Intramuscular Preadipocytes Research Articles

FGF1 promotes the differentiation of goat intramuscular and subcutaneous preadipocytes

Fibroblast growth factor 1(FGF1) has been proved to bind to specific signal molecules and activate intracellular signal transduction, leading to proliferation or differentiation of cells. However, the role of FGF1 in goat adipocytes is still unclear. Here, we investigated its role in lipogenesis of goats, which depends on the activation of FGFRs. In goat intramuscular and subcutaneous adipocytes, we observed that adipocytes accumulation was inhibited by interfering of FGF1, the expression of C/EBPα, C/EBPβ, LPL, Pref-1, PPARγ, AP2, KLF4, KLF6, KLF10 and KLF17 were significantly down-regulated (p < 0.05). When the FGF1 was up-regulated, the opposite result was found, while the expression of C/EBPβ, LPL, PPARγ, SREBP1, AP2, KLF4, KLF7, KLF15, KLF16 and KLF17 were increased significantly (p < 0.05) in goat intramuscular and subcutaneous adipocytes. The expression level of FGFR1 was significantly and decreased with the interference of FGF1, and increased with the overexpression of FGF1. But in goat subcutaneous adipocytes, only the expression of FGFR2 was consistent with the expression of FGF1. Interference methods confirmed that FGFR1 or FGFR2 and FGF1 have the similarly promoting function in adipocytes differentiation. With the co-transfection technology, we confirmed that FGF1 promoted the differentiation of intramuscular and subcutaneous adipocytes might via FGFR1 or FGFR2, respectively.

Knockdown of KLF7 inhibits the differentiation of both intramuscular and subcutaneous preadipocytes in goat

KLF7 belongs to the Krüppel-like factors (KLFs) family, which function as transcriptional regulators controlling a number of basic cellular processes, involving proliferation, differentiation, and migration. Here, we reveal insights into the differentiated expression of KLF7 in different goat tissues and different stages of growth, and the inhibition role of KLF7 knockdown to differentiation by using goat intramuscular and subcutaneous preadipocytes. We demonstrate that KLF7 expression is obviously changed during the differentiation of preadipocytes into mature adipocytes. Knockdown of KLF7 inhibited lipid droplet accumulation, reduced the expression of adipogenic markers both in intramuscular and subcutaneous preadipocytes in goats, suggesting that KLF7 is a novel regulator of adipogenesis. KLF7 expression changed also up or down-regulation the other KLF family members, but there were differences between these two types of cells. Investigation into the mechanism that KLF7 regulates preadipocyte differentiation revealed that KLF family members KLF1, KLF5, KLF6, KLF8, KLF11, KLF12, KLF16, KLF17 and adipogenic markers C/EBPα and SREBP1 promoter region present KLF7 transcriptional binding sites. Altogether, the data here identify KLF7 as a novel regulator of adipogenesis.

LKB1 Regulates Goat Intramuscular Adipogenesis Through Focal Adhesion Pathway.

Intramuscular fat (IMF) deposition is one of the most important factors to affect meat quality in livestock and induce insulin resistance and adverse metabolic phenotypes for humans. However, the key regulators involved in this process remain largely unknown. Although liver kinase B1 (LKB1) was reported to participate in the development of skeletal muscles and classical adipose tissues. Due to the specific autonomic location of intramuscular adipocytes, deposited between or within muscle bundles, the exact roles of LKB1 in IMF deposition need further verified. Here, we cloned the goat LKB1 coding sequence with 1,317 bp, encoding a 438 amino acid peptide. LKB1 was extensively expressed in detected tissues and displayed a trend from decline to rise during intramuscular adipogenesis. Functionally, knockdown of LKB1 by two individual siRNAs enhanced the intramuscular preadipocytes differentiation, accompanied by promoting lipid accumulation and inducing adipogenic transcriptional factors and triglyceride synthesis-related genes expression. Conversely, overexpression of LKB1 restrained these biological signatures. To further explore the mechanisms, the RNA-seq technique was performed to compare the difference between siLKB1 and the control group. There were 1,043 differential expression genes (DEGs) were screened, i.e., 425 upregulated genes and 618 downregulated genes in the siLKB1 group. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis predicted that the DEGs were mainly enriched in the focal adhesion pathway and its classical downstream signal, the PI3K-Akt signaling pathway. Specifically, knockdown of LKB1 increased the mRNA level of focal adhesion kinase (FAK) and vice versa in LKB1-overexpressed cells, a key component of the activated focal adhesion pathway. Convincingly, blocking this pathway by a specific FAK inhibitor (PF573228) rescued the observed phenotypes in LKB1 knockdown adipocytes. In conclusion, LKB1 inhibited goat intramuscular adipogenesis through the focal adhesion pathway. This work expanded the genetic regulator networks of IMF deposition and provided theoretical support for improving human health and meat quality from the aspect of IMF deposition.

