Subcutaneous Preadipocytes Research Articles

EGCG alleviates heat-stress-induced fat deposition by targeting HSP70 through activation of AMPK-SIRT1-PGC-1α in porcine subcutaneous preadipocytes

Obesity has emerged as a prominent global health concern, with heat stress posing a significant challenge to both human health and animal well-being. Despite a growing interest in environmental determinants of obesity, very few studies have examined the associations between heat stress-related environmental factors and adiposity. Consequently, there exists a clear need to understand the molecular mechanisms underlying the obesogenic effects of heat stress and to formulate preventive strategies. This study focused on culturing porcine subcutaneous preadipocytes at 41.5 ℃ to induce heat stress, revealing that this stressor triggered apoptosis and fat deposition. Analysis demonstrated an upregulation in the expression of HSP70, BAX, adipogenesis-related genes (PPARγ, AP2, CEBPα and FAS), the p-AMPK/AMPK ratio and SIRT1, PGC-1α in the heat stress group compared to the control group (P < 0.05). Conversely, the expression of lipid lysis-related genes (ATGL, HSL and LPL) and Bcl-2 decreased in the heat stress group compared to the control group (P < 0.05). Furthermore, subsequent activator and/or inhibitor experiments validated that heat stress modulated HSP70 and AMPK signalling pathways to enhance lipogenesis and inhibit lipolysis in porcine subcutaneous preadipocytes. Importantly, this study reveals, for the first time, that EGCG mitigates heat-stress-induced fat deposition by targeting HSP70 through the activation of AMPK-SIRT1-PGC-1α in porcine subcutaneous preadipocytes. These findings elucidate the molecular mechanisms contributing to heat stress-induced obesity and provide a foundation for the potential clinical utilisation of EGCG as a preventive measure against both heat stress and obesity.

Systems genetics analysis of human body fat distribution genes identifies adipocyte processes.

Excess abdominal fat is a sexually dimorphic risk factor for cardio-metabolic disease and is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Whereas this trait is highly heritable, few causal genes are known. We aimed to identify novel drivers of WHRadjBMI using systems genetics. We used two independent cohorts of adipose tissue gene expression and constructed sex- and depot-specific Bayesian networks to model gene-gene interactions from 8,492 genes. Using key driver analysis, we identified genes that, in silico and putatively in vitro, regulate many others. 51-119 key drivers in each network were replicated in both cohorts. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We overexpressed or down-regulated seven key driver genes in human subcutaneous pre-adipocytes. Key driver genes ANAPC2 and RSPO1 inhibited adipogenesis, whereas PSME3 increased adipogenesis. RSPO1 increased Wnt signaling activity. In differentiated adipocytes, MIGA1 and UBR1 down-regulation led to mitochondrial dysfunction. These five genes regulate adipocyte function, and we hypothesize that they regulate fat distribution.

MiR‐429 inhibits palmitic acid‐induced apoptosis of porcine subcutaneous preadipocytes by targeting Sox5

AbstractMicroRNAs (miRNAs) can regulate several physiological processes of cells after transcription, such as cell proliferation, differentiation, and apoptosis. In this study, we found that 500 μM of palmitic acid (PA) could significantly induce the apoptosis of porcine subcutaneous preadipocytes (p &lt; 0.05). The overexpression of miR‐429 decreased the apoptotic rate of porcine preadipocytes and inhibited the expression of the proapoptotic gene P53 (p &lt; 0.05). In addition, miR‐429 can specifically bind to the 3′ untranslated region of Sox5, and the upregulation of miR‐429 downregulated Sox5 expression. However, Sox5 overexpression promoted the apoptosis of porcine preadipocytes (p &lt; 0.01); the co‐transfection of miR‐429 and pcDNA3.1‐Sox5 into preadipocytes could reverse the inhibition of PA‐induced apoptosis by miR‐429. In conclusion, the present results provide a theoretical basis for elucidating the molecular mechanisms by which miR‐429 and Sox5 regulate the apoptosis of porcine subcutaneous preadipocytes.

