Molecular modelling, synthesis, and biological evaluations of a 3,5-disubstituted isoxazole fatty acid analogue as a PPARα-selective agonist.

The peroxisome proliferator activated receptors (PPARs) are important drug targets in treatment of metabolic and inflammatory disorders. Fibrates, acting as PPARα agonists, have been widely used lipid-lowering agents for decades. However, the currently available PPARα targeting agents show low subtype-specificity and consequently a search for more potent agonists have emerged. In this study, previously isolated oxohexadecenoic acids from the marine algae Chaetoceros karianus were used to design a PPARα-specific analogue. Herein we report the design, synthesis, molecular modelling studies and biological evaluations of the novel 3,5-disubstituted isoxazole analogue 6-(5-heptyl-1,2-oxazol-3-yl)hexanoic acid (1), named ADAM. ADAM shows a clear receptor preference and significant dose-dependent activation of PPARα (EC50 = 47 µM) through its ligand-binding domain (LBD). Moreover, ADAM induces expression of important PPARα target genes, such as CPT1A, in the Huh7 cell line and primary mouse hepatocytes. In addition, ADAM exhibits a moderate ability to regulate PPARγ target genes and drive adipogenesis. Molecular modelling studies indicated that ADAM docks its carboxyl group into opposite ends of the PPARα and -γ LBD. ADAM interacts with the receptor-activating polar network of amino acids (Tyr501, His447 and Ser317) in PPARα, but not in PPARγ LBD. This may explain the lack of PPARγ agonism, and argues for a PPARα-dependent adipogenic function. Such compounds are of interest towards developing new lipid-lowering remedies.

Synthesis and biological evaluations of marine oxohexadecenoic acids: PPARα/γ dual agonism and anti-diabetic target gene effects.

Obesity and associated disorders such as metabolic syndrome and type 2 diabetes (T2D) have reached epidemic proportions. Several natural products have been reported as Peroxisome Proliferator-Activated Receptor (PPAR) agonists, functioning as lead compounds towards developing new anti-diabetic drugs due to adverse side effects of existing PPAR drugs. We recently isolated and identified (7E)-9-oxohexadec-7-enoic acid (1) and (10E)-9-oxohexadec-10-enoic acid (2) from the marine algae Chaetoceros karianus. Herein we report the total synthesis, pharmacological characterization, and biological evaluations of these naturally occurring oxo-fatty acids (oFAs). The syntheses of 1 and 2 afforded sufficient material for extensive biological evaluations. Both oFAs show an appreciable dose-dependent activation of PPARα and -γ, with EC50 values in the micromolar range, and an ability to regulate important PPAR target genes in hepatocytes and adipocytes. Moreover, both 1 and 2 are able to drive adipogenesis when evaluated in the Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocyte cell model, but with lowered expression of adipocyte markers and reduced lipid accumulation compared to the drug rosiglitazone. This seems to be caused by a transient upregulation of PPARγ and C/EBPα expression. Importantly, whole transcriptome analysis shows that both compounds induce anti-diabetic gene programs in adipocytes by upregulating insulin-sensitizing adipokines and repressing pro-inflammatory cytokines.

Two Isomeric C16 Oxo-Fatty Acids from the Diatom Chaetoceros karianus Show Dual Agonist Activity towards Human Peroxisome Proliferator-Activated Receptors (PPARs) α/γ.

The peroxisome proliferator-activated receptors (PPARs) function as ligand-activated transcription factors that convert signals in the form of lipids to physiological responses through the activation of metabolic target genes. Due to their key roles in lipid and carbohydrate metabolism, the PPARs are important drug targets. However, for several of the PPAR drugs currently in use, adverse side effects have been reported. In an effort to identify compounds from marine organisms that may serve as molecular scaffolds for the development of novel and safer PPAR-targeting drugs, we performed a bioassay-guided screening of organic extracts made from organisms supplied by the Norwegian Biobank of Arctic Marine Organisms (Marbank). Among several interesting hits, we identified two poorly described isomeric oxo-fatty acids from the microalgae Chaetoceros karianus for which we provide the first evidence that they might display dual specificity towards human PPARα and PPARγ. Principal component analysis showed that C. karianus stood out from other Chaetoceros species, both with respect to the metabolic profile and the PPAR activity. The isolation of these compounds holds the potential of uncovering a PPAR pharmacophore with tunable activity and specificity.

LXRα Regulates Hepatic ChREBPα Activity and Lipogenesis upon Glucose, but Not Fructose Feeding in Mice.

