The insulin induced gene 1 (INSIG1) plays a regulatory role in fat metabolism and adipocyte differentiation. In 1993, INSIG1 gene was originally identified and characterized from regenerating liver (Diamond et al., Reference Diamond, Du, Lee, Mohn, Haber, Tewari and Taub1993). The study showed that INSIG1 could effectively block proteolytic activation of sterol-regulatory element binding protein (SREBP) family members (Carobbio et al., Reference Carobbio, Hagen, Lelliott, Slawik, Medina-Gomez, Tan, Sicard, Atherton, Barbarroja, Bjursell, Bohlooly-Y, Virtue, Tuthill, Lefai, Laville, Wu, Considine, Vidal, Langin, Oresic, Tinahones, Fernandez-Real, Griffin, Seth, López and Vidal-Puig2013). INSIG1 is a membrane protein that resides in the endoplasmic reticulum (ER) and contains six membrane-spanning helices (Feramisco et al., Reference Feramisco, Goldstein and Brown2004). Recent studies have shown that the expression of INSIG1 gene is regulated by insulin, free fatty acids, glucose and other nutrients (Dong and Tang, Reference Dong and Tang2010).
As an important transcription factor, SREBP is involved in the regulation of sterol and fatty acid synthesis in mammals, and directly involved in the regulation of the expression of more than 30 genes related to the biosynthesis and uptake of cholesterol, triglycerides, fatty acids and phospholipids (Horton et al., Reference Horton, Goldstein and Brown2002, Reference Horton, Shah, Warrington, Anderson, Park, Brown and Goldstein2003). With the participation of intracellular sterols, INSIG1 regulates the activation of SREBP, thereby feedback-regulating the uptake of exogenous cholesterol in cells (Gong et al., Reference Gong, Lee, Lee, Goldstein, Brown and Ye2006; Liou et al., Reference Liou, Bai, Lin, Chen, Chen, Hong and Tsai2012). SREBP can only be transported from ER to the Golgi by an escort protein called SREBP cleavage activating protein (SCAP), which forms a SCAP/SREBP complex immediately after their synthesis (Tsushima et al., Reference Tsushima, Tang, Puviindran, Hsu, Nadesan, Yu, Zhang, Mirando, Hilton and Alman2018). INSIG1 prevents the transport of SREBP/SCAP complex to Golgi by binding to SCAP in the presence of sterols, causing ER retention of the SREBP. In this way, the suppression of SREBP causes a decrease of lipogenic enzyme's gene expression which consequently results in lower rate of fatty acid synthesis in animals. However, in the sterol-depleted cell, SCAP undergoes a conformational change that leads it to dissociate from INSIG1, SCAP escorts SREBP to the Golgi where the SREBP is processed by two membrane-bound proteases that release the active fragment of SREBP, which can enter the nucleus and regulate the transcription of a variety of target genes. Thus, SREBP can play a transcriptional regulatory function and promote the expression of related genes in the process of sterol and fatty acid biosynthesis (Espenshade and Hughes, Reference Espenshade and Hughes2007; Owen et al., Reference Owen, Zhang, Bae, Farooqi, Liang, Hammer, Goldstein and Brown2012). Finally, free INSIG1 protein is degraded rapidly by ubiquitination. When cells are incubated with sterols, INSIG1 is stabilized by binding to SCAP, blocking the degradation of INSIG1 (Lee et al., Reference Lee, Gong, Zhang and Ye2006).
The synthesis of milk fat has been widely studied, and the main aspects of milk fat metabolism, including de novo synthesis and fatty acids uptake from blood, are defined and quantified (Osorio et al., Reference Osorio, Lohakare and Biona2016). Milk fat is primarily composed of triglycerides (TAG) and a small proportion of other lipids. It is an important energy source in milk. In ruminants, about half of the milk fat is synthesized de novo (Harvatine et al., Reference Harvatine, Boisclair and Bauman2009). A network summarizing 45 genes related to milk fat synthesis was previously constructed, and it is suggested that SREBP is at the core of the milk fat synthesis network in the bovine mammary gland (Bionaz and Loor, Reference Bionaz and Loor2008). They also suggested that INSIG1 was an essential protein regulating SREBP. Therefore, INSIG1 also plays an important role in the synthesis of milk fat in bovine. However, the regulatory function and the molecular mechanism of INSIG1 gene in milk fat synthesis of buffalo are not known. The objective of the present study was to examine the role of INSIG1 in the regulation of milk fat synthesis for buffalo. To assess the role of INSIG1, both lentivirus-mediated RNA interference and overexpression were performed in buffalo mammary epithelial cells (BMECs).
