Pro-adipogenic Transcription Factors Research Articles

The lack of EphB3 receptor prevents bone loss in mouse models of osteoporosis.

Bone homeostasis is a complex process in which some Eph kinase receptors and their ephrin ligands appear to be involved. In the present study, we address this issue by examining, both in vitro and in vivo, the role of EphB2 and EphB3 in mesenchymal stromal/stem cell (MSC) differentiation into bone tissue. This was first evaluated by quantitative reverse transcription PCR (RT-qPCR) and histological staining in MSCs cultured in specific mediums revealing that although EphB2-/- MSCs mainly expressed pro-adipogenic transcription factors, EphB3-/- MSCs showed abundant osteogenic transcripts, such as Runx2, Msx2, and Sp7. To clarify the underlying molecular mechanisms, we found that the lack of EphB3 signaling alters the genetic profile of differentiating MSCs, reducing the expression of many inhibitory molecules and antagonists of the BMP signaling pathway, and increasing Bmp7 expression, a robust bone inductor. Then, to confirm the osteogenic role of EphB3 in vivo, we studied the condition of 2 mouse models of induced bone loss (ovariectomy or long-term glucocorticoid treatment). Interestingly, in both models, both WT and EphB2-/- mice equally developed the disease but EphB3-/- mice did not exhibit the typical bone loss, nor an increase in urine Ca2+ or blood serum CTX-1. This phenotype in EphB3-KO mice could be due to their significantly higher proportions of osteoprogenitor cells and preosteoblasts, and their lower number of osteoclasts, as compared with WT and EphB2-KO mice. Thus, we conclude that EphB3 acts as a negative regulator of the osteogenic differentiation, and its absence prevents bone loss in mice subjected to ovariectomy or dexamethasone treatment.

Abstract PO3-08-07: Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers

Abstract Background: Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Further, they do not prevent ER- breast cancer. Effective alternative strategies with lower toxicity are needed. Previously, we have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. These data led us to hypothesize that LicA creates a tumor preventive environment in the breast by reprogramming metabolism and antioxidant/anti-inflammatory responses in the breast leading to decreased proliferation and tumor suppression. We now report on the breast tumor preventive effects of LicA in xenograft models, its oral bioavailability, and its biologic effects on human breast microstructures from women at increased risk of breast cancer. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified and analyzed by gene ontology and pathway membership. The RNA-seq data was also utilized to conduct metabolism flux analysis. Combined enrichment scores > 4 and FDR < 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Xenograft models of MCF-7aro and MDA-MB-231 tumors were established in female nude mice and the animals treated for 28 days with vehicle or LicA (80 mg/kg.day, s.c.). We measured the rate of tumor growth. We also conducted a PK/PD study with oral LicA (100 mg/kg) in intact BALB/c female mice. Results: We observed significant (FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses < 10 µM. LicA significantly reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) in vivo models (unpaired t-test with Welch’s correction for unequal variances). Promising serum and breast bioavailability, equivalent to low micromolar concentrations sufficient to show efficacy was demonstrated as well. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention in both ER+ and ER- breast cancers through reprogramming metabolism and antioxidant pathways leading to decreased proliferation. We will study LicA in intraductal models of ER+ and ER- precancer lesions in immunocompetent mice and will monitor their progression to invasive breast cancer to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Susan Clare, Seema Khan. Licochalcone A as a risk reducing agent against luminal and non-luminal breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-08-07.

Abstract 7306: Licochalcone A is an excellent candidate for preventing luminal and non-luminal breast cancers

