Preadipocyte Proliferation Research Articles

Sangyod rice extract inhibits adipocyte growth and differentiation via mTOR, Akt, and AMPK pathways

This study aimed to evaluate Sangyod rice extract (SRE) in two aspects: its effect on 3T3-L1 cell proliferation during early adipocyte differentiation and its anti-adipogenic properties. Initially, SRE reduced 3T3-L1 preadipocyte proliferation by downregulating mTOR and Akt expression. In later stages, it significantly inhibited adipogenesis, lowering lipid accumulation and triglyceride levels. SRE achieved these effects by suppressing key adipogenic transcription factors (PPAR-γ, C/EBPα, C/EBPβ, SREBP-1c, SREBP-2) and lipogenic genes (perilipin, aP2, ACC, FasN, LPL, HSL, ATGL), as well as adipokine genes (leptin, resistin, AdipoQ, AdipoQ-R1, AdipoQ-R2). AMPK activation played a crucial role in SRE's mechanism, hindering adipogenesis. Moreover, SRE was found to decrease glucose uptake by suppressing the gene expression of glucose transporter 4 (Glut4) and the insulin receptor (IsnR). This research highlights the potential of SRE as an anti-obesity agent, particularly in alleviating insulin resistance and obesity. This suggests that SRE could be formulated into a functional food product with these advantageous properties.

Date Seed Polyphenol Pills as Renewable Raw Materials Showed Anti-Obesity Effects with High Digestible Antioxidants in 3T3-L1 Cells

Natural polyphenol-rich plant resources, such as agricultural waste, were proven to diminish insulin resistance and weight gain in rats on a high-fat diet. To test whether date seed polyphenol pills (DSPPs) might lower adipose tissue accumulation by precisely affecting adipocytes, we explored the impacts of DSPPs on cell proliferation, differentiation, and lipolysis in 3T3-L1 cells. We utilized tablets made commercially from date seed polyphenols that were mostly composed of epicatechin (45.9 g/kg). The total polyphenol and antioxidant capacities of the digested and non-digested DSPPs were also evaluated. DSPPs at doses of 25, 50, and 100 µg/mL hindered the proliferation of both pre-confluent preadipocytes and mature post-confluent adipocytes. DSPPs decreased the quantity of viable cells in completely developed adipocytes. Treatment with 100 µg/mL of DSPPs decreased the basal lipolysis of completely differentiated adipocytes but modestly boosted epinephrine-induced lipolysis. A significant transcription factor for the adipogenic gene, the peroxisome proliferator-activated receptor (PPAR), was repressed by DSPPs, which significantly decreased lipid buildup. The total polyphenol and antioxidant capacities were also increased after digestion with a good bubble Pearson correlation between both. DSPPs may have anti-obesity and anti-diabetic characteristics by inhibiting adipocyte development and basal lipolysis, which could be commercially industrialized.

Cloning and functional characterization of porcine AACS revealing the regulative roles for fat deposition in pigs.

Fat deposition is a quantitative trait controlled by multiple genes in pigs. Using transcriptome sequencing, we previously reported that AACS is differentially expressed in the subcutaneous fat tissue of Dingyuan pigs with divergent backfat thickness. Therefore, with the aim of further characterizing this gene and its protein, we cloned the entire 3286-bp mRNA sequence of the porcine AACS, and the encoded AACS protein is a hydrophilic protein without a signal peptide or transmembrane sequence. Our findings suggested that among various tissues and pig breeds, AACS was highly expressed in subcutaneous fat. We have identified three completely linked SNP loci in the AACS gene: A-1759C, C-1683T, and A-1664G. The double luciferase activity test in the 5' flanking region indicated that the flanking region of AACS contained several active regulatory elements. The three linked SNPs that were identified in one of the critical active elements, and might serve as important molecular markers regulating backfat thickness. Finally, we observed that AACS overexpression inhibited the proliferation and differentiation of subcutaneous preadipocytes. Collectively, our results suggest that AACS inhibits subcutaneous fat deposition in pigs. This study provides a new molecular marker for understanding the mechanism of porcine fat deposition.

Exposure to Chlorpyrifos Alters Proliferation, Differentiation and Fatty Acid Uptake in 3T3-L1 Cells.