Materials and methods
All procedures for sample collection were performed in accordance with the Guide for Animal Care and Use of Experimental Animals and approved by the Yunnan Provincial Experimental Animal Management Committee under Contract 2007-0069.
Buffalo mammary gland tissue collection
Six adult female Binglangjiang buffalo with the same management conditions, of which three were in non-lactating stage (the dry-off period) and another three were in the peak of lactation, were euthanized and mammary gland tissue samples were collected. Total protein was isolated from each tissue sample using RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China) according to the manufacturer's instructions. Protein concentrations were determined using the BCA protein kit (Beyotime, Shanghai, China) according to the manufacturer's instructions.
Vector construction
The lentiviral construct that expressed buffalo INSIG1 was pLVX-IRES-ZsGreen1 bicistronic shuttle vector (CLONTECH Laboratories, Inc.). Buffalo INSIG1 cDNA (Accession no. JX853922) was prepared by reverse transcription polymerase chain reaction (RT-PCR). The primers used for cDNA cloning are presented in online Supplementary Table S1. The open reading frame sequence of buffalo INSIG1 gene was inserted into the vector through XhoI and BamHI sites to obtain the recombinant pLVX-INSIG1-IRES-ZsGreen1 (Lv-INSIG1).
To knock down buffalo INSIG1, three short hairpin RNA sequences (shRNA103, shRNA547 and shRNA681) were designed by the BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/) using the buffalo INSIG1 gene sequence (Accession no. JX853922). We selected the shRNA sequences with the highest interference efficiency and those sequences were synthesized with the EcoRI and AgeI restriction sites (Sangon Biotech Co. Ltd., Shanghai, China; Supplementary Table S2). The shRNA sequences were constructed into the pLKO.1-TRC cloning vector through EcoRI and AgeI sites to generate pLKO.1-shRNA recombinants (Lv-shINSIG1). Meanwhile, the INSIG1 gene open reading frame sequence was amplified and inserted into the vector through XhoI and HandIII sites to get pEGFP-INSIG1-N1 (CLONTECH Laboratories, Inc.) construct. Detailed primer information is described in online Supplementary Table S1.
Cell culture
The details of 293T cell culture were previously described (Zu et al., Reference Zu, Yan, Robbins, Krishack, Liao and Cao2007). Briefly, the cells were cultured in basal Dulbecco's modified Eagle medium/F12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 2% penicillin/streptomycin (Gibco). Routine cultures were incubated at 37 °C in 5% CO2 and medium was changed every 24 h.
The BMECs were isolated from peak lactation buffalo and identified by cytokeratin 18 (Sigma, USA) as described previously (Hu et al., Reference Hu, Wang, Bu, Wei, Zhou, Li and Loor2009; Shi et al., Reference Shi, Shi, Luo, Wang, Haile, Xu and Li2014). The purified BMECs were cultured in basal DMEM/F12 supplemented with 10% FBS and 2% penicillin/streptomycin. To promote the synthesis of milk protein and fat, the induction medium was basal medium containing 5 μg/ml insulin (Sigma), 5 μg/ml Holotransferrin (Sigma), 5 μg/ml hydrocortisone (Sigma), 1 μg/ml epidermal growth factor (Sigma). The BMECs were incubated at 37 °C in 5% CO2 and medium was changed every 24 h.
Preliminary screening of shRNA sequences
In order to obtain the most effective shRNA for targeting INSIG1 gene, we screened the shRNA sequences in 293T cells. The 239T cells at about 80% confluence in 6 plates were transiently transfected with 4 μg of three pLKO.1-shRNAs with pEGFP-INSIG1-N1, at a ratio of 3 : 1 using TransIntroTM EL Transfection Reagent (TransGen Biotech, Beijing, China) in accordance with the manufacturer's protocol. The pLKO.1-TRC control (Lv-shNC) and pEGFP-INSIG1-N1 also were transfected together as a negative control in the same amount as above. The green fluorescent protein was observed using fluorescent microscope (Nikon, Japan). Meanwhile, the expression of INSIG1 gene was further verified by real-time quantitative PCR (RT-qPCR).
