PPARγ Transcriptional Activity Research Articles

SMYD2-Methylated PPARγ Facilitates Hypoxia-Induced Pulmonary Hypertension by Activating Mitophagy.

Hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) and consequent pulmonary vascular remodeling are the crucial pathological features of pulmonary hypertension (PH). Protein methylation has been shown to be critically involved in PASMC proliferation and PH, but the underlying mechanism remains largely unknown. PH animal models were generated by treating mice/rats with chronic hypoxia for 4 weeks. SMYD2-vTg mice (vascular smooth muscle cell-specific suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2 transgenic) or wild-type rats and mice treated with LLY-507 were used to investigate the function of SMYD2 (suppressor of variegation, enhancer of zeste, trithorax and myeloid Nervy DEAF-1 domain-containing protein 2) on PH development in vivo. Primary cultured rat PASMCs with SMYD2 knockdown or overexpression were used to explore the effects of SMYD2 on proliferation and to decipher the underlying mechanism. We demonstrated that the expression of the lysine methyltransferase SMYD2 was upregulated in the smooth muscle cells of pulmonary arteries from patients with PH and hypoxia-exposed rats/mice and in the cytoplasm of hypoxia-induced rat PASMCs. More importantly, targeted inhibition of SMYD2 by LLY-507 significantly attenuated hypoxia-induced pulmonary vascular remodeling and PH development in both male and female rats in vivo and reduced rat PASMC hyperproliferation in vitro. In contrast, SMYD2-vTg mice exhibited more severe PH phenotypes and related pathological changes than nontransgenic mice after 4 weeks of chronic hypoxia treatment. Furthermore, SMYD2 overexpression promoted, while SMYD2 knockdown suppressed, the proliferation of rat PASMCs by affecting the cell cycle checkpoint between S and G2 phases. Mechanistically, we revealed that SMYD2 directly interacted with and monomethylated PPARγ (peroxisome proliferator-activated receptor gamma) to inhibit the nuclear translocation and transcriptional activity of PPARγ, which further promoted mitophagy to facilitate PASMC proliferation and PH development. Furthermore, rosiglitazone, a PPARγ agonist, largely abolished the detrimental effects of SMYD2 overexpression on PASMC proliferation and PH. Our results demonstrated that SMYD2 monomethylates nonhistone PPARγ and inhibits its nuclear translocation and activation to accelerate PASMC proliferation and PH by triggering mitophagy, indicating that targeting SMYD2 or activating PPARγ are potential strategies for the prevention of PH.

Sulfonylureas exert antidiabetic action on adipocytes by inhibition of PPARγ serine 273 phosphorylation

ObjectiveSulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic β-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice. MethodsPrimary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated. ResultsSUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes. ConclusionsOur findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.

ACSS2 controls PPARγ activity homeostasis to potentiate adipose-tissue plasticity

The appropriate transcriptional activity of PPARγ is indispensable for controlling inflammation, tumor and obesity. Therefore, the identification of key switch that couples PPARγ activation with degradation to sustain its activity homeostasis is extremely important. Unexpectedly, we here show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) critically controls PPARγ activity homeostasis via SIRT1 to enhance adipose plasticity via promoting white adipose tissues beiging and brown adipose tissues thermogenesis. Mechanistically, ACSS2 binds directly acetylated PPARγ in the presence of ligand and recruits SIRT1 and PRDM16 to activate UCP1 expression. In turn, SIRT1 triggers ACSS2 translocation from deacetylated PPARγ to P300 and thereafter induces PPARγ polyubiquitination and degradation. Interestingly, D-mannose rapidly activates ACSS2-PPARγ-UCP1 axis to resist high fat diet induced obesity in mice. We thus reveal a novel ACSS2 function in coupling PPARγ activation with degradation via SIRT1 and suggest D-mannose as a novel adipose plasticity regulator via ACSS2 to prevent obesity.

