Postlipolytic insulin-dependent remodeling of micro lipid droplets in adipocytes

Despite the lipolysis-lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. We characterized mLD formation in cultured adipocytes, and in primary adipocytes isolated from mouse epididymal fat pads, in response to acute activation of lipolysis. Using real-time quantitative imaging and electron tomography, we show that formation of mLDs in cultured adipocytes occurs throughout the cell to increase total LD surface area by ~30% but does not involve detectable fission from large LDs. Peripheral mLDs are monolayered structures with a neutral lipid core and are sites of active lipolysis. Electron tomography reveals preferential association of mLDs with the endoplasmic reticulum. Treatment with insulin and fatty acids results in the reformation of macroLDs and return to the basal state. Insulin-dependent reformation of large LDs involves two distinct processes: microtubule-dependent homotypic fusion of mLDs and expansion of individual mLDs. We identify a physiologically important role for LD fusion that is involved in a reversible lipolytic cycle in adipocytes.     

Lipid-Overloaded Enlarged Adipocytes Provoke Insulin Resistance Independent of Inflammation

In obesity, adipocyte hypertrophy and proinflammatory responses are closely associated with the development of insulin resistance in adipose tissue. However, it is largely unknown whether adipocyte hypertrophy per se might be sufficient to provoke insulin resistance in obese adipose tissue. Here, we demonstrate that lipid-overloaded hypertrophic adipocytes are insulin resistant independent of adipocyte inflammation. Treatment with saturated or monounsaturated fatty acids resulted in adipocyte hypertrophy, but proinflammatory responses were observed only in adipocytes treated with saturated fatty acids. Regardless of adipocyte inflammation, hypertrophic adipocytes with large and unilocular lipid droplets exhibited impaired insulin-dependent glucose uptake, associated with defects in GLUT4 trafficking to the plasma membrane. Moreover, Toll-like receptor 4 mutant mice (C3H/HeJ) with high-fat-diet-induced obesity were not protected against insulin resistance, although they were resistant to adipose tissue inflammation. Together, our in vitro and in vivo data suggest that adipocyte hypertrophy alone may be crucial in causing insulin resistance in obesity.     

Remodeling of lipid droplets during lipolysis and growth in adipocytes

Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.     

Impact of perturbed pyruvate metabolism on adipocyte triglyceride accumulation

This study aimed to test the hypothesis that adipocyte TG accumulation could be altered by specifically perturbing pyruvate metabolism. We treated cultured 3T3-L1 adipocytes with chemical inhibitors of lactate dehydrogenase (LDH) and pyruvate carboxylase (PC), and characterized their global effects on intermediary metabolism using metabolic flux and isotopomer analysis. Inhibiting the enzymes over several days did not alter the adipocyte differentiation program as assessed by the expression levels of peroxisome proliferator-activated receptor-γ and glycerol-3-phosphate dehydrogenase. The main metabolic effects were to up-regulate intracellular lipolysis and decrease TG accumulation. Inhibiting PC also up-regulated glycolysis. Flux estimates indicated that the reduction in TG was due to decreased de novo fatty acid synthesis. Exogenous addition of free fatty acids dose-dependently increased the cellular TG level in the inhibitor-treated adipocytes, but not in untreated control cells. The results of this study support our hypothesis regarding the critical role of pyruvate reactions in TG synthesis.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

Mechanism for Endogenously Expressed ApoE Modulation of Adipocyte Very Low Density Lipoprotein Metabolism: ROLE IN ENDOCYTIC AND LIPASE-MEDIATED METABOLIC PATHWAYS*

Triglyceride-rich lipoproteins distribute energy in the form of fatty acids to peripheral tissues. We have previously shown that the absence of endogenous adipocyte apoE expression impairs adipocyte triglyceride acquisition from apoE-containing triglyceride-rich lipoproteins in vitro and in vivo. Studies were performed to evaluate the mechanism(s) for this impairment. We excluded a role for secreted apoE in accounting for the difference in very low density lipoprotein (VLDL)-induced adipocyte triglyceride accumulation using cross-incubation studies to show that secreted apoE did not enhance triglyceride synthesis in apoE knockout (EKO) adipocytes incubated with apoE-containing VLDL. Subsequent experiments established that both endocytic and lipase-mediated pathways for lipid acquisition from VLDL were impaired in EKO adipocytes. Binding and internalization of VLDL to EKO adipocytes were significantly lower due to decreased expression or redistribution of low density lipoprotein receptor family proteins. An important role for the VLDL receptor for contributing to differences in VLDL binding between wild-type and EKO adipocytes was identified. Lipoprotein lipase-dependent adipocyte lipogenesis was also significantly decreased in EKO adipocytes even though they secreted as much or more lipolytic activity. This decrease was related to impaired fatty acid internalization in EKO cells. Evaluation of potential mechanisms revealed reduced caveolin-1 and plasma membrane raft expression in EKO adipocytes. Increasing caveolin expression in EKO adipocytes increased fatty acid internalization. Our results establish a role for endogenous adipocyte apoE in VLDL-induced adipocyte lipogenesis by impacting both endocytic and lipoprotein lipase-mediated metabolic pathways. Reduced adipocyte apoE expression, for example that accompanying obesity, will suppress adipocyte acquisition of lipid from apoE-containing VLDL.     