Lentivirus generation and transduction
The 239T cells in log-phase growth were transiently transfected with 8 μg of pLKO.1-shRNA with psPAX2 (packaging plasmid) and pMD2.G (envelope plasmid), at a ratio of 5 : 2 : 3 in accordance with the manufacturer's protocol. The pLKO.1-TRC control was used as a negative control in the same amount as above. Analogously, the 293T cells were transfected by sequencing-verified pLVX-INSIG1-IRES-ZsGreen1 for packaging lentiviral particles. Lentivirus containing green fluorescent protein pLVX-IRES-ZsGreen1 (Lv-GFP) was used as a negative control. These lentivirus particles were directly collected and concentrated from cell culture media 52 h after transduction by multi-steps of ultracentrifugation (50 000 × g, 2 h at 4 °C). The BMECs at about 80% confluence were transfected with lentivirus supernatant (Lv-INSIG1, Lv-GFP, Lv-shINSIG1 and Lv-shNC) as described previously (Zhou et al., Reference Zhou, Lin, Wang, Yao, Leong, Tan and Huang2017). The transfected BMECs were harvested after 52 h of culture for total RNA extraction and TAG analysis.
RT-qPCR and western blot
The process of total RNA extraction and cDNA synthesis was described previously (Song et al., Reference Song, Ou-Yang, Huo, Zhang, Yu, Liu, Teng and Miao2016). The RT-qPCR was performed according to the manufacturer's instruction using SYBR Green (TaKaRa, Dalian, China). Specific primers were designed for several genes related to the synthesis of milk fat: INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6 (Supplementary Table S3). The insulin induced gene 2 (INSIG2) and INSIG1 belong to the same family. The peroxisome proliferator-activated receptor-γ (PPARG) plays a crucial role in fatty acid (FA) metabolism through regulation of stearoyl-CoA desaturase (SCD) gene expression, which is the rate-limiting enzyme for the biosynthesis of monounsaturated FA (Shi et al., Reference Shi, Luo, Yao, Zhu, Xu, Shi and Loor2013). The glycerol-3-phosphate acyltransferase (GPAM), diacylglycerol acyltransferase 2 (DGAT2) and 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6) are related to TAG synthesis (Shi et al., Reference Shi, Wu, Zhu, Zhang, Yao, Luo and Loor2017). The beta-actin (ACTB) was selected as an internal control gene. All the RT-qPCR reactions were performed in a Mastercycler ep realplex (Eppendorf, Germany) Real-time qPCR detector and data were normalized to ACTB.
For Western blot, the proteins extracted from mammary tissue were separated by SDS-PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and probed with the primary antibodies polyclonal rabbit anti-INSIG1 (1 : 500; Absin, Shanghai, China; Lot: abs125485a) and monoclonal mouse anti-ACTB (1 : 5000; TransGen Biotech; Lot: #K20912). Polyclonal goat anti-rabbit IgG (1 : 5000; Millipore; Lot: #2491145) and polyclonal goat anti-mouse IgG (1 : 5000; Millipore; Lot: #2517746) were used as secondary antibody. All antibodies were used according to the manufacturer's recommendations. The results were visualized by the chemiluminescent ECL Western blot detection system (Pierce, USA).
Quantification of cellular TAG
Cellular TAG was extracted according to the manufacturer's instructions of GPO-Trinder triglyceride assay kit (Applygen Technologies, Beijing, China). The concentrations were calculated by the equation obtained from a linear regression of the standard curve, which was established according to the manufacturer's procedure. And TAG content was normalized by cellular protein concentration assessed using a BCA protein assay kit.
Data analysis
All the treatments were executed in triplicates and all the experimental data are presented as means ± standard error of the mean (sem). Data of RT-qPCR were analyzed using the 2−ΔΔCt method. Significance of mRNA expression, cellular TAG content after INSIG1 overexpression or knock down and Western blot results was determined via Student's t-test. Significance was declared at P < 0.05.
Results
Insig1 expression level at 2 stages of lactation
The expression of INSIG1 protein in mammary tissue during peak lactation and dry-off period was detected. Relative to the dry-off period, the expression level of INSIG1 at peak lactation decreased significantly (P < 0.05) (Fig. 1 and Fig. S1).
INSIG1 expression in buffalo mammary gland tissue. a. representative immunblots; b. corresponding mean gray values of INSIG1 protein. Protein abundance was calibrated with β-actin. Values are presented as means ± sem; *P < 0.05.