Transcriptional modulation of heat shock proteins and adipogenic regulators in bovine adipocytes following heat exposure

This research endeavored to elucidate the transcriptional modulation of heat shock proteins and adipogenic regulators in bovine subcutaneous adipocytes following thermal exposure. Post-differentiation, mature adipocytes were subjected to three treatments of control (CON), moderate (MHS), and extreme (EHS) heat stress. These treatments consist of thermal conditions at temperatures of 38 °C (CON), 39.5 °C (MHS), or 41 °C (EHS) along with of 3 or 12 h. There was no statistically significant variations observed in the gene expressions of HSP27 and HSP70 when comparing CON with MHS across both exposures. Contrastingly, when comparing CON with EHS, an upregulation (P < 0.01) in HSP27 gene expression was evident for both 3 and 12 h of incubation, while HSP70 gene expression exhibited elevation (P < 0.01) at the 3-h mark, with no change observed at 12 h. Protein quantification, however, revealed an elevation (P < 0.01) in HSP27 and HSP70 for both CON vs. MHS and CON vs. EHS at the 12-h exposure. This trend in protein level mirrored (P < 0.05) that of proliferator-activated receptor-gamma (PPARγ). Elevated (P < 0.05) protein levels of fatty acid synthase (FAS) were exclusively discernible in the CON vs. MHS. Increased (P < 0.01) transcriptional activity of PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα), stearoyl-CoA desaturase (SCD), and FAS was evident in the CON vs. EHS comparison. Complementary to these molecular findings, an augmented lipid droplet accumulation was observed (P < 0.01) in EHS-exposed adipocytes progressively from day 6 through day 9. Our current study highlights how different levels and lengths of heat stress can impact the activity of important heat-related proteins and factors that play a role in fat development in beef cattle. These findings can help guide strategies to manage how beef cattle are exposed to heat, which can affect fat storage and ultimately the quality of the meat's marbling.

Nuclear pore protein POM121 regulates subcellular localization and transcriptional activity of PPARγ

Manipulation of the subcellular localization of transcription factors by preventing their shuttling via the nuclear pore complex (NPC) emerges as a novel therapeutic strategy against cancer. One transmembrane component of the NPC is POM121, encoded by a tandem gene locus POM121A/C on chromosome 7. Overexpression of POM121 is associated with metabolic diseases (e.g., diabetes) and unfavorable clinical outcome in patients with colorectal cancer (CRC). Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor with anti-diabetic and anti-tumoral efficacy. It is inhibited by export from the nucleus to the cytosol via the RAS-RAF-MEK1/2-ERK1/2 signaling pathway, a major oncogenic driver of CRC. We therefore hypothesized that POM121 participates in the transport of PPARγ across the NPC to regulate its transcriptional activity on genes involved in metabolic and tumor control. We found that POM121A/C mRNA was enriched and POM121 protein co-expressed with PPARγ in tissues from CRC patients conferring poor prognosis. Its interactome was predicted to include proteins responsible for tumor metabolism and immunity, and in-silico modeling provided insights into potential 3D structures of POM121. A peptide region downstream of the nuclear localization sequence (NLS) of POM121 was identified as a cytoplasmic interactor of PPARγ. POM121 positivity correlated with the cytoplasmic localization of PPARγ in patients with KRAS mutant CRC. In contrast, POM121A/C silencing by CRISPR/Cas9 sgRNA or siRNA enforced nuclear accumulation of PPARγ and activated PPARγ target genes promoting lipid metabolism and cell cycle arrest resulting in reduced proliferation of human CRC cells. Our data suggest the POM121-PPARγ axis as a potential drugable target in CRC.

Lactobacillus amylovorus KU4 induces adipose browning in obese mice by regulating PP4C.

We previously reported that Lactobacillus amylovorus KU4 (LKU4) promotes adipocyte browning in mice fed a high-fat diet (HFD mice) in part by remodeling the PPARγ transcription complex. However, the mechanism through which LKU4 enables PPARγ to drive adipocyte browning remains elusive. Here, we report that LKU4 inhibits the expression of PP4C in inguinal white adipose tissue of HFD mice and in insulin-resistant 3T3-L1 adipocytes, which promotes SIRT1-dependent PPARγ deacetylation by activating AMPK, leading to the browning of adipocytes. Consistently, the silencing of PP4C further enhances this pathway. Furthermore, we observed that lactate, a key LKU4 metabolite, reduces insulin-induced PP4C expression and suppresses PP4C inhibition of PPARγ deacetylation and transcriptional activity via AMPK-SIRT1, thereby facilitating the browning of adipocytes. Together, these data demonstrate that LKU4 promotes the AMPK-SIRT1-PPARγ pathway by inhibiting PP4C, thereby facilitating adipocyte browning in HFD mice.