Mitochondrial Ca2+ signaling is a hallmark of specific adipose tissue-cancer crosstalk

Obesity is associated with increased risk and worse prognosis of many tumours including those of the breast and of the esophagus. Adipokines released from the peritumoural adipose tissue promote the metastatic potential of cancer cells, suggesting the existence of a crosstalk between the adipose tissue and the surrounding tumour. Mitochondrial Ca2+ signaling contributes to the progression of carcinoma of different origins. However, whether adipocyte-derived factors modulate mitochondrial Ca2+ signaling in tumours is unknown. Here, we show that conditioned media derived from adipose tissue cultures (ADCM) enriched in precursor cells impinge on mitochondrial Ca2+ homeostasis of target cells. Moreover, in modulating mitochondrial Ca2+ responses, a univocal crosstalk exists between visceral adipose tissue-derived preadipocytes and esophageal cancer cells, and between subcutaneous adipose tissue-derived preadipocytes and triple-negative breast cancer cells. An unbiased metabolomic analysis of ADCM identified creatine and creatinine for their ability to modulate mitochondrial Ca2+ uptake, migration and proliferation of esophageal and breast tumour cells, respectively.

The myokine CCL5 recruits subcutaneous preadipocytes and promotes intramuscular fat deposition in obese mice.

Intramuscular fat (IMF) refers to the lipid stored in skeletal muscle tissue. The number and size of intramuscular adipocytes are the primary factors that regulate IMF content. Intramuscular adipocytes can be derived from either in situ or ectopic migration. In this study, it was discovered that the regulation of IMF levels is achieved through the chemokine (C-C motif) ligand 5 (CCL5)/chemokine (C-C motif) receptor 5 (CCR5) pathway by modulating adipocyte migration. In coculture experiments, C2C12 myotubes were more effective in promoting the migration of 3T3-L1 preadipocytes than C2C12 myoblasts, along with increasing CCL5. Correspondingly, overexpressing the CCR5, one of the receptors of CCL5, in 3T3-L1 preadipocytes facilitated their migration. Conversely, the application of the CCL5/CCR5 inhibitor, MARAVIROC (MVC), reduced this migration. In vivo, transplanted experiments of subcutaneous adipose tissue (SCAT) from transgenic mice expressing green fluorescent protein (GFP) provided evidence that injecting recombinant CCL5 (rCCL5) into skeletal muscle promotes the migration of subcutaneous adipocytes to the skeletal muscle. The level of CCL5 in skeletal muscle increased with obesity. Blocking the CCL5/CCR5 axis by MVC inhibited IMF deposition, whereas elevated skeletal muscle CCL5 promoted IMF deposition in obese mice. These results establish a link between the IMF and the CCL5/CCR5 pathway, which could have a potential application for modulating IMF through adipocyte migration.NEW & NOTEWORTHY C2C12 myotubes attract 3T3-L1 preadipocyte migration regulated by the chemokine (C-C motif) ligand 5 (CCL5)/ chemokine (C-C motif) receptor 5 (CCR5) axis. High levels of skeletal muscle-specific CCL5 promote the migration of subcutaneous adipocytes to skeletal muscle and induce the intramuscular fat (IMF) content.

Silencing ANGPTL8 reduces mouse preadipocyte differentiation and insulin signaling

ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.

κ-Carrageenan/konjac glucomannan composite hydrogel-based 3D porcine cultured meat production