Abstract Breast cancer (BC) risk reducing drugs have minimal impact due to their adverse effects and low acceptance by risk-eligible women. Further, they are ineffective against hormone receptor negative (HR-) BC. Novel agents with reduced toxicity and sufficient efficacy that extends beyond HR+ BC are needed. We showed previously that licochalcone A (LicA) from licorice has antioxidant/anti-inflammatory effects, inhibits aromatase, and blocks estrogen genotoxic metabolism. We hypothesize that LicA prevents HR+ and HR- BC through reprogramming metabolism and antioxidant pathways. We prepared microstructures from the fresh tissue of contralateral unaffected breast of 6 postmenopausal women with unilateral BC. After exposing them to DMSO (control) or LicA (5 µM), we performed total RNA sequencing followed by differential gene expression, pathway identification, and metabolism flux analyses. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of HR- and HR+ pre-malignant and malignant, as well as BRCA mutated cells. Xenograft models of HR+ and HR- tumors were established in female nude mice followed by 28-day treatment with vehicle or LicA (80 mg/kg.day, s.c.) and monitoring the tumor growth. Using LC-MS/MS we measured LicA in the blood and various tissues of intact BALB/c female mice receiving oral LicA (100 mg/kg) and assessed PK using Phoenix Platform. We observed significant (combined enrichment scores > 4 and FDR < 0.05) upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system. This was accompanied by significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of PI3K-AKT genes and the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis, transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and antiproliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cell lines at doses <10 µM. It reduced tumor growth in luminal (P = 0.008) and triple negative (P = 0.001) xenograft models. Oral bioavailability in serum and breast was in the low micromolar range and was sufficient to show efficacy.Our data suggest that LicA is an excellent candidate for both HR+ and HR- BC prevention by reprogramming metabolism and antioxidant pathways leading to decreased proliferation. Our next study will test an optimized oral formulation of LicA in intraductal models of HR+ and HR- precancer lesions in immunocompetent mice to further establish its preventive efficacy. Citation Format: Atieh Hajirahimkhan, Elizabeth T. Bartom, Sriram Chandrasekaran, Ruohui Chen, Jeremy J. Johnson, Susan E. Clare, Seema A. Khan. Licochalcone A is an excellent candidate for preventing luminal and non-luminal breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7306.

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

The inhibitory effects of Xiao-Gao-Jiang-Zhuo-containing serum on adipogenesis in 3T3-L1 preadipocytes.

Obesity and related metabolic diseases are becoming a worldwide epidemic, leading to increased mortality and heavy medical costs. Our Chinese herbal formula Xiao-Gao-Jiang-Zhuo (XGJZ) has remarkable effects on curing obese patients in the clinic, but the cellular and molecular basis remains unknown. This study aimed to reveal the molecular mechanism involved in adipogenesis in vitro. Chinese herbal formula XGJZ-containing serum was prepared from XGJZ-treated obesity model rats. The function of XGJZ-containing serum was validated in 3T3-L1 preadipocytes. Oil O staining was performed to determine intracellular lipid accumulation in differentiated 3T3-L1 cells. The expression of pro-adipogenic transcription factors was measured to further validate the adipogenesis of 3T3-L1 adipocytes. The contents of triglyceride (TG), free fatty acid (FFA), and glycerin, along with the activities of lipid metabolism-related enzymes (including FAT, FATP1, DGAT, GPAT, ATGL, and HSL) were measured to study the lipogenesis in 3T3-L1 adipocytes. XGJZ-containing serum inhibited 3T3-L1 differentiation, decreased intracellular lipid accumulation, and suppressed the expression of pro-adipogenic transcription factors in differentiated 3T3-L1 cells. The contents of TG, FFA, and glycerin were decreased when treated with XGJZ-containing serum, which also modulated lipid metabolism-related enzyme activities. The activities of fatty acid transporters (FAT, FATP1) and lipid mobilization enzymes (ATGL, HSL) were promoted, while activities of triglyceride biosynthesis enzymes (DGAT, GPAT) were attenuated in differentiated 3T3-L1 cells. XGJZ-containing serum has inhibitory effects on adipogenesis in 3T3-L1 preadipocytes, affirming the effect of XGJZ in treating obesity. It provides evidence for the mechanism of obesity.

Abstract 5263: Licochalcone A is a candidate for breast cancer prevention through its reprogramming of metabolic and antioxidant pathways