(E)-2-Methoxy-4-(3-(4-Methoxyphenyl) Prop-1-en-1-yl) Phenol Suppresses Breast Cancer Progression by Dual-Regulating VEGFR2 and PPARγ.

In cancer treatment, multi-target approach has paid attention to a reasonable strategy for the potential agents. We investigated whether (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP) could exert an anticancer effect by dual-regulating VEGFR2 and PPARγ. MMPP showed modulating effects in TNBC type (MDA-MB-231 and MDA-MB-468) and luminal A type (MCF7) breast cancer cell lines. MMPP enhanced PPARγ transcriptional activity and inhibited VEGFR2 phosphorylation. MMPP-induced signaling by VEGFR2 and PPARγ ultimately triggered the downregulation of AKT activity. MMPP exhibited anticancer effects, as evidenced by growth inhibition, inducement of apoptosis, and suppression of migration and invasion. At the molecular level, MMPP activated pro-apoptotic proteins (caspase3, caspase8, caspase9, and bax), while inhibiting the anti-apoptotic proteins (bcl2). Additionally, MMPP inhibited the mRNA expressions of EMT-promoting transcription factors. Therefore, our findings showed molecular mechanisms of MMPP by regulating VEGFR2 and PPARγ, and suggested that MMPP has potential to treat breast cancer.

Adipocyte Subpopulations Mediate Growth Hormone-induced Lipolysis and Glucose Tolerance in Male Mice.

In adipose tissue, growth hormone (GH) stimulates lipolysis, leading to an increase in plasma free fatty acid levels and a reduction in insulin sensitivity. In our previous studies, we have found that GH increases lipolysis by reducing peroxisome proliferator-activated receptor γ (PPARγ) transcription activity, leading to a reduction of tat-specific protein 27 (FSP27, also known as CIDEC) expression. In previous studies, our laboratory uncovered 3 developmentally distinct subpopulations of white adipocytes. In this manuscript, we show that one of the subpopulations, termed type 2 adipocytes, has increased GH-induced signaling and lipolysis compared to other adipocyte subtypes. To assess the physiological role of GH-mediated lipolysis mediated by this adipocyte subpopulation, we specifically expressed human FSP27 (hFSP27) transgene in type 2 adipocytes (type2Ad-hFSP27tg mice). Systemically, male type2Ad-hFSP27tg mice displayed reduced serum glycerol release and nonesterified fatty acids levels after acute GH treatment, and improvement in acute, but not chronic, GH-induced glucose intolerance. Furthermore, we demonstrate that type2Ad-hFSP27tg mice displayed improved hepatic insulin signaling. Taken together, these results indicate that this adipocyte subpopulation is a critical regulator of the GH-mediated lipolytic and metabolic response. Thus, further investigation of adipocyte subpopulations may provide novel treatment strategies to regulate GH-induced glucose intolerance in patients with growth and metabolic disorders.

Nutritional supplement of Lepidium sativum L. seeds alleviates metabolic disorders and inflammatory responses in high-fat diet-induced obese rats via modulating AMPK/SREBP-1c of PPARγ signaling pathway