MiR-10a-5p Inhibits the Differentiation of Goat Intramuscular Preadipocytes by Targeting KLF8 in Goats.

Intramuscular fat contributes to the improvement of meat quality of goats. MicroRNAs (miRNAs) have been reported to regulate adipocyte differentiation and maturation. The aim of our study was to clarify whether miR-10a-5p regulates goat intramuscular preadipocyte (GIPC) differentiation and its direct downstream signaling pathway. GIPCs were isolated from longissimus dorsi, whose miR-10a-5p level was measured at different time point of differentiation induction. Adipogenic differentiation of the GIPCs was evaluated by Oil Red O and BODIPY staining, and the expression changes of adipogenic genes like ACC, ATGL, CEBPβ, PPARγ, etc. Related mechanisms were verified by qPCR, a bioinformatic analysis, a dual-luciferase reporter assay, overexpression, and siRNA transfection. Oil Red O and BODIPY staining both with adipogenic gene detection showed that miR-10a-5p suppressed the accumulation of lipid droplets in GIPCs and inhibited its differentiation. The dual-luciferase reporter assay experiment revealed that miR-10a-5p regulates GIPC differentiation by directly binding to KLF8 3’UTR to regulate its expression. Thus, the results indicated that miR-10a-5p inhibits GIPC differentiation by targeting KLF8 and supply a new target for fat deposition and meat quality improvement.

KLF4 Inhibits the Differentiation of Goat Intramuscular Preadipocytes Through Targeting C/EBPβ Directly.

Intramuscular fat (IMF) deposition is a complicated process, and most of the underlying regulators of this biological process are unknown. Here, we cloned the intact CDS of KLF4 gene, investigated the role of KLF4 by gaining or losing function in vitro and further explored the pathways of KLF4 regulating differentiation of intramuscular preadipocytes in goat. Our results show that goat KLF4 gene consists of 1,536 bp encoding a protein of 486 amino acids. The expression of KLF4 is higher in the lung while lower in the heart and muscle in goat. Knockdown of KLF4 mediated by siRNA technique significantly promotes intramuscular preadipocyte lipid accumulation and upregulates mRNA expression of adipogenic related genes including C/EBPα, C/EBPβ, and PPARγ in vivo cultured cells. Consistently, overexpression of KLF4 inhibits intramuscular adipocyte lipid accumulation and significantly downregulation gene expression of C/EBPβ, PPARγ, aP2, and Pref-1. Further, we found that other members of KLFs were upregulated or downregulated after interference or overexpression of KLF4, including KLF2 and KLF5–7. We also found that C/EBPβ was a potential target of KLF4, because it had an opposite expression pattern with KLF4 during the differentiation of intramuscular preadipocytes and had putative binding sites of KLF4. The dual-luciferase reporter assay indicated that overexpression of KLF4 inhibited the transcriptional activity of C/EBPβ. These results demonstrate that KLF4 inhibits the differentiation of intramuscular preadipocytes in goat by targeting C/EBPβ.

LncIMF2 promotes adipogenesis in porcine intramuscular preadipocyte through sponging MiR-217

Intramuscular fat is positively related to meat quality including tenderness, flavor, and juiciness. Long noncoding RNA (LncRNA) plays a vital role in regulating adipogenesis. However, it is largely unknown about lncRNAs associated with porcine intramuscular adipocyte adipogenesis. In the present study, we focus on a novel LncRNA, which is named lncIMF2, associated with adipogenesis by our previous RNA-sequence analysis and bioinformatics analysis. We demonstrated LncIMF2 knockdown inhibited the proliferation of porcine intramuscular adipocytes while expression of cell cycle-related genes was decreased. Besides, we found LncIMF2 knockdown inhibited expression of adipogenic differentiation marker genes including PPARγ (Peroxisome proliferator-activated reporter gamma) and ATGL (Adipose triglyceride lipase). Similarly, overexpression of LncIMF2 promotes proliferation and differentiation of porcine intramuscular preadipocytes. Moreover, we proved that IncIMF2 acts as a molecular sponge for MicroRNA-217 (miR-217), which has been found associated with adipogenesis, thereby affecting the expression of the miR-217 target gene. Collectively, our findings will contribute to a deeper understanding of the role of LncRNA in pig IMF deposition for the improvement of meat quality.