Abstract Background: Increased adiposity is a risk factor for postmenopausal breast cancer. It is accompanied by protumorigenic effects: chronic low-grade inflammation and elevated levels of reactive oxygen species. Breast cancer risk reducing drugs with proven efficacy have adverse side effects, significantly minimizing their uptake and impact. Effective alternative strategies with lower toxicity are needed. We have shown that licochalcone A (LicA) suppresses aromatase expression and activity, enhances the activity of detoxifying enzymes, and reduces estrogen genotoxic metabolism in cell lines and animal models. However, no previous data exist on the breast tissue of women at substantial risk of breast cancer. We hypothesize that LicA creates a tumor preventive environment in the breast by modulating antioxidant/anti-inflammatory responses in the breast and adipogenesis leading to decreased proliferation. Methods: We prepared microstructures from the fresh tissue of contralateral unaffected mastectomy specimens of 6 postmenopausal women with incident unilateral breast cancer. After exposing them to DMSO (control) and LicA (5 µM), we performed total RNA sequencing. Differentially expressed genes were identified, and analyzed by gene ontology and pathway membership. The RNA-seq data was utilized also to conduct metabolism flux analysis. Combined enrichment scores > 4 and FDR < 0.05 was considered significant. The NanoString metabolism panel was employed in 6 additional subjects. We performed live cell imaging to monitor proliferation of pre-malignant DCIS.COM, DCIS.COM/ER+ PR+; and malignant MDA-MB-231 (ER- PR-), MCF-7 (ER+ PR+), MCF-7aro, and BRCA1 defective HCC-1937, and HCC3153 cells. Results: We observed upregulation of antioxidant genes (up to 8-fold), consistent with upregulation of NRF2 and the thioredoxin system, the major regulators of antioxidant pathways. This was accompanied with the significant downregulation of RELA- and NF-kB1-dependent inflammatory pathways. In addition, we observed decreased expression of the pro-adipogenic transcription factors SREBF1 and SREBF2, which may explain the downregulation (4 to 32-fold) of cholesterol biosynthesis and transport, and lipid metabolism genes. Metabolism studies confirmed these data and demonstrated a robust increase in the pentose phosphate shunt and NAD(P)H generation without enhancing ribose 5 phosphate formation, suggesting an antioxidant and anti-proliferative environment. LicA also suppressed proliferation of pre-malignant and malignant cells, with sustained effects on aggressive cells at doses < 10 µM. Conclusion: Our data suggest that LicA is a good candidate for breast cancer prevention through modulation of metabolic and antioxidant pathways leading to decreased proliferation. Our ongoing in vivo study will further demonstrate the efficacy of LicA for breast cancer prevention. Citation Format: Atieh Hajirahimkhan, Elizabeth Bartom, Sriram Chandrasekaran, Xiaoling Xuei, Susan Clare, Seema Khan. Licochalcone A is a candidate for breast cancer prevention through its reprogramming of metabolic and antioxidant pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5263.

The Beneficial Effect of Salicornia herbacea Extract and Isorhamnetin-3-O-Glucoside on Obesity

Salicornia herbacea is a halophyte indigenous to marine coastal areas and salt fields and has been used as a traditional remedy for diarrhea, abdominal pain, constipation, and indigestion. Its component isorhamnetin-3-O-glucoside (IR3G) may have antioxidant, anti-inflammatory, and anti-adipogenic properties. In the present study, we aimed to investigate the anti-obesity effect of S. herbacea extract and IR3G on mouse 3T3-L1 adipocytes and db/db obesity mice. S. herbacea extract and IR3G inhibited lipase in a concentration-dependent manner. Oil Red O staining disclosed that S. herbacea extract and IR3G significantly suppressed lipid accumulation and adipogenesis and also inhibited the expression of the C/EBPα in the 3T3-L1 adipocytes. In experiments using db/db mice, administering of S. herbacea extract limited body weight gain and significantly reduced feed efficiency and adipose tissue weight. Moreover, analyzing blood triglycerides, total cholesterol, high-density lipoprotein, and low-density lipoprotein, it was confirmed that LDL was significantly decreased and total cholesterol slightly reduced by S. herbacea extract. However, there was no significant change by S. herbacea extract in the changes in blood levels of leptin and adiponectin. Taken together, these results suggest that S. herbacea extract and IR3G inhibit adipogenesis by suppressing the pro-adipogenic transcription factors in 3T3-L1 preadipocytes and prevent obesity by regulating the blood lipid profile as well as the weight of adipose tissue.