BackgroundObesity is a multifactorial chronic non-communicable disease that affects more than one- third of the world population. It represents a burden on human health in both developed and developing countries. Nowadays there is an urgent need for effective natural alternatives to manage obesity. This study aimed at exploring the fundamental molecular processes and contributive pathways of the hydroalcoholic extract of Lepidium sativum, L. seeds (LP) to manage weight gain with its accompanied metabolic complications in a high-fat diet-induced obesity animal model. MethodsDifferent doses of the hydroalcoholic extract were investigated for adipogenesis inhibition in liver tissues through Peroxisome-proliferator-activated receptor-gamma (PPARγ) transcriptional activity and mitochondrial phosphorylation of 5′AMP-activated protein kinase (AMPK) using western blot. Sterol regulatory element-binding protein (SREBP)-1c which plays a role in regulating cellular free fatty acid homeostasis via fatty acid oxidation and lipogenesis was evaluated using RT-qPCR gene analysis. Furthermore, Analyses of alterations in body weight and serum biomarkers such as triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), insulin, leptin and adiponectin were used to assess the anti-obesity effect. The protective seeds’ impact on hepatic tissues was further assessed by histopathological examination. The bioactive metabolites profiling was conducted via HPLC/ESI/PDA/MS-MS. ResultsLP effectively modulated PPARγ transcriptional activity via activation of mitochondrial phosphorylation of AMPK. Moreover, RT-qPCR gene analysis presented suppression of nuclear expression of SREBP-1c in dose-dependent method related to control group. Furthermore, it was revealed that LP moderated serum lipid profile, glycemic profile, leptin, and adiponectin. In addition, it reduced liver injury through decreasing ALT and AST enzymes in serum, upregulating liver antioxidant enzyme glutathione, and downregulating oxidative stress manifested in decreased malondialdehyde (MDA) levels. The anti-inflammatory activity was confirmed by declining in proinflammatory cytokine leukotriene B4 (LT-B4). ConclusionThis study is the first to report the potential impact of nutritional supplementation of Lepidium sativum seeds (400 mg/kg) to alleviate metabolic disorders and inflammatory responses in high-fat diet-induced obese rats via modulating AMPK/SREBP-1c of the PPARγ signaling Pathway.

Class III Alcohol Dehydrogenase Plays a Key Role in the Onset of Alcohol-Related/-Associated Liver Disease as an S-Nitrosoglutathione Reductase in Mice.

Lipid accumulation in the liver due to chronic alcohol consumption (CAC) is crucial in the development of alcohol liver disease (ALD). It is promoted by the NADH/NAD ratio increase via alcohol dehydrogenase (ADH)-dependent alcohol metabolism and lipogenesis increase via peroxisome proliferator-activated receptor γ (PPARγ) in the liver. The transcriptional activity of PPARγ on lipogenic genes is inhibited by S-nitrosylation but activated by denitrosylation via S-nitrosoglutathione reductase (GSNOR), an enzyme identical to ADH3. Besides ADH1, ADH3 also participates in alcohol metabolism. Therefore, we investigated the specific contribution of ADH3 to ALD onset. ADH3-knockout (Adh3-/-) and wild-type (WT) mice were administered a 10% ethanol solution for 12 months. Adh3-/- exhibited no significant pathological changes in the liver, whereas WT exhibited marked hepatic lipid accumulation (p < 0.005) with increased serum transaminase levels. Adh3-/- exhibited no death during CAC, whereas WT exhibited a 40% death. Liver ADH3 mRNA levels were elevated by CAC in WT (p < 0.01). The alcohol elimination rate measured after injecting 4 g/kg ethanol was not significantly different between two strains, although the rate was increased in both strains by CAC. Thus, ADH3 plays a key role in the ALD onset, likely by acting as GSNOR.

234-LB: Cell-State Dependent Regulation of PPARg Signaling by Zbtb9 in Adipocytes

