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J. Anim. Sci. 2002. 80:2840-2849
© 2002 American Society of Animal Science

Coordinate regulation of ovine adipose tissue gene expression by propionate

S. H. Lee and K. L. Hossner1

Department of Animal Sciences, Colorado State University, Fort Collins 80523

1 Correspondence:
phone: 970-491-6667; fax: 970-491-5326; E-mail:
khossner{at}agsci.colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The current study examined the acute effects of intravenous propionate infusion on plasma hormones and metabolites and the expression of adipose tissue lipogenic genes. Four yearling rams were assigned to one of two groups (saline or propionate infusion) in a crossover design. All sheep were cannulated in both jugular veins and infused with 1.2 M propionate at a rate of 64 µmol•min-1•kg BW-1 for 30 min. Blood samples were collected at -10, 0, 5, 10, 20, 30, 60, and 120 min after initiation of infusion. Subcutaneous adipose tissue biopsies were obtained from the tailhead at 0 and 2 h after propionate infusion and analyzed for gene expressions of lipoprotein lipase, acetyl CoA carboxylase, fatty acid synthase, peroxisome proliferator-activated receptor {gamma}, leptin, and uncoupling protein-2 using a nonisotopic ribonuclease protection assay. The partial cDNA of the enoyl reductase region of ovine fatty acid synthase was cloned and sequenced from s.c. adipose tissue of sheep. The deduced amino acid sequence (210 amino acids) was 86% identical to human, 88% identical to rat, 88% identical to mouse, and 72% identical to chicken. Plasma glucose and insulin concentrations abruptly increased 5 min after beginning propionate infusion and further increased up until 30 min but were unaffected in saline-infused sheep (P < 0.05). Plasma concentration of NEFA decreased (P < 0.05) during propionate infusion, whereas IGF-I levels were unaltered. The amounts of lipoprotein lipase, acetyl CoA carboxylase, fatty acid synthase, peroxisome proliferator-activated receptor {gamma}, and leptin mRNA increased (P < 0.05) in s.c. adipose tissue of propionate-infused sheep compared with those of saline-infused sheep. However, uncoupling protein-2 mRNA decreased (P < 0.05) in propionate-infused sheep. This study demonstrates that an acute nutrient challenge, in the form of i.v. propionate, can stimulate or inhibit the expression of various adipose tissue genes involved with lipogenesis and adipose tissue metabolism.

Key Words: Adipose Tissue • Complementary DNA • Fatty Acid Synthase • Lipogenesis • Propionate • Sheep


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
In contrast to nonruminants, VFA, the products of ruminal fermentation, are the primary energy source in ruminants. Infusion of the VFA propionate, but not acetate, stimulates glucagon and insulin release in ruminants (Sano et al., 1993; Harmon, 1992). These hormones stimulate gluconeogenesis and glucose transport, respectively. Insulin also is an important regulator of lipid metabolism. Thus, mediation of nutrient stimuli occurs via hormonal pathways. Utilization of nutrients only begins, of course, with cellular uptake, as nutrients are subsequently used to make more complex molecules, such as glycogen, proteins, and lipids.

