Harry J. Mersmann

Expression of porcine adipocyte transcripts: tissue distribution and differentiation in vitro and in vivo.

Transcription factor transcripts implicated in adipocyte differentiation (peroxisome proliferator-activated receptor gamma (PPAR gamma), retinoid x receptor alpha (RXR alpha), adipocyte determination and differentiation-dependent factor 1 (ADD1), and CCAAT/enhancer binding protein alpha (C/EBP alpha)) and adipocyte-characteristic protein transcripts (lipoprotein lipase (LPL) and adipocyte fatty acid binding protein (aP2)) were measured in pig tissues. Transcripts for PPAR gamma, ADD1, and aP2 were localized in porcine subcutaneous and perirenal adipose tissues; transcripts for C/EBP alpha and LPL were detected in other tissues, but the greatest concentrations were in the adipose tissues. In porcine stromal-vascular cells (S/V cells) differentiating in vitro, transcripts for PPAR gamma and aP2 increased gradually, transcripts for ADD1, and LPL increased early and transcripts for C/EBP alpha increased late. In pigs, adipose tissue transcripts for PPAR gamma, ADD1, and LPL were minimal at birth and increased to 28 days postpartum, transcripts for C/EBP alpha were low until 28 days and transcripts for aP2 were at high levels, regardless of age. Although transcript development was somewhat different in vitro and in vivo, the data suggest PPAR gamma (and ADD1 are involved in regulation of transcripts for LPL and that there may be more partially differentiated precursor cells in S/V cells at day 0 than in adipose tissue at birth.

Expression of porcine adipocyte transcripts during differentiation in vitro and in vivo.

Transcript concentrations for the transcription factors, CCAAT enhancer binding protein beta and alpha (C/EBPbeta and C/EBPalpha), plus the adipocyte-characteristic proteins, fatty acid synthase (FAS), glucose transporter 4 (Glut 4), hormone-sensitive lipase (HSL), insulin receptor (InsR), lipoprotein lipase (LPL), and leptin were measured during differentiation of porcine stromal-vascular (S/V) cells in vitro. These same transcripts, excluding FAS and InsR, were measured in porcine adipose tissue from birth to 7 weeks of age. In S/V cells, C/EBPbeta and InsR were continuously elevated. At day 0, C/EBPalpha was approximately 20% of the day 9 value. The LPL increased gradually from day 0 to 9, whereas most other transcripts had a lag period of several days. In tissue, C/EBPbeta was substantial at birth and increased gradually. The C/EBPalpha was relatively low at birth and increased at day 17. The LPL and leptin increased continuously. The Glut 4 was low at birth and increased at day 28. The HSL was relatively low at birth, increased at day 10, and plateaued at day 28. Transcripts in porcine S/V cells develop somewhat differently from adipocyte differentiation models established in clonal cells, but the porcine cells represent a model that should be more applicable to pigs.

Fatty acids modulate porcine adipocyte differentiation and transcripts for transcription factors and adipocyte-characteristic proteins*

Porcine stromal-vascular cells (S/V cells) differentiate into adipocytes in vitro when presented with appropriate hormones and growth factors. Porcine S/V cells were differentiated in vitro in serum-free media with or without fatty acids to determine the effect of fatty acids on differentiation and on transcripts for peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), lipoprotein lipase (LPL) and adipocyte fatty acid binding protein (aP2). Differentiation was measured by Oil Red O staining and transcript concentrations were measured by Northern analysis using porcine riboprobes. Addition of 100 µM oleic acid (C18:1) for 5 days increased differentiation and the mRNA levels for PPARgamma, C/EBPalpha, LPL and aP2. Other medium- and long-chain fatty acids were less active. Adipocyte differentiation and transcript concentrations for PPARgamma, C/EBPalpha, LPL and aP2 were increased by C18:1 in a dose-related manner. Differentiation was greater at 10 days than at 5 days than at 1 day, and C18:1 increased differentiation at each time. Transcript concentrations were increased by C18:1 at 1 and 5 days, but not at 10 days. These results suggest that the main effect of C18:1 is on regulating gene expression (an acute or drug-like effect) rather than changing the membrane fluidity as a result of changing membrane fatty acid composition (a chronic or nutrient-like effect). Taken together, these results indicate that selected fatty acids modulate porcine adipocyte differentiation and transcripts for adipocyte differentiation-related proteins such as PPARgamma, C/EBPalpha, LPL and aP2.

Effects of isomers of conjugated linoleic acid on porcine adipocyte growth and differentiation.

