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1.
Methods Mol Biol ; 2783: 93-107, 2024.
Article in English | MEDLINE | ID: mdl-38478227

ABSTRACT

Murine models of obesity or reduced adiposity are a valuable resource for understanding the role of adipocyte dysfunction in metabolic disorders. Adipose tissue stromal vascular cells or primary adipocytes derived from murine adipose tissue and grown in culture are essential tools for studying the mechanisms underlying adipocyte development and function. Herein, we describe methods for the isolation, expansion, and long-term storage of murine adipose-derived stromal/stem cells, along with protocols for inducing adipogenesis to white or beige adipocytes in this cell population and osteogenic differentiation. Isolation of the adipose stromal vascular fraction cells for flow cytometric analysis is also described.


Subject(s)
Adipogenesis , Adiposity , Mice , Humans , Animals , Flow Cytometry/methods , Osteogenesis , Adipocytes , Adipose Tissue , Cell Differentiation , Obesity/metabolism , Stem Cells
2.
Front Endocrinol (Lausanne) ; 14: 1106544, 2023.
Article in English | MEDLINE | ID: mdl-36896177

ABSTRACT

Brown adipocytes is a specialized fat cell that dissipates nutrient-derived chemical energy in the form of heat, instead of ATP synthesis. This unique feature provides a marked capacity for brown adipocyte mitochondria to oxidize substrates independent of ADP availability. Upon cold exposure, brown adipocytes preferentially oxidize free fatty acids (FFA) liberated from triacylglycerol (TAG) in lipid droplets to support thermogenesis. In addition, brown adipocytes take up large amounts of circulating glucose, concurrently increasing glycolysis and de novo FA synthesis from glucose. Given that FA oxidation and glucose-derived FA synthesis are two antagonistic mitochondrial processes in the same cell, it has long been questioned how brown adipocytes run FA oxidation and FA synthesis simultaneously. In this review, I summarize mechanisms regulating mitochondrial substrate selection and describe recent findings of two distinct populations of brown adipocyte mitochondria with different substrate preferences. I further discuss how these mechanisms may permit a concurrent increase in glycolysis, FA synthesis, and FA oxidation in brown adipocytes.


Subject(s)
Adipocytes, Brown , Adipose Tissue, Brown , Adipose Tissue, Brown/metabolism , Lipid Metabolism , Fatty Acids/metabolism , Glucose/metabolism
3.
Front Physiol ; 12: 780312, 2021.
Article in English | MEDLINE | ID: mdl-34899399

ABSTRACT

Brown and beige adipocytes are specialized to dissipate energy as heat. Sgk2, encoding a serine/threonine kinase, has been identified as a brown and beige adipocyte-specific gene in rodents and humans; however, its function in brown/beige adipocytes remains unraveled. Here, we examined the regulation and role of Sgk2 in brown/beige adipose tissue thermogenesis. We found that transcriptional coactivators PGC-1α and NT-PGC-1α activated by the ß3 adrenergic receptor-cAMP-PKA pathway are recruited to the Sgk2 promoter, triggering Sgk2 transcription in response to cold. SGK2 elevation was closely associated with increased serine/threonine phosphorylation of proteins carrying the consensus RxRxxS/T phosphorylation site. However, despite cold-dependent activation of SGK2, mice lacking Sgk2 exhibited normal cold tolerance at 4°C. In addition, Sgk2+/+ and Sgk2-/- mice induced comparable increases in energy expenditure during pharmacological activation of brown and beige adipose tissue with a ß3AR agonist. In vitro loss- and gain-of-function studies further demonstrated that Sgk2 ablation or activation does not alter thermogenic gene expression and mitochondrial respiration in brown adipocytes. Collectively, our results reveal a new signaling component SGK2, although dispensable for cold-induced thermogenesis that adds an additional layer of complexity to the ß3AR signaling network in brown/beige adipose tissue.

