Stard 3 (Steroidogenic Acute Regulatory-Related Lipid Transfer Domain-Containing Protein 3) Play Important Role in Preadipocyte Differentiation

Aim: To evaluate Stard 3’s effects and relative mechanisms in preadipocyto differentiation by vitro study. Materials and Methods: The 3T3-L1 cell were divided into 5 groups as NC, si-Stard 3, ROS agonist, ROS inhibitor and si-Stard 3+ROS agonist groups. The cell of different groups were evaluated by Oil red O staining and Triglyceride. Evaluating ROS production by DHE and NBT assay. Using RT-qPCR and WB methods to evaluate gene and protein expressions. Results: Compared with NC group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly down-regulation in si-Stard 3 and ROS inhibitor groups (P < 0.001, respectively), and were significantly up-regulation in ROS agonist group (P < 0.001, respectively); However, with si-Stard 3 transfection and ROS agonist treatment, compared with si-Stard 3 group, Triglyceride, DHE fluorescence intensity and NBT positive rate were significantly increased in si-Stard 3+ROS agonist group (P < 0.001, respectively). With RT-qPCR and WB assay, Compared with NC group, Stard 3 gene and protein expressions of si-Stard 3 and si-Stard 3+ROS agonist group were significantly depressed (P < 0.001, respectively), AMPK, PPARγ, CEBPα and FABP4 gene expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively) and p-AMPK, PPARγ, CEBPα and FABP4 protein expressions were significantly differences in si-Stard 3, ROS agonist and ROS inhibitor groups (P < 0.001, respectively), with si-Stard 3 transfection and ROS agonist the relative gene and protein expressions were significantly resumed compared with si-Stard 3 group (P < 0.001, respectively). Conclusion: Stard 3 knockdown had effects to suppress 3T3-L1 cells transformation into adipocytes in vitro study.

Activation of the aryl hydrocarbon receptor by clozapine induces preadipocyte differentiation and contributes to endothelial dysfunction

Background: The superior therapeutic benefit of clozapine is often associated with metabolic disruptions as obesity, insulin resistance, tachycardia, higher blood pressure, and even hypertension. Aims: These adverse vascular/ metabolic events under clozapine are similar to those caused by polycyclic aromatic hydrocarbons (PAHs), and clozapine shows structural similarity to well-known ligands of the aryl hydrocarbon receptor (AhR). Therefore, we speculated that the side effects caused by clozapine might rely on AhR signaling. Methods: We examined clozapine-induced AhR activation by luciferase reporter assays in hepatoma HepG2 cells and we proved upregulation of the prototypical AhR target gene Cyp1A1 by realtime-PCR (RT-PCR) analysis and enzyme activity. Next we studied the physiological role of AhR in clozapine’s effects on human preadipocyte differentiation and on vasodilatation by myography in wild-type and AhR-/- mice. Results: In contrast to other antipsychotic drugs (APDs), clozapine triggered AhR activation and Cyp1A1 expression in HepG2 cells and adipocytes. Clozapine induced adipogenesis via AhR signaling. After PGF2α-induced constriction of mouse aortic rings, clozapine strongly reduced the maximal vasorelaxation under acetylcholine in rings from wild-type mice, but only slightly in rings from AhR-/- mice. The reduction was also prevented by pretreatment with the AhR antagonist CH-223191. Conclusion: Identification of clozapine as a ligand for the AhR opens new perspectives to explain common clozapine therapy-associated adverse effects at the molecular level.

miR-214-5p Regulating Differentiation of Intramuscular Preadipocytes in Goats via Targeting KLF12

Intramuscular fat (i.m.) is an adipose tissue that is deposited between muscle bundles. An important type of post-transcriptional regulatory factor, miRNAs, has been observed as an important regulator that can regulate gene expression and cell differentiation through specific binding with target genes, which is the pivotal way determining intramuscular fat deposition. Thus, this study intends to use RT-PCR, cell culture, liposome transfection, real-time fluorescent quantitative PCR (qPCR), dual luciferase reporter systems, and other biological methods clarifying the possible mechanisms on goat intramuscular preadipocyte differentiation that is regulated by miR-214-5p. Ultimately, our results showed that the expression level of miR-214-5p peaked at 48 h after the goat intramuscular preadipocytes were induced for adipogenesis. Furthermore, after inhibition of the expression of miR-214-5p, the accumulation of lipid droplets and adipocyte differentiation in goat intramuscular adipocytes were promoted by the way of up-regulation of the expression level of lipoprotein lipase (LPL) (p &lt; 0.05) and peroxisome proliferator-activated receptor gamma (PPARγ) (p &lt; 0.01) but inhibited the expression of hormone-sensitive lipase (HSL) (p &lt; 0.01). Subsequently, our study confirmed that Krüppel-like factor 12 (KLF12) was the target gene of miR-214-5p. Inhibition of the expression of KLF12 promoted adipocyte differentiation and lipid accumulation by upregulation of the expression of LPL and CCAAT/enhancer binding protein (C/EBPα) (p &lt; 0.01). Overall, these results indicated that miR-214-5p and its target gene KLF12 were negative regulators in progression of goat preadipocyte differentiation. Our research results provided an experimental basis for finally revealing the mechanism of miR-214-5p in adipocytes.