Alternative technology for cultured meat production holds the potential to alleviate ethical, environmental, and public health concerns stemming from conventional husbandry. As a key food component, fat plays a crucial role in the sensory characteristics and nutritional value of meat products. However, current adipose tissue engineering protocols have so far proven unsuitable for edible cultured fat production. In this study, hybrid hydrogels based on Κ-Carrageenan (Car) and konjac glucomannan (KGM) were designed as a culture scaffold for adipocytes. The edible hydrogel with excellent biocompatibility, food safety, and low cost was demonstrated to support cell proliferation and allow the formation of tissue-like cell spheres. Furthermore, both porcine subcutaneous pre-adipocytes (PreAs) and fibro-adipogenic progenitors (FAPs) acquired mature adipocyte characteristics and higher lipid accumulation in Car-KGM (CK) hydrogel, suggesting its superior ability to support in vitro adipogenesis than conventional monolayer culture and alginate (Alg)-based 3D culture. Taken together, the excellent nature of CK hydrogel makes it a versatile platform for improving the molecular and cellular phenotypes of a variety of adipocyte models. The established spheroid model opens possibilities for broad applications in cultivated fat and represents a viable strategy for the manufacture of ‘snowflake pork’.

Effect of DHCR7 on adipocyte differentiation in goats

Cholesterol is regarded as a signaling molecule in regulating the metabolism and function of fat cells, in which 7-Dehydrocholesterol reductase (DHCR7) is a key enzyme that catalyzes the conversion of 7-dehydrocholesterol to cholesterol, however, the exact function of DHCR7 in goat adipocytes remains unknown. Here, the effect of DHCR7 on the formation of subcutaneous and intramuscular fat in goats was investigated in vitro, and the result indicated that the mRNA level of DHCR7 showed a gradual downward trend in subcutaneous adipogenesis, but an opposite trend in intramuscular adipogenesis. In the process of subcutaneous preadipocytes differentiation, overexpression of DHCR7 inhibited the expression of adipocytes differentiation marker genes (CEBP/α, CEBP/β, SREBP1 and AP2), lipid metabolism-related genes (AGPAT6, FASN, SCD1 and LPL), and the lipid accumulation. However, in intramuscular preadipocyte differentiation, DHCR7 overexpression showed a promoting effect on adipocyte differentiation marker genes (CEBP/α, CEBP/β, PPARγ and SREBP1) and lipid metabolism-related genes (GPAM, AGPAT6, DGAT1 and SCD1) expression, and on lipid accumulation. In summary, our work demonstrated that DHCR7 played an important role in regulating adipogenic differentiation and lipid metabolism in preadipocytes in goats, which is of great significance for uncovering the underlying molecular mechanism of adipocyte differentiation and improving goat meat quality.

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.

Bioactive peptides PDBSN improve mitochondrial function and suppression the oxidative stress in human adiposity cells

ABSTRACT Introduction: Mitochondria are essential for generating cellular energy and are significant in the pathogenesis of obesity. Peptide PDBSN has been demonstrated to inhibit the adipogenic differentiation of adipocytes in vitro and improves metabolic homoeostasis in vivo. Therefore, in this study, we further investigated the effects of PDBSN on the morphology, synthesis, and function of adipocyte mitochondria. Methods: Human visceral and subcutaneous primary preadipocytes (HPA-v and HPA-s) were cultured into mature adipocytes. Intracellular triglyceride content was assessed using oil-red O staining and tissue triglyceride determination. Gene and protein levels associated with mitochondrial synthesis were detected using real-time quantitative polymerase chain reaction and western blotting. Mitochondrial membrane potentials and ROS were detected using fluorescent indicators. Morphological changes were observed by electron microscopy. Results: PDBSN significantly increased mitochondrial membrane potential (MMP), while decreasing intracellular triglyceride (TG) and intracellular reactive oxygen species (ROS) levels. On the other hand, the transcription and protein levels of genetic marker genes PGC1-α and MTFA were significantly up-regulated after PDBSN administration. Further studies showed that transcriptional and protein levels of mitochondrial fusion and fission genetic markers MFN1, MFN2, NRF1, and DRP1 increased. Conclusion: PDBSN significantly reduces intracellular TG and ROS levels and increases MMP. The maximum respiratory capacity in adults significantly increases after PDBSN administration, and ROS levels are significantly reduced. This suggests that PDBSN improves mitochondrial function to some extent, which not only provides an essential basis for the pathophysiology of obesity but also provides insights for the development of new drugs to treat obesity and metabolic diseases.