4.
BMC Genomics ; 22(1): 500, 2021 Jul 03.
Article in English | MEDLINE | ID: mdl-34217204

ABSTRACT

BACKGROUND: Brown adipose tissue (BAT) is specialized to dissipate energy in the form of heat. BAT-mediated heat production in rodents and humans is critical for effective temperature adaptation of newborns to the extrauterine environment immediately after birth. However, very little is known about whether and how fetal BAT development is modulated in-utero in response to changes in maternal thermal environment during pregnancy. Using BL6 mice, we evaluated the impact of different maternal environmental temperatures (28 °C and 18 °C) on the transcriptome of the placenta and fetal BAT to test if maternal cold exposure influences fetal BAT development via placental remodeling. RESULTS: Maternal weight gain during pregnancy, the average number of fetuses per pregnancy, and placental weight did not differ between the groups at 28 °C and 18 °C. However, the average fetal weight at E18.5 was 6% lower in the 18 °C-group compared to the 28 °C-group. In fetal BATs, cold exposure during pregnancy induced increased expression of genes involved in de novo lipogenesis and lipid metabolism while decreasing the expression of genes associated with muscle cell differentiation, thus suggesting that maternal cold exposure may promote fetal brown adipogenesis by suppressing the myogenic lineage in bidirectional progenitors. In placental tissues, maternal cold exposure was associated with upregulation of genes involved in complement activation and downregulation of genes related to muscle contraction and actin-myosin filament sliding. These changes may coordinate placental adaptation to maternal cold exposure, potentially by protecting against cold stress-induced inflammatory damage and modulating the vascular and extravascular contractile system in the placenta. CONCLUSIONS: These findings provide evidence that environmental cold temperature sensed by the mother can modulate the transcriptome of placental and fetal BAT tissues. The ramifications of the observed gene expression changes warrant future investigation.


Subject(s)
Adipose Tissue, Brown , Cold Temperature , Animals , Female , Fetus , Mice , Placenta , Pregnancy , Thermogenesis , Transcriptome
5.
Physiology (Bethesda) ; 36(4): 246-255, 2021 07 01.
Article in English | MEDLINE | ID: mdl-34159808

ABSTRACT

Obesity research progresses in understanding neuronal circuits and adipocyte biology to regulate metabolism. However, the interface of neuro-adipocyte interaction is less studied. We summarize the current knowledge of adipose tissue innervation and interaction with adipocytes and emphasize adipocyte transitions from white to brown adipocytes and vice versa. We further highlight emerging concepts for the differential neuronal regulation of brown/beige versus white adipocyte and the interdependence of both for metabolic regulation.


Subject(s)
Adipocytes, Beige , Thermogenesis , Adipocytes, Brown , Adipose Tissue , Energy Metabolism , Humans , Obesity
6.
Sci Rep ; 11(1): 1323, 2021 01 14.
Article in English | MEDLINE | ID: mdl-33446719

ABSTRACT

Transcriptional coactivator PGC-1α and its splice variant NT-PGC-1α regulate metabolic adaptation by modulating many gene programs. Selective ablation of PGC-1α attenuates diet-induced obesity through enhancing fatty acid oxidation and thermogenesis by upregulation of NT-PGC-1α in brown adipose tissue (BAT). Recently, we have shown that selective ablation of NT-PGC-1α reduces fatty acid oxidation in BAT. Thus, the objective of this study was to test our hypothesis that NT-PGC-1α-/- mice would be more prone to diet-induced obesity. Male and female NT-PGC-1α+/+ (WT) and NT-PGC-1α-/- mice were fed a regular chow or 60% high-fat (HF) diet for 16 weeks. Contrary to our expectations, both male and female NT-PGC-1α-/- mice fed HFD were protected from diet-induced obesity, with more pronounced effects in females. This lean phenotype was primarily driven by reduced dietary fat intake. Intriguingly, HFD-fed female, but not male, NT-PGC-1α-/- mice further exhibited decreased feed efficiency, which was closely associated with increased fecal fat excretion and decreased uptake of fatty acids by the intestinal enterocytes and adipocytes with a concomitant decrease in fatty acid transporter gene expression. Collectively, our results highlight the role for NT-PGC-1α in regulating whole body lipid homeostasis under HFD conditions.