To study the interaction between nutrients, hormones, and tissue metabolism, we examined the effects of an acute propionate infusion on ovine adipose tissue expression of six genes involved with lipid metabolism and lipogenesis. These include the nuclear hormone receptor peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), which regulates the expression of genes involved in lipid metabolism, energy balance, and adipocyte differentiation (Rosen and Spiegelman, 2001). In fact, PPAR{gamma} may act as the body’s "fatty acid sensor," and mimics many of insulin’s actions (Kliewer et al., 2001). Lipoprotein lipase (LPL) provides fatty acids to adipose tissue (Auwerx et al., 1992), while acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS) catalyze irreversible and rate-limiting steps of lipogenesis (Abu-Elheiga et al., 2001; Wakil, 1989). In addition, we assessed the effects of propionate infusion on the expression of two other genes that regulate metabolism: uncoupling protein-2 (UCP2), which uncouples oxidative phosphorylation from energy production (Ricquer et al., 2000), and leptin, which is secreted by adipocytes, is proportional to fat mass, and inhibits feed intake (Hossner, 1998). The objective of the current study was to examine the acute nutritional and hormonal regulation of adipose tissue genes that are involved in ruminant lipid metabolism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals, Diets, and Experimental Procedure
Four yearling Southdown rams, weighing 65.9 ± 3.3 kg, were used in a crossover design. All sheep were housed indoors in metabolic crates and fed 520 g of alfalfa hay and 212 g of concentrate to meet 80% of total nutritional needs for maintenance (NRC, 1985) at 0900 and 1700 h. Water was available ad libitum. The Colorado State University Animal Care and Use Committee approved all animal procedures. Each period of the crossover design consisted of 5 d for adjustment and 1 d for infusion and sampling.

Sheep were fitted with cannulas in both jugular veins the day before infusion. Jugular veins were cannulated with a 12-gauge needle and 100 cm of polypropylene tubing (i.d.: 0.040 in., o.d.: 0.070 in.) (Norton Co., Akron, OH) under local anesthesia with 2% lidocaine HCl (Abbott Laboratories, North Chicago, IL). Cannulae were sutured to the skin of the animal adjacent to the jugular vein and filled with sterile 3.8% trisodium citrate solution to prevent clotting when not in use. The patency of cannulae was checked by withdrawing blood and by flushing the tubing with fresh trisodium citrate solution every morning. Liquids for jugular infusion (1.2 M propionic acid adjusted to pH 7.4 with NaOH and 0.9% NaCl) were sterilized by passage through a 0.2-µm filter before infusion. On the afternoon before infusion, diets were withdrawn and sheep were allotted into saline control and propionate infusion groups, based on paired body weights. Propionate solution was infused into the jugular cannula at the rate of 64 µmol•min-1•kg-1 BW for 30 min, whereas control animals were infused with sterile saline. Infusions were done with a multichannel peristaltic pump (Harvard Apparatus, Mills, MA). Blood samples were withdrawn from the contralateral jugular cannula using a syringe 10 min prior to infusion, immediately before infusion, and 5, 10, 20, 30, 60, and 120 min after beginning infusion. Blood samples were placed in chilled heparinized vacutainer tubes, centrifuged (5,000 x g, 4°C) for 15 min, and plasma was stored in small aliquots at -20°C before analysis.

Adipose tissue biopsies (~1 g) were taken from alternating sides of the tailhead immediately prior to and 2 h after infusion under lidocaine anaesthesia. Adipose tissue was frozen immediately in liquid nitrogen, transported to the laboratory, and stored at -70°C until analyzed. Incisions were closed with sterile #2 nylon suture, and sheep were treated with 20 mg/kg BW tetracycline (Boehringer Ingelheim, Marburg, Germany) after last biopsies.

Cloning and Sequence Analysis of Partial cDNA for Ovine Fatty Acid Synthase
Degenerate PCR.
Total RNA was isolated from s.c. adipose tissue of sheep using Trizol reagent (Sigma, St. Louis, MO). Reverse transcription reactions were performed in a 25-µL reaction using MMLV-reverse transcriptase (Promega, Madison, WI). Random hexamer (500 ng) and 1 µg of total cellular RNA were heated to 70°C for 10 min and quick-chilled on ice. The reverse transcription reactions were performed with 500 µM deoxynucleotide triphosphates (dNTPs), 20 U of RNase inhibitor, and 200 U of MMLV-reverse transcriptase at 37°C for 1 h. This pool of cDNA was used as template in the PCR with degenerate primers. Degenerate primers were designed based on two conserved segments of enoyl reductase region from human, mouse, rat, and chicken (Schweizer et al., 1989; Holzer et al., 1989; Jayakumar et al., 1995; Ueno, 2000). Polymerase chain reaction was performed in a 50-µL reaction containing 200 µM dNTPs, 2.5 mM MgCl2, 1.2 µM of each degenerate primer