250 The Impact of Altering Fatty Acid Combinations in Vitro on Bovine Preadipocyte Hyperplasia

Abstract In the cattle industry, fat deposited on a carcass can have advantages and disadvantages depending on location and amount. Excess subcutaneous fat deposition reduces carcass red meat yield, while intramuscular fat (IMF) deposition, or marbling, increases carcass value and enhances tenderness and flavor. Two distinct adipocyte growth cycles can be attributed to total fat deposition: hypertrophy and hyperplasia. Hyperplasia refers to the adipogenic side of growth where there is an increase in preadipocyte cell number before cell differentiation. This study aimed to investigate the effects of altering fatty acid composition of cell culture media in vitro on preadipocyte hyperplasia. Subcutaneous fat was harvested from Angus-cross yearling steers (n = 4). Subcutaneous preadipocytes were isolated and propagated for a series of six passages. Preadipocytes were subsequently seeded, and eight different treatment mixtures with combinations of different fatty acids (C16:0, C18:0, C18:1cis-9, and C18:2cis-9,12) at varying ratios were added to the basal media (DMEM F12, 10% bovine fetal serum, 1% HEPES, 1% AB/AM) for a period of four days. Cell culture treatment media was changed daily. Each individual media mix was applied at the same total molar concentration (200 ug). At the end of the four-day treatment period, cells were harvested for RNA extraction and fatty acid analysis. The expression of critical adipogenic and lipogenic genes was evaluated. Treatments displayed differences (P &amp;lt; 0.05) in total fatty acid content (ug), which would suggest that the addition of fatty acids to media could push primary bovine preadipocytes into differentiation. Mix 8 (75% 18:1cis-9 - 25% 18:2cis-9,12) exhibited the greatest (P &amp;lt; 0.05) fatty acid content (ug), while mix 5 (50% C16:0- 50% C18:0) had the least (P &amp;lt; 0.05) fatty acid amount (ug). The expression of fatty acid binding protein-4 (FABP4) and stearoyl Co-A desaturase-1 (SCD-1) differed (P &amp;lt; 0.05) across treatments. The expression of peroxisome proliferating-activated receptor gamma (PPARy), sterol-regulatory element-binding protein (SREB-1c), and SREBP cleavage-activating protein (SCAP) were all downregulated (P &amp;lt; 0.05) in the fully saturated fatty acid mix, mix 5 (50% C16:0- 50% C18:0), compared with all other treatments. Based on the data, it appears that the addition of fatty acids at different ratios to media can affect molecular mechanisms, hyperplastic growth, and potentially the differentiation of primary bovine adipocytes. Additional examination of fatty acid supplementation in vitro could provide valuable insight into their effects on adipocyte growth and how it could be applied to alter fat deposition in vivo.

Comparative alternative polyadenylation profiles in differentiated adipocytes of subcutaneous and intramuscular fat tissue in cattle

Alternative polyadenylation (APA) is a key molecular mechanism involved in the post-transcriptional regulation of gene expression, which has been proven to play a critical role in cell differentiation. In the present study, we performed IVT-SAPAS sequencing to profile the dynamic changes of APA sites in bovine subcutaneous preadipocytes and intramuscular preadipocytes during adipogenesis. A total of 52621 high quality APA sites were identified in preadipocytes and adipocytes. Compared with preadipocytes, the increased usage of canonical AATAAA was observed in the cell-biased APA sites of adipocytes. Furthermore, 1933 and 2140 differentially expressed APA (DE-APA) sites, as well as 341 and 337 untranslated region-APA (UTR-APA) switching genes were identified in subcutaneous preadipocytes and intramuscular preadipocytes during adipogenesis, respectively. The UTR-APA switching genes showed divergent trends in preadipocytes, among which UTR-APA switching genes in intramuscular preadipocytes tended to use shorter 3′UTR for differentiation into mature adipocytes. APA events mediated by UTR-APA switching in intramuscular adipocytes were enriched in lipid synthesis and adipocyte differentiation. TRIB3, WWTR1, and INSIG1 played important roles in the differentiation of intramuscular preadipocytes. Briefly, our results provided new insights into understanding the mechanisms of bovine adipocyte differentiation.

