Expression Patterns of SREBF1 and SREBF2 in Liver and Adipose Tissues of Two Fat-Tailed Sheep Breeds


 Background

The fat-tailed sheep is raised largely in China and other countries due to its high adaptability to nutritionally challenging environments and disease resistance as well as fat deposition in tails. So the purpose of the study is to explore the expression pattern and regulation mechanism of sterol regulatory element binding proteins (SREBPs) in liver and adipose tissues of fat-tailed sheep bases on its role in fat metabolism and deposition with sheep grown and development.
Methods

Two representative Chinese fat-tailed sheep breeds i.e. Guangling Large Tailed (GLT) and Small Tailed Han (STH) were employed to evaluate the ontogenetic expressions of SREBF1 and SREBF2 from seven different adipose tissues and liver at 4, 6, 8, 10 and 12 month by real-time PCR. Five serum indicators were detected, and the function speculation of SREBP1 and SREBP2 were evaluated by bioinformatics approaches.
Results

Serum indicators, especially total cholesterol TC and non-esterified fatty acid (NEFA) showed obvious differences and dynamical changes with the different development ages, gender factors only had a significant effect on serum NEFA concentrations. Both SREBF1 and SREBF2 mRNA expression in sheep were significantly affected by breed, tissue, age factors, and show a significant positive correlation (r = 0.286). Merely the expression of SREBF1 gene in STH is significantly higher than that in GLT, but SREBF2 gene expression is opposite. The expressions of SREBF1/2 in liver are extremely higher than that in seven adipose tissues, the interaction of two factors between breed and month age, breed and tissue, and tissue and gender also significantly affects its expression. Subcellular locations and function prediction imply that SREBP1/2 expressions are closely related with metabolism processes in cells.
Conclusions

The mRNA expression profiling of SREBF1 and SREBF2 showing a breed-specific, gender-specific, and temporal and spatial expressions differences, which imply that SREBF1/2 play a crucial role in lipid metabolism regulation during growth and development of two fat-tailed sheep. This study provides a phenotypic basis for further revealing the genetic mechanism in lipid metabolism and fat deposition that causes differences in ovine tail types, which also provides a novel insight for improving quality of meat.


Background
Page 3/25 Domesticated sheep are economically important livestock species, and provide many daily necessaries for human consumption including meat, milk, and wool worldwide. 1 High-quality domesticated sheep breed is a valuable genetic resource for the global animal husbandry. 2 The fat-tailed sheep is widely bred in China and other countries, due to its high adaptability to different climatic conditions, disease resistance and high production in poor nutritional situations. 3 Among, the fat deposition in the tails plays a crucial role for the sheep to adapt to nutritionally challenging environments. 4 It is known that traits such as fatty acid content and lipid deposition are vital for sensory, nutritional and technological properties of mutton. 5 Lipid metabolism is also an extremely important physiological process maintaining nutrient adjustment, homeostasis, and animal health. [6][7][8] Thus, exploring the mechanism of fat metabolism and deposition of fat-tailed sheep has profound meaning to improve meat quality in food and agriculture elds.
Sterol regulatory element binding proteins (SREBPs) can regulate the lipid homeostasis by regulating its target genes, which are crucial for the cholesterol and fatty acid metabolism. 9,10 The mature forms of SREBPs are transcriptionally active and trans-located into the nucleus where bind to the promoters of SREBPs target genes, most of which are involved in lipid metabolism. 11 For example, SREBP1c which is encoded by SREBF1 mainly promotes the fatty acid synthesis by activating many genes involved in lipid metabolism in fat tissue and liver. 9 SREBP2, another member of the SREBPs nuclear transcription factor family, which is encoded by SREBF2, activates the target gene transcription and the gene expressions on the cholesterol biosynthesis pathway by binding to the sterol regulator and promoter/enhancer in the lipid synthetic enzyme gene. 12,13 However, the role of the transcriptional regulation of SREBPs in fat-tailed sheep is unclear.
Therefore, two representative Chinese fat-tailed sheep breeds were employed in this study. Five serum lipid metabolism indicators, including triglyceride (TG), total cholesterol (TC), non-esteri ed fatty acid (NEFA), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC), were determined based on their body size and weight measurement traits. More importantly, the transcriptional levels and function speculation of SREBP1 and SREBP2 in the various adipose tissues and liver with the development stages were evaluated by qRT-PCR and bioinformatics approaches. All these investigations will provide a new insight for revealing the regulation function of SREBP1 and SREBP2 in fat metabolism and deposition, as well as in meat quality from the different fat-tailed sheep.