Organophosphorus pesticides (OPs) are important factors in the etiology of many diseases, including obesity and type 2 diabetes mellitus. The aim of this study was to investigate the effect of a representative of OPs, chlorpyrifos (CPF), on viability, proliferation, differentiation, and fatty acid uptake in 3T3-L1 cells. The effect of CPF exposure on preadipocyte proliferation was examined by the MTT, NR, and BrdU assays. The impact of CPF exposure on the differentiation of preadipocytes into mature adipocytes was evaluated by Oil Red O staining and RT-qPCR. The effect of CPF on free fatty acid uptake in adipocytes was assessed with the fluorescent dye BODIPY. Our experiments demonstrated that exposure to CPF decreased the viability of 3T3-L1 cells; however, it was increased when the cells were exposed to low concentrations of the pesticide. Exposure to CPF inhibited the proliferation and differentiation of 3T3-L1 preadipocytes. CPF exposure resulted in decreased lipid accumulation, accompanied by down-regulation of the two key transcription factors in adipogenesis: C/EBPα and PPARγ. Exposure to CPF increased basal free fatty acid uptake in fully differentiated adipocytes but decreased this uptake when CPF was added during the differentiation process. Increased free fatty acid accumulation in fully differentiated adipocytes may suggest that CPF leads to adipocyte hypertrophy, one of the mechanisms leading to obesity, particularly in adults. It can therefore be concluded that CPF may disturb the activity of preadipocytes and adipocytes, although the role of this pesticide in the development of obesity requires further research.

249 The Effects of Breed and Early Supplementation on Bovine Intramuscular Fat Deposition, Adipogenic and Lipogenic Gene Expression

Abstract Wagyu cattle are known to deposit greater amounts of intramuscular fat, which can directly increase quality grade and carcass value. Supplementation can positively influence animal production and overall nutrient acquisition when used strategically. The objectives of this study were to evaluate 1) the effect of breed and early supplementation on intramuscular and subcutaneous fat deposition in Wagyu and Hereford cross calves, and 2) changes in adipogenic and lipogenic gene expression in longissimus muscle (LM) biopsies from Wagyu and Hereford cross calves during an early supplementation period. Angus cows (n = 34) were artificially inseminated with semen from a Wagyu bull. A Hereford bull was utilized as a clean-up bull. From this breeding, Wagyu cross steers (n = 8) and Hereford cross steers (n = 9) were produced and were placed on study on day 100 of age. At this time, calves were allowed access to four SuperSmart Feeder stalls and were individually supplemented with steam-rolled corn at 0.5% body weight (BWT). Calves were ultrasounded on day 0, day 43, and day 86 of the study to evaluate intramuscular fat deposition and backfat thickness. An longissimus muscle (LM) biopsy was taken on day 0 and day 86 of the study to evaluate gene expression. Supplemented steers tended (P < 0.10) to have more backfat than non-supplemented steers. Intramuscular fat amount differed (P < 0.05) by breed, while ribeye area tended (P < 0.10) to differ by breed. Wagyu exhibited a greater (P < 0.05) amount of intramuscular fat at the beginning and at the end of the study. Breed did not (P > 0.05) influence backfat thickness. Neither breed nor supplementation affected (P > 0.05) average daily gain or final body weights. Platelet-derived growth factor alpha (PDGRFA) and SREBP cleavage-activating protein (SCAP) differed (P < 0.05) between biopsy phases. Fatty acid binding protein-4 (FABP4) and stearoyl-coA desaturase-1 (SCD1) did not differ (P > 0.05) between breed or biopsy phase. In conclusion, changes in gene expression could indicate enhanced preadipocyte proliferation during this stage of calf development. Overall, supplementation altered backfat deposition but did not alter intramuscular fat levels. Opposingly, sire breed influenced intramuscular fat levels at an early age. A deeper understanding of the physical and molecular effects of supplementation and sire breed could provide additional avenues for manipulating adipose tissue.