FRI018 A Novel Truncating Variant Of EBF2 Disrupts Human Adipocyte Differentiation In Lipodystrophy Syndromes: An Example Of A Discovery From A Clinical Translational Pipeline

Abstract Disclosure: M.C. Foss de Freitas: Advisory Board Member; Self; PTC Therapeutics. N. Wys: None. M. Udler: None. L. Pais: None. A. Monteiro da Rocha: None. O.A. MacDougald: None. E.A. Oral: Consulting Fee; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Aegerion Pharmaceuticals, Ionis Pharmaceuticals Inc., Novo Nordisk, Fractyl Laboratories, Rhythm pharmaceuticals, GID Dynamics. Other; Self; Aegerion Pharmaceuticals. T. Chun: None. Objective/Goals: Partial Lipodystrophy is characterized by the gradual loss of adipose tissues, which progresses after birth. The genetic and molecular underpinning of many cases of partial lipodystrophy have not been fully understood. Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy, we initiated deep sequencing for our patients with partial lipodystrophy syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. Methods: We performed loss-of-function and gene rescue experiments using 3T3-L1 and human primary subcutaneous preadipocytes. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen). After lentivirus transfection (Full-length EBF2 or truncated variant EBF2), cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone, insulin and isobutyl methylxanthine. Total RNA was extracted using RNeasy Mini Kit (Qiagen), and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. Results: The patient was found to carry a heterozygous nonsense mutation in exon 6 of 16 (NM_022659.4) of EBF2, causing the premature termination of the protein at amino acid position 165 (EBF2 8:26033143 C&amp;gt;A (hg38), c.493G&amp;gt;T, p.Glu165Ter). Adipogenesis was significantly suppressed in 3T3-L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed a marked reduction of intracellular lipid content and Pparg and Fabp4 expression (&amp;gt;80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 p.Glu165Ter did not. Of note, 3T3-L1 cells transfected with the EBF2 variant impaired adipogenesis, suggesting an inhibitory effect of the EBF2 variant on adipogenesis. We next confirmed the inhibition in adipogenesis with the EBF2 variant in primary human preadipocytes from subcutaneous adipose tissues. Using lentiviral gene transfer in human preadipocytes, we observed that the full-length EBF2 increased the gene expression of PPARg and FABP4 while the truncated EBF2 variant did not. Conclusion: Our data suggest that EBF2 is indispensable for adipogenesis of mouse and human preadipocytes. The truncating variant of EBF2 p.Glu165Ter likely plays a pathogenic role in the partial lipodystrophy presentation of this patient. Whole genome (or exome) sequencing undertaken in patients with partial lipodystrophy and subsequent biological functional analysis may lead to identification of novel mechanisms driving novel discoveries. Presentation: Friday, June 16, 2023

γδ T cells control murine skin inflammation and subcutaneous adipose wasting during chronic Trypanosoma brucei infection

African trypanosomes colonise the skin to ensure parasite transmission. However, how the skin responds to trypanosome infection remains unresolved. Here, we investigate the local immune response of the skin in a murine model of infection using spatial and single cell transcriptomics. We detect expansion of dermal IL-17A-producing Vγ6+ cells during infection, which occurs in the subcutaneous adipose tissue. In silico cell-cell communication analysis suggests that subcutaneous interstitial preadipocytes trigger T cell activation via Cd40 and Tnfsf18 signalling, amongst others. In vivo, we observe that female mice deficient for IL-17A-producing Vγ6+ cells show extensive inflammation and limit subcutaneous adipose tissue wasting, independently of parasite burden. Based on these observations, we propose that subcutaneous adipocytes and Vγ6+ cells act in concert to limit skin inflammation and adipose tissue wasting. These studies provide new insights into the role of γδ T cell and subcutaneous adipocytes as homeostatic regulators of skin immunity during chronic infection.