Animals and Ethical Statement
Total 80 individuals of Guangling Large Tailed sheep (GLT) and Small Tailed Han sheep (STH) with half for each breed were selected according to the genders and ages in this study. GLT, a local outstanding breed distributed in the mountain regions of northern Shanxi province, is typically of large tails for fat deposit and good mutton quality but low fecundity. STH is characterized by small fat tails but higher fecundity in the plain regions of Shandong and Hebei provinces. The disparity of fat deposition in tail between GLT and STH is obvious in different development stages. The experimental animals are fed and managed as described before. 14  This study was carried out under the guidelines for the care and use of animals for scienti c purposes. The protocol was approved by the Institutional Animal Care and Use Ethics Committee of Shanxi Agricultural University.

Body measurement
The body size and weight measurement traits of all sheep, i.e. body height at withers (BH), body length (BL), heart girth (HG), cannon circumference (CC), tail length (TL), tail width (TW), live weight (LW), carcass weight (CW), and absolute tail fat weight (ATW) were measured by using measuring tape and platform scale according to the standard procedure. Relative tail fat weight (RTW) and dressing percentage (DP) were calculated as follows: RTW = ATW/CW; DP = CW/LW.

Serum and tissues collection
Four males and four females for each breed were randomly selected and slaughtered at 4, 6, 8, 10 and 12 months of age, respectively. Blood samples were collected from external jugular vein. Blood was allowed to clot, followed by centrifugation at 3000 rpm for 10 min. The serum was collected and sent to measurement of serum biochemical parameters. Adipose tissues, i.e. tail fat (TA), great omental fat (GO), subcutaneous fat (SC), small omentum fat (SO), perirenal fat (PR), retroperitoneal fat (RP), mesenteric fat (MT) and liver (LV), were rapidly dissected, weighed and placed in liquid nitrogen and stored at − 80 °C.

Measurement of serum biochemical parameters
Serum samples were separated from all sheep to measure the contents of key biochemical indicators related to lipid metabolism and deposition. The indicators included triglyceride (TG), total cholesterol (TC), non-esteri ed fatty acid (NEFA), high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC). The concrete operations were conducted according to the manufacturer's manual of kits (Nanjing Jiancheng Bioengineering Institute, China).

RNA Extraction and Real-Time RT-PCR
Total RNA was extracted from liver and adipose in tail, subcutaneous, great and small omental, retroperitoneal, mesenteric, perirenal tissues by Trizol (Invitrogen) following manufacturer's instructions. All of the RNA samples were treated with DNase I, followed by standard reverse transcription using SYBR® PrimeScript™ RT-PCR Kit. Real-Time qPCR was carried out using SYBR® Green PCR Master Mix (Applied Biosystems). The programs for the ampli cation were as following: activation of polymerase at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. The analysis of dissociation curves was always performed after 45 cycles. Primers were designed by Primer 3 Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and checked by Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LOC=BlastHome). The primers are listed as followed:

Statistical analysis
The data of body size measurements, serum biochemical parameters, and relative mRNA expressions were grouped and subjected to statistical analysis by T-test and one-way ANOVA using GraphPad Prism 8.0 software (San Diego, CA, USA). In addition, the potential interaction relationships between SREBP1/2 expression levels and other traits with breed, age, gender were analyzed by the software SPSS (version 26.0, IBM, Chicago, USA) according to the following general linear model: where, y ijkl is the observation for body size measurements and serum biochemical parameters, µ is the overall mean for each trait, B i , M j and G k are the main effect of breed, month of age and gender, respectively, BM ij , BG ik and MG jk are the interaction effect of breed × month of age, breed × gender and month of age × gender, respectively, and e ijkl is the random error. Associations between SREBF1/2 mRNA expression in eight tissues and slaughter and tail traits in sheep were analyzed by SPSS (version 26.0, IBM, Chicago, USA).