Subject(s)
Diet, High-Fat/adverse effects , Eating , Intestinal Absorption , Obesity/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Animals , Feces , Female , Male , Mice , Mice, Knockout , Obesity/chemically induced , Obesity/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism
7.
Front Neurosci ; 14: 592947, 2020.
Article in English | MEDLINE | ID: mdl-33335471

ABSTRACT

The central nervous system controls feeding behavior and energy expenditure in response to various internal and external stimuli to maintain energy balance. Here we report that the newly identified transcription factor zinc finger and BTB domain containing 16 (Zbtb16) is induced by energy deficit in the paraventricular (PVH) and arcuate (ARC) nuclei of the hypothalamus via glucocorticoid (GC) signaling. In the PVH, Zbtb16 is expressed in the anterior half of the PVH and co-expressed with many neuronal markers such as corticotropin-releasing hormone (Crh), thyrotropin-releasing hormone (Trh), oxytocin (Oxt), arginine vasopressin (Avp), and nitric oxide synthase 1 (Nos1). Knockdown (KD) of Zbtb16 in the PVH results in attenuated cold-induced thermogenesis and improved glucose tolerance without affecting food intake. In the meantime, Zbtb16 is predominantly expressed in agouti-related neuropeptide/neuropeptide Y (Agrp/Npy) neurons in the ARC and its KD in the ARC leads to reduced food intake. We further reveal that chemogenetic stimulation of PVH Zbtb16 neurons increases energy expenditure while that of ARC Zbtb16 neurons increases food intake. Taken together, we conclude that Zbtb16 is an important mediator that coordinates responses to energy deficit downstream of GCs by contributing to glycemic control through the PVH and feeding behavior regulation through the ARC, and additionally reveal its function in controlling energy expenditure during cold-evoked thermogenesis via the PVH. As a result, we hypothesize that Zbtb16 may be involved in promoting weight regain after weight loss.

8.
Methods Mol Biol ; 1966: 7-16, 2019.
Article in English | MEDLINE | ID: mdl-31041735

ABSTRACT

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) plays a central role in the response and adaptation to environmental and nutritional stimuli by initiating tissue-specific transcriptional reprogramming. Since its discovery in 1998, the field of PGC-1α biology has grown exponentially and a large body of research has elucidated the diverse roles of PGC-1α in brown adipose tissue thermogenesis, fatty acid oxidation, muscle fiber type switching, hepatic gluconeogenesis, and circadian clock regulation, etc. In addition, recent research has identified a splice variant(s) of PGC-1α in humans and rodents. The common misconception relating to PGC-1α is that it migrates at a predicted molecular weight of ~90 kDa by SDS-PAGE gel electrophoresis. However, several recent studies have provided solid evidence that the biologically relevant molecular weight of PGC-1α is ~110 kDa. In this chapter, we describe an optimized immunoblotting protocol that is developed to detect the low abundance protein PGC-1α and its alternatively spliced isoform named NT-PGC-1α in various rodent tissues. We also describe an optimized immunoprecipitation protocol that can isolate and concentrate endogenous PGC-1α and NT-PGC-1α. The protocols presented here will hopefully allow investigators to report accurate and reliable data regarding PGC-1α isoforms.


Subject(s)
Immunoblotting/methods , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/analysis , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Alternative Splicing , Animals , Mice , Protein Isoforms/analysis , Rats , Rodentia/metabolism
9.
J Lipid Res ; 59(9): 1660-1670, 2018 09.
Article in English | MEDLINE | ID: mdl-30026188

ABSTRACT

Transcriptional coactivator PPAR γ coactivator (PGC)-1α and its splice variant N-terminal (NT)-PGC-1α mediate transcriptional regulation of brown adipose tissue (BAT) thermogenesis in response to changes in ambient temperature. PGC-1α is dispensable for cold-induced BAT thermogenesis as long as NT-PGC-1α is present. However, the functional significance of NT-PGC-1α in BAT has not been determined. In the present study, we generated NT-PGC-1α-/- mice to investigate the effect of NT-PGC-1α deficiency on adaptive BAT thermogenesis. At thermoneutrality, NT-PGC-1α-/- mice exhibited abnormal BAT phenotype with increased accumulation of large lipid droplets concomitant with marked downregulation of FA oxidation (FAO)-related genes. Consistent with transcriptional changes, mitochondrial FAO was significantly diminished in NT-PGC-1α-/- BAT. This alteration, in turn, enhanced glucose utilization within the NT-PGC-1α-/- BAT mitochondria. In line with this, NT-PGC-1α-/- mice had higher reliance on carbohydrates. In response to cold or ß3-adrenergic receptor agonist, NT-PGC-1α-/- mice transiently exhibited lower thermogenesis but reached similar thermogenic capacities as their WT littermates. Collectively, these findings demonstrate that NT-PGC-1α is an important contributor to the maintenance of FAO capacity in BAT at thermoneutrality and provide deeper insights into the relative contributions of PGC-1α and NT-PGC-1α to temperature-regulated BAT remodeling.