LncRNA MSTRG4710 Promotes the Proliferation and Differentiation of Preadipocytes through miR-29b-3p/IGF1 Axis

Obesity, a major global health issue, is increasingly associated with the integral role of long non-coding RNA (lncRNA) in adipogenesis. Recently, we found that lncRNA-MSTRG4710 was highly expressed in the liver of rabbits fed a high-fat diet, but whether it is involved in lipid metabolism remains unclear. A series of experiments involving CCK-8, EDU, qPCR, and Oil Red O staining demonstrated that the overexpression of MSTRG4710 stimulated the proliferation and differentiation of preadipocytes while its knockdown inhibited these processes. Bioinformatics analysis showed that miR-29b-3p was a potential target gene of MSTRG4710, and IGF1 was a downstream target gene of miR-29b-3p. Luciferase reporter gene analysis and qPCR analysis confirmed that miR-29b-3p was a potential target gene of MSTRG4710, and miR-29b-3p directly targeted the 3′UTR of IGF1. The overexpression of miR-29b-3p was observed to regulate IGF1 protein and mRNA levels negatively. Additionally, a total of 414 known differentially expressed genes between the miR-29b-3p mimic, miR-29b-3p negative control (NC), siMSTRG4710, and siMSTRG4710-NC group were screened via transcriptome sequencing technology. The GO- and KEGG-enriched pathways were found to be related to lipid metabolism. The study also established that miR-29b-3p targets IGF1 to inhibit preadipocyte proliferation and differentiation. Notably, IGF1 knockdown significantly reduced preadipocyte proliferation and differentiation. Furthermore, co-transfection of pcDNA3.1(+)-MSTRG4710 and mimics into rabbit preadipocytes revealed that the mimics reversed the promotional effect of pcDNA3.1(+)-MSTRG4710. In conclusion, these results uncover that MSTRG4710 positively regulated cell proliferation and adipogenesis by the miR-29b-3p/IGF1 axis. Our findings might provide a new target for studying adipogenesis in rabbit preadipocytes and obesity.

Low concentrations of antimony impair adipogenesis and endoplasmic reticulum homeostasis during 3T3-L1 cells differentiation

Antimony (Sb) is a metalloid widely present in plastics used for food contact packaging, toys and other household items. Since Sb can be released by these plastics and come into contact with humans, health concerns have been highlighted. The effect of Sb on human tissues is yet controversial, and biochemical mechanisms of toxicity are lacking. In the present study, the effect of very low nanomolar concentrations of Sb(III), able to mimicking chronic human exposure, was evaluated in 3T3-L1 murine cells during the differentiation process. Low nanomolar Sb exposure (from 0.05 to 5 nM) induced lipid accumulation and a marked increase in C/EBP-β and PPAR-γ levels, the master regulators of adipogenesis. The Sb-induced PPAR-γ was reverted by the estrogen receptor antagonist ICI 182,780. Additionally, Sb stimulated preadipocytes proliferation inducing G2/M phase of cell cycle and this effect was associated to reduced cell-cycle inhibitor p21 levels. In addition to these metabolic dysfunctions, Sb activated the proinflammatory NF-κB pathway and altered endoplasmic reticulum (ER) homeostasis inducing ROS increase, ER stress markers XBP-1s and pEIF2a and downstream genes, such as Grp78 and CHOP. This study, for the first time, supports obesogenic effects of low concentrations exposure of Sb during preadipocytes differentiation.

Bovine C-X-C Motif Chemokine Ligand 14 Expression Is Regulated by Alternative Polyadenylation and MicroRNAs.

Alternative polyadenylation (APA), including APA that occurs only in the 3' UTR (3' UTR-APA), is an important post-transcriptional regulatory mechanism that leads to distinct 3' UTRs for some genes, increasing the complexity of the transcriptome. The post-transcriptional events regulating the expression of bovine, the C-X-C motif chemokine ligand 14 (CXCL14) gene, remains largely unknown. Here, we find that the bovine CXCL14 gene produces two different lengths of mRNA isoforms due to 3' UTR-APA, and the short and long 3' UTR is 126 bp and 1155 bp, respectively. We found that the expression level of the short isoform was significantly higher than that of the long isoform by luciferase assays and overexpression of different CXCL14 3' UTR-APA isoforms. Moreover, using luciferase assay and site-directed mutagenesis experiments, the results showed that the long CXCL14 3' UTR-APA isoform is downregulated by miR-17-5p, miR-150, and miR-217. However, because the short isoform lacks the true target of miR-17-5p, miR-150, and miR-217 in its 3' UTR and thus escapes the inhibitory effect of these microRNAs, its expression level is significantly higher than that of the long isoform. Finally, we demonstrate that the short CXCL14 3' UTR-APA isoform promotes preadipocyte proliferation by cell counting kit 8 (CCK8) assays. Collectively, our results show that the CXCL14 gene is post-transcriptionally regulated through APA and microRNAs.