PSUN83 Exploring the Role of PARP1-mediated ADP-ribosylation in Diet-induced Obesity

Abstract Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification that regulates various cellular processes, including the differentiation of preadipocytes. PARylation involves the covalent attachment of a chain of ADP-ribose units to proteins by poly(ADP-ribose) polymerases (PARPs), mainly PARP1. We previously found that PARP1 activity decreases precipitously upon differentiation of preadipocytes, including 3T3-L1 cells, a preadipocyte cell line. Upon knockout or knockdown of Parp1, or chemical inhibition of PARP1, adipogenesis is dramatically increased. We have previously used a mouse lineage tracing model to show that knockout of Parp1 in preadipocytes promotes adipogenesis by expanding the pool of precursors, ultimately leading to accumulation of fat. PARP1 controls adipogenesis, in part, by PARylating two sites (E135A and E139A) in the regulatory domain of C/EBPβ, a proadipogenic transcription factor. Mutation of these sites enhances chromatin binding and transcriptional activity of C/EBPβ, as well as promotes a proadipogenic gene expression program and differentiation, in 3T3-L1 cells. However, we still do not know the role of PARP1-mediated site-specific PARylation in diet-induced obesity. We recently created a set of PAR-Trackers (PAR-Ts) that can detect changes in PAR levels during physiological processes in cells. We used PAR-T NanoLuc to observe dynamic changes in PAR levels during the differentiation of 3T3-L1 cells. We are now modifying the system to investigate PAR levels in the tissues of live animals. To investigate the physiological impact of loss of C/EBPβ PARylation in adipocytes, we have generated a knock-in mouse model for inducible tissue-specific expression of a C/EBPβ PARylation site mutant (Cebpb/E135A/E139A or C/EBPβ PAR mut). Using this model, we will investigate the impact of site-specific PARylation of C/EBPβ in mice fed a high fat diet. With these studies, we hope to reveal the specific roles of PARP1-mediated ADP-ribosylation in diet-induced obesity. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

A STAT5-Smad3 dyad regulates adipogenic plasticity of visceral adipose mesenchymal stromal cells during chronic inflammation

Adipogenic differentiation of visceral adipose tissue-resident multipotent mesenchymal stromal cells (VA-MSC) into adipocytes is metabolically protective. Under chronic inflammatory stress, this neoadipogenesis process is suppressed by various pro-inflammatory cytokines and growth factors. However, the underlying mechanism(s) regulating VA-MSC plasticity remains largely unexplored. Using an adipogenic differentiation screen, we identified IFNγ and TGFβ as key inhibitors of primary human VA-MSC differentiation. Further studies using human and mouse VA-MSCs and a chronic high-fat diet-fed murine model revealed that IFNγ/JAK2-activated STAT5 transcription factor is a central regulator of VA-MSC differentiation under chronic inflammatory conditions. Furthermore, our results indicate that under such conditions, IFNγ-activated STAT5 and TGFβ-activated Smad3 physically interact via Smad4. This STAT5–Smad4-Smad3 complex plays a crucial role in preventing the early adipogenic commitment of VA-MSCs by suppressing key pro-adipogenic transcription factors, including CEBPδ, CEBPα, and PPARγ. Genetic or pharmacological disruption of IFNγ-TGFβ synergy by inhibiting either STAT5 or Smad3 rescued adipogenesis under chronic inflammatory stress. Overall, our study delineates a central mechanism of MSC plasticity regulation by the convergence of multiple inflammatory signaling pathways.

Effect of Metformin and Simvastatin in Inhibiting Proadipogenic Transcription Factors.

Obesity is a multifactorial chronic disease characterized by the excessive accumulation of fat in adipose tissue driven by hypertrophy and hyperplasia of adipocytes through adipogenesis. Adipogenesis plays a key role in the development of obesity and related metabolic disorders, which makes it potential target for the therapeutic approach to obesity. An increasing number of studies confirm the pleiotropic action of the combined treatment with metformin and statins, suggesting their anti-hypertensive, anti-inflammatory, and anti-adipogenic effect. The aim of this study was to analyze the effect of different doses of metformin (MET) and simvastatin (SIM) on the expression of key transcription factors of adipogenesis. Mouse 3T3-L1 preadipocytes were induced to differentiation in adipogenic medium with sustained MET and SIM treatment to assess the effect on adipogenesis. Nine days after initiating adipogenesis, the cells were prepared for further experiments, including Oil Red O staining, RT-PCR, Western blotting, and immunocytochemistry. Treating the cells with the combination of MET and SIM slightly reduced the intensity of Oil Red O staining compared with the control group, and down-regulated mRNA and protein expression of PPARγ, C/EBPα, and SREBP-1C. In conclusion, the inhibitory effect of MET and SIM on adipocyte differentiation, as indicated by decreased lipid accumulation, appears to be mediated through the down-regulation of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding pro-tein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP-1C).