MiR-125a-3p regulates the expression of FSTL1, a pro-inflammatory factor, during adipogenic differentiation, and inhibits adipogenesis in mice.

Adipogenesis is tightly regulated by various factors, including genes and microRNAs. Excessive fat deposition is the key feature of obesity, which is a low-grade chronic inflammatory disease. Follistatin-like 1 (FSTL1) has been reported to be an important mediator involved in various inflammatory diseases. However, the underlying mechanism of FSTL1 in preadipocyte differentiation and inflammatory response is still unclear. The current study was designed to explore the biological function and potential mechanism of FSTL1 in mouse subcutaneous preadipocyte differentiation. We found that FSTL1 was highly expressed in the early stage of differentiation and subsequently decreased sharply, suggesting that FSTL1 played a possible role in adipogenesis. Meanwhile, the gain- and loss-of-function assays showed that FSTL1 was not only involved in the inflammatory response by inducing the expression of pro-inflammatory factors IL-1β and CCL2 but also significantly attenuated preadipocyte differentiation, as evidenced by the reduction of lipid accumulation and the levels of adipogenic genes, including PPARγ and FABP4. In addition, the target gene prediction and luciferase reporter assay validated that miR-125a-3p targeted the 3' UTR region of FSTL1. These results demonstrated that miR-125a-3p negatively regulated the expression of FSTL1 at the mRNA and protein levels. Furthermore, overexpressing miR-125a-3p in preadipocytes dramatically accelerated adipogenic differentiation and downregulated the levels of IL-1β and CCL2, which were in accordance with the knockdown of FSTL1. On the contrary, treatment with miR-125a-3p inhibitors attenuated adipogenesis but induced the expression of inflammatory genes. In summary, this study suggests a positive function of FSTL1 in adipocyte-induced inflammation and negatively regulates preadipocyte differentiation. Further studies demonstrated that miR-125a-3p could reverse the effect by targeting FSTL1, which might provide a better understanding of treating obesity-related inflammatory diseases.

5-OR: Adipocyte ANGPTL8 Regulates Insulin Sensitivity and Thermogenic Programing

ANGPTL8 (A8), expressed mainly in the liver and adipocytes, regulates lipoprotein lipase (LPL) and lipid metabolism. Hepatic A8 and ANGPTL3 form a complex that inhibits LPL. However, the precise intracellular role of A8 in adipocytes is yet to be explored. We observed that RNAi-knockdown of A8 in mouse primary subcutaneous (SC) pre-adipocytes reduces adipocyte differentiation, glycerol, and fatty acid release at day 7 post-differentiation. RNAseq analysis of A8 siRNA or control siRNA transfected SC pre-adipocytes on days 0, 2, 4 and 7 of differentiation showed that A8 silencing impeded adipogenic and oxidative phosphorylation pathways and reduced expression of insulin signaling pathway genes IRS1, IRS2, PPARG, mTOR, early during differentiation, suggesting a role for A8 in the regulation of insulin sensitivity. Next, we investigated the functional role of adipose A8 by generating conditional adipocyte-specific A8 Knockout (AT-A8-KO) mice. AT-A8-KO mice on 45% high fat diet (HFD) with 30% fructose for 20 weeks showed a decrease in body weight gain, glycemia, brown fat mass, plasma TG and smaller cell size of SC adipocytes, while increased rectal temperature compared to controls. Interestingly, OGTT revealed AT-A8-KO mice in control diet or in HFD have significantly lower insulinemia. ITT results confirmed significant improvement of insulin sensitivity of AT-A8-KO mice on HFD. In addition, upon exposure to 6°C for 24h, AT-A8-KO mice exhibited elevated body temperature and energy expenditure compared to control mice, indicating that adipose tissue A8 negatively modulates cold-induced thermogenesis. Our results reveal that A8 regulates in vitro adipocyte differentiation, probably by modulating insulin signaling and mitochondrial respiration, and that deletion of adipocyte A8 in mice enhances insulin sensitivity, cold induced body temperature and energy expenditure. Therefore, targeting adipose specific A8 may be beneficial in promoting insulin sensitivity and preventing obesity. Disclosure A.Ghosh: None. I.Al-khairi: None. J.Abubaker: None. F.Al-mulla: None. M.Abu-farha: None. M.Prentki: None. Y.H.Leung: None. J.C.Y.Yu: None. R.M.Sladek: None. I.Chenier: None. A.Oppong: None. M.Peyot: None. M.Madiraju: None. Funding Dasman Diabetes Institute (731)