The discovery of new genes involved in adipocyte development and function can shed light on the pathophysiology of obesity and T2D and provide new therapeutic targets for these disorders. Towards this end, GWAS and rare variant association studies in the T2D Knowledge Portal revealed associations between ZBTB9 and T2D, BMI, and hip circumference. However, the function of ZBTB9 in organismal physiology was not known. We found that Zbtb9 plays a key role in adipocyte biology and differentiation via cell-state dependent regulation of PPARγ signaling. Zbtb9 directly interacts with the PPARγ/RXRα complex as shown by co-IP, and acts as a positive regulator of PPARγ transcriptional activity as shown by a PPARγ reporter assay, together establishing Zbtb9 as a new PPARγ cofactor. In line with these findings, endogenous PPARγ target gene expression was decreased in the context of Zbtb9 deficiency in adipocytes. Given the central role of PPARγ in adipogenesis, we also sought to investigate the role of Zbtb9 in this process. Surprisingly, loss of Zbtb9 in preadipocytes increased adipogenesis and the expression of adipogenic genes, including PPARγ. Transcriptome analysis prior to and during adipogenesis revealed enhanced expression of E2F target genes prior to the initiation of differentiation and later induction of adipogenic gene expression. Treatment of Zbtb9 deficient preadipocytes with an E2F1 inhibitor (HLM006474) blocked the effects of Zbtb9 deficiency on adipogenic gene expression and adipogenesis. Collectively, these results demonstrate that Zbtb9 inhibits adipogenesis as a negative regulator of PPARγ expression via altered E2F1 signaling. We further demonstrated that E2F1 activation by Zbtb9 is associated with increased phosphorylation of Rb, which is known to cause dissociation of the Rb/E2F complex, and activation of E2F. Our findings revealed cell-state dependent roles of Zbtb9 in adipocytes, identifying a new molecule that may be important in the pathogenesis and treatment of obesity and T2D. Disclosure X. Xu: None. D. Buchner: None. Funding National Institutes of Health (DK119305)

310-OR: FBF1 Deficiency Promotes Ciliary Cold-Sensing TRP Channel Activation in Thermogenesis

Brown adipose tissue (BAT) dissipates energy as heat to maintain optimal thermogenesis and promotes metabolic health. Exposure to cold conditions strongly induces both hyperplasia and activity of brown fat by distinct but poorly understood mechanisms. The proliferation of preadipocytes and their subsequent differentiation is proposed to be a major contributor to BAT hyperplasia. Primary cilia are sensory organelles emanating from the surface of most eukaryotic cells. We previously discovered that primary cilia mediate hyperplasia and beiging of the white fat tissue in mice by FBF1-dependent manner. WAT preadipocytes transiently form primary cilia during induced differentiation. FBF1 deficiency promotes proliferation, differentiation, and beiging of WAT preadipocytes and thus leads to expansion of metabolically healthy fat tissue. Here, we discovered that BAT preadipocytes dynamically produce sensory cilia during differentiation. FBF1 deficiency enhances brown preadipocyte differentiation and expansion by stimulating PPARγ transcriptional activity. Consistently, Fbf1tm1a/tm1a mice exhibit BAT hyperplasia. We further discovered that FBF1 deficiency promotes the ciliary recruitment of cold-sensing TRP channels upon cold exposure, which likely explains the mechanism underlying cilia-mediated BAT hyperplasia. Collectively, our data provide molecular insights into coldness-induced BAT hyperplasia and highlight the importance of cilia-mediated TRP pathway in thermogenesis regulation. Disclosure Y.Zhang: None. B.Liu: None. J.Hao: None. Y.Huang: None. K.Ling: None. J.Hu: None.

Tumor necrosis factor-α reduces adiponectin production by decreasing transcriptional activity of peroxisome proliferator-activated receptor-γ in calf adipocytes

