Natural human genetic variation determines basal and inducible expression of PM20D1, an obesity-associated gene

PM20D1 is a candidate thermogenic enzyme in mouse fat, with its expression cold-induced and enriched in brown versus white adipocytes. Thiazolidinedione (TZD) antidiabetic drugs, which activate the peroxisome proliferator-activated receptor-γ (PPARγ) nuclear receptor, are potent stimuli for adipocyte browning yet fail to induce Pm20d1 expression in mouse adipocytes. In contrast, PM20D1 is one of the most strongly TZD-induced transcripts in human adipocytes, although not in cells from all individuals. Two putative PPARγ binding sites exist near the gene’s transcription start site (TSS) in human but not mouse adipocytes. The −4 kb upstream site falls in a segmental duplication of a nearly identical intronic region +2.5 kb downstream of the TSS, and this duplication occurred in the primate lineage and not in other mammals, like mice. PPARγ binding and gene activation occur via this upstream duplicated site, thus explaining the species difference. Furthermore, this functional upstream PPARγ site exhibits genetic variation among people, with 1 SNP allele disrupting a PPAR response element and giving less activation by PPARγ and TZDs. In addition to this upstream variant that determines PPARγ regulation of PM20D1 in adipocytes, distinct variants downstream of the TSS have strong effects on PM20D1 expression in human fat as well as other tissues. A haplotype of 7 tightly linked downstream SNP alleles is associated with very low PMD201 expression and correspondingly high DNA methylation at the TSS. These PM20D1 low-expression variants may account for human genetic associations in this region with obesity as well as neurodegenerative diseases.

Synthesis and Investigation on the Antidiabetic Effect of 3-aryl-1-(5-methylisoxazol-3-ylamino)-1-(4-nitrophenyl) Propan-1-one

Background: Diabetes mellitus is the third-largest non-communicable chronic disease worldwide. There are many effective drugs, but the long-term use of these clinical drugs may cause various side effects. Therefore, it is urgent to develop new antidiabetic molecules with higher efficacy and lower toxicity. Methods: Fifteen new 3-aryl-1-(5-methylisoxazol-3-ylamino)-1-(4-nitrophenyl)propan-1-one were synthesized directly through the Mannich reaction of 4-nitroacetophenone, 3-amino-5- methylisoxazole and aromatic aldehydes catalyzed by concentrated hydrochloric acid. The molecular structures of the products were fully characterized by 1H NMR, 13C NMR, ESI MS and HRMS. The peroxisome proliferator-activated receptor (PPAR) response element and α-glucosidase inhibitory activity of these compounds were evaluated in vitro. Molecular docking, molecular physical parameters calculation, and molecular toxicity prediction were performed to analyze the structure- activity relationship and evaluate the druggability of these compounds theoretically. Results: All compounds exhibited weak antidiabetic activities, but compound 15 showed promising as a high performance, dual-target antidiabetic lead compound with peroxisome proliferatoractivated receptor (PPAR) response element relative agonist activity of 99.55% at 27.2 nmol·mL−1 and α-glucosidase inhibitory activity of 35.21% at 13.6 nmol·mL−1. All compounds obtained may have no cardiotoxicity, no acute toxicity, no carcinogenic, and within safe range of mutagenic risk. Conclusion: This study identified a potential PPAR lead molecule and presented an unusual strategy for antidiabetic drug development.

HIV-1 Vpr Enhances PPARβ/δ-Mediated Transcription, Increases PDK4 Expression, and Reduces PDC Activity

HIV infection and its therapy are associated with disorders of lipid metabolism and bioenergetics. Previous work has suggested that viral protein R (Vpr) may contribute to the development of lipodystrophy and insulin resistance observed in HIV-1–infected patients. In adipocytes, Vpr suppresses mRNA expression of peroxisomal proliferator-activating receptor-γ (PPARγ)-responsive genes and inhibits differentiation. We investigated whether Vpr might interact with PPARβ/δ and influence its transcriptional activity. In the presence of PPARβ/δ, Vpr induced a 3.3-fold increase in PPAR response element-driven transcriptional activity, a 1.9-fold increase in pyruvate dehydrogenase kinase 4 (PDK4) protein expression, and a 1.6-fold increase in the phosphorylated pyruvate dehydrogenase subunit E1α leading to a 47% decrease in the activity of the pyruvate dehydrogenase complex in HepG2 cells. PPARβ/δ knockdown attenuated Vpr-induced enhancement of endogenous PPARβ/δ-responsive PDK4 mRNA expression. Vpr induced a 1.3-fold increase in mRNA expression of both carnitine palmitoyltransferase I (CPT1) and acetyl-coenzyme A acyltransferase 2 (ACAA2) and doubled the activity of β-hydroxylacyl coenzyme A dehydrogenase (HADH). Vpr physically interacted with the ligand-binding domain of PPARβ/δ in vitro and in vivo. Consistent with a role in energy expenditure, Vpr increased state-3 respiration in isolated mitochondria (1.16-fold) and basal oxygen consumption rate in intact HepG2 cells (1.2-fold) in an etomoxir-sensitive manner, indicating that the oxygen consumption rate increase is β-oxidation–dependent. The effects of Vpr on PPAR response element activation, pyruvate dehydrogenase complex activity, and β-oxidation were reversed by specific PPARβ/δ antagonists. These results support the hypothesis that Vpr contributes to impaired energy metabolism and increased energy expenditure in HIV patients.