A Comprehensive Review of Phytonutrients as a Dietary Therapy for Obesity.

Obesity is a complex medical condition mainly caused by eating habits, genetics, lifestyle, and medicine. The present study deals with traditional diets like the Mediterranean diet, Nordic diet, African Heritage diet, Asian diet, and DASH, as these are considered to be sustainable diets for curing obesity. However, the bioavailability of phytonutrients consumed in the diet may vary, depending on several factors such as digestion and absorption of phytonutrients, interaction with other substances, cooking processes, and individual differences. Hence, several phytochemicals, like polyphenols, alkaloids, saponins, terpenoids, etc., have been investigated to assess their efficiencies and safety in the prevention and treatment of obesity. These phytochemicals have anti-obesity effects, mediated via modulation of many pathways, such as decreased lipogenesis, lipid absorption, accelerated lipolysis, energy intake, expenditure, and preadipocyte differentiation and proliferation. Owing to these anti-obesity effects, new food formulations incorporating these phytonutrients were introduced that can be beneficial in reducing the prevalence of obesity and promoting public health.

New role for the anandamide metabolite prostaglandin F2α ethanolamide: Rolling preadipocyte proliferation

White adipose tissue regulation is key to metabolic health, yet still perplexing. The chief endocannabinoid anandamide metabolite, prostaglandin F2α ethanolamide (PGF2αEA), inhibits adipogenesis, that is, the formation of mature adipocytes. We observed that adipocyte progenitor cells—preadipocytes—following treatment with PGF2αEA yielded larger pellet sizes. Thus, we hypothesized that PGF2αEA might augment preadipocyte proliferation. Cell viability MTT and crystal violet assays, cell counting, and 5-bromo-2′-deoxyuridine incorporation in cell proliferation ELISA analyses confirmed our prediction. Additionally, we discovered that PGF2αEA promotes cell cycle progression through suppression of the expression of cell cycle inhibitors, p21 and p27, as shown by flow cytometry and qPCR. Enticingly, concentrations of this compound that showed no visible effect on cell proliferation or basal transcriptional activity of peroxisome proliferator-activated receptor gamma could, in contrast, reverse the anti-proliferative and peroxisome proliferator-activated receptor gamma-transcription activating effects of rosiglitazone (Rosi). MTT and luciferase reporter examinations supported this finding. The PGF2αEA pharmaceutical analog, bimatoprost, was also investigated and showed very similar effects. Importantly, we suggest the implication of the mitogen-activated protein kinase pathway in these effects, as they were blocked by the selective mitogen-activated protein kinase kinase inhibitor, PD98059. We propose that PGF2αEA is a pivotal regulator of white adipose tissue plasticity, acting as a regulator of the preadipocyte pool in adipose tissue.

MiR-383-5p Regulates Preadipocyte Proliferation and Differentiation by Targeting RAD51AP1.

Obesity has become a major health problem worldwide, and increasing evidence supports the importance of microRNAs (miRNAs) in its pathogenesis. Recently, we found that miR-383-5p_1 is highly expressed in the perirenal fat of high-fat-fed rabbits, but it is not yet known whether miR-383-5p is involved in lipid metabolism. Here, we used transcriptome sequencing technology to screen 1642 known differentially expressed genes between miR-383-5p mimic groups and miR-383-5p negative control groups. Gene Ontology Resource (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched in the pathway related to lipid metabolism, and glycine biosynthesis, the NOD receptor signal pathway and nonalcoholic fatty liver were significantly enriched. Afterwards, our research results indicated that miR-383-5p can promote the proliferation and differentiation of rabbit preadipocytes, and there is a direct targeting relationship with RAD51AP1. Mechanistically, miR-383-5p directly interacts with the lipid metabolism and participates in adipogenesis and lipid accumulation by targeting RAD51AP1. In conclusion, our data highlight a physiological role for miRNA in lipid metabolism and suggest the miR-383-5p/RAD51AP1 axis may represent a potential mechanism for controlling lipid accumulation in obesity.