Isolation of mammary gland epithelial cells
Buffalo mammary epithelial cells were isolated by tissue culture method. The process of cell separation is shown in online Supplementary Fig. S2. After the tissue was cultured for 7 d, primary fibroblasts were first migrated out from mammary tissue (Fig. S2A). Primary epithelial cells were emigrated from mammary tissue after 10 d in culture. Fibroblast and epithelial cells were observed in the dish, most being mixed together (Fig. S2B). The BMECs were separated after about 3–5 passages and purified. The resulting BMECs were present as a cellular island (Fig. S2C) and cobblestone form (Fig. S2D). Furthermore, we identified the purified cells by cytokeratin 18. The results showed that purified cells exhibited intense positive staining of the cytoplasmic meshwork of cytokeratin fibrils when incubated with the antibody, indicating that the purified cells were buffalo mammary gland epithelial cells (Fig. S3).
Overexpression of INSIG1 inhibits expression of lipogenic genes and synthesis of intracellular TAG
Compared with controls (Lv-GFP), the mRNA abundance of INSIG1 gene increased markedly in BMECs incubated with Lv-INSIG1 (Fig. 2a). Overexpression of INSIG1 dramatically downregulated the mRNA levels of genes related to synthesis of milk fat (Fig. 2b). As a member of the same family of INSIG1, the expression of INSIG2 decreased by 80.2% (P < 0.01). The relative expressions of SREBP (P < 0.05), PPARG (P < 0.05) and SCD (P < 0.05) were also significantly decreased. Meanwhile, overexpression of INSIG1 decreased the transcription of genes measured involved in TAG synthesis, with 70.9% decrease for GPAM, 65.0% decrease for DGAT2, and 45.1% decrease for AGPAT6. Accordingly, the TAG content of BMECs transfected with Lv-INSIG1 was decreased significantly (P < 0.05) compared with the negative control (Fig. 3).
Overexpression of INSIG1 altered genes related to milk fat synthesis. The buffalo mammary epithelial cells were transfected with a lentivirus expressing INSIG1 (Lv-INSIG1) or the control (Lv-GFP) for 52 h and then extracted RNA. a. mRNA expression of INSIG1; b. mRNA expression of genes related to milk fat synthesis (INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6). Values are means ± sem from 3 individual cultures; *P < 0.05, **P < 0.01.
INSIG1 inhibited the synthesis of cellular TAG. The buffalo mammary epithelial cells were transfected with a lentivirus expressing INSIG1 (Lv-INSIG1) or the control (Lv-GFP) and collected at 52 h for cellular TAG analysis. Values are means ± sem from 3 individual cultures. *P < 0.05 compared with control.
Preliminary shRNA screening
To obtain the highest knockdown of INSIG1 transcript, three lentiviruses with shINSIG1 sequence were co-transfected in 293T cells with pEGFP-INSIG1-N1, respectively. The GFP protein on the pEGFP-INSIG1-N1 vector was used to assess the interference efficiency via intensity of green fluorescence inside the cells (Fig. 4), and in addition the mRNA of INSIG1 gene was verified via RT-qPCR in the cells (Fig. 5). The shRNAs that were specific for INSIG1 would inhibit green fluorescence expression, in other words, they would inhibit the expression of INSIG1 gene. Thus, the interference efficiency of shRNA was assessed by the disappearance of green fluorescence inside the cells and mRNA expression level of INSIG1. As shown in Fig. 5, shRNA103 and shRNA681 were more efficient than shRNA547 to down-regulate INSIG1 expression. Compared with shRNA103 (98.8%), the shRNA681 (99.7%) was more efficient in knocking down buffalo INSIG1 by the RT-qPCR detecting (Fig. 5).
Efficiency screening of the three designed shRNA via images analysis. The pLKO.1-TRC control vector was co-transfected with pEGFP-INSIG1-N1 vector as a control (a). The three tested shRNA (shRNA103, shRNA547 and shRNA681) as pLKO.1-shRNA vector were co-transfected with pEGFP-INSIG1-N1 (b–d). Shown are representative images of the INSIG1 expression (in green) after a 48 h co-transfection. The images show that the shRNA103 and shRNA681 had a higher effect on INSIG1 vector expression (B and D).
Efficiency screening of the three designed shRNA via RT-qPCR. The efficiency of Lv-shINSIG1 (co-transfected with pEGFP-INSIG1-N1 for 52 h) in decreasing INSIG1 expression in 293T cells was assessed by RT-qPCR. The data revealed that Lv-shRNA681 had the highest knockdown of INSIG1 transcript; thus, it was used in the subsequent experiments.