Liver X receptors (LXRα/ß) and carbohydrate response element-binding proteins (ChREBPα/ß) are key players in the transcriptional control of hepatic de novo lipogenesis. LXRα/ß double knockout (LXRα-/-/ß-/-) mice have reduced feeding-induced nuclear O-linked N-acetylglucosamine (O-GlcNAc) signaling, ChREBPα activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXRα knockout (LXRα-/-) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXRα-/- mice. This was associated with lower expression of liver pyruvate-kinase (L-pk) and Chrebpß, indicating reduced ChREBPα activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the L-pk promoter was increased in fructose fed LXRα-/- mice, concomitant with increased glucose-6-phosphatase (G6pc) expression and O-GlcNAc modified LXRß, suggesting a role for LXRß in regulating ChREBPα activity upon fructose feeding. In conclusion, we propose that LXRα is an important regulator of hepatic lipogenesis and ChREBPα activity upon glucose, but not fructose feeding in mice.

Cysteine deprivation prevents induction of peroxisome proliferator-activated receptor gamma-2 and adipose differentiation of 3T3-L1 cells.

Plasma cysteine is strongly associated with body fat mass in human cohorts and diets low in cysteine prevents fat accumulation in mice. It is unclear if plasma cysteine affects fat development or if fat accumulation raises plasma cysteine. To determine if cysteine affects adipogenesis, we differentiated 3T3-L1 preadipocytes in medium with reduced cysteine. Cells incubated in media with 10-20µM cysteine exhibited reduced capacity to differentiate into triacylglycerol-storing mature adipocytes compared with cells incubated with 50µM cysteine. Low cysteine severely reduced expression of peroxisome proliferator-activated receptor gamma2 (Pparγ2) and its target genes perlipin1 (Plin1) and fatty acid binding protein-4 (Fabp4). Expression of stearoyl-CoA desaturase-1 (Scd1), known to be repressed with cysteine depletion, was also reduced with low cysteine. Medium depletion of the essential amino acids leucine, valine, and isoleucine had only a modest effect on adipocyte specific gene expression and differentiation. Stimulation with the PPARγ agonist BRL-49653 or addition of a hydrogen sulfide donor enhanced differentiation of 3T3-L1 cells cultured in low cysteine. This demonstrates that the ability to induce PPARγ expression is preserved when cells are cultured in low cysteine. It therefore appears that cysteine depletion inhibits adipogenesis by specifically affecting molecular pathways required for induction of PPARγ expression, rather than through a general reduction of global protein synthesis. In conclusion, we show that low extracellular cysteine reduces adipocyte differentiation by interfering with PPARγ2 and PPARγ target gene expression. Our results provide further evidence for the hypothesis that plasma cysteine is a casual determinant for body fat mass.

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity.

Liver X receptor (LXR)α and LXRß play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked ß-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/ß(+/+) and LXRα/ß(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPß. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

Fatty acids regulate perilipin5 in muscle by activating PPARδ.

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.

Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor α in mouse liver reveals extensive sharing of binding sites.

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

Liver X receptor protects against liver injury in sepsis caused by rodent cecal ligation and puncture.

BACKGROUND: Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP). METHODS: Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. RESULTS: Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαß-deficient mice compared with wild-type and LXRß-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model. CONCLUSIONS: Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.

Suggestive evidence of associations between liver X receptor ß polymorphisms with type 2 diabetes mellitus and obesity in three cohort studies: HUNT2 (Norway), MONICA (France) and HELENA (Europe).

BACKGROUND: The liver X receptors (LXR) α and ß regulate lipid and carbohydrate homeostasis and inflammation. Lxrß⁻/⁻ mice are glucose intolerant and at the same time lean. We aimed to assess the associations between single nucleotide polymorphisms (SNPs) in LXRß and risk of type 2 diabetes mellitus (T2DM), obesity and related traits in 3 separate cohort studies. METHODS: Twenty LXRß SNPs were identified by sequencing and genotyped in the HUNT2 adult nested case-control study for T2DM (n = 835 cases/1986 controls). Five tag-SNPs (rs17373080, rs2695121, rs56151148, rs2303044 and rs3219281), covering 99.3% of the entire common genetic variability of the LXRß gene were identified and genotyped in the French MONICA adult study (n = 2318) and the European adolescent HELENA cross-sectional study (n = 1144). In silico and in vitro functionality studies were performed. RESULTS: We identified suggestive or significant associations between rs17373080 and the risk of (i) T2DM in HUNT2 (OR = 0.82, p = 0.03), (ii) obesity in MONICA (OR = 1.26, p = 0.05) and (iii) overweight/obesity in HELENA (OR = 1.59, p = 0.002). An intron 4 SNP (rs28514894, a perfect proxy for rs17373080) could potentially create binding sites for hepatic nuclear factor 4 alpha (HNF4α) and nuclear factor 1 (NF1). The C allele of rs28514894 was associated with ~1.25-fold higher human LXRß basal promoter activity in vitro. However, no differences between alleles in terms of DNA binding and reporter gene transactivation by HNF4α or NF1 were observed. CONCLUSIONS: Our results suggest that rs17373080 in LXRß is associated with T2DM and obesity, maybe via altered LXRß expression.