Effects of HOXC8 on the Proliferation and Differentiation of Porcine Preadipocytes.

Transcription factor Homeobox C8 (HOXC8) is identified as a white adipose gene as revealed by expression profile analysis in fat tissues. However, the specific role of HOXC8 in fat accumulation remains to be identified. This study was designed to reveal the effects of HOXC8 on preadipocyte proliferation and differentiation. We first make clear that the expression of HOXC8 is associated with fat contents in muscles, highlighting a role of HOXC8 in fat accumulation. Next, it is demonstrated that HOXC8 promotes the proliferation and differentiation of preadipocytes through gain- and loss-of-function assays in primary cultured porcine preadipocytes. Then, mechanisms underlying the regulation of HOXC8 on preadipocyte proliferation and differentiation are identified with RNA sequencing, and a number of differentially expressed genes (DEGs) in response to HOXC8 knockdown are identified. The top GO (Gene Ontology) terms enriched by DEGs involved in proliferation and differentiation, respectively, are identical. IL-17 signaling pathway is the common one significantly enriched by DEGs involved in preadipocyte proliferation and differentiation, respectively, indicating its importance in mediating fat accumulation regulated by HOXC8. Additionally, we find that the inhibition of proliferation is one of the main processes during preadipocyte differentiation. The results will contribue to further revealing the mechanisms underlying fat accumulation regulated by HOXC8.

Bta-miR-484 Targets SFRP1 and Affects Preadipocytes Proliferation, Differentiation, and Apoptosis.

MicroRNAs (miRNAs) are essential regulators of numerous biological processes in animals, including adipogenesis. Despite the abundance of miRNAs associated with adipogenesis, their exact mechanisms of action remain largely unknown. Our study highlights the role of bta-miR-484 as a major regulator of adipocyte proliferation, apoptosis, and differentiation. Here, we demonstrated that the expression of bta-miR-484 initially increased during adipogenesis before decreasing. Overexpression of bta-miR-484 in adipocytes ultimately inhibited cell proliferation and differentiation, reduced the number of EdU fluorescence-stained cells, increased the number of G1 phase cells, reduced the number of G2 and S phase cells, and downregulated the expression of proliferation markers (CDK2 and PCNA) and differentiation markers (CEBPA, FABP4, and LPL). Additionally, overexpression of bta-miR-484 promoted the expression of apoptosis-related genes (Caspase 3, Caspase 9, and BAX), and increased the number of apoptotic cells observed via flow cytometry. In contrast, bta-miR-484 inhibition in adipocytes yielded opposite effects to those observed during bta-miR-484 overexpression. Moreover, luciferase reporter assays confirmed SFRP1 as a target gene of bta-miR-484, and revealed that bta-miR-484 downregulates SFRP1 mRNA expression. These findings offer compelling evidence that bta-miR-484 targets SFRP1, inhibits proliferation and differentiation, and promotes apoptosis. Therefore, these results offer novel insights into the bta-miR-484 regulation of adipocyte growth and development.

Pleiotrophin and metabolic disorders: insights into its role in metabolism.

Pleiotrophin (PTN) is a cytokine which has been for long studied at the level of the central nervous system, however few studies focus on its role in the peripheral organs. The main aim of this review is to summarize the state of the art of what is known up to date about pleiotrophin and its implications in the main metabolic organs. In summary, pleiotrophin promotes the proliferation of preadipocytes, pancreatic β cells, as well as cells during the mammary gland development. Moreover, this cytokine is important for the structural integrity of the liver and the neuromuscular junction in the skeletal muscle. From a metabolic point of view, pleiotrophin plays a key role in the maintenance of glucose and lipid as well as whole-body insulin homeostasis and favors oxidative metabolism in the skeletal muscle. All in all, this review proposes pleiotrophin as a druggable target to prevent from the development of insulin-resistance-related pathologies.