Molecular mechanism of anti-adipogenic effect of vitexin in differentiating hMSCs.

In this study, we evaluated a detailed molecular mechanism of anti-adipogenic activity of vitexin, apigenin flavone glucoside, present in germinated fenugreek seeds, in differentiating human mesenchymal stem cells (hMSCs). The lipid content of differentiated adipocytes was estimated by ORO staining. Effect on mitotic clonal expansion was checked by cell cycle analysis. Expression of early and terminal adipocyte differentiation markers, anti- and pro-adipogenic transcription factors and signalling intermediates regulating them was evaluated at RNA and protein level. We found vitexin to be non-cytotoxic up to 20 μM at which intracellular lipid accumulation was significantly decreased. Cell cycle analysis suggested that vitexin does not affect mitotic clonal expansion. Expression of early and late differentiation markers, such as CEBPα, CEBPβ, PPARγ, FABP4, perilipin, adiponectin and Glut4 was significantly reduced in the presence of vitexin. Expression of KLF4 and KLF15, positive regulators of PPARγ, was decreased, whereas that of negative regulators, namely KLF2, GATA2, miR20a, miR27a, miR27b, miR128, miR130a, miR130b, miR182 and miR548 increased with vitexin treatment. This effect was mediated by the activation of the AMP-activated protein kinase (AMPK) pathway via the activation of LepR and additionally by inhibiting ROS. Thus, our results showed that vitexin regulates the expression of PPARγ and inhibits adipogenesis of hMSCs at an early stage of differentiation.

Dillenia Indica Fruit Extract Suppressed Diet-induced Obesity in Rats by Down-regulating the mRNA Level of Proadipogenic Transcription Factors and Lipid Metabolizing Enzymes

Background: Dillenia indica (Family: Dilleniaceae) is an antioxidant-rich edible fruitbearing medicinal plant. The fruit of this plant (known as elephant apple) has many uses in traditional medicine. Objective: By considering its antioxidant content and ameliorating effects, this study aimed to evaluate the anti-adipogenic effects of D. indica fruit extract (DIFE) on high-fat diet (HFD) fed obese rats. Methods: Male Wistar rats were fed with a standard diet (SD), or high-fat diet (HFD), or HFD with 100 mg/kg or 200 mg/kg or 400 mg/kg DIFE for 8 weeks. The fruit extract was given orally by feeding gavage. The body weight, liver weight, visceral fat weight, plasma lipids, and oxidative stress-related parameters were measured. The mRNA level of different adipogenesis related transcription factors, lipogenic and lipolytic enzymes was also evaluated. Results: Consumption of DIFE daily (400 mg/kg) for 8 weeks resulted in a significant reduction of high-fat diet-induced body weight, liver weight, visceral fat weight, total cholesterol, and LDL-cholesterol level. High-fat diet-mediated elevation of oxidative stress markers was also lowered, with a parallel augmentation of the activities of antioxidant enzymes, due to 400 mg/kg DIFE feeding. DIFE also down-regulated the mRNA level of important pro-adipogenic factors like PPARγ, LXRα, and SREBP1c which consequently downregulated the transcript levels of lipogenic enzymes: ACC, FAS, HMGCR, and DGAT. The transcript level of lipolytic enzyme, HSL was also down-regulated in 400 mg/kg DIFE-fed rats. Conclusion: These findings indicate that the antioxidant-rich ethanolic extract of D. indica fruit can down-regulate the gene expression of pro-adipogenic transcription factors and lipid metabolizing enzymes and thus can suppress diet-induced obesity in Wistar rat.

The low molecular weight protein tyrosine phosphatase promotes adipogenesis and subcutaneous adipocyte hypertrophy.

Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.

Zc3h10 regulates adipogenesis by controlling translation and F-actin/mitochondria interaction.