Comparison of lipid deposition of intramuscular preadipocytes in Tan sheep co-cultured with satellite cells or alone.

The purpose of this study was to investigate the effect of the skeletal muscle satellite cells (SMSCs) on the lipid deposition of the intramuscular preadipocytes (IMPs) in a co-culture system of the Tan sheep cells. The SMSCs and IMPs from Tan sheep were separated and cultured. After the two kinds of cells were separated and cultured, they were inoculated onto a transwell cell chamber co-culture plate for co-cultivation. When the cell density reached more than 90%, the cells were induced to differentiate. After the induction of the SMSCs differentiation for 8days, the level of the IMPs differentiation and the expression levels of the differentiation marker genes and the key enzymes of the lipid metabolism were assessed. The results showed that the number and area of the lipid droplets in the IMPs in the co-culture system were significantly reduced compared to those in the IMPs culture alone (p<0.05). Meanwhile, the expression levels of the PPARγ, c/EBPα, ACC, FAS mRNA in the IMPs were significantly decreased (p<0.05); the expression level of aP2mRNA was decreased, but the difference was not significant (p>0.05).These findings indicate that the SMSCs of the Tan sheep in the co-culture system inhibited the lipid deposition by the IMPs.

Regulatory Effects of Berberine on Adipogenic Differentiation in Porcine Intramuscular Preadipocytes Through the Multi-Gene Pattern

Regulatory Effects of Berberine on Adipogenic Differentiation in Porcine Intramuscular Preadipocytes Through the Multi-Gene Pattern

TMT-based quantitative proteomics analysis reveals the key proteins related with the differentiation process of goat intramuscular adipocytes

BackgroundIntramuscular adipocytes differentiation is a complex process, which is regulated by various transcription factor, protein factor regulators and signal transduction pathways. However, the proteins and signal pathways that regulates goat intramuscular adipocytes differentiation remains unclear.ResultIn this study, based on nanoscale liquid chromatography mass spectrometry analysis (LC-MS/MS), the tandem mass tag (TMT) labeling analysis was used to investigate the differentially abundant proteins (DAPs) related with the differentiation process of goat intramuscular adipocytes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes enrichment and protein-protein interaction network analyses were performed for the characterization of the identified DAPs. The candidate proteins were verified by parallel reaction monitoring analysis. As a result, a total of 123 proteins, 70 upregulation proteins and 53 downregulation proteins, were identified as DAPs which may be related with the differentiation process of goat intramuscular adipocytes. Furthermore, the cholesterol metabolism pathway, glucagon signaling pathway and glycolysis / gluconeogenesis pathway were noticed that may be the important signal pathways for goat Intramuscular adipocytes differentiation.ConclusionsBy proteomic comparison between goat intramuscular preadipocytes (P_IMA) and intramuscular adipocytes (IMA), we identified a series protein that might play important role in the goat intramuscular fat differentiation, such as SRSF10, CSRP3, APOH, PPP3R1, CRTC2, FOS, SERPINE1 and AIF1L, could serve as candidates for further elucidate the molecular mechanism of IMF differentiation in goats.

Angiopoietin-like protein 8, molecular cloning and regulating lipid accumulation in goat intramuscular preadipocytes

This study aimed to clone the full-length open reading frame (ORF) of goat ANGPTL8 gene sequence, reveal its molecular and expression characteristics, and explore its effect on the differentiation of goat intramuscular preadipocytes. The full-length ORF sequence of goat ANGPTL8 gene was cloned by RT-PCR technology, and bioinformatics analysis was performed by related biological software. RT-qPCR was used to detect the expression of ANGPTL8 mRNA in goat tissues. Further use of RNA interference to study the effect of ANGPTL8 on the differentiation of goat intramuscular preadipocytes. The total length of the ANGPTL8 gene nucleotide sequence is 717 bp, including 597 bp of ORF, encoding 198 amino acids. Goat ANGPTL8 has the closest relationship with sheep, it was widely expressed in different tissues, and relatively enriched in liver. The silence of ANGPTL8 inhibited the accumulation of lipid droplets by 5.76% in goat intramuscular preadipocytes (p > 0.05) and significantly suppressed the expression of the genes related to preadipocytes differentiation, fatty acid synthesis and transport (p＜0.05 or p＜0.01). These data illuminate the speculation that ANGPTL8 may involve in the lipid accumulation regulation via the control of PPARγ and C/EBPβ in goat adipocytes.