Subject(s)
Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Fatty Acids/metabolism , Mitochondria/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/chemistry , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/deficiency , Adipose Tissue, White/metabolism , Animals , Gene Expression Regulation , Lipolysis , Mice , Mutation , Oxidation-Reduction , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Temperature , Thermogenesis
10.
Sci Rep ; 8(1): 7876, 2018 05 18.
Article in English | MEDLINE | ID: mdl-29777200

ABSTRACT

Transcriptional coactivator PGC-1α and its splice variant NT-PGC-1α play crucial roles in regulating cold-induced thermogenesis in brown adipose tissue (BAT). PGC-1α and NT-PGC-1α are highly induced by cold in BAT and subsequently bind to and coactivate many transcription factors to regulate expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, respiration and thermogenesis. To identify the complete repertoire of PGC-1α and NT-PGC-1α target genes in BAT, we analyzed genome-wide DNA-binding and gene expression profiles. We find that PGC-1α-/NT-PGC-1α binding broadly associates with cold-mediated transcriptional activation. In addition to their known target genes in mitochondrial biogenesis and oxidative metabolism, PGC-1α and NT-PGC-1α additionally target a broad spectrum of genes involved in diverse biological pathways including ubiquitin-dependent protein catabolism, ribonucleoprotein complex biosynthesis, phospholipid biosynthesis, angiogenesis, glycogen metabolism, phosphorylation, and autophagy. Our findings expand the number of genes and biological pathways that may be regulated by PGC-1α and NT-PGC-1α and provide further insight into the transcriptional regulatory network in which PGC-1α and NT-PGC-1α coordinate a comprehensive transcriptional response in BAT in response to cold.


Subject(s)
Adipose Tissue, Brown/metabolism , Gene Regulatory Networks , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Protein Isoforms/metabolism , Animals , Binding Sites , Chromatin/chemistry , Chromatin/metabolism , Cold Temperature , DNA/chemistry , DNA/metabolism , Male , Mice , Mice, Inbred C57BL , Organelle Biogenesis , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/chemistry , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Thermogenesis/genetics , Transcriptional Activation , Transcriptome
11.
Sci Rep ; 8(1): 6672, 2018 04 27.
Article in English | MEDLINE | ID: mdl-29704006

ABSTRACT

Release of fatty acids from lipid droplets upon activation of the sympathetic nervous system (SNS) is a key step in nonshivering thermogenesis in brown adipose tissue (BAT). However, intracellular lipolysis appears not to be critical for cold-induced thermogenesis. As activation of the SNS increases glucose uptake, we studied whether intracellular glycolysis plays a role in BAT thermogenesis. To stimulate BAT-innervating sympathetic nerves in vivo, we expressed channelrhodopsin-2 (ChR2) in catecholaminergic fibers by crossbreeding tyrosine hydroxylase-Cre mice with floxed-stop ChR2 mice. Acute optogenetic stimulation of sympathetic efferent fibers of BAT increased body temperature and lowered blood glucose levels that were completely abolished by the ß-adrenergic receptor antagonist. Knockdown of the Ucp1 gene in BAT blocked the effects of optogenetic stimulation on body temperature and glucose uptake. Inhibition of glucose uptake in BAT and glycolysis abolished optogenetically induced thermogenesis. Stimulation of sympathetic nerves upregulated expression of the lactate dehydrogenase-A and -B genes in BAT. Optogenetic stimulation failed to induce thermogenesis following treatment with the LDH inhibitor. Pharmacological blockade and genetic deletion of the monocarboxylate transporter 1 completely abolished the effects of sympathetic activation. Our results suggest that intracellular glycolysis and lactate shuttle play an important role in regulating acute thermogenesis in BAT.