The commitment of mesenchymal stem cells to preadipocytes is stimulated by hormonal induction. Preadipocytes induced to differentiate repress protein synthesis, remodel their cytoskeleton, and increase mitochondrial function to support anabolic pathways. These changes enable differentiation into mature adipocytes. Our understanding of the factors that coordinately regulate the early events of adipocyte differentiation remains incomplete. Here, by using multipronged approaches, we have identified zinc finger CCCH-type containing 10 (Zc3h10) as a critical regulator of the early stages of adipogenesis. Zc3h10 depletion in preadipocytes resulted in increased protein translation and impaired filamentous (F)-actin remodeling, with the latter detrimental effect leading to mitochondrial and metabolic dysfunction. These defects negatively affected differentiation to mature adipocytes. In contrast, Zc3h10 overexpression yielded mature adipocytes with remarkably increased lipid droplet size. Overall, our study establishes Zc3h10 as a fundamental proadipogenic transcription factor that represses protein synthesis and promotes F-actin/mitochondria dynamics to ensure proper energy metabolism and favor lipid accumulation.

The role of Zfp467 in mediating the pro-osteogenic and anti-adipogenic effects on bone and bone marrow niche.

Conditional deletion of the PTH receptor (Pth1r) in mesenchymal progenitors reduces osteoblast differentiation and bone mass while enhancing adipogenesis and bone marrow adipose tissue. Mechanistically, PTH suppresses the expression of Zfp467, a pro-adipogenic zinc finger transcription factor. Consequently, Pth1r deficiency in mesenchymal progenitors leads to increased Zfp467 expression. Based on these observations, we hypothesized that genetic loss of Zfp467 would lead to a shift in marrow progenitor cell fate towards osteogenesis and increased bone mass. To test this hypothesis, we generated Zfp467-/- mice. Zfp467-/- mice (-/-) were significantly smaller than Zfp467+/+ mice (+/+). μCT showed significantly higher trabecular bone and cortical bone area in -/- vs. +/+, and histomorphometry showed higher structural and dynamic formation parameters in -/- mice vs. +/+. Femoral gene expression including Alpl, Sp7, and Acp5 were increased in -/-mice, whereas Adiponectin, Cebpa, Lepr, and Ppraγ mRNA were lower in -/- mice. Similarly, Fabp4 and Lep in the inguinal depot were also decreased in -/- mice. Moreover, marrow adipocyte numbers were reduced in -/- vs +/+ mice (p<0.007). In vitro, COBs and BMSCs-/- showed more positive ALP and Alizarin Red staining and a decrease in ORO droplets. Pth1r mRNA and protein levels were increased in COBs and BMSCs from -/- mice vs +/+ (p<0.02 for each parameter, -/- vs. +/+). -/- cells also exhibited enhanced endogenous levels of cAMP vs. control cells. Moreover, in an ovariectomy (OVX) mouse model, Zfp467-/- mice had significantly lower fat mass but similar bone mass compared to OVX +/+ mice. In contrast, in a high fat diet (HFD) mouse model, in addition to reduced adipocyte volume and adipogenesis related gene expression in both peripheral and bone marrow fat tissue, greater osteoblast number and higher osteogenesis related gene expression were also observed in -/- HFD mice vs. +/+ HFD mice. Taken together, these results demonstrate that ZFP467 negatively influences skeletal homeostasis and favors adipogenesis. Global deletion of Zfp467 increases PTHR1, cAMP and bone turnover, hence its repression is a component of PTH signaling and its regulation. These data support a critical role for Zfp467 in early lineage allocation and provide a novel potential mechanism by which PTH acts in an anabolic manner on the bone remodeling unit.

SIAH2 is Expressed in Adipocyte Precursor Cells and Interacts with EBF1 and ZFP521 to Promote Adipogenesis.

