Julieta Díaz-Delfín

Peroxisome Proliferator-activated Receptor α (PPARα) Induces PPARγ Coactivator 1α (PGC-1α) Gene Expression and Contributes to Thermogenic Activation of Brown Fat INVOLVEMENT OF PRDM16

Background: PPARα is a distinctive marker of the brown-versus-white fat phenotype. Results: PPARα induces PGC-1α gene transcription in brown adipocytes through mechanisms involving PRDM16. Conclusion: PPARα regulates brown fat thermogenesis via induction of PGC-1α and PRDM16 gene expression. Significance: Activation of PGC-1α by PPARα provides a molecular mechanism for concerted induction of thermogenic genes (UCP1, mitochondrial genes, and lipid oxidation genes) in brown fat. Peroxisome proliferator activated receptor α (PPARα) is a distinctive marker of the brown fat phenotype that has been proposed to coordinate the transcriptional activation of genes for lipid oxidation and for thermogenic uncoupling protein 1 in brown adipose tissue. Here, we investigated the involvement of PPARα in the transcriptional control of the PPARγ coactivator (PGC)-1α gene. Treatment with PPARα agonists induced PGC-1α mRNA expression in brown fat in vivo and in primary brown adipocytes. This enhancement of PGC-1α transcription was mediated by PPARα binding to a PPAR-responsive element in the distal PGC-1α gene promoter. PGC-1α gene expression was decreased in PPARα-null brown fat, both under basal conditions and in response to thermogenic activation. Moreover, PPARα- and cAMP-mediated pathways interacted to control PGC-1α transcription. PRDM16 (PRD1-BF1-RIZ1 homologous domain-containing 16) promoted PPARα induction of PGC-1α gene transcription, especially under conditions in which protein kinase A pathways were activated. This enhancement was associated with the interaction of PRDM16 with the PGC-1α promoter at the PPARα-binding site. In addition, PPARα promoted the expression of the PRDM16 gene in brown adipocytes, and activation of PPARα in human white adipocytes led to the appearance of a brown adipocyte pattern of gene expression, including induction of PGC-1α and PRDM16. Collectively, these results suggest that PPARα acts as a key component of brown fat thermogenesis by coordinately regulating lipid catabolism and thermogenic gene expression via induction of PGC-1α and PRDM16.

Peroxisome Proliferator-activated Receptor-γ Coactivator-1α Controls Transcription of the Sirt3 Gene, an Essential Component of the Thermogenic Brown Adipocyte Phenotype

Sirt3 (silent mating type information regulation 2, homolog 3), a member of the sirtuin family of protein deacetylases with multiple actions on metabolism and gene expression is expressed in association with brown adipocyte differentiation. Using Sirt3-null brown adipocytes, we determined that Sirt3 is required for an appropriate responsiveness of cells to noradrenergic, cAMP-mediated activation of the expression of brown adipose tissue thermogenic genes. The transcriptional coactivator Pgc-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) induced Sirt3 gene expression in white adipocytes and embryonic fibroblasts as part of its overall induction of a brown adipose tissue-specific pattern of gene expression. In cells lacking Sirt3, Pgc-1α failed to fully induce the expression of brown fat-specific thermogenic genes. Pgc-1α activates Sirt3 gene transcription through coactivation of the orphan nuclear receptor Err (estrogen-related receptor)-α, which bound the proximal Sirt3 gene promoter region. Errα knockdown assays indicated that Errα is required for full induction of Sirt3 gene expression in response to Pgc-1α. The present results indicate that Pgc-1α controls Sirt3 gene expression and this action is an essential component of the overall mechanisms by which Pgc-1α induces the full acquisition of a brown adipocyte differentiated phenotype.