Intracellular nicotinamide phosphoribosyltransferase protects against hepatocyte apoptosis and is down-regulated in nonalcoholic fatty liver disease.

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.

LXRbeta deficient mice have reduced hepatic insulin clearance during hyperinsulinemic euglucemic clamp.

The present study addresses the insulin sensitivity in mice deficient in LXRbeta (LXRbeta(-/-)) as well as in wild type (wt) mice assessed by hyperinsulinemic euglycemic clamp. Wt and LXRbeta(-/-) mice were fed either a normal chow diet or a high fat and high cholesterol diet (HFCD), and insulin sensitivity was assessed by hyperinsulinemic euglycemic clamps. We show that LXRbeta(-/-) mice have reduced insulin clearance during hyperinsulinemic clamps upon feeding both HFCD and a regular chow diet. Moreover we also observed reduced hepatic inflammation in LXRbeta(-/-) mice compared to wt mice upon feeding an HFCD, despite equal levels of hepatic steatosis. In summary, our results indicate that LXRbeta(-/-) mice have reduced insulin clearance during hyperinsulinemic euglycemic clamps and also reduced hepatic inflammation upon feeding an HFCD for 26weeks.

Nuclear receptor liver X receptor is O-GlcNAc-modified in response to glucose.

Post-translational modification of nucleocytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has for the last 25 years emerged as an essential glucose-sensing mechanism. The liver X receptors (LXRs) function as nutritional sensors for cholesterol-regulating lipid metabolism, glucose homeostasis, and inflammation. LXRs are shown to be post-translationally modified by phosphorylation, acetylation, and sumoylation, affecting their target gene specificity, stability, and transactivating and transrepressional activity, respectively. In the present study, we show for the first time that LXRalpha and LXRbeta are targets for glucose-hexosamine-derived O-GlcNAc modification in human Huh7 cells. Furthermore, we observed increased hepatic LXRalpha O-GlcNAcylation in vivo in refed mice and in streptozotocin-induced refed diabetic mice. Importantly, induction of LXRalpha O-GlcNAcylation in both mouse models was concomitant with increased expression of the lipogenic gene SREBP-1c (sterol regulatory element-binding protein 1c). Furthermore, glucose increased LXR/retinoic acid receptor-dependent activation of luciferase reporter activity driven by the mouse SREBP-1c promoter via the hexosamine biosynthetic pathway in Huh7 cells. Altogether, our results suggest that O-GlcNAcylation of LXR is a novel mechanism by which LXR acts as a glucose sensor affecting LXR-dependent gene expression, substantiating the crucial role of LXR as a nutritional sensor in lipid and glucose metabolism.

Regulation of hepatic fatty acid elongase 5 by LXRalpha-SREBP-1c.

Dietary essential fatty acids linoleic acid and alpha-linolenic acid are converted to arachidonic-, eicosapentaenoic-, and docosahexaenoic acid under tight regulation by nutritional status and hormones. Hepatic fatty acid elongase 5 (Elovl5) elongates C18-20 polyunsaturated fatty acids (PUFAs) and is important for biosynthesis of C20-22 PUFAs. We demonstrate that Liver X Receptor alpha (LXRalpha) and sterol regulatory binding protein-1c (SREBP-1c) regulate hepatic Elovl5 expression. LXRalpha and LXRbeta play different roles in maintenance of basal expression of Elovl5. LXRalpha is necessary for basal as well as LXR agonist induced Elovl5 transcription. Promoter studies revealed that the mouse Elovl5 gene is a direct SREBP-1c target. The up-regulation of Elovl5 expression by LXR agonist is likely secondary to the induction of SREBP-1c. PUFAs repress expression of SREBP-1c and Elovl5, but when combined with LXR ligand stimulation, which increases SREBP-1c mRNA and nuclear SREBP-1c, Elovl5 mRNA levels are restored to normal. Our studies suggest that an LXRalpha-SREBP-1c pathway plays a regulatory role in hepatic biosynthesis of PUFAs through transcriptional activation of Elovl5 as well as other desaturases. The stimulatory role of LXRalpha-SREBP-1c in the production of PUFAs enables the possibility for a feedback regulation of hepatic lipogenesis through PUFA mediated repression of SREBP-1c expression.