Conjugated linoleic acids (CLAs) decrease fat deposition in mammals, including pigs. To determine mechanisms for CLA effects on adipocyte growth, porcine stromal-vascular cells (preadipocytes) were isolated and plated in medium containing 10% fetal bovine serum. After 24 h, differentiation factors (insulin + hydrocortisone + transferrin) were added. Oleic acid (200 microM) was added to some plates as a positive control. One of two isomers of CLA (50 microM cis 9, trans 11 or >50 microM trans 10, cis 12), or a mixture of the two isomers (25 microM each) was added to other plates. The cell number increased 7+ times in 7 days after initiation of differentiation, and was not different among treatment groups. By 7 days, Oil Red O-stained material (OROSM), expressed per cell, increased 10+ times in control cells and 64 times in oleic acid-treated cells. Addition of either isomer of CLA or the mixture caused OROSM/cell to increase 10+ times at 2 days, with no further increase at later times. In CLA-treated cells there was no increase in peroxisome proliferator-activated receptor gamma (PPARgamma) or lipoprotein lipase mRNA concentrations. The increased OROSM/cell may represent triacylglycerol synthesis from medium CLA using existing biosynthetic capacity or provision of a limiting ligand for PPARgamma already present. The results are different from those observed with rodent-derived clonal cells (3T3-L1 cells), wherein proliferation and differentiation are inhibited by CLAs, and the active isomer is trans 10, cis 12-CLA. The results suggest distinctions between clonal and primary preadipocytes, or species differences.

Adiponectin receptor 1 regulates bone formation and osteoblast differentiation by GSK-3β/β-Catenin signaling in mice

Adiponectin and its receptors are expressed in bone marrow-derived osteoblasts. Previous studies in vivo and in vitro have produced controversial results. The purpose of this study was to use porcine adiponectin receptor 1 transgenic mice (pAdipoR1) as a model to evaluate the role of AdipoR1 on bone physiology at different ages. pAdipoR1 transgenic mice had higher bone mineral density than wild-type mice in both genders at 56 weeks of age. The bone volume and trabecular number, measured by micro-computed tomography (μCT) was significantly greater in transgenic than in wild-type female mice at both 8 and 56 weeks of age. ELISA analysis revealed that both serum osteocalcin and osteoprotegerin (OPG) were significantly increased in 8-week old pAdipoR1 transgenic mice of both genders. Furthermore, serum OPG was elevated at 32 and 56 weeks of age in female and male pAdipoR1 transgenic mice. Serum TRAP5b concentration was reduced in 8 and 56 weeks old male pAdipoR1 mice compared with wild-type male mice. Knock-down of AdipoR1 significantly decreased gene expression of osteocalcin, OPG, alkaline phosphatase and msh homeobox 2 and the mineralization in MC3T3-E1 cells and mesenchymal stem cells. In addition, pathscan analysis and real-time PCR analysis suggest AdipoR1 regulates osteoblast differentiation through GSK-3 β and β-Catenin signaling. Consequently, the lack of AdipoR1 impaired osteoblast differentiation and bone formation. We conclude that AdipoR1 is a critical factor for the osteoblast differentiation and bone homeostasis.

MODULATION OF ADIPOCYTE DETERMINATION AND DIFFERENTIATION-DEPENDENT FACTOR 1 BY SELECTED POLYUNSATURATED FATTY ACIDS

SummaryThe transcription factor, sterol regulatory binding protein 1c (also called adipocyte determination and differentiation-dependent factor 1), stimulates transcription of the messenger ribonucleic acids (mRNAs) for lipid synthesis enzymes. Hepatic ADD1 transcripts are reduced by polyunsaturated fatty acids (PUFAs). The ADD1 transcripts are expressed to a considerable extent in porcine adipocytes. Consequently, it was of interest to examine the effects of several PUFAs on ADD1 in a tissue wherein several long-chain fatty acids (FAs) increase adipocyte differentiation. The effects of arachidonic acid (C20∶4), docosahexaenoic acid (C22∶6), and cis 9, trans 11-conjugated linoleic acid (9,11-CLA) on differentiating preadipocyte ADD1 mRNA and protein and on preadipocyte differentiation were determined. Porcine stromal-vascular cells were plated in serum-containing medium and differentiated in serum-free medium containing insulin, hydrocortisone, and transferrin ± an individual FA. After 24-h differentiation ± FA, plates were stained with Oil Red O as an indicator of differentiation or total RNA was extracted or a nuclear fraction was isolated for protein measurement. Addition of C20∶4 or 9,11-CLA increased the number of Oil Red O-stained cells or the Oil Red O-stained material, whereas C22∶6 did not. Addition of C20∶4, C22∶6, or 9,11-CLA decreased the concentration of the mRNA and protein for ADD1. Thus, although all three FAs decreased the ADD1 mRNA and protein concentrations, C20∶4 and 9,11-CLA increased differentiation, measured by Oil Red O staining, whereas C22∶6 did not. The data suggest that the regulation of differentiation and mRNAs by individual FAs may involve distinct mechanisms.

Docosahexaenoic acid regulates serum amyloid A protein to promote lipolysis through down regulation of perilipin.