Subject(s)
Adipose Tissue, Brown/metabolism , Glycolysis , Thermogenesis , Animals , Animals, Genetically Modified , Blood Glucose , Body Temperature , Channelrhodopsins/analysis , Channelrhodopsins/genetics , Gene Knockdown Techniques , Lactates/metabolism , Mice , Optogenetics , Uncoupling Protein 1/biosynthesis
12.
PLoS One ; 13(3): e0195007, 2018.
Article in English | MEDLINE | ID: mdl-29566074

ABSTRACT

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of brown adipocyte differentiation and thermogenesis. The PPARγ gene produces two isoforms, PPARγ1 and PPARγ2. PPARγ2 is identical to PPARγ1 except for additional 30 amino acids present in the N-terminus of PPARγ2. Here we report that the C-terminally truncated form of PPARγ2 is predominantly present in the mitochondrial matrix of brown adipocytes and that it binds to the D-loop region of mitochondrial DNA (mtDNA), which contains the promoter for mitochondrial electron transport chain (ETC) genes. Expression of mitochondrially targeted MLS-PPARγ2 in brown adipocytes increases mtDNA-encoded ETC gene expression concomitant with enhanced mitochondrial respiration. These results suggest that direct regulation of mitochondrially encoded ETC gene expression by mitochondrial PPARγ2, in part, underlies the isoform-specific role for PPARγ2 in brown adipocytes.


Subject(s)
Adipocytes, Brown/metabolism , Mitochondria/metabolism , Oxygen Consumption , PPAR gamma/metabolism , PPAR gamma/physiology , Animals , Cell Respiration/genetics , Codon, Nonsense , DNA, Mitochondrial/metabolism , Electron Transport/genetics , Electron Transport Chain Complex Proteins/genetics , HeLa Cells , Humans , Mice , Mice, Inbred C57BL , Oxygen Consumption/genetics , PPAR gamma/genetics , Protein Binding , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Transport
13.
J Biol Chem ; 292(24): 9958-9966, 2017 06 16.
Article in English | MEDLINE | ID: mdl-28473468

ABSTRACT

Brown adipose tissue dissipates energy as heat, a process that relies on a high abundance of mitochondria and high levels of electron transport chain (ETC) complexes within these mitochondria. Two regulators of mitochondrial respiration and heat production in brown adipocytes are the transcriptional coactivator PGC-1α and its splicing isoform NT-PGC-1α, which control mitochondrial gene expression in the nucleus. Surprisingly, we found that, in brown adipocytes, some NT-PGC-1α localizes to mitochondria, whereas PGC-1α resides in the nucleus. Here we sought to investigate the role of NT-PGC-1α in brown adipocyte mitochondria. Immunocytochemistry, immunotransmission electron microscopy, and biochemical analyses indicated that NT-PGC-1α was located in the mitochondrial matrix in brown adipocytes. NT-PGC-1α was specifically enriched at the D-loop region of the mtDNA, which contains the promoters for several essential ETC complex genes, and was associated with LRP130, an activator of mitochondrial transcription. Selective expression of NT-PGC-1α and PGC-1α in PGC-1α-/- brown adipocytes similarly induced expression of nuclear DNA-encoded mitochondrial ETC genes, including the key mitochondrial transcription factor A (TFAM). Despite having comparable levels of TFAM expression, PGC-1α-/- brown adipocytes expressing NT-PGC-1α had higher expression of mtDNA-encoded ETC genes than PGC-1α-/- brown adipocytes expressing PGC-1α, suggesting a direct effect of NT-PGC-1α on mtDNA transcription. Moreover, this increase in mtDNA-encoded ETC gene expression was associated with enhanced respiration in NT-PGC-1α-expressing PGC-1α-/- brown adipocytes. Our findings reveal a previously unappreciated and isoform-specific role for NT-PGC-1α in the regulation of mitochondrial transcription in brown adipocytes and provide new insight into the transcriptional control of mitochondrial respiration.