CircDOCK7 facilitates the proliferation and adipogenic differentiation of chicken abdominal preadipocytes through the gga-miR-301b-3p/ACSL1 axis

BackgroundAbdominal fat deposition depends on both the proliferation of preadipocytes and their maturation into adipocytes, which is a well-orchestrated multistep process involving many regulatory molecules. Circular RNAs (circRNAs) have emergingly been implicated in mammalian adipogenesis. However, circRNA-mediated regulation in chicken adipogenesis remains unclear. Our previous circRNA sequencing data identified a differentially expressed novel circRNA, 8:27,886,180|27,889,657, during the adipogenic differentiation of chicken abdominal preadipocytes. This study aimed to investigate the regulatory role of circDOCK7 in the proliferation and adipogenic differentiation of chicken abdominal preadipocytes, and explore its molecular mechanisms of competing endogenous RNA underlying chicken adipogenesis.ResultsOur results showed that 8:27,886,180|27,889,657 is an exonic circRNA derived from the head-to-tail splicing of exons 19–22 of the dedicator of cytokinesis 7 (DOCK7) gene, abbreviated as circDOCK7. CircDOCK7 is mainly distributed in the cytoplasm of chicken abdominal preadipocytes and is stable because of its RNase R resistance and longer half-life. CircDOCK7 is significantly upregulated in the abdominal fat tissues of fat chickens compared to lean chickens, and its expression gradually increases during the proliferation and adipogenic differentiation of chicken abdominal preadipocytes. Functionally, the gain- and loss-of-function experiments showed that circDOCK7 promoted proliferation, G0/G1- to S-phase progression, and glucose uptake capacity of chicken abdominal preadipocytes, in parallel with adipogenic differentiation characterized by remarkably increased intracellular lipid droplet accumulation and triglyceride and acetyl coenzyme A content in differentiated chicken abdominal preadipocytes. Mechanistically, a pull-down assay and a dual-luciferase reporter assay confirmed that circDOCK7 interacted with gga-miR-301b-3p, which was identified as an inhibitor of chicken abdominal adipogenesis. Moreover, the ACSL1 gene was demonstrated to be a direct target of gga-miR-301b-3p. Chicken ACSL1 protein is localized in the endoplasmic reticulum and mitochondria of chicken abdominal preadipocytes and acts as an adipogenesis accelerator. Rescue experiments showed that circDOCK7 could counteract the inhibitory effects of gga-miR-301b-3p on ACSL1 mRNA abundance as well as the proliferation and adipogenic differentiation of chicken abdominal preadipocytes.ConclusionsCircDOCK7 serves as a miRNA sponge that directly sequesters gga-miR-301b-3p away from the ACSL1 gene, thus augmenting adipogenesis in chickens. These findings may elucidate a new regulatory mechanism underlying abdominal fat deposition in chickens.

Rna M6 a Methylation Regulates Glycolysis of Beige Fat and Contributes to Systemic Metabolic Homeostasis.

N6-methyladenosine (m6 A) modification has been implicated in the progression of obesity and metabolic diseases. However, its impact on beige fat biology is not well understood. Here, via m6 A-sequencing and RNA-sequencing, this work reports that upon beige adipocytes activation, glycolytic genes undergo major events of m6 A modification and transcriptional activation. Genetic ablation of m6 A writer Mettl3 in fat tissues reveals that Mettl3 deficiency in mature beige adipocytes leads to suppressed glycolytic capability and thermogenesis, as well as reduced preadipocytes proliferation via glycolytic product lactate. In addition, specific modulation of Mettl3 in beige fat via AAV delivery demonstrates consistently Mettl3's role in glucose metabolism, thermogenesis, and beige fat hyperplasia. Mechanistically, Mettl3 and m6 A reader Igf2bp2 control mRNA stability of key glycolytic genes in beige adipocytes. Overall, these findings highlight the significance of m6 A on fat biology and systemic energy homeostasis.

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.

1505-P: CD44 Knockdown in Human Adipocytes—Effect on Adipocyte Proliferation and Differentiation