Docosahexaenoic acid (DHA) increases lipolysis and decreases lipogenesis through several pathways. DHA also enhances the expression of serum amyloid A protein (SAA), a possible lipid metabolism related gene. The question of whether DHA regulates the expression of SAA to affect lipid metabolism and increase lipolysis needs to be demonstrated in human adipocytes. We designed experiments to determine the role of SAA in regulating lipid metabolism in HepG2 cells using microarray technology. In human hepatocytes, recombinant human SAA1 (hSAA1) inhibited the expression of genes related to lipogenesis and promoted the expression of those involved in lipolysis. When human breast adipocytes were treated with hSAA1 or DHA in vitro, the expression of peroxisome proliferator-activated receptor gamma and other lipogenic genes was decreased, whereas the expression of several lipolytic genes was increased. Glycerol release was increased by both SAA and DHA treatments, suggesting that they increased lipolytic activity in human adipocytes. The expression of perilipin, a lipid droplet-protective protein, was decreased, and hormone-sensitive lipase was increased by both of hSAA1 and DHA treatment. We speculate that the mechanism of lipolysis by DHA or SAA is at least partially the result of increased expression of hormone-sensitive lipase and decreased expression of perilipin. Whereas DHA treatment increased expression of hSAA1 in human adipocytes, the DHA-mediated reduction in expression of lipogenesis genes and enhancement of lipolysis may be through the activity of hSAA1. These results may be useful in developing new approaches to reduce body fat deposition.

Docosahexaenoic acid suppresses the expression of FoxO and its target genes

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has previously been shown to ameliorate obesity-associated metabolic syndrome. To decipher the mechanism responsible for the beneficial effects of DHA on energy/glucose homeostasis and the metabolic syndrome, 30 weaned cross-bred pigs were randomly assigned to three groups and fed ad libitum with a standard diet supplemented with 2% of beef tallow, soybean oil or DHA oil for 30 days, and the gene expression profile of various tissues was evaluated by quantitative real-time polymerase chain reaction. The DHA-supplemented diets reduced the expression of forkhead box O transcription factor (FoxO) 1 and FoxO3 in the liver and adipose tissue. DHA treatments also decreased the expression of FoxO1 and FoxO3 in human hepatoma cells, SK-HEP-1 and human and porcine primary adipocytes. In addition, DHA also down-regulated FoxO target genes, such as microsomal triacylglycerol transfer protein (MTP), glucose-6-phosphatase, apolipoprotein C-III (apoC-III) and insulin-like growth factor binding-protein 1 in the liver, as well as reduced total plasma levels of cholesterol and triacylglycerol in the pig. Transcriptional suppression of FoxO1, FoxO3, apoC-III and MTP by DHA was further confirmed by reporter assays with each promoter construct. Taken together, our study indicates that DHA modulates lipid and glucose homeostasis in part by down-regulating FoxO function. The down-regulation of genes associated with triacylglycerol metabolism and very low density lipoprotein assembly is likely to contribute to the beneficial effects of DHA on the metabolic syndrome.

Heritability of Plasma Cholesterol and Triglyceride Concentrations in Swine

Abstract Three-hundred and eighteen female swine representing contemporary commercial swine breeds (34 Chester White, 43 Large White, 42 Landrace, 40 Yorkshire, and 159 four-breed crossbreeds) were used to evaluate genetic variation between and within breeds for levels of plasma cholesterol and plasma triglycerides. Blood was sampled from all pigs after a 16-hr fast at 154 days of age. Plasma cholesterol was measured in all pigs and triglycerides were measured in 232 pigs. Paternal half-sib heritabilities (h 2) for plasma cholesterol and plasma triglycerides were 0.45 ± 0.23 and 1.04 ± 0.32, respectively. Breed differences were not apparent for either trait. Phenotypic and paternal half-sib genetic correlations between the two traits were 0.16 and 0.66, respectively. Neither body weight nor backfat depth were important in affecting the estimate of h 2 for either trait. The relatively high h 2 of total plasma cholesterol and of total triglycerides offers the possibility of developing, through selection, populations of hypercholesterolemic or hypertriglyceridemic swine useful as models for studies directed toward further understanding of human cardiovascular disease.

Adrenergic control of lipolysis in swine adipose tissue

Most potential adrenergic compounds did not stimulate lipolysis in swine adipose tissue slices. Most of the sympatholytic agents antagonized lipolysis. Most beta 1- and beta 2-adrenergic agonists were not active but many were active with rat adipose tissue. Catecholamines (epinephrine, norepinephrine and isoproterenol), the resorcinol containing beta 2-agonists (terbutaline, metaproterenol and fenoterol) and the beta 1-agonist, dobutamine were active. The beta 1-antagonists were generally more potent and efficacious than the beta 2-antagonists. The swine adipose tissue adrenoceptor was not readily classified as either beta 1- or beta 2-specific.