Subject(s)
Adipocytes, Brown/metabolism , DNA, Mitochondrial/metabolism , Electron Transport Chain Complex Proteins/agonists , Mitochondria/metabolism , Neoplasm Proteins/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Promoter Regions, Genetic , Adipocytes, Brown/cytology , Adipocytes, Brown/ultrastructure , Adipogenesis , Alternative Splicing , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Electron Transport Chain Complex Proteins/genetics , Electron Transport Chain Complex Proteins/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Mice, Inbred C57BL , Mitochondria/ultrastructure , Neoplasm Proteins/genetics , Oxidative Phosphorylation , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Response Elements
14.
Physiol Rep ; 4(20)2016 10.
Article in English | MEDLINE | ID: mdl-27798359

ABSTRACT

The transcriptional coactivator PGC-1α plays a central role in hepatic gluconeogenesis. We previously reported that alternative splicing of the PGC-1α gene produces an additional transcript encoding the truncated protein NT-PGC-1α NT-PGC-1α is co-expressed with PGC-1α and highly induced by fasting in the liver. NT-PGC-1α regulates tissue-specific metabolism, but its role in the liver has not been investigated. Thus, the objective of this study was to determine the role of hepatic NT-PGC-1α in the regulation of gluconeogenesis. Adenovirus-mediated expression of NT-PGC-1α in primary hepatocytes strongly stimulated the expression of key gluconeogenic enzyme genes (PEPCK and G6Pase), leading to increased glucose production. To further understand NT-PGC-1α function in hepatic gluconeogenesis in vivo, we took advantage of a previously reported FL-PGC-1α-/- mouse line that lacks full-length PGC-1α (FL-PGC-1α) but retains a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α254). In FL-PGC-1α-/- mice, NT-PGC-1α254 was induced by fasting in the liver and recruited to the promoters of PEPCK and G6Pase genes. The enrichment of NT-PGC-1α254 at the promoters was closely associated with fasting-induced increase in PEPCK and G6Pase gene expression and efficient production of glucose from pyruvate during a pyruvate tolerance test in FL-PGC-1α-/- mice. Moreover, FL-PGC-1α-/- primary hepatocytes showed a significant increase in gluconeogenic gene expression and glucose production after treatment with dexamethasone and forskolin, suggesting that NT-PGC-1α254 is sufficient to stimulate the gluconeogenic program in the absence of FL-PGC-1α Collectively, our findings highlight the role of hepatic NT-PGC-1α in stimulating gluconeogenic gene expression and glucose production.


Subject(s)
Gluconeogenesis/genetics , Glucose/metabolism , Liver/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Adenoviridae/genetics , Animals , Hepatocytes/metabolism , Male , Mice , Transcription Factors/metabolism
15.
PLoS One ; 11(7): e0159990, 2016.
Article in English | MEDLINE | ID: mdl-27454177

ABSTRACT

The ß3-adrenergic receptor (AR) signaling pathway is a major component of adaptive thermogenesis in brown and white adipose tissue during cold acclimation. The ß3-AR signaling highly induces the expression of transcriptional coactivator PGC-1α and its splice variant N-terminal (NT)-PGC-1α, which in turn activate the transcription program of adaptive thermogenesis by co-activating a number of transcription factors. We previously reported that NT-PGC-1α is able to increase mitochondrial number and activity in cultured brown adipocytes by promoting the expression of mitochondrial and thermogenic genes. In the present study, we performed genome-wide profiling of NT-PGC-1α-responsive genes in brown adipocytes to identify genes potentially regulated by NT-PGC-1α. Canonical pathway analysis revealed that a number of genes upregulated by NT-PGC-1α are highly enriched in mitochondrial pathways including fatty acid transport and ß-oxidation, TCA cycle and electron transport system, thus reinforcing the crucial role of NT-PGC-1α in the enhancement of mitochondrial function. Moreover, canonical pathway analysis of NT-PGC-1α-responsive genes identified several metabolic pathways including glycolysis and fatty acid synthesis. In order to validate the identified genes in vivo, we utilized the FL-PGC-1α-/- mouse that is deficient in full-length PGC-1α (FL-PGC-1α) but expresses a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α254). The ß3-AR-induced increase of NT-PGC-1α254 in FL-PGC-1α-/- brown and white adipose tissue was closely associated with elevated expression of genes involved in thermogenesis, mitochondrial oxidative metabolism, glycolysis and fatty acid synthesis. Increased adipose tissue thermogenesis by ß3-AR activation resulted in attenuation of adipose tissue expansion in FL-PGC-1α-/- adipose tissue under the high-fat diet condition. Together, the data strengthen our previous findings that NT-PGC-1α regulates mitochondrial genes involved in thermogenesis and oxidative metabolism in brown and white adipocytes and further suggest that NT-PGC-1α regulates a broad spectrum of genes to meet cellular needs for adaptive thermogenesis.