Adiponectin (encoded by ADIPOQ) is an adipokine that orchestrates energy homeostasis by modulating glucose and fatty acid metabolism in peripheral tissues. During the periparturient period, dairy cows often develop adipose tissue inflammation and decreased plasma adiponectin levels. Proinflammatory cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role in regulating the endocrine functions of adipocytes, but whether it affects adiponectin production in calf adipocytes remains obscure. Thus, the present study aimed to determine whether TNF-α could affect adiponectin production in calf adipocytes and to identify the underlying mechanism. Adipocytes isolated from Holstein calves were differentiated and used for (1) BODIPY493/503 staining; (2) treatment with 0.1 ng/mL TNF-α for different times (0, 8, 16, 24, or 48 h); (3) transfection with peroxisome proliferator-activated receptor-γ (PPARG) small interfering RNA for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h; and (4) overexpression of PPARG for 48 h followed by treatment with or without 0.1 ng/mL TNF-α for 24 h. After differentiation, obvious lipid droplets and secretion of adiponectin were observed in adipocytes. Treatment with TNF-α did not alter mRNA abundance of ADIPOQ but reduced the total and high molecular weight (HMW) adiponectin content in the supernatant of adipocytes. Quantification of mRNA abundance of endoplasmic reticulum (ER)/Golgi resident chaperones involved in adiponectin assembly revealed that ER protein 44 (ERP44), ER oxidoreductase 1α (ERO1A), and disulfide bond-forming oxidoreductase A-like protein (GSTK1) were downregulated in TNF-α-treated adipocytes, while 78-kDa glucose-regulated protein and Golgi-localizing γ-adaptin ear homology domain ARF binding protein-1 were unaltered. Moreover, TNF-α diminished nuclear translocation of PPARγ and downregulated mRNA abundance of PPARG and its downstream target gene fatty acid synthase, suggesting that TNF-α suppressed the transcriptional activity of PPARγ. In the absence of TNF-α, overexpression of PPARG enhanced the total and HMW adiponectin content in supernatant and upregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. However, knockdown of PPARG reduced the total and HMW adiponectin content in supernatant and downregulated the mRNA abundance of ADIPOQ, ERP44, ERO1A, and GSTK1 in adipocytes. In the presence of TNF-α, overexpression of PPARG decreased, while knockdown of PPARG further exacerbated TNF-α-induced reductions in total and HMW adiponectin secretion and gene expression of ERP44, ERO1A, and GSTK1. Overall, TNF-α reduces adiponectin assembly in the calf adipocyte, which may be partly mediated by attenuation of PPARγ transcriptional activity. Thus, locally elevated levels of TNF-α in adipose tissue may be one reason for the decrease in circulating adiponectin in periparturient dairy cows.

Rosiglitazone-induced PPARγ activation promotes intramuscular adipocyte adipogenesis of pig

Intramuscular fat (IMF) positively influences various aspects of meat quality, while the subcutaneous fat (SF) has negative effect on carcass characteristics and fattening efficiency. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipocyte differentiation, herein, through bioinformatic screen for the potential regulators of adipogenesis from two independent microarray datasets, we identified that PPARγ is a potentially regulator between porcine IMF and SF adipogenesis. Then we treated subcutaneous preadipocytes (SA) and intramuscular preadipocytes (IMA) of pig with RSG (1 µmol/L), and we found that RSG treatment promoted the differentiation of IMA via differentially activating PPARγ transcriptional activity. Besides, RSG treatment promoted apoptosis and lipolysis of SA. Meanwhile, by the treatment of conditioned medium, we excluded the possibility of indirect regulation of RSG from myocyte to adipocyte and proposed that AMPK may mediate the RSG-induced differential activation of PPARγ. Collectively, the RSG treatment promotes IMA adipogenesis, and advances SA lipolysis, this effect may be associated with AMPK-mediated PPARγ differential activation. Our data indicates that targeting PPARγ might be an effective strategy to promote intramuscular fat deposition while reduce subcutaneous fat mass of pig.

Single-cell Expression Atlas Reveals Cell Heterogeneity in the Creeping Fat of Crohn's Disease.

Creeping fat (CrF) has been recognized to play a positive role in Crohn's disease (CD) progression, yet the cellular compositions within mesenteric adipose tissue (MAT) and their potential mechanism in CrF formation are poorly understood. Analysis of 10X single-cell RNA sequencing was performed on 67 064 cells from 3 pairs of surgically resected samples of CrF and their uninvolved MAT. The results were validated in another cohort with 6 paired MAT samples by immunofluorescence. All samples manifested excellent consistency and repeatability in our study, and 10 cell types from the transcriptome atlas, including 20 clusters, were identified. In CrF, a specific vascular endothelial cell subpopulation highly expressing lipoprotein lipase was first identified, with a significantly increased proportion. This vascular endothelial cell subpopulation manifested robust peroxisome proliferator-activated receptor γ (PPARγ) transcription activity and an upregulated PPAR signaling pathway and was involved in lipid metabolism and the antibacterial response. A novel fibroblast subpopulation (FC3) with remarkable GREM1 and RFLNB expression was identified and validated to predominantly accumulate in the CrF. The FC3 was annotated as inflammation-associated fibroblasts, which are characterized by inflammatory responses and the regulation of Smad phosphorylation related to intestinal fibrosis. The trajectory of fibroblasts revealed their pro-inflammatory and profibrotic conversion tendency during CrF formation with corresponding gene dynamics. Additionally, we unprecedently dissected the different origins and functions of 6 macrophage subclusters within the myeloid compartment. Our results uncover the cellular heterogeneity in the MAT of CD and the role of these various cellular compositions in CrF development. This comprehensive understanding of CrF provides future directions for in-depth research on and potential targets for MAT-based treatment.