100 ng of cDNA template, and 2.5 U of Taq DNA polymerase (Sigma, St. Louis, MO). The PCR conditions included a single 3-min 93°C cycle, three cycles at 93°C for 1 min, 37°C for 1 min, 72°C for 2.5 min, 35 cycles of 93°C for 1 min, 53°C for 1 min, and 72°C for 2.5 min with a final extension at 72°C for 5 min. Amplified PCR products were purified by separation in a 2% agarose gel and checked for an expected band size of 630 bp.

Cloning and Sequence Analysis.
Polymerase chain reaction products were subcloned into the 2.1-TOPO plasmid vector using the TA cloning kit (Invitrogen, Carlsbad, CA). The transformation procedure was performed according to manufacturer’s directions with TOP10 cells (Invitrogen). The positive clones (live colonies) were selected and subjected to an alkaline lysis miniprep procedure (Davis et al., 1986). The plasmids were cut with 12 U of EcoRI (Promega, Madison, WI) for 2 h at 37°C and identified with 2% agarose gel electrophoresis. The plasmids (with the expected 630-bp PCR product) were sequenced by Macromolecular Resources (Colorado State University) using a Perkin-Elmer 2400 thermal cycler (Norwalk, CT) with both M13 forward and reverse primers. The sequence was compared with those of known species through the GenBank database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD) by using the GCG Blast program.

Analytical Procedures
Plasma glucose was determined spectrophotometrically by the glucose oxidase-peroxidase method (Sigma, St. Louis, MO). Plasma insulin and IGF-I concentrations were analyzed using double-antibody sandwich enzyme immunoassay kits (Diagnostic Systems Laboratories, Webster, TX). Serum-binding proteins for IGF were removed by acid-ethanol precipitation (Daughaday et al., 1982). Serial dilutions of serum were parallel to the IGF-I standard curve. Nonesterified fatty acid (NEFA) concentrations were analyzed using an enzymatic colorimetric method (Wako Chemicals, Richmond, VA).

Ribonuclease Protection Assay
Antisense biotin-labeled riboprobes were synthesized using reverse-transcription PCR. Two micrograms of total adipose-tissue RNA were reverse-transcribed with 200 U of reverse transcriptase for 1 h at 37°C. The cDNA from this reaction was subjected to PCR using the primer pairs in which the bacteriophage T7 promoter sequences were added to the 5' flanking region of antisense primers (Kain et al., 1991) as described in Table 1Go. The PCR reactions were performed with 100 ng of cDNA, 2.5 mM MgCl2, 200 µM dNTPs, 400 nM each of sense and antisense primer, and 2.5 U of Taq DNA polymerase (Sigma) with 35 cycles in the thermal conditions described in Table 1Go. An in vitro transcription was carried out using a MAXIscript labeling kit (Ambion, Inc., Austin, TX) in a 20-µL reaction volume containing 5 µL of PCR products; 1x buffer; T7 RNA polymerase; 0.5 mM each of ATP, GTP, and UTP with 0.3 mM CTP; and 0.2 mM biotin-labeled 14-CTP (Gibco BRL, Grand Island, NY). The reaction was performed at 37°C for 1 h. After incubation with DNase I for 15 min at 37°C, transcripts were denatured (94°C for 3 min) and separated on 5% acrylamide/8 M urea denaturing polyacrylamide gels. After staining with 2.0 µg/mL of acridine orange for 15 min, the full-length riboprobe was eluted from the gel overnight at 37°C, precipitated, and quantified at 260/280 nm.