Immunohistochemical identification of the uncoupling protein in human hibernoma

The expression of the uncoupling protein (UCP), a protein unique to brown adipocyte mitochondria, was studied in sections of a human hibernoma by means of immunohistochemistry. Multilocular, but not unilocular, adipocytes expressed the UCP in the tissue. Further, the immunostaining was not uniform in multilocular cells, because small adipocytes with finely multivacuolar or scanty lipid deposit showed more intense staining. This pattern is similar to that found in brown adipose tissue. Ultrastructural investigation confirmed that a majority of proliferating cells had the morphological characteristics of brown adipocyte. Results indicate that adipocytes in hibernoma may be very close to brown adipocytes both morphologically and immunocytochemically.     

Adipocyte-specific Disruption of Fat-specific Protein 27 Causes Hepatosteatosis and Insulin Resistance in High-fat Diet-fed Mice

White adipose tissue (WAT) functions as an energy reservoir where excess circulating fatty acids are transported to WAT, converted to triglycerides, and stored as unilocular lipid droplets. Fat-specific protein 27 (FSP27, CIDEC in humans) is a lipid-coating protein highly expressed in mature white adipocytes that contributes to unilocular lipid droplet formation. However, the influence of FSP27 in adipose tissue on whole-body energy homeostasis remains unclear. Mice with adipocyte-specific disruption of the Fsp27 gene (Fsp27^ΔAd) were generated using an aP2-Cre transgene with the Cre/LoxP system. Upon high-fat diet feeding, Fsp27^ΔAd mice were resistant to weight gain. In the small WAT of these mice, small adipocytes containing multilocular lipid droplets were dispersed. The expression levels of the genes associated with mitochondrial abundance and brown adipocyte identity were increased, and basal lipolytic activities were significantly augmented in adipocytes isolated from Fsp27^ΔAd mice compared with the Fsp27^F/F counterparts. The impaired fat-storing function in Fsp27^ΔAd adipocytes and the resultant lipid overflow from WAT led to marked hepatosteatosis, dyslipidemia, and systemic insulin resistance in high-fat diet-treated Fsp27^ΔAd mice. These results demonstrate a critical role for FSP27 in the storage of excess fat in WAT with minimizing ectopic fat accumulation that causes insulin-resistant diabetes and non-alcoholic fatty liver disease. This mouse model may be useful for understanding the significance of fat-storing properties of white adipocytes and the role of local FSP27 in whole-body metabolism and estimating the pathogenesis of human partial lipodystrophy caused by CIDEC mutations.     

Adipocyte lipid storage and adipokine production are modulated by lipoxygenase-derived oxylipins generated from 18-carbon fatty acids

Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6 days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30 nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production.     

Sequential ordered fatty acid alpha oxidation and Delta9 desaturation are major determinants of lipid storage and utilization in differentiating adipocytes

Herein, we exploit the power of global lipidomics to identify the critical role of peroxisomal processing of fatty acids in adipocyte lipid storage and metabolism. Remarkably, 3T3-L1 differentiating adipocytes rapidly acquired the ability to alpha oxidize unbranched fatty acids, which is manifested in the accumulation of odd chain length unbranched fatty acids in all major lipid classes. Moreover, in differentiating adipocytes, unsaturated odd chain length fatty acids in TAG molecular species contained exclusively Delta9 olefinic linkages. Unsaturated fatty acids (e.g., oleic and palmitoleic acids) were not subject to alpha oxidation, resulting in the absence of Delta8 unsaturated odd chain length fatty acids. This highly selective substrate utilization resulted in the obligatory sequential ordering of alpha oxidation prior to Delta9 desaturation. On the basis of these results, a putative type 2 peroxisomal localization sequence was identified at the N-terminus of mouse stearoyl-CoA desaturase I (SCD I) comprised of (30)KVKTVPLHL(38). Kinetic analysis demonstrated that the rate of alpha oxidation of exogenously administered [9,10-(3)H]palmitic acid increased 4-fold during differentiation. Similarly, quantitative PCR demonstrated a 4-fold increase in phytanoyl-CoA alpha hydroxylase (PAHX) and fatty acyl-CoA oxidase (FACO) mRNA levels during differentiation. Collectively, these results underscore the role of peroxisomal fatty acid processing as an important determinant of the metabolic fate of fatty acids in the differentiating adipocyte.     