Subject(s)
Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Protein Interaction Domains and Motifs , Trans-Activators/metabolism , Transcriptional Activation , Transcriptome , Adipocytes/metabolism , Adipose Tissue, Brown/drug effects , Adipose Tissue, White/drug effects , Adrenergic beta-3 Receptor Agonists/pharmacology , Animals , Energy Metabolism/genetics , Gene Expression Profiling , Gene Expression Regulation/drug effects , Gene Regulatory Networks , Male , Mice , Mice, Knockout , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/chemistry , Receptors, Adrenergic, beta-3/metabolism , Signal Transduction , Thermogenesis/drug effects , Thermogenesis/genetics , Trans-Activators/chemistry
16.
Proc Natl Acad Sci U S A ; 112(19): 6074-9, 2015 May 12.
Article in English | MEDLINE | ID: mdl-25918410

ABSTRACT

EWS (Ewing sarcoma) encodes an RNA/ssDNA binding protein that is frequently rearranged in a number of different cancers by chromosomal translocations. Physiologically, EWS has diverse and essential roles in various organ development and cellular processes. In this study, we uncovered a new role of EWS in mitochondrial homeostasis and energy metabolism. Loss of EWS leads to a significant decrease in mitochondria abundance and activity, which is caused by a rapid degradation of Peroxisome proliferator-activated receptor γ Coactivator (PGC-1α), a central regulator of mitochondria biogenesis, function, and cellular energy metabolism. EWS inactivation leads to increased ubiquitination and proteolysis of PGC-1α via proteasome pathway. Complementation of EWS in Ews-deficient cells restores PGC-1α and mitochondrial abundance. We found that expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased in the absence of Ews and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice. Furthermore, expression of mitochondrial biogenesis, respiration and fatty acid ß-oxidation genes is significantly reduced in the liver of Ews-null mice. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability, and further implicate altered mitochondrial and energy metabolism in cancers harboring the EWS translocation.


Subject(s)
Mitochondria/metabolism , RNA-Binding Protein EWS/antagonists & inhibitors , Transcription Factors/metabolism , Adipose Tissue, Brown/metabolism , Animals , DNA, Mitochondrial/metabolism , Energy Metabolism , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7 , Fatty Acids/chemistry , Fatty Acids/metabolism , Gene Expression Profiling , HEK293 Cells , Homeostasis , Humans , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Muscle, Skeletal/metabolism , Oxygen/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Protein Conformation , RNA-Binding Protein EWS/metabolism , Ubiquitin/chemistry , Ubiquitin-Protein Ligases/metabolism
17.
Biomed Res Int ; 2014: 402175, 2014.
Article in English | MEDLINE | ID: mdl-25136584

ABSTRACT

PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b) and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3(') splicing between exon 6 and exon 7, we have investigated isoform-specific expression of NT-PGC-1α mRNA in mouse skeletal muscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a) is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and ß 2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC-1α-b (PGC-1α4) and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I, MHC II a) and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B). In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise.


Subject(s)
Alternative Splicing/physiology , Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , Physical Conditioning, Animal , RNA, Messenger/biosynthesis , Transcription Factors/biosynthesis , Animals , Male , Mice , Mitochondria, Muscle/genetics , Mitochondria, Muscle/metabolism , Muscle Proteins/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA, Messenger/genetics , Transcription Factors/genetics
18.
Diabetes ; 63(11): 3615-25, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24848065