Expression of zinc finger protein 423 (ZFP423), a key proadipogenic transcription factor in adipocyte precursor cells, is regulated by interaction of the proadipogenic early B-cell factor 1 (EBF1) and antiadipogenic ZFP521. The ubiquitin ligase seven-in-absentia homolog 2 (SIAH2) targets ZFP521 for degradation. This study asked whether SIAH2 is expressed in adipocyte precursor cells and whether SIAH2 interacts with ZFP521 and EBF1 to regulate ZFP521 protein levels during adipogenesis. SIAH2 expression in precursor cells was assessed in primary cells and tissues from wild-type and SIAH2 null mice fed a control or high-fat diet. Primary cells, 3T3-L1 preadipocytes, and HEK293T cells were used to analyze Siah2, Ebf1, and Zfp521 expression and SIAH2-mediated changes in ZFP521 and EBF1 protein levels. Siah2 is expressed in platelet-derived growth factor receptor α (PDGFRα)+ and stem cell antigen-1 (SCA1)+ adipocyte precursor cells. SIAH2 depletion reduces Ebf1 gene expression and increases EBF1 protein levels in early but not late adipogenesis. In early adipogenesis, SIAH2 forms a protein complex with EBF1 and ZFP521 to enhance SIAH2-mediated ubiquitylation and degradation of ZFP521 while increasing EBF1 protein levels. Siah2 is expressed in PDGFRα+ adipocyte precursor cells and is linked to precursor cell commitment to adipogenesis by interacting with EBF1 and ZFP521 proteins to target the antiadipogenic ZFP521 for degradation.

Antioxidant-rich Tamarindus indica L. leaf extract reduced high-fat diet-induced obesity in rat through modulation of gene expression

BackgroundDifferent parts of the medicinal plant Tamarindus indica L. are full of phytochemicals that are able to reduce elevated blood pressure, blood sugar and lipids. These pharmacological effects are due to the presence of antioxidant type compounds in those parts of the plant. This study was aimed to explore the molecular mechanism of anti-obesity effects of ethanolic extract of T. indica L. leaves (TILE) through the evaluation of biochemical parameters and gene expression analysis in high-fat diet (HFD) consuming Wistar rats.MethodsMale Wistar rats were supplied with a standard diet (SD), or HFD, or HFD with 100 mg/kg or 200 mg/kg or 400 mg/kg TILE for 8 weeks. The body weight, liver weight, fat weight, plasma lipids, and oxidative stress-related parameters were measured. The transcript levels of different adipogenesis related transcription factors, lipogenic enzymes, and lipolytic enzymes were also evaluated by quantitative real-time PCR.ResultPhytochemical analysis demonstrated that TILE is enriched with a substantial level of polyphenols (287.20 ± 9.21 mg GAE/g extract) and flavonoids (107.52 ± 11.12 mg QE/g extract) which might be the reason of significant antioxidant and radical scavenging activities. Feeding of TILE (400 mg/kg/day) to HFD-fed rats increased activity of superoxide dismutase and catalase which is reflected as a significant reduction of oxidative stress markers like nitric oxide and malondialdehyde. TILE (400 mg/kg/day) feeding also down-regulated the mRNA levels of proadipogenic transcription factors including liver X receptor alpha (LXRα), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol regulatory element-binding protein 1c (SREBP1c) in diet-induced obese rats. As a consequence of this, the mRNA level of lipogenic enzymes like acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), diacylglycerol acyltransferase (DGAT), and HMG-CoA reductase was down-regulated with a parallel up-regulation of the transcript level of lipolytic enzyme, hormone-sensitive lipase (HSL).ConclusionObservations from this study indicate that antioxidant-rich TILE can reduce HFD-induced body weight, fat weight and liver weight as well as blood lipids through down-regulating the gene expression of proadipogenic transcription factors and lipogenic enzymes with a concerted diminution of the gene expression of lipolytic enzyme, HSL.

Tricyclic antipsychotics promote adipogenic gene expression to potentiate preadipocyte differentiation in vitro.

Antipsychotic-induced weight gain is a well-established but poorly understood clinical phenomenon. New mechanistic insights into how antipsychotics modulate adipose physiology are sorely needed, in hopes of either devising a therapeutic intervention to ameliorate weight gain or contributing to improved design of future agents. In this study, we have hypothesized that the weight gain-associated tricyclic antipsychotics clozapine and chlorpromazine directly impact adipose tissue by potentiating adipogenic differentiation of preadipocytes. Utilizing a well-established in vitro model system (3T3-L1 preadipocyte cell line), we demonstrate that, when applied specifically during induction of adipogenic differentiation, both clozapine and chlorpromazine significantly potentiate in vitro adipogenesis, observed as morphological changes and increased intracellular lipid accumulation. These persistent effects, observed at endpoints well after the end of antipsychotic exposure, are accompanied by increased transcript- and protein-level expression of the mature adipocyte marker perilipin-1, as indicated by RT-qPCR and Western blotting, but not by further upregulation of pro-adipogenic transcription factors versus positive controls. Our findings point to a possible physiological mechanism of antipsychotic-induced hyperplasia, with potentiated expression of mature adipocyte markers enhancing the differentiation and maturation of preadipocytes.