Ghrelin is a signal to facilitate the utilization of fatty acids and save glucose by the liver,skeletal muscle,and adipose tissues in chicks

Ghrelin, classically known as a central appetite-stimulating hormone, has recently been recognized to play an important role in peripheral tissue energy metabolism. In chicken, contrary to mammal, ghrelin acts as an anorexia signal, increased by fasting and further elevated after refed. In the present study, the effect of ghrelin on glucose/lipid utilization by peripheral tissues was investigated. Injection of exogenous acyl ghrelin reduced plasma triglyceride and glucose levels of chickens at both fasting and fed status. In the in vitro cultured chicken primary hepatocytes, adipocytes, and myoblasts, ghrelin suppressed glucose uptake, stimulated fatty acids uptake and oxidation, and decreased TG content. In hepatocyte, ghrelin increased the activities of LPL and HL, and upregulated the expression levels of gene ACC, CPT1, and PPARα. Ghrelin treatment markedly increased the protein level of p-ACC, PPARγ, PGC1α, and CPT1 in hepatocytes, adipocytes and myoblasts. Inhibition of AMPK activity by Compound C had no influence on glucose uptake by hepatocyte, adipocyte, and myoblast, but further amplified the stimulated fatty acid uptake of adipocyte by ghrelin. The present result demonstrates that ghrelin facilitates the uptake and oxidation of fatty acid and cut down the utilization of glucose by the liver, muscle, and adipose tissues. The result suggests that ghrelin functions as a signal of fatty acid oxidation. The study provides a vital framework for understanding the intrinsic role of ghrelin as a crucial factor in the concerted regulation of metabolic substrate of hepatocytes, adipocytes, and myoblasts.     

Adipocyte protein S3-12 coats nascent lipid droplets

Most animals store lipid intracellularly in protein-coated droplets. The protein coat usually contains at least one member of the PAT (perilipin, adipose differentiation-related protein, and TIP47) family. Evidence suggests that PAT proteins control access to the lipid they enclose. The protein S3-12, which has sequence similarity to the PAT proteins, was found in a screen for adipocyte-specific proteins. The adipocyte expression of S3-12 and its similarity to the PAT proteins suggest that S3-12 is involved in adipocyte lipid storage. To test this hypothesis, we supplemented 3T3-L1 adipocytes with fatty acids and assessed the distribution of S3-12 by immunofluorescence microscopy. Prior to fatty acid incubation, S3-12 was distributed diffusely throughout the cytoplasm on punctate structures of heterogeneous size. After 10 min of lipid loading, S3-12 localized to 500-nm structures concentrated at the adipocyte periphery. After longer incubations, S3-12 coated the surface of lipid droplets up to several micrometers in diameter. Initially, these droplets were distinct from those droplets surrounded by perilipin; but by 240 min, most perilipin-coated droplets had some S3-12 on the surface as well. We additionally report that the formation of S3-12-coated droplets 1) required glucose and fatty acids that can be incorporated into triacylglycerol, 2) was blocked by an inhibitor of triacylglycerol synthesis, and 3) was insulin-dependent. This study reports for the first time the early morphological events in the genesis and maturation of adipocyte lipid droplets.     

Oleate promotes differentiation of chicken primary preadipocytes in?vitro

In addition to providing energy and constituting cell membrane, fatty acids also play an important role in adipocyte differentiation and lipid metabolism. As an important member of monounsaturated fatty acids, oleate, together with other components, is widely used to induce chicken preadipocyte differentiation. However, it is not clear whether oleate alone can induce chicken preadipocyte differentiation. In the present study, four different treatments were designed to test this question: basal medium, IDX [insulin, dexamethasone and IBMX (isobutylmethylxanthine)], oleate and IDX plus oleate. Cytoplasmic lipid droplet accumulation and mRNA expression for adipogenesis-related genes were monitored. After treatment of oleate on chicken preadipocytes, apparent lipid droplet formation and lipid accumulation were observed, accompanied by increasing expression of PPARγ (peroxisome proliferator-activated receptor-γ) and AFABP (adipocyte fatty acid-binding protein), but decreasing level of GATA2 (GATA-binding protein 2). In contrast, for cells cultured in the basal medium with or without IDX supplementation, lipid droplet barely occurred. These results suggest that exogenous oleate alone can act as an inducer of preadipocyte differentiation into adipocytes.     