Body size and weight in the two fat-tailed breeds
To evaluate the breed characteristics between GLT and STH, the parameters of body size and body weight were evaluated. As shown in Fig. 1. The mean body height of GLT is higher signi cantly than that of STH (P < 0.01), while the length and width of tail are less than that of GLT (P < 0.001). No signi cant differences were observed in body length, heart girth and cannon circumference (Fig. 1A). Although the live weight of STH was slightly lower compared to that of GLT (P < 0.05), the total carcass weights of the two breeds have no signi cant differences (Fig. 1B). These differences fully re ect the characteristics of these two breeds, especially in tail fat deposits.

Dynamical changes of serum lipid metabolism indicators on fat-tailed sheep
To explore the dynamic changes and lipid metabolic differences between GLT and STH, ve serum biochemical parameters involved in lipid metabolism were examined between two fat-tailed breeds at different development stages of 4, 6, 8, 10, 12 months. The results showed that the average of serum TC, and NEFA at ve time points are obvious different, while other parameters have no signi cant difference between breeds. Interestingly, only serum NEFA in ewe is signi cantly higher than that in rams' values (Table S1).
The changes of these indicators with the age of month are relatively complicated (Fig. 2). In STH, serum TG concentration was the highest at 4 months of age, signi cantly higher than other months of age, and decreased with the increase of age; TC, NEFA, HDLC, and LDLC concentrations in serum did not change signi cantly with age between 4 and 10 months, but there was a tendency to decrease or increase (LDLC) at the age of 12 months. Comparatively, in GLT, TG did not change signi cantly between 4 and 12 months with a tendency to decrease at 12 months. TC, HDLC, and LDLC show the similar changes which rstly increase signi cantly and then decreases from 4 to 12 months of age. The concentration of NEFA was the highest at 4 months which was signi cantly higher than that at 6 and 8 months, and there was a tendency to increase at 10 months and decreased again at 12 months. Those dynamical changes indicate that the lipid metabolism pattern of STH and GLT maybe different with developmental ages.
The mRNA expression pro le of SREBF1 in liver and fat tissues To determinate the role of SREBF1 in fat metabolism regulation of fat-tailed sheep, the relative mRNA expression pro le of SREEP1 in liver and adipose tissues in two breeds with different ages were detected by real-time RT-PCR (qPCR). The result showed that the global mRNA expression was signi cantly different between GLT and STH (P < 0.05, Fig. 3A), but no signi cant gender difference was found (Fig. 3B, Table S2). Merely, in female, the mRNA expresses in STH higher than those in GLT (P < 0.05, Fig. 3C). During development age, SREBF1 expresses relatively stable in either male or female or combination in GLT ( Fig. 3D and 3E). However, the total mRNA level was the highest at 10 months of age, and signi cantly higher than that at 8 months of age (P < 0.05, Table S2). In male STH, the expression at 10-month is signi cantly higher than that both at 6-and 8-month (P < 0.01, Fig. 3E).
We also examined SREBF1 expression levels in liver and adipose tissues, which are involved in adipogenesis and lipid metabolism. The expression in liver was extremely higher than adipose tissues including tail (TA), great omental (GO), subcutaneous (SC), small omentum (SO), perirenal (PR), retroperitoneal (RP), and mesenteric (MT) fats in both GLT and STH (Fig. 3F, G). Similar results also were observed in males of GLT and STH. The difference is that the female of GLT have not presented a tissuespeci c expression, except in MT. In STH females, the expression in MT was lower than that in liver signi cantly (P < 0.05).