TNF-α Represses β-Klotho Expression and Impairs FGF21 Action in Adipose Cells: Involvement of JNK1 in the FGF21 Pathway

Fibroblast growth factor 21 (FGF21) is a member of the FGF family that reduces glycemia and ameliorates insulin resistance. Adipose tissue is a main target of FGF21 action. Obesity is associated with a chronic proinflammatory state. Here, we analyzed the role of proinflammatory signals in the FGF21 pathway in adipocytes, evaluating the effects of TNF-α on β-Klotho and FGF receptor-1 expression and FGF21 action in adipocytes. We also determined the effects of rosiglitazone on β-Klotho and FGF receptor-1 expression in models of proinflammatory signal induction in vitro and in vivo (high-fat diet-induced obesity). Because c-Jun NH(2)-terminal kinase 1 (JNK1) serves as a sensing juncture for inflammatory status, we also evaluated the involvement of JNK1 in the FGF21 pathway. TNF-α repressed β-Klotho expression and impaired FGF21 action in adipocytes. Rosiglitazone prevented the reduction in β-Klotho expression elicited by TNF-α. Moreover, β-Klotho levels were reduced in adipose tissue from high-fat diet-induced obese mice, whereas rosiglitazone restored β-Klotho to near-normal levels. β-Klotho expression was increased in white fat from JNK1(-/-) mice. The absence of JNK1 increased the responsiveness of mouse embryonic fibroblast-derived adipocytes and brown adipocytes to FGF21. In conclusion, we show that proinflammatory signaling impairs β-Klotho expression and FGF21 responsiveness in adipocytes. We also show that JNK1 activity is involved in modulating FGF21 effects in adipocytes. The impairment in the FGF21 response machinery in adipocytes and the reduction in FGF21 action in response to proinflammatory signals may play important roles in metabolic alterations in obesity and other diseases associated with enhanced inflammation.

Human dendritic cell activities are modulated by the omega-3 fatty acid, docosahexaenoic acid, mainly through PPARγ:RXR heterodimers: comparison with other polyunsaturated fatty acids

There is accumulating evidence that omega‐3 fatty acids may modulate immune responses. When monocytes were differentiated to dendritic cells (DCs) in the presence of docosahexaenoic acid (DHA), the expression of costimulatory and antigen presentation markers was altered in a concentration‐dependent way, positively or negatively, depending on the markers tested and the maturation stage of the DCs. Changes induced by eicosapentaenoic acid and linoleic acid were similar but less intense than those of DHA, whereas oleic acid had almost no effect. DHA‐treated, mature DCs showed inhibition of IL‐6 expression and IL‐10 and IL‐12 secretion, and their lymphoproliferative stimulation capacity was impaired. The phenotypic alterations of DCs induced by DHA were similar to those already reported for Rosiglitazone (Rosi), a peroxisome proliferator‐activated receptor γ (PPARγ) activator, and the retinoid 9‐cis‐retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPARγ target genes pyruvate dehydrogenase kinase‐4 and aP‐2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPARγ inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPARγ:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.

Effects of nevirapine and efavirenz on human adipocyte differentiation, gene expression, and release of adipokines and cytokines

The non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and efavirenz are drugs of choice for initial antiretroviral treatment for HIV-1 infection. Although NNRTIs have not traditionally been associated with the appearance of adipose alterations, recent data suggest that efavirenz may contribute to adipose tissue alterations in antiretroviral-treated patients, consistent with its ability to impair differentiation of adipocytes in cell cultures. No such effects have been reported for nevirapine, the other most commonly used NNRTI. In this study, we determined the effects of nevirapine on differentiation, gene expression and release of regulatory proteins (adipokines and cytokines) in differentiating human adipocytes, and compared them with those of efavirenz. Efavirenz caused a dose-dependent repression of adipocyte differentiation that was associated with down-regulation of the master adipogenesis regulator genes SREBP-1, PPARγ and C/EBPα, and their target genes encoding lipoprotein lipase, leptin and adiponectin, which are key proteins in adipocyte function. In contrast, nevirapine does not affect adipogenesis and causes a modest but significant coordinate increase in the expression of SREBP-1, PPARγ and C/EBPα and their target genes only at a concentration of 20 μM. Whereas efavirenz caused a significant increase in the release of pro-inflammatory cytokines (interleukin [IL]-8, IL-6, monocyte chemoattractant protein-1), plasminogen activator inhibitor type-1 and hepatocyte growth factor (HGF), nevirapine either had no effect on these factors or decreased their release (IL-6 and HGF). Nevirapine significantly increased adiponectin release, whereas efavirenz strongly repressed it. Moreover, nevirapine inhibited preadipocyte endogenous reverse transcriptase activity, whereas efavirenz did not alter it. It is concluded that, in contrast with the profound anti-adipogenic and pro-inflammatory response elicited by efavirenz, nevirapine does not impair adipogenesis.