Methylmercury Induces Metabolic Alterations in Caenorhabditis elegans: Role for C/EBP Transcription Factor.

Methylmercury (MeHg) is a well-known neurotoxicant; however, its role in metabolic diseases has been gaining wider attention. We have previously shown that MeHg causes metabolic alterations in Caenorhabditis elegans, leading to decreased nicotinamide adenine dinucleotide cofactor, mitochondrial dysfunction, and oxidative stress. We were, therefore, interested in whether MeHg also affects nutrient metabolism, particularly lipid homeostasis, which may contribute to the development of metabolic conditions such as obesity or metabolic syndrome (MS). RNA from wild-type worms exposed to MeHg was collected immediately after treatment and used for gene expression analysis by DNA microarray. MeHg differentially regulated 215 genes, 17 genes involved in lipid homeostasis, and 12 genes involved in carbohydrate homeostasis. Of particular interest was cebp-1, the worm ortholog to human C/EBP, a pro-adipogenic transcription factor implicated in MS. MeHg increased the expression of cebp-1 as well as pro-adipogenic transcription factors sbp-1 and nhr-49, triglyceride synthesis enzyme acl-6, and lipid transport proteins vit-2 and vit-6. Concurrent with the altered gene expression, MeHg increased triglyceride levels, lipid storage, and feeding behaviors. Worms expressing mutant cebp-1 were protected from MeHg-induced alterations in lipid content, feeding behaviors, and gene expression, highlighting the importance of this transcription factor in the worm's response to MeHg. Taken together, our data demonstrate that MeHg induces biochemical, metabolic, and behavioral changes in C. elegans that can lead to metabolic dysfunction.

Twenty Novel Disease Group-Specific and 12 New Shared Macrophage Pathways in Eight Groups of 34 Diseases Including 24 Inflammatory Organ Diseases and 10 Types of Tumors.

The mechanisms underlying pathophysiological regulation of tissue macrophage (Mφ) subsets remain poorly understood. From the expression of 207 Mφ genes comprising 31 markers for 10 subsets, 45 transcription factors (TFs), 56 immunometabolism enzymes, 23 trained immunity (innate immune memory) enzymes, and 52 other genes in microarray data, we made the following findings. (1) When 34 inflammation diseases and tumor types were grouped into eight categories, there was differential expression of the 31 Mφ markers and 45 Mφ TFs, highlighted by 12 shared and 20 group-specific disease pathways. (2) Mφ in lung, liver, spleen, and intestine (LLSI-Mφ) express higher M1 Mφ markers than lean adipose tissue Mφ (ATMφ) physiologically. (3) Pro-adipogenic TFs C/EBPα and PPARγ and proinflammatory adipokine leptin upregulate the expression of M1 Mφ markers. (4) Among 10 immune checkpoint receptors (ICRs), LLSI-Mφ and bone marrow (BM) Mφ express higher levels of CD274 (PDL-1) than ATMφ, presumably to counteract the M1 dominant status via its reverse signaling behavior. (5) Among 24 intercellular communication exosome mediators, LLSI- and BM- Mφ prefer to use RAB27A and STX3 than RAB31 and YKT6, suggesting new inflammatory exosome mediators for propagating inflammation. (6) Mφ in peritoneal tissue and LLSI-Mφ upregulate higher levels of immunometabolism enzymes than does ATMφ. (7) Mφ from peritoneum and LLSI-Mφ upregulate more trained immunity enzyme genes than does ATMφ. Our results suggest that multiple new mechanisms including the cell surface, intracellular immunometabolism, trained immunity, and TFs may be responsible for disease group-specific and shared pathways. Our findings have provided novel insights on the pathophysiological regulation of tissue Mφ, the disease group-specific and shared pathways of Mφ, and novel therapeutic targets for cancers and inflammations.