CREBRF p.Arg457Gln Promotes Obesity and Improves Insulin Sensitivity by Promoting Preadipocyte Differentiation and Reducing Oxidative Metabolism in Gene-modified Pig Models

Obesity is one of the most important risk factors for type 2 diabetes (T2DM). The CREBRF missense allele of rs373863828 (p.Arg457Gln) is associated with increased body mass index (BMI), yet reduced risk of T2DM in people with Pacific ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation p.Arg457Gln into porcine genome by using a CRISPR/Cas9-mediated homologous recombination (HR)-dependent approach. The CREBRF p.Arg457Gln pig models displayed dramatically increased fat deposition, yet improved sensitivity to insulin. Transcriptome and metabolome analyses of subcutaneous white adipose tissues showed that the CREBRF p.Arg457Gln mutation promoted preadipocyte differentiation, which indicated that obesity was caused by increased number (hyperplasia) rather than size (hypertrophy) of adipocytes. In addition, the oxidative capacity decreased in the adipose tissue of pigs with CREBRF p.Arg457Gln variant. The pre-oxidative metabolite content (4-HNE and MDA) significantly decreased, while activity of antioxidant enzymes (GPX, SOD, and CAT) increased, thereby repressing oxidative metabolism of adipose tissue and reducing level of reactive oxygen species (ROS). The low reactive oxygen species could prevent insulin resistance and reduce risk of obesity-induced type 2 diabetes. This study provides further mechanistic insights into favourable adiposity resulting from CREBRF p.Arg457Gln.

The Effect of FATP1 on Adipocyte Differentiation in Qinchuan Beef Cattle

FATP1 plays an important role in the regulation of fatty acid metabolism and lipid accumulation. In this study, we investigated the patterns of FATP1 expression in various tissues obtained from calf and adult Qinchuan cattle, and in differentiating adipocytes. Next, we investigated the effect of FATP1 expression on preadipocyte differentiation in Qinchuan cattle using overexpression and interference assays. We also identified the differentially expressed genes (DEGs) and pathways associated with FATP1 overexpression/interference. Our results reveal that FATP1 was broadly expressed in heart, kidney, muscle, small intestine, large intestine, and perirenal fat tissues. While FATP1 overexpression promoted preadipocyte differentiation, fat deposition, and the expression of several genes involved in fat metabolism, FATP1 interference had the opposite effects on adipocyte differentiation. Following FATP1 overexpression and FATP1 interference in adipocytes, RNA-seq analysis was performed to identify DEGs related to fat metabolism. The DEGs identified include SLPI, STC1, SEMA6A, TNFRSF19, SLN, PTGS2, ADCYP1, FADS2, and SCD. Pathway analysis revealed that the DEGs were enriched in the PPAR signaling pathway, AMPK signal pathway, and Insulin signaling pathway. Our results provide an in-depth understanding of the function and regulation mechanism of FAPT1 in fat metabolism.

Impaired mRNA splicing and proteostasis in preadipocytes in obesity-related metabolic disease

Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we aimed at identifying the pathogenic processes underlying impaired adipocyte differentiation in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). We report that down-regulation of a key member of the major spliceosome, PRFP8/PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8/PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of endoplasmic reticulum (ER)-associated protein degradation (ERAD) hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals.

MicroRNA Profiling Reveals an Abundant ssc-miRNA-148a-3p that Promotes Proliferation and Differentiation during Intramuscular Adipogenesis in Chinese Guizhou Congjiang Pigs Breeds by targeting PPARGC1A