LSDP5 is a PAT protein specifically expressed in fatty acid oxidizing tissues.

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.

22-Hydroxycholesterols regulate lipid metabolism differently than T0901317 in human myotubes.

The nuclear liver X receptors (LXRalpha and beta) are regulators of lipid and cholesterol metabolism. Oxysterols are known LXR ligands, but the functional role of hydroxycholesterols is at present unknown. In human myotubes, chronic exposure to the LXR ligand T0901317 promoted formation of diacylglycerol (DAG) and triacylglycerol (TAG), 22-R-hydroxycholesterol (22-R-HC) had no effect, and 22-S-hydroxycholesterol (22-S-HC) reduced the formation. In accordance with this, 22-HC and T0901317 regulated the expression of fatty acid transporter CD36, stearoyl-CoA desaturase-1, acyl-CoA synthetase long chain family member 1 and fatty acid synthase (FAS) differently; all genes were increased by T0901317, 22-R-HC did not change their expression level, while 22-S-HC reduced it. Transfection studies confirmed that the FAS promoter was activated by T0901317 and repressed by 22-S-HC through an LXR response element in the promoter. Both 22-R-HC and T0901317 increased gene expression of LXRalpha, sterol regulatory element-binding protein 1c and ATP-binding cassette transporter A1, while 22-S-HC had little effect. In summary, 22-R-HC regulated lipid metabolism and mRNA expression of some LXR target genes in human myotubes differently than T0901317. Moreover, 22-S-HC did not behave like an inactive ligand; it reduced synthesis of complex lipids and repressed certain genes involved in lipogenesis and lipid handling.

PPARalpha activators and fasting induce the expression of adipose differentiation-related protein in liver.

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.

Effects of structural changes of fatty acids on lipid accumulation in adipocytes and primary hepatocytes.

Conjugated linoleic acids (CLAs), tetradecylthioacetic acid (TTA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are all shown to differently affect lipid homeostasis. Additionally, previous studies have shown that introducing a methyl group in the molecule potentiates the hypolipidemic effect of EPA. The objective of this study was to determine how cis9,trans11 CLA, trans10,cis12 CLA, TTA, EPA and DHA affect lipid accumulation in 3T3-L1 adipocytes and in cultured primary rat hepatocytes, and to what extent changes in cis/trans configuration or introducing a methyl group in the molecules influence their way of affecting lipid accumulation in these cells. Our results show that trans10,cis12 CLA is highly specific in preventing lipid accumulation in adipocytes, and that small structural changes in the molecule (changing to trans/trans or introducing an alpha-methyl group) totally abolish this effect and up-regulate the expression levels of adipogenic marker genes towards control levels. Furthermore, all the fatty acids increased hepatic lipid accumulation, whereas the lipid content was normalized after adding an alpha-methyl group into the molecules. Taken together, our data demonstrate that the various fatty acids are highly specialized molecules, and that small structural changes markedly alter their way of affecting lipid accumulation in adipocytes and hepatocytes.

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.

Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid.

The resistin gene is expressed in adipocytes and encodes a protein proposed to link obesity and type 2 diabetes. Increased plasma FFA is associated with insulin resistance. We examined the effect of separate FFAs on the expression of resistin mRNA in cultured murine 3T3-L1 adipocytes. The FFAs tested did not increase resistin expression, whereas both arachidonic acid (AA) and eicosapentaenoic acid (EPA) reduced resistin mRNA levels. AA was by far the most potent FFA, reducing resistin mRNA levels to approximately 20% of control at 60-250 muM concentration. Selective inhibitors of cyclooxygenase-1 and of mitogen-activated protein kinase kinase counteracted AA-induced reduction in resistin mRNA levels. Transient overexpression of sterol-regulatory element binding protein-1a (SREBP-1a) activated the resistin promoter, but there was no reduction in the abundance of approximately 65 kDa mature SREBP-1 after AA exposure. Actinomycin D as well as cycloheximide abolished the AA-induced reduction of resistin mRNA levels, indicating dependence on de novo transcription and translation. Our data suggest that reductions in resistin mRNA levels involve a destabilization of the resistin mRNA molecule. An inhibitory effect of AA and EPA on resistin expression may explain the beneficial effect of ingesting PUFAs on insulin sensitivity.