ABSTRACT

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and its splice variant N terminal (NT)-PGC-1α regulate adaptive thermogenesis by transcriptional induction of thermogenic and mitochondrial genes involved in energy metabolism. We previously reported that full-length PGC-1α (FL-PGC-1α) is dispensable for cold-induced nonshivering thermogenesis in FL-PGC-1α(-/-) mice, since a slightly shorter but functionally equivalent form of NT-PGC-1α (NT-PGC-1α(254)) fully compensates for the loss of FL-PGC-1α in brown and white adipose tissue. In the current study, we challenged FL-PGC-1α(-/-) mice with a high-fat diet (HFD) to investigate the effects of diet-induced thermogenesis on HFD-induced obesity. Despite a large decrease in locomotor activity, FL-PGC-1α(-/-) mice exhibited the surprising ability to attenuate HFD-induced obesity. Reduced fat mass in FL-PGC-1α(-/-) mice was closely associated with an increase in body temperature, energy expenditure, and whole-body fatty acid oxidation (FAO). Mechanistically, FL-PGC-1α(-/-) brown adipose tissue had an increased capacity to oxidize fatty acids and dissipate energy as heat, in accordance with upregulation of thermogenic genes UCP1 and DIO2. Furthermore, augmented expression of FAO and lipolytic genes in FL-PGC-1α(-/-) white adipose tissue was highly correlated with decreased fat storage in adipose tissue. Collectively, our data highlight a protective effect of NT-PGC-1α on diet-induced obesity by enhancing diet-induced thermogenesis and FAO.


Subject(s)
Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/metabolism , Diet, High-Fat/adverse effects , Obesity/metabolism , Transcription Factors/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Adipose Tissue/cytology , Adipose Tissue/metabolism , Animals , Blotting, Western , Body Composition , Calorimetry, Indirect , Cells, Cultured , Energy Metabolism/genetics , Energy Metabolism/physiology , Male , Mice , Mice, Knockout , Obesity/etiology , Oxidation-Reduction , Oxygen Consumption/physiology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Real-Time Polymerase Chain Reaction , Transcription Factors/genetics , Triglycerides/metabolism
19.
Methods Mol Biol ; 952: 163-73, 2013.
Article in English | MEDLINE | ID: mdl-23100231

ABSTRACT

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a nuclear transcriptional coactivator that regulates the genes involved in energy metabolism. Recent evidence has been provided that alternative splicing of PPARGC1A gene produces a functional but predominantly cytosolic isoform of PGC-1α (NT-PGC-1α). We have demonstrated that transcriptional coactivation capacity of NT-PGC-1α is directly correlated with its nuclear localization in a PKA phosphorylation-dependent manner. In this chapter, we describe quantitative imaging analysis methods that are developed to measure the relative fluorescence intensity of the protein of interest in the nucleus and cytoplasm in a single cell and the frequency distribution of nuclear/cytoplasmic intensity ratios in the population of cells, respectively. This chapter also describes transient cotransfection and dual-luciferase reporter gene assay that examine the ability of coactivators to activate the transcriptional activity of transcription factors.


Subject(s)
Cytosol/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Animals , CHO Cells , Cricetinae , Cricetulus , Fluorescent Antibody Technique , Genes, Reporter/genetics , Luciferases/genetics , Microscopy , Phosphorylation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , Transcription Factors/genetics , Transfection
20.
PPAR Res ; 2012: 320454, 2012.
Article in English | MEDLINE | ID: mdl-23093952

ABSTRACT

Brown fat expresses two PGC-1α isoforms (PGC-1α and NT-PGC-1α) and both play a central role in the regulation of cellular energy metabolism and adaptive thermogenesis by interacting with a wide range of transcription factors including PPARγ, PPARα, ERRα, and NRF1. PGC-1α consists of 797 amino acids, whereas alternative splicing of the PGC-1α gene produces a shorter protein called NT-PGC-1α (aa 1-270). We report in this paper that transcriptional activity of PGC-1α and NT-PGC-1α is differently affected by the transcriptional regulator, Twist-1. Twist-1 suppresses PGC-1α but not NT-PGC-1α. The inhibition of PGC-1α activity by Twist-1 is mediated by direct interaction through the C-terminal region of PGC-1α (aa 353-797). Thus, the absence of the corresponding C-terminal domain in NT-PGC-1α allows NT-PGC-1α to be free from Twist-1-mediated inhibition. Overexpression of Twist-1 in brown adipocytes suppresses transcription of a subset of PGC-1α-target genes involved in mitochondrial fatty acid oxidation and uncoupling (CPT1ß, UCP1, and ERRα). In contrast, NT-PGC-1α-mediated induction of these genes is unaffected by Twist-1. These findings show that differences in inhibitory protein-protein interactions of PGC-1α and NT-PGC-1α with Twist-1 lead to differential regulation of their function by Twist-1.

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