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  		Table 1. The sequences of sense (S) and antisense (AS) primers used for synthesis of lipoprotein lipase (LPL), acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), leptin, uncoupling protein-2 (UCP2), and ß-actin riboprobes. The minimal T7 promoter region (27 nt) is underlined
 
Ten micrograms of total RNA and 400 pg of biotin-labeled riboprobe was denatured (94°C for 3 min), then allowed to hybridize at 45°C overnight with 30 µL of hybridization buffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, 40 mM PIPES, pH 6.8), and then treated with 5 U/mL of RNase A and 200 U/mL of RNase T1 for 30 min at 37°C. After inactivation of RNase with 20 µL of 10% SDS and 2.5 µL of 20 mg/mL proteinase K for 10 min at 37°C, protected RNA was collected by phenol/chloroform/isoamylalcohol (25:24:1) extraction, ethanol precipitation, and separated by electrophoresis through an 8 M urea, 5% polyacrylamide gel. The protected RNA was electrophoretically transferred onto a positively charged nylon membrane (Schleicher & Schuell Inc, Dassel, Germany) with 0.5x TBE at 400 mA for 1 h using Semi-dry Transblot (BioRad, Hercules, CA; Ishihara and Shikita, 1990). After UV-crosslinking, the membrane was incubated with alkaline phosphatase-conjugated strepavidin (Promega, Madison, WI) for 1 h, incubated with 0.25 mM of CDP-star (Roche Diagnostics Corp., Indianapolis, IN) for 10 min at room temperature and exposed to CL-X Posure film (Pierce, Rockford, IL) for 20 to 30 min at room temperature. Autoradiographs were scanned (ScanJet 6100C, Hewlett Packard, Palo Alto, CA) and the image was analyzed using Scion Image software (Scion Corp., Frederick, MD). A ratio was calculated for the intensity of target band vs ß-actin standard bands on each lane of the gels and the ratio was compared between control- and propionate-infused groups.

Statistical Analysis
Statistical analyses of data were performed with GLM procedures of SAS (SAS Inst. Inc., Cary, NC). All variables were analyzed using a split-plot model. The model included sheep, period, treatment, and time effects. The whole-plot effects were period x animal x treatment, and the subplot effects were time and treatment x time interaction. If a sampling time effect was observed, means were compared with the 0-time sample. When significant treatment x time interactions were obtained, treatment means for each time were separated using a t-test at the P < 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
Cloning and Sequence Analysis of the Partial cDNA for Ovine Fatty Acid Synthase
The choice of the region for amplification was based on its conserved AA sequence between species and the relative paucity of R, L, and S amino acids. Initially, we tried to clone a sequence of the thioesterase region, but these degenerate primers provided artifactual results. Thus, we used the consensus sequence of the enoyl reductase region.

Degenerate PCR amplification resulted in a product of 630 bp. The cloned cDNA coded for 210 AA (Figure 1Go). As shown in Figure 2Go, the AA sequence shows 86% (182/210), 88% (185/210), 88% (185/210), and 72% (152/211) identity, respectively, with the AA sequences of the human (Jayakumar et al., 1995), mouse (Ueno, 2000), rat (Schweizer et al., 1989), and chicken (Holzer et al., 1989) FAS. We readily identified the highly conserved active sites of nucleotide binding sites and enoyl reductase in the deduced AA sequence (Figure 1Go). The nucleotide binding sites (Gly92, Gly94, Gly97) and pyridoxal phosphate binding sites (Lys120) for the enoyl reductase were identified (Figure 1Go). A consensus sequence for the active site of enoyl reductase was reported as Gly, Ser, Ala (Schweizer et al., 1989).



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  		Figure 1. The partial nucleotide and deduced amino acid sequence of the enoyl reductase portion of sheep fatty acid synthase. Nucleotides and amino acids are numbered to the left of the sequence. The deduced amino acid sequence is given in the one letter code. The amino acids surrounding the active center of nucleotide binding sites (Gly92, Gly94, Gly97) and pyridoxal phosphate binding sites (Lys120) are underlined. The consensus sequences for the active site of enoyl reductase are indicated.*

 


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  		Figure 2. Amino acid comparison of the enoyl reductase region of sheep fatty acid synthase to the amino acid sequences of humans, mice, rats, and chickens. Amino acids are numbered relative to the first amino acid of sequenced peptide. The identical sequences are indicated by bold letters.