Differential Effects of Efavirenz and Lopinavir/Ritonavir on Human Adipocyte Differentiation, Gene Expression and Release of Adipokines and Pro-Inflammatory Cytokines.

In the present study, a comparative assessment of the effects of efavirenz (EFV) and lopinavir/ritonavir (LPV/r; 4:1) on human adipocytes in culture has been performed. Human pre-adipocytes were treated with EFV or LPV/r during or after adipogenic differentiation. Acquisition of adipocyte morphology, expression of gene markers of mitochondrial toxicity, adipogenesis and inflammation, and release of adipokines and cytokines to the medium were measured. Results indicated that EFV and LPV/r impaired adipocyte differentiation in association with a reduction in transcript levels for adipogenic differentiation genes (adiponectin, lipoprotein lipase, leptin) and master regulators of adipogenesis (PPAR, C/EBP). The effects were greater with EFV than LPV/r. Both LPV/r and EFV induced increases in monocytechemoattactant protein-1 (MCP-1) mRNA levels, but the effect was greater with EFV. Similarly, the release of proinflammatory cytokines and other inflammation-related molecules (interleukins 6 and 8, MCP-1, PAI-1) was enhanced to a much higher degree by EFV than by LPV/r. Adiponectin and leptin release by adipocytes was reduced by both drugs, although to a higher extent by EFV. Neither drug affected mitochondrial DNA levels, transcripts encoding mitochondrial proteins or lactate release by adipocytes. In previously differentiated adipocytes, EFV caused a significant reduction in PPARγ and adiponectin expression, whereas LPV/r did not. We conclude that both EFV and LPV/r impair human adipogenesis, reduce adipokine release and increase the expression and release of inflammation-related cytokines, but the overall effects are greater with EFV. These findings may have implications for the pathogenesis of HIV-1-associated lipodystrophy and the development of HIV-1 therapies.

Effects of Rilpivirine on Human Adipocyte Differentiation, Gene Expression, and Release of Adipokines and Cytokines

ABSTRACT Rilpivirine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) recently developed as a drug of choice for initial antiretroviral treatment of HIV-1 infection. Disturbances in lipid metabolism and, ultimately, in adipose tissue distribution and function are common concerns as secondary effects of antiretroviral treatment. Efavirenz, the most commonly used NNRTI, causes mild dyslipidemic effects in patients and strongly impaired adipocyte differentiation in vitro. In this study, we provide the first demonstration of the effects of rilpivirine on human adipocyte differentiation, gene expression, and release of regulatory proteins (adipokines and cytokines) and compare them with those caused by efavirenz. Rilpivirine caused a repression of adipocyte differentiation that was associated with impaired expression of the master adipogenesis regulators peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT enhancer binding protein alpha (C/EBPα), and sterol regulatory element binding transcription factor 1 (SREBP-1) and their target genes encoding lipoprotein lipase and the adipokines leptin and adiponectin. Rilpivirine also repressed adiponectin release by adipocytes, but only at high concentrations, and did not alter leptin release. Rilpivirine induced the release of proinflammatory cytokines (interleukin-6 and -8, monocyte chemoattractant protein 1 [MCP-1], plasminogen activator inhibitor type 1 [PAI-1]) only at very high concentrations (10 μM). A comparison of the effects of rilpivirine and efavirenz at the same concentration (4 μM) or even at lower concentrations of efavirenz (2 μM) showed that rilpivirine-induced impairment of adipogenesis and induction of proinflammatory cytokine expression and release were systematically milder than those of efavirenz. It is concluded that rilpivirine causes an antiadipogenic and proinflammatory response pattern, but only at high concentrations, whereas efavirenz causes similar effects at lower concentrations.