Correlation analysis showed that the mRNA expressions between liver and Go were extremely signi cantly positively correlated in GLT (Table 1, r = 0.854, P < 0.01). In STH, TA and SC, PR and GO, PR and SO presented the positive correlation (r = 0.852, 0.915, or 0.979, P < 0.05). All results reveal that SREBF1 maybe plays a crucial role in fat metabolism regulation during growth and development of two breeds of fat-tailed sheep. The mRNA expression of SREBF2 in liver and fat tissues Transcriptional levels of SREBF2, another member of SREBFs family, in adipose tissues and liver were further examined and shown in Fig. 4. Different from SREBF1, SREBF2 expressed signi cantly higher in GLT than in STH (P < 0.001, Fig. 4A) and signi cantly higher in female than in male (P < 0.001, Fig. 4B), especially in female of GLT (P < 0.001, Fig. 4C). The age had no signi cant effects on the expression in GLT (Fig. 4D). However, it expressed signi cantly higher at 10-month-old females than in males at the same age in GLT (P < 0.05). In STH, it reduced to the lowest point at 8-month-old in comparison to 4month-old (P < 0.05) and then went up a little. The same changing tendency was observed in female STH (P < 0.01, Fig. 4E).
Similar to SREBF1, the expression of SREBF2 was enriched in liver than other adipose tissues. In GLT, SREBF2 mRNA expressions in liver were signi cantly higher than TA (P < 0.05), PR (P < 0.05) and MT (P < 0.01, Fig. 4F). In STH, SREBF2 mRNA expressions in liver were markedly higher than all the adipose tissues except GO (P < 0.001 or P < 0.0001, Fig. 4G). According to the analysis of gender, SREBF2 mRNA expressions in liver of male GLT were signi cantly higher than all adipose tissues. MT of GLT is the only tissue that SREBF2 mRNA expression differed signi cantly between male and female (P < 0.001). In female GLT, the SREBF2 mRNA levels was signi cantly higher in RP than in TA (P < 0.01), while there were no signi cant differences between any other tissues (Fig. 4H, Table S3). The correlation coe cients between SC and LV, MT and LV, PR and LV, PR and SC, PR and MT, SO and MT are signi cant( Table 2, P < 0.05 or P < 0.01). In STH, expression of SREBF2 was numerically but not signi cantly increased in liver of male STH, but the signi cantly expressions variant observed between adipose tissues, such as MT and TA, GO and RP, SC and PR (Fig. 4I). In female STH, liver showed higher expression levels than any other adipose tissues, and SREBF2 expression in MT was the lowest compared to liver and GO (P < 0.05). Further analysis results showed that the signi cantly positive correlation of SREBF2 expressions occurred in between GO and SO, PR and GO, PR and SC, PR and SO ( Table 2, P < 0.05). These results indicated that SREBF2 also played a crucial role in the regulation of lipid metabolism during growth and development of the two breeds of fat-tailed sheep.
Associations between SREBP1/2 expressions and slaughter and tail traits Associations between SREBF1/2 mRNA expression in eight tissues and slaughter and tail traits in sheep were analyzed. The results demonstrated that SREBF1 mRNA expressions in TA were signi cantly related to tail-type traits in GLT, such as absolute tail fat weight (ATW) and relative tail fat weight (RTW). The signi cant correlation also were found between LV and tail length (TL), LV and RTW, GO and TL, respectively. While in STH, signi cant correlation only occurred between SREBF1 expressions in PR and body weight (BW), as well as carcass weight (CW), which was not related to tail-type traits (Table 3). Relative SREBF2 mRNA abundances in liver were signi cantly related negatively to tail-type traits including TL, TW, and ATW in GLT. There were also signi cant negatively relationships between SC and TW, as well as between GO and ATW or CW. While the only signi cant positively correlation to CW was SREBF2 mRNA abundances in SC of STH (Table 4). According to SREBP1/2 expression levels in different tissues, age, and breeds, the location and function of SREBP1/2 in cell were analyzed by bioinformatics approaches. The subcellular locations results showed that SREBP1 and SREBP2 working mainly in different parts inside cells (Fig. 5A). About 78.30% of SREBP1 distributed in nucleus, and the rest in cytoplasm, vesicles of secretory system, plasma membrane, and endoplasmic reticulum. Unlike in SREBP1, only 30.40% of SREBP2 can be predicted in nucleus and 39.1% in the endoplasmic reticulum. In addition, it also distributed in vacuole, cytoplasm, and vesicles of secretory system, Golgi and mitochondria.
Functional prediction shows that both SREBP1 and SREBP2 play roles in many biological processes (Fig. 5B). SREBP1 mainly functioned in purines and pyrimidines, replication and transcription, and regulatory function, while SREBP2 mainly functioned in the process of transport and binding, purines and pyrimidines, translation and central intermediary metabolism.