MiR-421 regulates goat intramuscular preadipocytes differentiation via targeting FGF13

As a member of the MicroRNA s (miRNAs) family, miR-421 has been widely studied in regulating the proliferation and apoptosis of cancer cells a. However, there are still no reports on miR-421 in regulating adipocyte differentiation and its related mechanisms. Accordingly, this study aimed to investigate the potential involvement of miR-421 in goat intramuscular preadipocytes (P_IMA). The expression level of miR-421 was measured via quantitative real-time PCR during goat P_IMA differentiation. And the effects of miR-421 on goat P_IMA differentiation were studied by liposome transfection, Oil red O staining and qRT-PCR. Furthermore, the miR-421 target was searched and the underlying mechanism was clarified by luciferase reporter assay and rescue experiment. Our results showed that inhibition of miR-421 could accumulation of lipid droplets by upregulation the expression level of AP2, LPL, C/EBPα and SREBP1. Further studies showed that fibroblast growth factor 13 (FGF13) was the direct target of miR-421. Knocking down of FGF13 expression could inhibit lipid droplet formation and down-regulated the expression of key adipogenic regulatory genes. In addition, the rescue experiment revealed that FGF13 is involved in miR-421-induced differentiation of goat P_IMA as a key factor. Overall, these findings indicate that miR-421 is a negative regulator in the progression of differentiation of goat P_IMA by inhibiting the expression of FGF13.

Identification of the molecular regulation of differences in lipid deposition in dedifferentiated preadipocytes from different chicken tissues

BackgroundA body distribution with high intramuscular fat and low abdominal fat is the ideal goal for broiler breeding. Preadipocytes with different origins have differences in terms of metabolism and gene expression. The transcriptome analysis performed in this study of intramuscular preadipocytes (DIMFPs) and adipose tissue-derived preadipocytes (DAFPs) aimed to explore the characteristics of lipid deposition in different chicken preadipocytes by dedifferentiation in vitro.ResultsCompared with DAFPs, the total lipid content in DIMFPs was reduced (P < 0.05). Moreover, 72 DEGs related to lipid metabolism were screened, which were involved in adipocyte differentiation, fatty acid transport and fatty acid synthesis, lipid stabilization, and lipolysis. Among the 72 DEGs, 19 DEGs were enriched in the PPAR signaling pathway, indicating its main contribution to the regulation of the difference in lipid deposition between DAFPs and DIMFPs. Among these 19 genes, the representative APOA1, ADIPOQ, FABP3, FABP4, FABP7, HMGCS2, LPL and RXRG genes were downregulated, but the ACSL1, FABP5, PCK2, PDPK1, PPARG, SCD, SCD5, and SLC27A6 genes were upregulated (P < 0.05 or P < 0.01) in the DIMFPs. In addition, the well-known pathways affecting lipid metabolism (MAPK, TGF-beta and calcium) and the pathways related to cell communication were enriched, which may also contribute to the regulation of lipid deposition. Finally, the regulatory network for the difference in lipid deposition between chicken DAFPs and DIMFPs was proposed based on the above information.ConclusionsOur data suggested a difference in lipid deposition between DIMFPs and DAFPs of chickens in vitro and proposed a molecular regulatory network for the difference in lipid deposition between chicken DAFPs and DIMFPs. The lipid content was significantly increased in DAFPs by the direct mediation of PPAR signaling pathways. These findings provide new insights into the regulation of tissue-specific fat deposition and the optimization of body fat distribution in broilers.

MiR-146a-5p targeting SMAD4 and TRAF6 inhibits adipogenensis through TGF-\u03b2 and AKT/mTORC1 signal pathways in porcine intramuscular preadipocytes

BackgroundIntramuscular fat (IMF) content is a vital parameter for assessing pork quality. Increasing evidence has shown that microRNAs (miRNAs) play an important role in regulating porcine IMF deposition. Here, a novel miRNA implicated in porcine IMF adipogenesis was found, and its effect and regulatory mechanism were further explored with respect to intramuscular preadipocyte proliferation and differentiation.ResultsBy porcine adipose tissue miRNA sequencing analysis, we found that miR-146a-5p is a potential regulator of porcine IMF adipogenesis. Further studies showed that miR-146a-5p mimics inhibited porcine intramuscular preadipocyte proliferation and differentiation, while the miR-146a-5p inhibitor promoted cell proliferation and adipogenic differentiation. Mechanistically, miR-146a-5p suppressed cell proliferation by directly targeting SMAD family member 4 (SMAD4) to attenuate TGF-β signaling. Moreover, miR-146a-5p inhibited the differentiation of intramuscular preadipocytes by targeting TNF receptor-associated factor 6 (TRAF6) to weaken the AKT/mTORC1 signaling downstream of the TRAF6 pathway.ConclusionsMiR-146a-5p targets SMAD4 and TRAF6 to inhibit porcine intramuscular adipogenesis by attenuating TGF-β and AKT/mTORC1 signaling, respectively. These findings provide a novel miRNA biomarker for regulating intramuscular adipogenesis to promote pork quality.