Impaired lipid accumulation by trans10, cis12 CLA during adipocyte differentiation is dependent on timing and length of treatment

Conjugated linoleic acids (CLAs) are a group of polyunsaturated fatty acids found in ruminant products, where the predominant isomers are cis9, trans11 (c9,t11) and trans10, cis12 (t10,c12) CLA. We have previously shown that t10,c12 CLA prevents lipid accumulation in mature adipocytes in part by acting as a peroxisome proliferator-activated receptor gamma (PPAR gamma) modulator. The objective of this study was to further establish the molecular mechanisms underlying the attenuating effect on lipid accumulation by t10,c12 CLA, with focus on time point and duration of treatment during adipogenesis. We have shown that t10,c12 CLA treatment has its most attenuating effect early (day (D) 0-6) during differentiation. Treatment during this period is sufficient to prevent lipid accumulation in mature adipocytes. The adipogenic marker genes PPAR gamma and CCAAT/enhancer binding protein alpha (C/EBP alpha) are both down-regulated after treatment within the period from D0-6, while additional treatment also down-regulates the expression of sterol regulatory element binding protein-1c (SREBP-1c), liver X receptor alpha (LXR alpha), fatty acid binding protein (aP2), fatty acid translocase (CD36) and insulin-sensitive glucose transporter 4 (GLUT4). These effects of t10,c12 CLA reflect the subsequent attenuation of lipid accumulation observed in mature adipocytes. Interestingly, the early B-cell factor (O/E-1), which is known to promote adipogenesis and to be involved in control of genes important for terminal adipocyte differentiation, is unaffected by treatment of t10,c12 CLA. Taken together, our data indicate that inhibition of lipid accumulation induced by t10,c12 CLA treatment during adipocyte differentiation is associated with a tight regulatory cross-talk between early (PPAR gamma and C/EBP alpha) and late (LXR alpha, aP2 and CD36) adipogenic marker genes.     

Intermediary metabolism of adipose tissue.

Metabolism of ruminant adipocytes involves the synthesis and mobilization of lipids. Rates of lipid synthesis from the uptake of preformed fatty acids (via lipoprotein lipase) and de novo synthesis of fatty acids are related to the energy balance. Acetate is the major carbon source for fatty acid synthesis with NADPH originating from the pentose cycle and the isocitrate cycle. Ruminant adipose tissue lacks the ability to utilize for lipogenesis those substrates that generate mitochondrial acetyl CoA because of an absence of ATP citrate-lyase and NADP-malate dehydrogenase. Lipid mobilization in ruminant adipocytes is apparently regulated via cAMP levels and a summary of the compounds investigated for lipolytic responses is presented. The control of lipid synthesis and mobilization is interrelated in ruminant adipose tissue. The coordinated manner in which these two functions are regulated is examined with regard to adipocyte responses to insulin and epinephrine. In both lipid synthesis and lipid mobilization, ruminant adipocytes are uniquely different from nonruminant adipose tissue. The physiological significance and possible basis for these species differences in adipose metabolism are discussed.     

Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells.     

Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids,GLUT4 Level,and Lipogenesis in 3T3-L1 Adipocytes

Straight chain fatty acid α-oxidation increases during differentiation of 3T3-L1 adipocytes, leading to a marked accumulation of odd chain length fatty acyl moieties. Potential roles of this pathway in adipocyte differentiation and lipogenesis are unknown. Mammalian fatty acid 2-hydroxylase (FA2H) was recently identified and suggested to catalyze the initial step of straight chain fatty acid α-oxidation. Accordingly, we examined whether FA2H modulates adipocyte differentiation and lipogenesis in mature adipocytes. FA2H level markedly increases during differentiation of 3T3-L1 adipocytes, and small interfering RNAs against FA2H inhibit the differentiation process. In mature adipocytes, depletion of FA2H inhibits basal and insulin-stimulated glucose uptake and lipogenesis, which are partially rescued by the enzymatic product of FA2H, 2-hydroxy palmitic acid. Expression of fatty-acid synthase and SCD1 was decreased in FA2H-depleted cells, and levels of GLUT4 and insulin receptor proteins were reduced. 2-Hydroxy fatty acids are enriched in cellular sphingolipids, which are components of membrane rafts. Accelerated diffusional mobility of raft-associated lipids was shown to enhance degradation of GLUT4 and insulin receptor in adipocytes. Consistent with this, depletion of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B. Moreover, the enhanced recovery rates were partially reversed by treatment with 2-hydroxy palmitic acid. In conclusion, our findings document the novel role of FA2H in adipocyte lipogenesis possibly by modulation of raft fluidity and level of GLUT4.     

Activation of peroxisome proliferator-activated receptor-α enhances fatty acid oxidation in human adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPARα in adipocytes have been unclarified. We examined the functions of PPARα using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPARα by GW7647, a potent PPARα agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPARγ, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPARα activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPARγ is activated. On the other hand, PPARα activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPARα-dependent manner. Moreover, PPARα activation increased the production of CO₂ and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPARα stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPARα agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPARα activation are very valuable for managing diabetic conditions accompanied by obesity, because PPARγ agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.