Discussion
The fat-tailed sheep is raised largely in many countries due to its high adaptability to nutritionally challenging environments and disease resistance as well as fat deposition in tails. 4,15,16 The tail fat plays a crucial role during growth and development of different breed sheep. 17 Therefore, as two typical fattailed breeds, Guangling Large Tailed (GLT) and Small Tailed Han (STH) sheep in China were selected in this study. GLT is typically of large tails for fat deposit and good meat quality but low fecundity, and STH is characterized by small tails but higher fecundity in the plain regions. Our study rstly revealed the dynamical changes of serum lipid metabolism indicators, and the expression patterns of key genes SREBF1 and SREBF2 in liver and adipose tissues of GLT and STH.
Mutton is becoming attractive and alternative meats due to the low percentage of fat and good source of essential fatty acids. 5,18 It is of great importance to improve meat production traits such as live weight and carcass weight in sheep breeding. 19 In general, live and carcass weights have the same variation tendencies. 20 In this study, in spite of obvious difference in live weight between the two breeds, there was no signi cant difference in carcass weight. If GLT is to achieve a carcass weight similar to that of STH, it needs to be much heavier. In other words, GLT may need to consume more feed and forage than STH with comparable carcass weight. However, the feed and forage is not only converted into meat but also into tail fat, which can be derived from the result that GLT with higher live weight also has larger fat tail than STH. It is obviously not economical in sheep production. 3,21 Therefore, regulating tail fat deposition is a bene cial attempt to improve meat production performance and economic bene ts.
Serum biochemical parameters are the crucial indicators for lipid metabolism which is closely correlative with production property and physiological process maintaining nutrient adjustment, homeostasis, and sheep healthy. [6][7][8]22 For example, triglyceride (TG) is a fat molecule formed by the condensation of longchain fatty acids and glycerol. Most tissues can be powered by ATP produced by the oxidative decomposition of fat. 23,24 High density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) are important part of total cholesterol (TC) which affects normal lipid metabolism and resulting in metabolic disorders. 25,26 As a hydrolyzed product of triglycerides, non-esteri ed fatty acid (NEFA) is stored in adipose tissue and also a source of animal energy. 27 In the present study, serum indicators, especially TC and NEFA showed obvious differences and dynamical changes with the different development ages, gender factors only had a signi cant effect on serum NEFA concentrations.
Studies have shown that TC content is positively correlated with sebum rate and liver fat rate. 28 The signi cantly increased serum NEFA concentration in starved cows was reported by previous study. 29 So those results implied that the fat metabolism regulation pattern in STH and GLT may be distinguishing which provide the phenotypic changes basis for revealing the genetic mechanism leading to the difference of ovine tail type.
Lipid metabolism and deposition is composed of many enzymatic reactions and often are regulated by many molecules. 9,30 In our previous studies, the roles of Lpin2/3, angiopoietin-like protein 4, miR-124-3p, and other genes and non-coding RNAs in the regulation of fat deposition from two fat-tailed sheep breeds had been discussed. [31][32][33][34] Therefore, this study mainly focuses on the crucial role of sterol regulatory element binding proteins (SREBPs) in fat-tailed sheep base on the dynamical changes of serum lipid metabolism indicators. SREBPs including SREBP-1a, SREBP-1c and SREBP2 are mainly responsible for regulating cellular lipogenesis and lipid homeostasis. 35 SREBP-1a and SREBP-1c are encoded by SREBF1, which involves in the synthesis of fatty acid and triglyceride, while SREBP2 is encoded by SREBF2, which mostly regulates the cholesterol gene expression. 