Effect of CXCL17 on Subcutaneous Preadipocytes Proliferation in Goats.

The presence or absence of subcutaneous adipose accumulation will affect the energy storage, insulation resistance and metabolism of animals. Proliferation and differentiation of preadipocytes play a significant role in lipid deposition. The objective of this study was to clone the goat CXCL17 gene and investigate its potential functions on goat subcutaneous preadipocytes' proliferation by gaining or losing function in vitro. The goat CXCL17 gene was cloned by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and bioinformatics analysis was performed. The expression of the CXCL17 gene in the different goat tissues and adipocytes at different differentiation stages was detected by real-time fluorescence quantitative PCR (qPCR). The results showed that the cloned sequence of goat CXCL17 gene is 728 bp and the CDS region is 357 bp, encoding 118 amino acids. CXCL17 protein is located in nucleus, cytoplasm, mitochondria and extracellular matrix. Tissue-expression profiles revealed that CXCL17 expressed in all of the examined tissues. In visceral tissues, the highest expression level was found in lung (p < 0.01); in muscle tissues, the highest CXCL17 expression level was found in the longissimus dorsi (p < 0.01) and in adipose tissues, the highest expression level was found in subcutaneous adipose (p <0.01). Compared with those cells before differentiation, CXCL17 expression levels upregulated at 48 h (p < 0.01), 72 h (p < 0.01), 120 h (p < 0.01) and downregulated at 96 h (p < 0.01). Furthermore, the results of crystal violet staining and semi-quantitative assay showed that transfection with 1 μg CXCL17 expression plasmid reduced the cell numbers in vitro. Meanwhile, the expression of CCND1 was significantly decreased. A similar consequence happened after interfering with CXCL17 expression. However, plasmid transfected with 2 μg pEGFPN1-CXCL17 increased the number of cells in vitro. These results suggest that CXCL17 is involved in the proliferation of goat subcutaneous preadipocytes.

Senescence markers in subcutaneous preadipocytes differ in childhood- versus adult-onset obesity before and after weight loss.

The aim of this study was to determine the effect of age of obesity onset on senescence-related markers in abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) before and after moderate (~10%) weight loss. AB and FEM SAT were collected from human females with childhood-onset obesity (CO) or adult-onset obesity (AO) before and after diet- and exercise-induced weight loss. Immunofluorescence analysis of γH2AX/RAD51 (DNA damage/repair markers) and p53/p21 (senescence markers) was conducted in cultured preadipocytes, and senescence-associated β-galactosidase (SA-β-gal) activity was measured in SAT. CO had proportionately more AB and FEM preadipocytes with DNA damage (γH2AX+ ) and senescence markers (p53+ and/or p21+ ) than AO at baseline. The proportion of γH2AX+ FEM preadipocytes declined with weight loss in CO and was similar between groups after weight loss. The number of γH2AX foci in γH2AX+ preadipocytes decreased similarly between groups and regions with weight loss in parallel with an increase in RAD51. The proportion of p53+ and p21+ preadipocytes and SA-β-gal+ cells in SAT did not change with weight loss, but the total p21 intensity in p53+ /p21+ FEM preadipocytes declined in AO. These results provide preliminary evidence that females with CO have an accelerated preadipocyte aging state that improves with weight loss in terms of DNA damage but not senescence.

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.

Cloning, identification and functional analysis of the goat transcription factor c-fos

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.