Knockdown of INSIG1 promotes expression of lipogenic genes
Based on the above results, the Lv-shRNA681 (Lv-shINSIG1) was selected to block the expression of INSIG1 in BMECs, and expression analysis of genes related to milk fat synthesis was evaluated (Fig. 6). Results demonstrated that the mRNA expression of INSIG1 decreased markedly (P < 0.05) in the Lv-shINSIG1 group compared with the Lv-shNC group. The expression of INSIG2 was increased significantly (P < 0.01) by the loss of INSIG1. INSIG1 gene silencing upregulated the mRNA expression levels of SREBP, PPARG and SCD (all P < 0.01) in the cells transfected with Lv-shINSIG1 compared with the cells transfected with Lv-shNC. Meanwhile, INSIG1 gene knockdown increased the transcription of all the genes measured involved in TAG synthesis, with a 6.5-fold increase for GPAM, 2.3-fold increase for DGAT2, and 2.3-fold increase for AGPAT6.
Effect of INSIG1 knockdown on genes involved in milk fat synthesis in BMECs. The buffalo mammary epithelial cells were transfected with lentivirus containing small hair RNA knocking down INSIG1 (Lv-shINSIG1) or the lentivirus containing negative control (Lv-GFP) for 52 h and then extracted RNA. a. mRNA expression of INSIG1; b. mRNA expression of genes related to milk fat synthesis (INSIG2, SREBP, PPARG, SCD, GPAM, DGAT2 and AGPAT6). Values are means ± sem from three individual cultures; *P < 0.05, **P < 0.01.
Discussion
In humans, the INSIG1 mRNA is highly expressed in the liver (Krapivner et al., Reference Krapivner, Popov, Chernogubova, Hellénius, Fisher, Hamsten and Van'T Hooft2008). The expression of INSIG1 mRNA in mouse is very high in mammary glands during lactation (Han et al., Reference Han, Li, Wang, Zhu, Wang, Guo, Lu, Wang and Yang2010), and the mRNA expression of buffalo INSIG1 is high in the mammary gland and liver and moderate in the abomasum, brain and muscle (Wu et al., Reference Wu, Liu, Huo, Li, Yuan, Yuan and Miao2014). To investigate the role of INSIG1, we first demonstrated that INSIG1 protein was expressed in buffalo mammary tissue, with expression level of dry-off period being higher than that of peak lactation. The enrichment of INSIG1 at the dry-off period may be a reflection of its physiological role in inhibiting the synthesis of milk fat. However, previous studies have shown that the change of INSIG1 mRNA expression during bovine lactation was >12-fold in magnitude compared with non-lactating period (Bionaz and Loor, Reference Bionaz and Loor2008). This is probably due to the post-transcriptional regulation. Furthermore, buffalo milk is richer in milk fat than cow milk, suggesting that there are differences in fat metabolism of the mammary gland between the two species. To understand milk fat regulation in buffalo mammary gland, a representative model of cells is vital. In this study, mammary epithelial cells from a peak lactation buffalo were obtained to meet the demand of further research in buffalo lactation.
Lipogenesis is regulated by sterols and insulin by regulating the expression and activation of SREBP, but this process is more sensitive to cholesterol and less sensitive to insulin (Attie, Reference Attie2004). In addition, the regulation of lipid homeostasis by insulin is mediated to some extent by the enhanced transcription of SREBP gene. The stable expression of INSIG1 enhances the ability of sterols to inhibit activation of SREBP, which activates transcription of genes involved in fatty acid synthesis (Leichner et al., Reference Leichner, Avner, Harats and Roitelman2009). In INSIGs deleted mutant Chinese hamster ovary cells, sterols could not inhibit the processing of SREBP, but sterol regulation of SREBP processing was fully restored when they were transfected with expression plasmids encoding INSIG1. These results reveal the absolute demand for INSIG1 in the regulatory system that regulates lipid homeostasis in animal cells.
Previous results from overexpression and knockdown of INSIG1 in mice indicated that INSIG1 protein could regulate the transcription of SREBP (Engelking et al., Reference Engelking, Kuriyama, Hammer, Horton, Brown, Goldstein and Liang2004; McFarlane et al., Reference McFarlane, Liang and Engelking2014). In this study, lentivirus-mediated overexpression and knockdown were used to successfully overexpress and knock down the INSIG1 in BMECs, which altered mRNA expression level of SREBP in a fashion consistent with data in transgenic mice (Engelking et al., Reference Engelking, Kuriyama, Hammer, Horton, Brown, Goldstein and Liang2004; McFarlane et al., Reference McFarlane, Liang and Engelking2014). In this study, the results that INSIG1 overexpression decreased the expression of SREBP mRNA confirmed the role of INSIG1 in inhibiting SREBP transcription in buffalo mammary cells. And significant increase of SREBP mRNA after knockdown of INSIG1 further underscored the function of INSIG1 in inhibiting SREBP processing.
