Performing Department
Animal Science
Non Technical Summary
The combination of increasing age of the population with an increasing incidence of obesity and type II diabetes has led to a muscle dysfunction commonly known as steatosis. Although steatosis can indicate simple accumulation of lipid within muscle fibers, more recent evidence has demonstrated that steatosis also includes the invasion of muscle fasciculi with newly formed adipocytes. In animal production, this is known as marbling and is considered highly desirable in beef and pork products consumed in the U.S., Asia, and Australia. We propose the novel concept that steatosis in humans, and marbling in livestock species, is caused in part by the over-production of oleic acid in adipocytes, which acts as a trans-acting factor to promote the trans-differentiation of muscle satellite cells into marbling adipocytes. To address this hypothesis, pigs will be made transgenic to the stearoyl-CoA desaturase (SCD) gene with a ubiquitous promoter as well as in pigs in which the SCD gene is silenced. We will use these pigs, as well as pigs that have tissue-specific, temporally regulated SCD gene expression, in experiments in which pigs are fed diets high in saturated and trans-fats (beef tallow) or diets high in oleic acid (sunflower oil). In this manner, we will be comparing conditions in which SCD gene expression is maximally activated (SCD-overexpressing pigs fed saturated fatty acids) and when SCD gene expression is maximally depressed (SCD-silenced pigs fed oleic acid). This livestock animal model will mimic conditions in humans exhibiting steatosis, both in outcome and mechanism of action. Additional metabolic effects will include hypertriglyceridemia (caused by overexpression of hepatic SCD) and promotion of preadipocyte differentiation in subcutaneous adipose tissue. This unique animal model will provide invaluable insight into the etiology of steatosis in humans, and also will further define the relationship between hepatic lipid metabolism and adiposity in livestock species.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
20%
Developmental
10%
Goals / Objectives
The following specific objectives will be addressed during proposed project: Hypothesis: We propose that overexpression of the SCD gene and concomitantly increased SCD catalytic activity will promote steatosis in porcine muscle. SCD overexpression may increase steatosis by promoting development of marbling adipose tissue. Objective 1: Establish pig lines in which SCD gene expression is ubiquitously overexpressed or silenced Objective 2: Document the effects of saturated and monounsaturated fatty acids on muscle steatosis and muscle, liver, and adipose tissue lipid metabolism in control, non-transgenic pigs Objective 3: Document the effects of saturated and monounsaturated fatty acids on muscle steatosis and muscle, liver, and adipose tissue lipid metabolism in pig lines in which SCD gene expression is overexpressed or silenced
Project Methods
Overall approach. The experiments described below are designed to establish conditions in which SCD gene expression is maximally activated (SCD-overexpressing pigs fed saturated fatty acids) and when SCD gene expression is maximally depressed (SCD-silenced pigs fed oleic acid), as well as intermediate levels of SCD gene expression. During the first 2 years, while SCD-transgenic pigs are being produced, we will establish the effects of dietary SFA and MUFA on SCD gene expression and catalytic activity in adipose tissue, muscle, and liver. Objective 1: Establish pig lines in which SCD gene expression is ubiquitously overexpressed or silenced We will produce pigs that ubiquitously overexpress SCD and pigs in which SCD gene expression is suppressed. The Porcine SCD previously has been cloned and sequence-verified in our laboratory. This construct as well as oligonucleotides expressing SCD will be subcloned into vectors . Although there is evidence that overexpression of SCD specifically in myoblasts (satellite cells) will cause trans-differentiation of myoblasts to adipocytes, it also is possible and also likely that overexpression of SCD in the fibroblasts of perimyseal connective tissue promote the differentiation of fibroblasts into intramuscular preadipocytes. For this reason, we will initiate development of pigs that ubiquitously overexpress SCD. At the same time, we will develop a line of pigs in which SCD expression will be suppressed. In phase 1 of this experiment, recombinant lentiviral vectors (rLV) containing an antibiotic resistance gene (not shown) will be used to transduce myocytes, hepatocytes, and adipocytes in vitro. Three days following transduction, cells will be evaluated for GFP expression and placed under antibiotic selection for 7 - 10 d. Selected populations of cells will be harvested and Quantitative Real-Time PCR will be utilized to assess the effectiveness of each shRNA at reducing endogenous SCD mRNA. To evaluate functional depletion or overexpression of SCD, we will analyze fatty acid composition, SCD activity, and total lipid profiles. We will select the shRNAs that exhibit at least a 90% reduction in SCD mRNA levels and appropriate functional expression of gene activity for future investigations. In phase 2 of this experiment, we will utilize the effective recombinant lentivirus proven during in vitro studies to produce