36 In the present study, both SREBF1 and SREBF2 gene mRNA expression in sheep were signi cantly affected by breed, tissue, and age factors. The expression of SREBF1 gene in STH is signi cantly higher than that in GLT, but SREBF2 gene expression is opposite. Merely the expression of SREBF1 and SREBF2 genes in sheep showed a signi cant positive correlation (r = 0.286). The mRNA expression pro ling of SREBF1 and SREBF2 are different in the two sheep breeds, which are coincident with previous studies in other fat-tailed sheep. 37,38 Another study indicates that there is a genetic basis for the phenotypic differences between fat-tailed and thin-tailed sheep. 39 In the comparative study of the lung cells, the expression levels of SREBFs on the rst day of birth was signi cantly higher than that on the rst 17.5 days of embryonic development. 40 In this study, both SREBF1 and SREBF2 expressions in liver and the different fat tissues at the ages of 4, 6, 8, 10 and 12 month are various. For instance, the expression of SREBF1 in liver is extremely higher than that in seven adipose tissues, but the difference in SREBF1 gene expression between different genders is not signi cant; the interaction of two factors between breed and month age, breed and tissue, and tissue and gender also signi cantly affects its expression. In contrast, the role of SREBF2 gene is not exactly the same. The expression of SREBF2 in the liver is also signi cantly higher than adipose tissue; the expression of female is signi cantly higher than that of male. Interesting, the expression of SREBF1 gene has spatio-temporal differences, while the expression of SREBF2 gene does not have this characteristic, but it has different roles in different genders. Those results further provide the new evidence for revealing the genetic mechanism leading to the difference of sheep tail type.
In addition, SREBPs are transcriptionally inactive when sterols are abundant, and synthesized and inserted into the endoplasmic reticulum (ER) as large precursor proteins. 41,42 The depletion of sterol can promote the release of mature SREBPs proteins and are transported from ER to the Golgi. 43 The reduction of fat mass, hepatic and plasma triglycerides was observed in SREBP1c-speci c knockout mice. 44 In the present study, the results from subcellular locations and function prediction imply that SREBP1 and SREBP2 expressions are closely related with metabolism processes in cells. The differences in the expression of SREBFs in sheep between different breeds (different fat tail types) may be a cause of the difference in tail types between the two sheep breeds. The data from the associations between SREBF1/2 expressions and slaughter and tail traits also further con rmed this fact. The role of transcription factor genes Srebf1 and Srebf2 in the phenotype of fat deposition and the relationship with metabolites were shown in Fig. 6.

Conclusions
Taken together, our study reveals that the serum concentrations of TC and NEFA in sheep have signi cant differences between breeds, and mostly indicators showed the dynamical changes with the different development ages. Importantly, the mRNA expression pro ling of SREBF1 and SREBF2 are different in two sheep breeds, gender, and adipose tissues, showing a breed-speci c, gender-speci c, and temporal and spatial expressions differences. All results implied that SREBF1/2 play a crucial role in lipid metabolism regulation during growth and development of two fat-tailed sheep, correspondingly, lipid metabolism regulation pattern in STH and GLT maybe distinguishing. This study provides a phenotypic basis for further revealing the genetic mechanism in lipid metabolism and fat deposition that causes differences in ovine tail types, which also provides a novel insight for improving quality of meat.    The mRNA expression pro les of SREBF1 in adipose tissues in two fat-tailed sheep with age by qRT-PCR.

List Of Abbreviations
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