Conservation of the Nuclear Receptor Response Element in HIV-1 LTRs: A Possible PPAR Response Element?

Infection with HIV-1 continues to be a threat to public health. Successful antiretroviral therapy has reduced the risk of developing AIDS but cannot fully eradicate the virus due to latent proviral sequences remaining in infected cells. The 5′-long terminal repeat (LTR) of HIV-1 is critical for the regulation of transcription of the viral RNA and subsequent production of new viral particles. Indeed, the regulation of transcription relies upon the binding of host cell transcription factors and associated regulatory proteins to the LTR. Recently, it has been found that the treatment of cells with ligands of a number of nuclear receptors (NRs) resulted in inhibition of HIV-1 replication. This inhibition likely occurs via effects on other proteins that bind to the 5′-LTR, notably NF-κB. Here, the possible binding site of one NR, the peroxisome proliferator-activated receptor (PPAR), in the HIV-1 5′-LTR is analysed within isolates of the virus. Given the high mutation rate of HIV-1, it is striking that this region remains conserved in more recent isolates from geographically distinct regions. This work provides a rationale for further study of the binding site recognised by PPAR in the HIV-1 5′-LTR.

PPAR-γ Activation Restores Pancreatic Islet SERCA2 Levels and Prevents β-Cell Dysfunction under Conditions of Hyperglycemic and Cytokine Stress

The maintenance of intracellular Ca2+ homeostasis in the pancreatic β-cell is closely regulated by activity of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump. Our data demonstrate a loss of β-cell SERCA2b expression in several models of type 2 diabetes including islets from db/db mice and cadaveric diabetic human islets. Treatment of 832/13 rat INS-1-derived cells with 25 mm glucose and the proinflammatory cytokine IL-1β led to a similar loss of SERCA2b expression, which was prevented by treatment with the peroxisome proliferator-activated receptor (PPAR)-γ agonist, pioglitazone. Pioglitazone was able to also protect against hyperglycemia and cytokine-induced elevations in cytosolic Ca2+ levels, insulin-secretory defects, and cell death. To determine whether PPAR-γ was a direct transcriptional regulator of the SERCA2 gene, luciferase assays were performed and showed that a −259 bp region is sufficient to confer PPAR-γ transactivation; EMSA and chromatin immunoprecipitation experiments confirmed that PPAR-γ directly binds a PPAR response element in this proximal region. We next sought to characterize the mechanisms by which SERCA2b was down-regulated. INS-1 cells were exposed to high glucose and IL-1β in time course experiments. Within 2 h of exposure, activation of cyclin-dependent kinase 5 (CDK5) was observed and correlated with increased serine-273 phosphorylation of PPAR-γ and loss of SERCA2 protein expression, findings that were prevented by pioglitazone and roscovitine, a pharmacological inhibitor of CDK5. We conclude that pioglitazone modulates SERCA2b expression through direct transcriptional regulation of the gene and indirectly through prevention of CDK5-induced phosphorylation of PPAR-γ.

The Peroxisomal3-keto-acyl-CoA thiolase BGene Expression Is under the Dual Control of PPARα and HNF4α in the Liver

PPARα and HNF4α are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARα or HNF4α, we show that the expression of the peroxisomal3-keto-acyl-CoA thiolase B(Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of theThbgene, by which PPARα but not HNF4α transactivates. Intriguingly, we found that HNF4α enhanced PPARα/RXRα transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4α was physically interacting with RXRα. RT-PCR performed with RNA from liver-specific HNF4α-null mice confirmed the involvement of HNF4α in the PPARα-regulated induction ofThbby Wy14,643. Overall, we conclude that HNF4α enhances the PPARα-mediated activation ofThbgene expression in part through interaction with the obligate PPARα partner, RXRα.