Introduction: eGWAS implicates the immune-cell receptor CD44 as the top candidate gene in the molecular pathogenesis of T2D1. We have shown that in human adipose tissue, CD44 expression is associated with inflammatory gene expression, M1 macrophage phenotype, and systemic insulin resistance2. Here we sought to determine whether CD44 directly impairs adipocyte function via siRNA knockdown in human preadipocytes with quantification of proliferation, differentiation, and gene expression. Methods: Abdominal subcutaneous adipose tissue was harvested from five healthy nondiabetic obese individuals during bariatric surgery. Preadipocytes were isolated via collagenase digestion and seeded at 1×10^4 cells/well and treated with siRNA-CD44 or mock condition (control) using siRNA Maxi Transfection Reagents (Invitrogen). Cell proliferation was assayed by MTS (Promega) on Days 1, 2, 3 post transfection. Differentiation was induced at confluence (as described3), and cells were harvested at days 3, 6, 12 after confluence for gene expression and at day 12 for oil red O staining (total lipid uptake) and immunofluorescence microscopy (% differentiation = bodipy/dapi*100). Results: CD44 knockdown decreased preadipocyte proliferation and increased adipocyte differentiation and lipid uptake. Conclusion: CD44 may contribute to human insulin resistance by impairing adipocyte differentiation and fat storage. Disclosure H.Chen: None. N.R.Saini: None. E.M.Ayhan: None. T.Mclaughlin: Board Member; January, Inc., Research Support; Merck & Co., Inc., Stock/Shareholder; Eiger Bio. Funding National Institutes of Health (1R21AI159024-01A1)

β-Hydroxy-β-methyl Butyrate Regulates the Lipid Metabolism, Mitochondrial Function, and Fat Browning of Adipocytes.

A growing number of in vivo studies demonstrated that β-hydroxy-β-methyl butyrate (HMB) can serve as a lipid-lowering nutrient. Despite this interesting observation, the use of adipocytes as a model for research is yet to be explored. To ascertain the effects of HMB on the lipid metabolism of adipocytes and elucidate the underlying mechanisms, the 3T3-L1 cell line was employed. Firstly, serial doses of HMB were added to 3T3-L1 preadipocytes to evaluate the effects of HMB on cell proliferation. HMB (50 µM) significantly promoted the proliferation of preadipocytes. Next, we investigated whether HMB could attenuate fat accumulation in adipocytes. The results show that HMB treatment (50 µM) reduced the triglyceride (TG) content. Furthermore, HMB was found to inhibit lipid accumulation by suppressing the expression of lipogenic proteins (C/EBPα and PPARγ) and increasing the expression of lipolysis-related proteins (p-AMPK, p-Sirt1, HSL, and UCP3). We also determined the concentrations of several lipid metabolism-related enzymes and fatty acid composition in adipocytes. The HMB-treated cells showed reduced G6PD, LPL, and ATGL concentrations. Moreover, HMB improved the fatty acid composition in adipocytes, manifested by increases in the contents of n6 and n3 PUFAs. The enhancement of the mitochondrial respiratory function of 3T3-L1 adipocytes was confirmed via Seahorse metabolic assay, which showed that HMB treatment elevated basal mitochondrial respiration, ATP production, H+ leak, maximal respiration, and non-mitochondrial respiration. In addition, HMB enhanced fat browning of adipocytes, and this effect might be associated with the activation of the PRDM16/PGC-1α/UCP1 pathway. Taken together, HMB-induced changes in the lipid metabolism and mitochondrial function may contribute to preventing fat deposition and improving insulin sensitivity.

The anti-obesity effects of resveratrol on the 3T3-L1 adipocytes.

Obesity is a severe health problem worldwide due to its association with various adverse health consequences. The present study aims to evaluate the anti-obesity effects of resveratrol, as a natural polyphenol, on the 3T3-L1 adipocytes. PubMed, Scopus, ScienceDirect, Web of Sciences, and Google Scholar databases were searched up to March 2022 using relevant keywords. All original articles, written in English, evaluating the anti-obesity effects of resveratrol on the 3T3-L1 adipocytes were eligible for this review. Initially, 4361 records were found in the electronic search databases. After removing duplicates and irrelevant studies according to the title and abstract, the full text of the 51 articles was critically screened and 38 in vitro studies were included in this review. Except for one case, all of these studies reported that different doses (ranged 1-200 μM) of resveratrol treatment have anti-obesity effects on 3T3L1 adipocytes through various mechanisms such as induction of apoptosis, a decrease of fat accumulation and adipogenesis, promotion of white adipocytes browning, inhibition of preadipocyte proliferation and consequent differentiation, and up-regulation of miRNA that involved in the antiadipogenic and triacylglycerol metabolism in white adipose tissue. The findings indicate that resveratrol has anti-obesity effects. Therefore, resveratrol treatment could be used to prevent and treat obesity and its related disorders. Well-designed randomized clinical trials with different doses of resveratrol are recommended to be performed on obese subjects.