 
Effect of Propionate Infusion on Plasma Hormones and Metabolites
Plasma glucose concentrations following saline or propionate infusion are presented in Figure 3AGo. Glucose concentration in propionate-infused sheep increased (P < 0.05) 5 min after beginning propionate infusion and plateaued at about twice the zero time levels for the remainder of the infusion period. Thereafter, glucose concentrations returned to preinfusion concentrations at 60 and 120 min after beginning propionate infusion. Glucose concentrations during saline infusion remained unchanged.



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  		Figure 3. Effect of intravenous propionate (–•–) or saline (–{circ}–) infusion in sheep for 30 min on plasma concentrations of (A) glucose, (B) insulin, (C) nonesterified fatty acid, and (D) IGF-I. Data are expressed as means ± SE for four sheep at each time relative to the 0 to 30 min infusion (black bar). Means with asterisks are different from saline-infused control (P < 0.05).

 
Plasma insulin concentration began to increase at 5 min, reached a maximum at 10 min, and returned to near baseline values by 60 and 120 min after propionate infusion (Figure 3BGo). Plasma insulin concentrations were not changed by saline infusion and remained constant at 1.1 to 3.0 µU/mL. Propionate-infused sheep had greater (P < 0.05) insulin concentrations than saline-infused sheep at 5, 10, 20, 30, and 60 min after infusion.

Concentrations of plasma NEFA during saline or propionate infusion are presented in Figure 3CGo. During propionate infusion, the concentration of NEFA reached a minimum at 30 min and gradually increased between 30 and 120 min. Saline-infused sheep had a higher (P < 0.05) concentration of plasma NEFA than propionate-infused sheep at 20, 30, and 60 min after infusion. Plasma glucose, insulin, and NEFA showed treatment x time interactions (P < 0.05). Plasma IGF-I concentrations were not influenced by infusion of propionate (Figure 3DGo).

Effect of Propionate Infusion on Adipose Gene Expression
The concentrations of mRNA for LPL, ACC, FAS, PPAR{gamma}, leptin, and UCP2 from s.c. adipose tissue of propionate- and saline-infused sheep were analyzed by ribonuclease protection assay (Figure 4Go). Of the adipose genes examined, LPL mRNA was the most affected by propionate as relative concentrations increased approximately fivefold over saline-infused controls at 2 h after propionate infusion. The FAS mRNA in the propionate-infused group was increased about twofold and ACC mRNA was increased about 80% when compared with the control group. The transcripts for PPAR{gamma} and leptin were increased by 55% and 45%, respectively, in the propionate-infused group. On the other hand, the steady state level of UCP2 mRNA in s.c. adipose tissue was reduced by approximately 35%. Saline infusion had no effect on the expression of any of the genes studied.



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  		Figure 4. Effect of intravenous propionate or saline infusion in sheep for 30 min on expression of lipoprotein lipase (LPL), acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), peroxisome proliferator activated receptor {gamma} (PPAR{gamma}), leptin, and uncoupling protein-2 (UCP2) in subcutaneous adipose tissue sampled 2 h after beginning infusion. Data are expressed as means ± SE for four sheep at each time postinfusion. Means with asterisks are different from saline-infused control (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
As ruminal fermentation results in degradation of dietary carbohydrates into VFA, ruminants are wholly dependent upon gluconeogenesis, using ruminally derived acetate and propionate as a source of circulating glucose (Young, 1977; Harmon, 1992). In addition, excess energy can be shunted into adipose tissue, where acetate and propionate are converted into lipids via lipogenesis. Regulation of these processes is coordinated by hormones such as glucagon and insulin, as well as by glucose itself, which interact with specific cell receptors to eventually produce additional amounts of requisite anabolic enzymes (Prior and Smith, 1982; Hillgartner et al., 1995). Differential induction of specific enzymes in skeletal muscle or adipose tissue results in differential energy use between tissues, and eventually, in differential tissue growth. This, of course, is the fundamental basis for carcass composition and energy utilization of domestic animals. In order to gain a better understanding of the fundamental processes that accompany lipogenesis in ruminants, we examined the effects of a bolus infusion of propionate into sheep on the induction of several genes involved with lipogenesis and adipose tissue metabolism.

