue dysfunction in cardio-metabolic disorders.

6J mice.

White Adipose Tissue Browning: A Double-edged Sword.

Central sympathetic innervations to visceral and subcutaneous white adipose tissue.

Fatty acid synthesis in vivo in brown adipose tissue, liver and white adipose tissue of the cold-acclimated rat.

Alternative mechanism for white adipose tissue lipolysis after thermal injury.

MitoNEET-mediated effects on browning of white adipose tissue.

Exercise Effects on White Adipose Tissue: Beiging and Metabolic Adaptations.

White adipose tissue resilience to insulin deprivation and replacement.

db) mice: developmental changes in brown adipose tissue, white adipose tissue and the liver.

The browning of white adipose tissue: some burning issues.

Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking.

Perivascular adipose tissue, potassium channels, and vascular dysfunction.