The Novel Inhibitory Effect of YM976 on Adipocyte Differentiation.

The pyrimidine derivative YM976 (4-(3-chlorophenyl)-1,7-diethylpyrido(2,3-d)-pyrimidin-2(1H)-one) exerts anti-inflammatory and anti-asthmatic effects. Considering that accumulation of lipids in adipose tissue is accompanied by inflammation, we investigated whether YM976 affects adipocyte differentiation. We found that YM976 significantly decreased lipid accumulation without cytotoxicity and reduced the expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) as well as their lipogenic regulators including fatty acid synthase (FASN) and fatty acid-binding protein 4 (FABP4) in 3T3-L1 cells induced for differentiation. YM976 mainly inhibited the early stage of adipocyte differentiation. Furthermore, intracellular cAMP level was elevated by YM976 resulting in increased phosphorylation of adenosine monophosphate-activated protein kinase (AMPK). Conversely, decreasing the levels of AMPK or treatment with Compound C, an AMPK inhibitor, lessened the suppressive effects of YM976 on PPARγ transcriptional activity and adipogenesis. Thus, our results suggest YM976 as a novel potential compound for controlling lipid accumulation and formation of adipocytes in obesity.

PPARγ activation inhibits endocytosis of claudin-4 and protects against deoxynivalenol-induced intestinal barrier dysfunction in IPEC-J2 cells and weaned piglets

The role of peroxisome proliferator activated receptor gamma (PPARγ) in the regulation of adipocyte differentiation has been well characterized. Besides adipose tissue, PPARγ is also highly expressed in the intestine. However, the functional role of PPARγ in the regulation of intestinal function still remains poorly understood. In the present study, we sought to understand the role of PPARγ activation on regulation of intestinal barrier function in intestinal porcine epithelial cells (IPEC-J2) and weaned piglets exposed to the mycotoxin, deoxynivalenol (DON). PPARγ activation by rosiglitazone and troglitazone, two pharmacological PPARγ ligands, increased the protein expression of tight junction proteins (TJP), claudin-3 and 4. PPARγ inhibition increased endocytosis of claudin-4 which was reversed by its activation with troglitazone. DON exposure decreased the protein expression of TJP, and also significantly suppressed PPARγ transcriptional activity. Interestingly, PPARγ activation reversed the reduction of claudin-3 and 4 caused by DON in vitro and in vivo. PPARγ activation also partially restored the transepithelial electrical resistance (TEER) and reduced the permeability of fluorescein isothiocyanate-dextran (FITC-dextran) that have been negatively impacted by DON. These effects were lost in the presence of a specific PPARγ antagonist or in PPARγ knockout cells, confirming the importance of PPARγ in the regulation of intestinal barrier function and integrity. Likewise, in weaned pigs exposed to DON, the PPARγ agonist pioglitazone mitigated the impaired villus-crypt morphology caused by DON. Therefore, pharmacological and natural bioactive compounds with PPARγ stimulatory activities could be effective in preventing DON-induced gut barrier dysfunction.