Cloning and Sequence Analysis of the Partial cDNA for Ovine Fatty Acid Synthase
Fatty acid synthase is a multifunctional protein (MW = 500,000) that exists as a homodimer organized in a head-to-tail fashion, generating two active catalytic centers. The seven partial activities are arranged on the multifunctional protein subunits from the amino to carboxyl termini in the following order: ß-ketoacyl synthase, acetyl- and malonyl-CoA transferase, dehydratase, enoyl reductase, ketoacyl reductase, acyl carrier protein, and thioesterase (Wakil, 1989). The cloned cDNA coding for 210 AA of the enoyl reductase portion of ovine FAS showed high levels of identity with other species (Figures 1Go and 2Go). Thus, this region was used to synthesize an antisense riboprobe specific to ovine FAS mRNA.

Effect of Propionate Infusion on Plasma Hormones and Metabolites
The current study was designed to examine the nutrient regulation of lipogenic and metabolic genes in the s.c. adipose tissue of sheep. In addition, we measured the concentrations of metabolites and metabolic hormones in plasma during propionate infusion. The experimental design was based on the studies of Sano et al. (1993), who examined the effects of the infusing 1 to 64 µmol•kg BW-1•min-1 propionate on plasma propionate, insulin, glucagon, and glucose in sheep. They observed a dose-dependent increase in all of these plasma metabolites in response to propionate infusion. We examined the levels of glucose and insulin during infusion and observed a doubling of glucose during the propionate infusion and an approximately 40-fold increase in insulin levels at 10 min after beginning propionate infusion. These values are similar to those observed by Sano et al. (1993) and provide us with the knowledge that gluconeogenesis and the insulin response were probably induced by the propionate treatment. Serum levels of glucose in the current study were about 40% higher than those of sheep fed a low roughage diet at twice energy maintenance (Evans et al., 1975), suggesting that the propionate dose we used induces glucose levels similar to those seen in animals fed in vivo.

Circulating IGF-I levels are proportionate to nutritional status in ruminants and are elevated by increased energy intake (Ellenberger et al., 1989) and by insulin treatment (McGuire et al., 1995). In the current study, there was no change in circulating concentrations of IGF-I in response to acute propionate infusion or to increased insulin. This suggests that the brief exposure to propionate was not sufficient to induce changes in IGF-I levels and that IGF-I levels are more related to long-term changes in nutrition. On the other hand, plasma levels of NEFA were rapidly reduced to about 70% of preinfusion levels 20 to 30 min after beginning the propionate infusion. This response is likely mediated by the increase in circulating insulin, which acts to increase fatty acid uptake, in part by stimulating lipoprotein lipase (Picard et al., 1999).

Effect of Propionate Infusion on Adipose Gene Expression
Lipogenesis is regulated by a wide array of interdependent factors, including nutrients, hormones, nuclear transcription factors, and lipogenic enzymes. In the current study, we investigated the effects of propionate on several adipose tissue genes involved in lipogenesis and lipid metabolism. All of these genes were examined 2 h after the initiation of propionate treatment. This time was chosen based on a preliminary experiment that suggested that the expression of several adipose genes was induced by propionate at this time. Although we noted an increase in the mRNA of most genes we studied at this time, one should bear in mind that this time is not necessarily optimal for maximal expression of any or all of these genes.