Lipotoxicity on the basis of metabolic syndrome and lipodystrophy in HIV-1-infected patients under antiretroviral treatment.

The development of efficacious antiretroviral drugs that minimize adverse effects is a current challenge in HIV-1 therapy. Metabolic alterations reminiscent of the metabolic syndrome and overt lipodystrophy appear often in HIV-1-infected patients undergoing antiretroviral treatment. The etiopathogenesis of these alterations is complex, but lipotoxicity has recently emerged as a key concept for explaining the metabolic syndrome in HIV-1-infected patients, similarly to what has been observed in diseases such as obesity and genetic lipodystrophies. Antiretroviral drugs from distinct drug families may directly elicit such lipotoxic phenomena, via increased lipolysis, enhanced adipocyte apoptosis and impaired adipogenesis, which collectively lead to a reduced capacity of subcutaneous adipose tissue to enlarge to meet fat storage requirements. Thus, fatty acids that cannot be properly stored as triglycerides in subcutaneous adipose tissue are expected to accumulate in visceral fat as well as in organs and tissues, such as the pancreas, muscle and liver, leading to the pattern of metabolic alterations associated with abnormal ectopic fat accumulation, mainly insulin resistance. Inflammatory responses, evoked by the combined effects of antiretroviral drugs and the underlying HIV-1 infection, also contribute to lipotoxicity, reflecting the action of pro-inflammatory cytokines that enhance lipolytic activity in adipose tissue and impair adipogenesis. Minimizing the lipotoxic action of antiretroviral drugs is ultimately essential in reducing metabolic alterations in treated patients. Moreover, pharmacological strategies that reduce lipotoxicity and promote adipose tissue expandability can be expected to ameliorate the overall metabolic abnormalities in HIV-1-infected, antiretroviral-treated patients.

Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation

Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patients with CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequently appear in HIV-infected patients under HAART, there is no information available on the effects of MVC on adipose tissue. Here we studied the effects of MVC during and after the differentiation of human adipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured the acquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity, adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally, we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed no alterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistent with this, there were no changes in the mRNA levels of PPARγ or SREBP-1c, two master regulators of adipogenesis. In addition, MVC caused a significant decrease in the gene expression and release of pro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflammation-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes in differentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rather shows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatory cytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissue development, metabolism, and inflammation, and thus could be a potentially beneficial component of antiretroviral therapy.

HIV-1 Tat protein impairs adipogenesis and induces the expression and secretion of proinflammatory cytokines in human SGBS adipocytes.

BACKGROUND HIV-1 Tat protein has been shown to play multiple roles in the pathogenesis of AIDS; however, there is no information currently available on its effects on adipose tissue alterations. We have studied the effects of Tat on SGBS adipocytes to gain insight on its role on the development of lipodystrophy. METHODS SGBS preadipocytes were exposed to Tat during and after differentiation. Acquisition of adipocyte morphology, expression of gene markers of adipogenesis and inflammation, release of adipokines and cytokines to the medium, and glucose uptake were measured. The action of Tat on tumour necrosis factor (TNF)-α-regulated messenger RNA expression was determined in differentiated adipocytes. The capacity of rosiglitazone, resveratrol and parthenolide to influence the action of Tat was also assessed. RESULTS Tat treatment reduced the number of SGBS preadipocytes that acquired adipocyte morphology. It also led to repression of adipogenic gene expression and induced the coordinate expression and release of proinflammatory cytokines in human adipose cells. Moreover, combined treatment with Tat and TNF-α produced an additive effect on the repression of adipocyte genes. The observed effects of Tat on gene transcription in adipocytes were due, in part, to TNF-α that was secreted as a consequence of intracellular exposure to Tat. CONCLUSIONS Tat impairs adipogenesis in human SGBS preadipocytes and increases the expression and release of proinflammatory cytokines. Positive crosstalk between Tat and TNF-α contributes to the anti-adipogenic and proinflammatory effects. HIV-1 Tat protein may play a role in the adipose tissue alterations that ultimately lead to lipoatrophy and systemic metabolic disturbances observed in HIV-1-infected patients.