MiR-26b-5p regulates the preadipocyte differentiation by targeting FGF21 in goats.

MicroRNAs are a class of highly conserved and widely distributed non-coding RNAs. It is known that miR-26b has a high abundance in adipose tissue and is considered to be an effective regulator of adipogenesis. However, it is unclear whether miR-26b-5p, the product of miR-26b precursor, has the same effect as miR-26b. In the present study, we explored the potential role of miR-26b-5p in preadipocyte differentiation of goats. We found that the expression of miR-26b-5p had dramatic change during goat intramuscular preadipocyte differentiation. Transfection and RT-qPCR revealed that overexpression of miR-26b-5p increased the level of adipogenic marker genes and lipid accumulation in goat preadipocyte, suggesting that miR-26b-5p positively regulates goat preadipocyte differentiation. Furthermore, bioinformatics analysis and dual fluorescein reporter assays were performed to predict and validate the targets of miR-26b-5p. The results showed that miR-26b-5p has a binding site in the 3'UTR of FGF21 and overexpression of miR-26b-5p significantly down-regulated the expression of FGF21 mRNA. Luciferase activity assays confirmed that miR-26b-5p is a positive regulator of goat intramuscular preadipocyte via targeting FGF21. These findings provide reference for further revealing of the regulatory networks of goat fat metabolism and contribute to a better understanding of intramuscular fat deposition in goats.

Identification and characterization of the adipogenesis in intramuscular and subcutaneous adipocytes of the goose (Anser cygnoides)

Chinese geese are domesticated from wild swan (Anser cygnoides), which have maintained a strong capacity of fat deposit. Fat mainly distributes subcutaneous, abdominal, intermuscular or intramuscular in poultry, and they display some special physiological and biochemical characteristics in different parts. This study aimed to characterize the adipogenesis in intramuscular (IM) and subcutaneous (SC) adipocytes of the goose. Here, IM and SC preadipocytes were isolated from the 26-day-old Yangzhou goose embryos, and subsequently induced them to differentiate into mature adipocytes. The results showed that SC preadipocytes grew a little faster than IM preadipocytes during the logarithmic multiplication phase (p < 0.05). Meanwhile, SC adipocytes accumulated more lipid than IM adipocytes during the differentiation process in vitro (p < 0.01). In addition, the expression level of key genes involved in adipogenesis, including peroxisome proliferator activated receptor γ/α (PPARγ/α), CCAAT/enhancer binding protein-α/β (C/EBPα/β), adipocyte fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) were detected. PPARγ, C/EBPα, FABP4, and LPL, were predominantly expressed in SC adipocytes, whereas C/EBPβ was highly expressed in IM adipocytes. Taken together, these results demonstrated that SC preadipocytes tended to grow faster and accumulate more lipid than IM adipocytes, and show greater potential for adipogenesis.

ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis.

Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at −1142 to −1129 bp, −831 to −826 bp, and −303 to −298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’UTR (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

MiR-25-3p regulates the differentiation of intramuscular preadipocytes in goat via targeting KLF4.

Adipocyte differentiation, which plays an important role in fat deposition, involves a complex molecular mechanism. MicroRNAs (miRNAs) are essential in this progress. Here, we showed that miR-25-3p expression had increased during goat intramuscular preadipocyte differentiation, which peaked at day 3. Using liposome transfection and qRT-PCR techniques, we found that knocking down miR-25-3p reduced the accumulation of lipid droplets by downregulating or upregulating the expression of LPL, PPAR , AP2, SREBP1, and C/EBP but upregulating the expression of KLF4. Overexpression of miR-25-3p results in the opposite. Furthermore, the dual luciferase assay showed that overexpression of miR-25-3p significantly inhibited luciferase activity of KLF4. These results showed that miR-25-3p has a binding site within the 3-UTR of KLF4 mRNA. Together, these findings indicate that miR-25-3p is a positive regulator of intramuscular preadipocyte differentiation via targeting to KLF4 in goats.