Central to the genes regulating lipid metabolism is PPAR{gamma}, a member of the steroid nuclear hormone receptor superfamily, which up-regulates several genes involved in lipogenesis and lipid metabolism. These include three of the genes examined in the current study: LPL, FAS, and ACC (Way et al., 2001). Two hours after initiation of propionate infusion, PPAR{gamma} mRNA was increased by approximately 55% compared with saline-infused animals. The expression of PPAR{gamma} is tightly regulated by insulin in vivo (Vidal-Puig et al., 1996), and PPAR{gamma} enhances lipogenesis in rodents (Spiegelman, 1998). In addition, free fatty acids act as activating ligands for PPAR{gamma} (Clarke et al., 1999), and the insulin-induced reduction of plasma NEFA in the current study would be expected to increase adipocyte free fatty acid levels (Picard et al., 1999).

The LPL mRNA increased approximately fivefold in response to propionate treatment. Activity of LPL in adipose tissue is proportional to plasma insulin levels (Picard et al., 1999). Bonnet et al. (2000) demonstrated that underfeeding sheep (22% of maintenance) suppressed blood insulin concentration and LPL mRNA in adipose and muscle tissue, which was restored by refeeding (190% of maintenance). It is believed that the extreme increase in LPL mRNA observed in our study is the result of the interaction of insulin, PPAR{gamma}, and cellular influx of free fatty acids reflected in the reduced plasma NEFA levels at 20 to 30 min after propionate infusion.

In the current study, mRNA levels of ACC increased by about 80% and FAS mRNA was more than doubled 2 h after cessation of propionate infusion. The activities of these enzymes, which catalyze the initial, rate-limiting steps of lipogenesis, are induced by propionate infusion into cattle (Prior and Scott, 1980). Travers et al. (1997) showed that ACC mRNA in ovine adipose tissue is increased by incubation in vitro with insulin and dexamethasone. We demonstrate that the genes for these enzymes are also activated in vivo by propionate infusion. We believe this is the first demonstration of nutrient-gene induction of these enzymes in ruminants. The induction of genes for these lipogenic enzymes by propionate is likely mediated by insulin and PPAR{gamma}. In addition, FAS gene transcription is regulated directly by glucose (Girard et al., 1997).

Leptin mRNA was also stimulated by propionate infusion. Increased leptin mRNA is in keeping with the response to an excess of energy, in the form of glucose, which would be expected to reduce feed intake and body weight and enhance energy expenditure (Hossner, 1998). In addition, it has been shown that insulin stimulates leptin secretion (Chen et al., 1999). Uncoupling proteins (UCP) are involved in the generation of heat via the uncoupling of mitochondrial oxidation from ATP generation and thus increase thermogenesis. One UCP subtype, UCP2, is widely expressed in different organs, is probably involved in body weight regulation, and is influenced by dietary intake (Fleury et al., 1997). The mRNA of this important regulatory factor was reduced by propionate infusion, suggesting that the adipose tissue response to a sudden influx of energy is to utilize that energy for cellular metabolism and not heat generation.

The current study provides information on the role of acute propionate treatment in stimulating the metabolic and molecular events of ruminant lipogenesis. The exact mechanism by which propionate induces gene expression of lipogenesis is not known, but glucose, insulin, and glucagon likely play important roles as mediators of these effects. The current study demonstrates the coordinated gene responses to an intravenous nutrient challenge in sheep and expands our understanding of the nutrient regulation of gene transcription in domestic animals.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
The current study demonstrates that a brief infusion of propionate (64 µmol•min-1•kg BW-1 for 30 min) into sheep can induce the subsequent expression of several subcutaneous adipose tissue genes involved with lipogenesis and lipid metabolism. This coordinate effect on multiple genes by nutrients reveals the relationship between nutrients, hormones, and gene expression. Application of this information to animal production systems, will, in the longterm, add to our knowledge of nutrient energy regulation and partitioning as they relate to body composition.

Received for publication February 25, 2002. Accepted for publication June 26, 2002.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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