BackgroundIntramuscular fat development is regulated by a series of complicated processes, and non-coding RNA (ncRNA) such as microRNA (miRNA) plays a critical role during intramuscular preadipocyte proliferation and differentiation development in pigs. In present research, we detected the expression profiles of miRNA during different differentiation stages, namely, day 0 (D0), day 4 (D4), and day 8 (D8), of intramuscular preadipocytes from the longissimus dorsi muscle of Chinese Guizhou Congjiang pigs to provide first insights into their potential involvement in intramuscular preadipocyte development. And we investigated the function of miR-148a-3p in adipocyte proliferation, apoptosis, and differentiation. ResultsA total of 67, 95, and 16 differentially expressed (DE) miRNAs were detected between D4 and D0, between D8 and D0, and between D8 and D4, respectively. We further characterized the role of miR-148a-3p which was differentially expressed and highest expressed abundance in D0, D4, and D8. To explore the role of miR-148a-3p in porcine intramuscular preadipocyte, miR-148a-3p mimics and inhibitors were used to perform miR-148a-3p overexpression and knockdown, respectively. Overexpression of miRNA-148a-3p increased the number of intramuscular preadipocytes in the S/G2 phase of the cell cycle and decreased the proportion of cells in the G0/G1 phase. Moreover, it promoted proliferation by regulation of cyclin B, cyclin G1, cyclin D1, CDK2, CDK3, and CDK4 and inhibited apoptosis of intramuscular preadipocyte by regulating the expression of Caspase-3, Bax, and Bcl-2. Meanwhile, the mimics of miR-148a-3p dramatically promoted intramuscular preadipocyte differentiation and upregulated the expression levels of adipogenic marker genes PPARγ, FASN, FABP4, HSL, APOE, LPL, and CEBPα. Furthermore, miR-148a-3p promoted intramuscular preadipocyte differentiation via restraining the AMPK/ACC/CPT1C signaling pathway. PPARGC1A was identified as a target gene of miR-148a-3p by luciferase activity and western blotting assays. ConclusionOur study provides novel insights into the regulatory mechanisms underlying intramuscular preadipocyte development and identified amount of miRNAs whose regulatory potential will need to be explored in the future. Our results establish that miR-148a-3p promoted adipocyte differentiation by targeting PPARGC1A.

Effect of the ACAA1 Gene on Preadipocyte Differentiation in Sheep

Acetyl-CoA acyltransferase 1 (ACAA1) functions as a key regulator of fatty acid β-oxidation in peroxisomes by catalyzing the cleavage of 3-ketoacyl-CoA to acetyl-CoA and acyl-CoA, which participate in the extension and degradation of fatty acids. Thus, ACAA1 is an important regulator of lipid metabolism and plays an essential role in fatty acid oxidation and lipid metabolism. Our previous study findings revealed that ACAA1 is closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways, which are involved in fat deposition in sheep, leading to our hypothesis that ACAA1 may be involved in fat deposition by regulating lipid metabolism. However, the associated molecular mechanism remains unclear. In the present study, to assess the potential function of ACAA1 in sheep preadipocyte differentiation, we knocked down and overexpressed ACAA1 in sheep preadipocytes and evaluated the pattern of ACAA1 gene expression during preadipocyte differentiation by qRT-PCR. ACAA1 was significantly expressed in the early stage of adipocyte differentiation, and then its expression decreased. ACAA1 deficiency increased lipid accumulation and the triglyceride content and promoted sheep preadipocyte differentiation, whereas ACAA1 overexpression inhibited adipogenesis and decreased lipid accumulation and the triglyceride content. Simultaneously, we demonstrated that ACAA1 deficiency upregulated the expressions of the adipogenic marker genes PPARγ and C/EBPα in sheep preadipocytes, but ACAA1 overexpression inhibited the expressions of these markers, indicating that ACAA1 affects lipid metabolism by regulating adipogenic marker genes. Our results may promote a better understanding of the regulation of adipogenesis by ACAA1.

miR-208b Regulates Rabbit Preadipocyte Proliferation and Differentiation

microRNAs (miRNAs) play an important role in gene regulation in animals by pairing with target gene mRNA. Many miRNAs are differentially expressed in the adipose tissue, often with conserved expression. In our study, we found that miR-208b expression was observed differently in the preadipocyte differentiation model. When miR-208b was overexpressed in the preadipocyte differentiation model, the overexpressed group displayed higher expression of PPARγ and FABP4—the markers of preadipocyte differentiation. Oil Red O staining revealed that the count of lipid droplets was increased in the overexpressed group. When the expression of miR-208b was inhibited, the above indicators showed an opposite trend. Moreover, results from both 5-ethynyl-2’-deoxyuridine (EDU) and cell counting kit (CCK) analysis showed that miR-208b promoted the proliferation of preadipocyte. Expression of gene CSNK2A2, a direct miR-208b target, was downregulated in the overexpressed group, providing a possible link to multiple signal pathways. Overall, our data indicate that miR-208b play a positive regulatory effect on the proliferation and differentiation of rabbit preadipocyte.