CircINSR Regulates Fetal Bovine Muscle and Fat Development.

The level of muscle development in livestock directly affects the production efficiency of livestock, and the contents of intramuscular fat (IMF) is an important factor that affects meat quality. However, the molecular mechanisms through which circular RNA (circRNA) affects muscle and IMF development remains largely unknown. In this study, we isolated myoblasts and intramuscular preadipocytes from fetal bovine skeletal muscle. Oil Red O and BODIPY staining were used to identify lipid droplets in preadipocytes, and anti-myosin heavy chain (MyHC) immunofluorescence was used to identify myotubes differentiated from myoblasts. Bioinformatics, a dual-fluorescence reporter system, RNA pull-down, and RNA-binding protein immunoprecipitation were used to determine the interactions between circINSR and the micro RNA (miR)-15/16 family. Molecular and biochemical assays were used to confirm the roles played by circINSR in myoblasts and intramuscular preadipocytes. We found that isolated myoblasts and preadipocytes were able to differentiate normally. CircINSR was found to serve as a sponge for the miR-15/16 family, which targets CCND1 and Bcl-2. CircINSR overexpression significantly promoted myoblast and preadipocyte proliferation and inhibited cell apoptosis. In addition, circINSR inhibited preadipocyte adipogenesis by alleviating the inhibition of miR-15/16 against the target genes FOXO1 and EPT1. Taken together, our study demonstrated that circINSR serves as a regulator of embryonic muscle and IMF development.

MicroRNA-378 regulates adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2

ABSTRACT Intramuscular fat, as one of the most important palatability attribute of beef carcase, is the primary determinant of beef quality. The research of adipogenesis mechanism would provide new insight into intramuscular fatty deposition. Here, the role of microRNA-378 was investigated during bovine adipogenic differentiation. It was revealed that miR-378 expression exists variably in bovine major tissue and organs by RT-qPCR. It was predicted that miR-378 targets CaMKK2, as an AMPKα kinase, by DIANA Tools. For better research, primary preadipocytes with stable transfection for up-/down-regulated expression of miR-378 were constructed by lentiviral vectors with GFP gene. The analyses of qPCR showed that PPARγ and adiponectin mRNA levels increased, but C/EBPβ, pref-1 and CaMKK2 mRNA levels decreased during adipogenic differentiation. When miR-378 was overexpressed, preadipocytes proliferation became slower, there are more cellular lipid droplets, and PPARγ and C/EBPβ mRNA levels were higher, but pref-1, adiponectin and CaMKK2 were lower than control groups. Luciferase assay and western blot analysis validated that miR-378 binds the nucleotide sites of the 3′- untranslated region of CaMKK2, which inhibits the mRNA and protein expression of CaMKK2. These findings suggest that miR-378 promotes adipogenic differentiation in bovine intramuscular preadipocytes by targeting CaMKK2 via AMPK signalling pathway.

Knockdown of adiponectin promotes the adipogenesis of goat intramuscular preadipocytes

Intramuscular fat (IMF) content determined by the intramuscular preadipocytes differentiation has a huge influence on the sensory quality traits of meats. It was reported that the adiponectin (ADIPOQ) gene could promote adipocytes differentiation, but the underlying molecular and functional characterization of the ADIPOQ for regulating goat IMF deposition remained unknown. Herein, the knockdown of ADIPOQ was mediated by siRNAs during goat intramuscular preadipocytes differentiation. Also, the qRT-PCR technique was performed to detect the mRNA levels of target genes in multiply experiment groups. These results showed that the ADIPOQ was expressed more than ∼400 folds in subcutaneous adipose tissue compared to that of heart tissue, and the mRNA level of ADIPOQ reached a peak at Hour 60 during the differentiation process, while at Hour 36 did ADIPOR1 and ADIPOR2. Moreover, the knockdown of ADIPOQ promoted the intramuscular preadipocytes differentiation and accelerated the lipid accumulation in the mature adipocytes with down-regulating the ADIPOR1 and preadipocyte factor 1 (Pref-1) mRNA levels and up-regulating the mRNA expression levels of the CAAT/enhancer-binding proteins (C/EBPs) and transcription factor peroxisomal proliferator-activated receptor γ (PPARγ), etc. Our study will provide a new opposite insight that the inhibition of ADIPOQ expression during intramuscular preadipocytes differentiation promotes goat IMF deposition.