transgenic pigs. Concentrated rLV containing the SCD overexpression, suppression, and null constructs will be injected in the previtelline space of porcine zygotes and transferred to synchronized recipients. Offspring will be evaluated visually for GFP expression and verified by tail docking at d 2 post-parturition and tissue used to verify transgene incorporation. These founder animals will be utilized for breeding to produce F1 offspring to evaluate the effectiveness of overexpression or suppression of SCD in all tissues. Objective 2: Document the effects of saturated and monounsaturated fatty acids on muscle steatosis and muscle, liver, and adipose tissue lipid metabolism in control pigs We propose that the dietary combination of palmitic and stearic acid will stimulate SCD gene expression, whereas dietary oleic acid will depress SCD expression. All animal experiments will test the effects of a diet high in palmitic and stearic acid (beef tallow) to a high in oleic acid (high-oleic sunflower oil). Commercial beef tallow contains 25% palmitic acid and 19% stearic acid, approximately 30% oleic acid and 5% trans fatty acids. High-oleic sunflower oil contains over 80% oleic acid and less than 8% total SFA. For Objective 2, using non-transgenic pigs, we will document SCD gene expression, lipid metabolism, tissue fatty acid composition, and muscle histology in response to specific dietary fats. Twenty pigs (initially 20 kg body weight) will have free access to a corn-soybean-meal-based diet containing 1.05% lysine. Pigs (n = 10 per treatment group) will be raised on this diet in individual pens until they reach starting weights for each experiment, approximately 50 kg. Pigs will be fed balanced diets containing either beef tallow (high in palmitic and stearic acid) or sunflower oil at 30% of their caloric intake. Diets will be fed from 50 kg to 80 kg final body weight, which is a time of rapid adipose tissue growth. If feed intake is altered in the SCD1-transgenic pigs, pair feeding of the control pigs will be initiated to control for feed intake. At the conclusion of the diet studies, pigs will be euthanized by exsanguination (severing of the jugular vein) while under a surgical plane of anesthesia (glycopyrrolate, 0.1 mg/kg body weight, and telazol, 6 - 8 mg/kg body weight). Portions of the fresh liver and longissimus muscle and subcutaneous adipose tissue at the 11th-12th thoracic rib interface will be placed in oxygenated, 37°C KHB containing 5 mM glucose and transported immediately to the laboratory, whereas other portions will be snap-frozen in liquid N2 for subsequent extraction of RNA, western blots, adiposity, and fatty acid composition. Samples of muscle will be frozen and -20°C for histological analyses. Objective 3: Document the effects of saturated and monounsaturated fatty acids on muscle steatosis and muscle, liver, and adipose tissue lipid metabolism in pig lines in which SCD gene expression is overexpressed or silenced For years 1 and 2, the study design will be a simple one-way analysis of variance, testing the effects of dietary fats on aspects of hepatic and adipose tissue lipid metabolism. Beginning in year 3, after the production of the SCD-transgenic pigs, the experimental design will be a 2 x 2 factorial design, testing the effects of two fats (tallow and sunflower) and two levels of SCD gene expression (SCD overexpressing and SCD silenced pigs) (n = 10 per treatment group). Dietary treatments are carcass endpoints will be as described for Objective 2. The following measurements will be made at the conclusion of all diet studies: 1. Lipogenesis in vitro. We expect that SCD overexpression will accelerate fatty acid and TAG biosynthesis is liver, muscle, and adipose tissue. Therefore, de novo fatty acid biosynthesis and TAG biosynthesis will be measured in fresh tissues. 2. Fatty acid composition. To confirm the biological effects of both the dietary treatments and the SCD transgene, lipids from adipose tissue, muscle, liver, and plasma will be measured. 3. Adiposity. Elevated SCD activity is associated with increased adiposity, so adipose tissues (subcutaneous and intramuscular) will be collected immediately postmortem from pigs for determination of cellularity (size and number of cells/g) by osmium fixation and counting. These data will indicate the effects of dietary treatments and the SCD transgene on adipocyte hypertrophy. 4. Stearoyl-CoA desaturase assay. A critical component of these experiments will be to demonstrate that overexpression of the SCD gene leads to elevated SCD activity. Subcutaneous adipose tissue, liver, and longissimus muscle microsomes will be isolated and SCD activity measured. 5. Histology. We anticipate increased abundance of intramuscular adipocytes in muscle in pigs overexpressing SCD. Histology of frozen blocks of longissimus muscle will be measured. 6. Specific gene expression. We predict that the expression of other genes associated with lipid metabolism with be profoundly influenced by the SCD transgene as well as by the dietary beef tallow and high-oleic sunflower oil. Accordingly, we will measure the mRNA levels for AMPKa, PGC-1a, PPARg, FAS, SCD, CPT1a and CPT1b, and RPS9 as a housekeeping gene. 7. Plasma lipids and metabolites. Plasma and serum samples will be analyzed for insulin, TAG, and lipoprotein cholesterol fractions by Spectracell (Houston, TX).