A Screening System for the Identification of Fragment Molecules toward the Creation of Nuclear Receptor Ligands

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily, which plays an important role in glucose and lipid metabolism as well as inflammation. The transcriptional activity of PPARγ is regulated by the binding of its ligand and the accompanied conformational change followed by the recruitment of cofactors. The ligand-binding pocket (LBP) of PPARγ comprises multiple sub-pockets and includes a large, Y-shaped cavity. In some cases, more than two ligands simultaneously occupy the LBP and cooperatively activate PPARγ transcription. Inspired by this peculiar character, the author proposed a strategy to create new PPARγ ligands in two steps: first, identifying a combination of ligands that cooperatively activate PPARγ, and second, designing and synthesizing their hybrid structure. Cooperative activation can be detected by a conventional cell-based assay using a reporter gene, which may provide advantages over the existing fragment-based drug discovery approach. Using this strategy, a plant-derived cinnamic acid derivative was found to synergistically activate PPARγ in combination with GW9662, an irreversible antagonist. The designed hybrid structure was synthesized and found to behave as a covalent agonist, which partially activates PPARγ transcription. Structure-activity studies revealed the importance of proximity and orientation in the linkage of the two units. The strategy discussed in this article may contribute to the development of a highly potent PPARγ agonist.

Antibiotics disrupt lipid metabolism in zebrafish (Danio rerio) larvae and 3T3-L1 preadipocytes

Antibiotics are emerging environmental contaminants with wide attention due to their high consumption and pseudo-persistence in the environment. They have been shown to induce obesity or obesity-related metabolic diseases in experimental animals, but the underlying toxicological mechanisms remain unclear. Here, the disruptive effects of four commonly used antibiotics, namely doxycycline (DC), enrofloxacin (ENR), florfenicol (FF) and sulfamethazine (SMT) on lipid metabolism were investigated in zebrafish (Danio rerio) larvae and murine preadipocyte cell line. Triglyceride (TG) content was reduced after 1 ng/L DC or ENR exposure but was increased at higher concentrations up to 100 mg/L. FF increased and SMT reduced TG content but did not show any concentration dependence. None of the antibiotics had any significant effect on total cholesterol (TC) content in zebrafish except 100 μg/L SMT. Expression levels of 8 lipid metabolism-related genes were also quantified. SMT was most disruptive by up-regulating six genes, followed by FF which up-regulated four genes and down-regulated one gene, whereas DC and ENR both up-regulated one gene. In 3T3-L1 preadipocytes, ENR, FF, and SMT in general increased TG content, while 100 mg/L FF reduced TG substantially. DC did not show any effect up to 10 mg/L, at which TG increased significantly. FF and SMT increased TC slightly at low concentrations but reduced it at high concentrations, whereas TC, DC and ENR had no effect at any tested concentrations. Gene expression measurement also indicated that SMT was most disruptive, followed by FF, DC, and ENR. Reporter gene assays showed that only SMT inhibited the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ). The above experimental results and clustering analysis demonstrate that the four antibiotics exerted disruption on lipid metabolism through different mechanisms, and one of the mechanisms for SMT may be inhibition of PPARγ transcriptional activity.

MMPP promotes adipogenesis and glucose uptake via binding to the PPARγ ligand binding domain in 3T3-L1 MBX cells.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor involved in adipogenesis, and its transcriptional activity depends on its ligands. Thiazolidinediones (TZDs), well-known PPARγ agonists, are drugs that improve insulin resistance in type 2 diabetes. However, TZDs are associated with severe adverse effects. As current therapies are not well designed, novel PPARγ agonists have been investigated in adipocytes. (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP) is known to have anti-arthritic, anti-inflammatory, and anti-cancer effects. In this study, we demonstrated the adipogenic effects of MMPP on the regulation of PPARγ transcriptional activity during adipocyte differentiation in vitro. MMPP treatment increased PPARγ transcriptional activity, and molecular docking studies revealed that MMPP binds directly to the PPARγ ligand binding domain. MMPP and rosiglitazone showed similar binding affinities to the PPARγ. MMPP significantly promoted lipid accumulation in adipocyte cells and increased the expression of C/EBPβ and the levels of p-AKT, p-GSK3, and p-AMPKα at an early stage. MMPP enhanced the expression of adipogenic markers such as PPARγ, C/EBPα, FAS, ACC, GLUT4, FABP4 and adiponectin in the late stage. MMPP also improved insulin sensitivity by increasing glucose uptake. Thus, MMPP, as a PPARγ agonist, may be a potential drug for type 2 diabetes and metabolic disorders, which may help increase adipogenesis and insulin sensitivity.