INSIG2 is a close homolog of INSIG1. Both INSIGs can bind SCAP in the endoplasmatic reticulum, inhibiting the SREBP processing (Krapivner et al., Reference Krapivner, Popov, Chernogubova, Hellénius, Fisher, Hamsten and Van'T Hooft2008). In primary cultures of rat hepatocytes, insulin-stimulated SREBP processing is mediated by selective depletion of INSIG2 by promoting decay of its homologous mRNA. Therefore, the decrease of INSIG2 protein induced by insulin leads to the enhancement of export of SCAP/SREBP complex from ER to the Golgi (Yellaturu et al., Reference Yellaturu, Deng, Park, Raghow and Elam2009). A previous study demonstrated that these two INSIG proteins have complementary roles in the regulation of the SREBP pathway (Engelking et al., Reference Engelking, Liang, Hammer, Takaishi, Kuriyama, Evers, Li, Horton, Goldstein and Brown2005), i.e. decrease of one INSIG protein is compensated for by an increase in the other INSIG protein. Consistent with that previous finding, we observed a significant decrease in the mRNA expression level of INSIG2 upon overexpression of INSIG1 in BMECs. Similarly, knockdown of INSIG1 increased the mRNA expression of buffalo INSIG2.
PPARG is an important central regulator of lipid metabolism in mammary cells. SCD involves desaturation in the de novo synthesis of fatty acids (Shi et al., Reference Shi, Luo, Yao, Zhu, Xu, Shi and Loor2013). INSIG1 is a PPARG target gene, and its regulation by PPARG can indirectly regulate SREBP protein activity (Kast-Woelbern et al., Reference Kast-Woelbern, Dana, Cesario, Sun, de Grandpre, Brooks, Osburn, Reifel-Miller, Klausing and Leibowitz2004). Furthermore, overexpression of SREBP significantly increased the expression of PPARG and SCD, indicating that they are target genes of SREBP (Xu et al., Reference Xu, Luo, Zhao, Yang, Tian, Shi and Bionaz2016). In this study, the change of INSIG1 significantly altered the mRNA expression level of PPARG and SCD. It is speculated that INSIG1 regulates PPARG and SCD by controlling SREBP. Thus, our study supported cross-talk between transcription factors in the regulation of milk fat synthesis (Bionaz and Loor, Reference Bionaz and Loor2008). However, INSIG1 interference had no effect on the expression of SCD in goat mammary epithelial cells (Li et al., Reference Li, Wang, Zhang, He, Shi, Luo and Loor2019), which may reflect a different molecular mechanism of milk fat metabolism in buffalo. Together, the data suggest that INSIG1 gene could affect desaturation of fatty acid.
TAG is a key component of milk fat, formed by long chain fatty acids and glycerol. In the process of TAG synthesis, there are three key enzymes: GPAM participates in the first step of catalytic reaction, AGPAT catalyzes the esterification of TAG sn-2 position, and DGAT makes diacylglycerol plus fatty acid acyl coenzyme A to form triacylglycerol, which is the rate-limiting enzyme for TAG synthesis (Abeni et al., Reference Abeni, Degano, Calza, Giangiacomo and Pirlo2005). In this study, overexpression of INSIG1 decreased the gene expression involved in the synthesis of TAG and knockdown of INSIG1 increased the gene expression. This is consistent with the results in rats that overexpression of INSIG1 can inhibit the expression of TAG synthesis genes (Takaishi et al., Reference Takaishi, Duplomb, Wang, Li and Unger2004). Thus, it is confirmed that a function of INSIG1 protein is to inhibit the TAG synthesis in buffalo. This idea is supported by the fact that the content of total TAG was decreased after overexpression of INSIG1.
In conclusion, we observed a significant difference in INSIG1 protein between peak lactation and dry-off period in buffalo mammary tissue. We successfully isolated the primary mammary epithelial cells from buffalo mammary tissue. The overexpression of INSIG1 decreased the expression of genes related to milk fat synthesis and cellular total TAG content, whereas knockdown increased the expression of genes. Our results provide a support for INSIG1 inhibiting SREBP processing. Furthermore, these results demonstrate a negative effect of INSIG1 on milk fat synthesis.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029920000710
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China (no. 31760659 and no. 31460582) and the Natural Science Foundation Key Project of Yunnan Province, China (no. 2014FA032 and no. 2007C0003Z).
