ABSTRACT
The surface of lipid droplets (LDs) in various cell types is coated with perilipin proteins encoded by the Plin genes. Perilipins regulate LD metabolism by selectively recruiting lipases and other proteins to LDs. We have studied the expression of perilipins in mouse muscle. The glycolytic fiber-enriched gastrocnemius muscle expresses predominantly Plin2-4. The oxidative fiber-enriched soleus muscle expresses Plin2-5. Expression of Plin2 and Plin4-5 is elevated in gastrocnemius and soleus muscles from mice fed a high-fat diet. This effect is preserved in peroxisome proliferator-activated receptor (PPAR)α-deficient mice. Mouse muscle derived C2C12 cells differentiated into glycolytic fibers increase transcription of these Plins when exposed to various long chain fatty acids (FAs). To understand how FAs regulate Plin genes, we used specific activators and antagonists against PPARs, Plin promoter reporter assays, chromatin immunoprecipitation, siRNA, and animal models. Our analyses demonstrate that FAs require PPARδ to induce transcription of Plin4 and Plin5. We further identify a functional PPAR binding site in the Plin5 gene and establish Plin5 as a novel direct PPARδ target in muscle. Our study reveals that muscle cells respond to elevated FAs by increasing transcription of several perilipin LD-coating proteins. This induction renders the muscle better equipped to sequester incoming FAs into cytosolic LDs.
Subject(s)
Fatty Acids/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/drug effects , PPAR delta/metabolism , Animals , Binding Sites/drug effects , Cells, Cultured , Fatty Acids/administration & dosage , Gene Silencing/drug effects , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle Proteins/chemistry , Muscle Proteins/genetics , Muscle, Skeletal/metabolism , PPAR delta/chemistry , PPAR delta/deficiency , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western and non-Western countries, but its pathogenesis is not fully understood. OBJECTIVE: Based on the role of nicotinamide phosphoribosyltransferase (NAMPT) in fat and glucose metabolism and cell survival, we hypothesized a role for NAMPT/visfatin in the pathogenesis of NAFLD-related disease. DESIGN AND SETTING: We conducted clinical studies at a referral medical center in well-characterized NAFLD patients (n = 58) and healthy controls (n = 27). In addition we performed experimental in vitro studies in hepatocytes. MAIN OUTCOME MEASURES: We examined 1) the hepatic and systemic expression of NAMPT/visfatin in patients with NAFLD and control subjects, 2) the hepatic regulation of NAMPT/visfatin, and 3) the effect of NAMPT/visfatin on hepatocyte apoptosis. RESULTS: Our main findings were as follows. 1) Patients with NAFLD had decreased NAMPT/visfatin expression both systemically in serum and within the hepatic tissue, with no difference between simple steatosis and nonalcoholic steatohepatitis. 2) By studying the hepatic regulation of NAMPT/visfatin in wild-type and peroxisome proliferators-activated receptor (PPAR)alpha(-/-) mice as well as in hepatocytes, we showed that PPARalpha activation and glucose may be involved in the down-regulation of hepatic NAMPT/visfatin expression in NAFLD. 4) Within the liver, NAMPT/visfatin was located to hepatocytes, and our in vitro studies showed that NAMPT/visfatin exerts antiapoptotic effects in these cells, involving enzymatic synthesis of nicotinamide adenine dinucleotide. CONCLUSION: Based on these findings, we suggest a role for decreased NAMPT/visfatin levels in hepatocyte apoptosis in NAFLD-related disease.
Subject(s)
Apoptosis/physiology , Fatty Liver/enzymology , Hepatocytes/physiology , Nicotinamide Phosphoribosyltransferase/physiology , Adult , Aged , Animals , Cell Line , Cells, Cultured , Down-Regulation , Fatty Liver/pathology , Female , Glucose/pharmacology , Humans , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Male , Mice , Middle Aged , Mitochondria, Liver/metabolism , NAD/metabolism , PPAR alpha/metabolism , RNA, Small Interfering , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
The PAT family (originally named for Perilipin, ADFP and TIP47) now includes four members: Perilipins, ADFP, TIP47 and S3-12. Significant primary sequence homology and the ability to associate with lipid storage droplets (LSDs) are well conserved within this family and across species. In this study, we have characterized a novel PAT protein, lipid storage droplet protein 5 (LSDP5) of 463 residues. A detailed sequence analysis of all murine PAT proteins reveals that LSDP5, TIP47 and ADFP share the highest order of sequence similarity, whereas perilipin and S3-12 have more divergent carboxyl- and amino-termini, respectively. Ectopically-expressed YFP-LSDP5 or flag-LSDP5 fusion proteins associate with LSDs. In accord with recent published data for perilipin, forced expression of LSDP5 in CHO cells inhibits lipolysis of intracellular LSDs. The LSDP5 gene is primarily transcribed in cells that actively oxidize fatty acids, such as heart, red muscle and liver. Expression of LSDP5 is stimulated by ligand activation of peroxisomal proliferator-activated receptor alpha (PPARalpha), and significantly reduced in liver and heart in the absence of this transcription factor. PPARalpha is generally required for regulation of fatty acid metabolism during fasting, but fasting induces LSDP5 mRNA in liver even in the absence of PPARalpha.
Subject(s)
Fatty Acids/metabolism , Phosphoproteins/metabolism , Proteins/genetics , Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Carrier Proteins , Chlorocebus aethiops , Chromosomes, Human, Pair 17 , Exons , Fasting/metabolism , Humans , Liver/metabolism , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Oxidation-Reduction , PPAR alpha/metabolism , Perilipin-1 , Perilipin-5 , Phosphoproteins/genetics , Sequence Homology, Amino Acid , Tissue DistributionABSTRACT
The adipose differentiation-related protein (ADFP)/adipophilin belongs to a family of PAT (for perilipin, ADFP, and TIP47) proteins that associate on the surface of lipid droplets (LDs). Except for LD association, a clear role for ADFP has not been found. We demonstrate that ADFP is transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) in mouse liver and rat and human hepatoma cells through a highly conserved direct repeat-1(DR-1) element. Although the ADFP mRNA is highly increased by a synthetic PPARalpha agonist, the ADFP protein is only substantially increased in cells containing LDs, such as hepatocytes incubated with fatty acids, and in livers of fasted mice. ADFP is induced by fasting even in the absence of a functional PPARalpha, in marked contrast to the PPARalpha target gene acyl-coenzyme A oxidase-1. Activation of LXRs, which stimulates LD formation through the activation of lipogenesis, does not affect ADFP mRNA levels. TIP47, another PAT member known to be expressed in liver, was unaffected by all treatments. This constitutively expressed PAT member seems to be less transcriptionally regulated than ADFP. These observations suggest that ADFP is primarily a fasting-induced protein in liver that coats the newly synthesized triacylglycerol-containing LDs formed during fasting.
