Ronald L. McNeel

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.

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.

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.

ISOMERS OF CONJUGATED LINOLEIC ACID MODULATE HUMAN PREADIPOCYTE DIFFERENTIATION

SummaryConjugated linoleic acids (CLAs) reduce fat deposition in several mammalian species. Among the proposed mechanisms for this effect are reduced preadipocyte proliferation and differentiation. We measured proliferation and differentiation of cultured human preadipocytes treated with CLAs. Preadipocytes were differentiated with insulin, hydrocortisone, transferrin, and 10% fetal bovine serum, with isobutyl-methylxanthine included for first 2 d. The differentiation medium contained 200 μM oleic acid (C18∶1), 50 μM cis-9, trans-11-CLA (9,11-CLA), or 50 μM trans-10,cis-12-CLA (10,12-CLA); the negative control medium contained no added fatty acid, and the cells did not differentiate. Cell number increased three to four times during the 17 d of differentiation, but was 30–35% lower in the CLA-treated cells than in the negative control cells. Compared with the negative control cells, differentiation was increased in the cells treated with C18∶1 (increased Oil Red O-stained material [OROSM], triacylglycerol, glycerol 3-phosphate dehydrogenase activity [GPDH], peroxisome proliferator-activated receptor-γ [PPARγ] messenger ribonucleic acid [mRNA], and lipoprotein lipase [LPL] mRNA). In effect, the C18∶1-treated cells act as a positive control to demonstrate the differentiation capacity of each cell lot. Both 9,11-CLA-and 10,12-CLA-treated cells had increased differentiation (increased OROSM, triacylglycerol, GPDH, PPARγ, and LPL) compared with the negative control cells. The data suggest that early in differentiation when de novo fatty acid (FA) synthesis is limited and competition for FAs by membrane and triacylglycerol synthetic pathways is great, human preadipocytes do not differentiate unless a PPARγ ligand is added. Either CLA isomer or C18∶1 can provide such a ligand.

CONJUGATED LINOLEIC ACID INCREASES THE DIFFERENTIATION OF PORCINE ADIPOCYTES IN VITRO

Abstract Individual long-chain fatty acids can modulate adipocyte differentiation. Conjugated linoleic acids (CLAs) either stimulate or inhibit 3T3-L1 clonal cell differentiation. We studied the effects of cis-9, trans-11 CLA (9,11-CLA), trans-10, cis-12 CLA (10, 12-CLA), and linoleic acid (LA) on differentiation of porcine stromal-vascular cells in vitro and on mRNA concentrations for adipocyte transcription factors and adipocyte-characterisitc proteins. Fatty acids were added to the differentiation medium at 0, 50, 100, or 300 μM, for 24 hours. The LA tended to, and 9,11-CLA increased differentiation. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA concentration was not changed by LA, 9,11-CLA, or 10,12-CLA. Lipoprotein lipase mRNA concentration was not changed by LA, but was increased by both CLA isomers. Adipocyte fatty acid binding protein (aP2) mRNA concentration was increased by LA and both isomers of CLA. In summary, CLA and LA increased differentiation of porcine stromal-vascular cells after 24 hours, but differentiation was not accompanied by increased PPARγ mRNA suggesting, at least in these experiments, that the primary action of the fatty acids was as ligands for PPARγ, rather than as inducers for PPARγ transcripts. The aP2 mRNA concentration was increased to a large extent by the various fatty acids.

Nutritional deprivation reduces the transcripts for transcription factors and adipocyte-characteristic proteins in porcine adipocytes.

For an organism to survive during nutritional deprivation, it must be able to regulate the genes involved in energy metabolism. White adipose tissue is an energy source during fasting conditions. In adipose tissue, transcription factors regulate several adipocyte-characteristic proteins involved in differentiation and energy metabolism. We investigated the transcript concentrations of two key transcription factors, as well as the transcript concentrations of several adipocyte-characteristic proteins, and genes involved in adipocyte energy metabolism in the adipose tissue of pigs fasted for 72 hours. Nutritional deprivation resulted in decreased transcript concentrations of the transcription factors, peroxisome proliferator-activated receptor gamma, and CCAAT-enhancer-binding protein alpha. The transcript concentrations of several adipocyte-characteristic proteins, fatty acid synthase, glucose transporter 4, lipoprotein lipase, leptin, and adipocyte fatty acid binding protein were also significantly reduced. The insulin receptor transcript concentration did not change. We conclude that these transcript concentration changes are aimed collectively at adjusting energy partitioning to conserve energy during nutritional deprivation, thereby enabling survival.

Abnormality of intracellular 5 alpha-dihydrotestosterone binding in simple hypospadias: studies on equilibrium steroid binding in sonicates of genital skin fibroblasts.

Summary: A method was developed for the measurement of the binding of [3H]5α-dihydrotestosterone, [3H]DHT and other steroids at equilibrium with intracellular androgen receptor of genital skin fibroblasts. This method utilized 0.2 M Na2MoO4 to stabilize the receptor and Sephadex G-25 chromatography to eliminate steroid metabolism. This binding protein showed the expected limited capacity, high affinity, and specificity of an androgen receptor.Using this method, penile skin cultures from 26 infants with simple hypospadias (HS) were compared with 18 controls. The [3H)DHT binding capacity (Bmax) was 10.1 ± 1.3 (±SE) fmol/mg protein for controls and 6.1 ± 1.7 for HS. The two populations were significantly different by Mann-Whitney test (P < 0.001). Equilibrium dissociation constant was similar for both groups. Surprisingly, there was no correlation between Bmax and the severity of the anatomic defect. Bmax was below the values seen in HS for two of three infants with male pseudohermaphroditism. In complete androgen insensitivity, DHT binding was unmeasurable.A subgroup exists in HS with an abnormality of intracellular androgen receptors. The lack of correlation between severity of hypospadias and Bmax suggests that additional factors, such as differences in physicochemical properties of the receptor or factors present in utero, contribute to the development of HS.

Cytosol androgen receptor (AR) in human skin fibroblasts: Characterization of the binding reaction and differentiation from androgen binding molecules of lower affinity

Androgen binding was studied in cytosol of human fibroblasts at 4 degrees C. When 5 alpha-dihydrotestosterone (DHT) was the ligand, a curvilinear Scatchard plot was seen, which was resolved into two components: I the androgen receptor (AR), Kd = 0.12-0.44 nM, and II a low affinity species, Kd = 6.3-28 nM. The same cytosol demonstrated only type I binding for 3H-methyltrienolone (MTr), Kd = 0.10-0.40 nM. The AR, i.e., 3H-MTr binding activity, eluted at 440,000 d by gel filtration chromatography in pre-labeling and post-labeling experiments. When the ligand was 3H-DHT, binding activity in the 10,000-45,000 d range was seen in addition to AR. Thus, saturable nonreceptor steroid binding was seen for DHT but not for MTr. The latter is the preferred ligand for the study of the AR in this system.

Influence of dietary fat on beta-adrenergic receptors and receptor-controlled metabolic function in porcine adipocytes**

In order to measure the effect of dietary fat on adipocyte plasma membrane function, 5-week-old pigs were fed either a low-fat diet, a high-fat diet with tallow (saturated fat), or a high-fat diet with corn oil (unsaturated fat) for approximately 5 weeks. Pigs fed the three diets gained the same amount of weight. Adipocytes isolated from the subcutaneous fat depot were larger in pigs fed the two high-fat diets than in pigs fed the low-fat diet. The fatty acid composition of the crude adipocyte membrane fraction (ghosts) was markedly different between the dietary groups. The affinity for the beta-adrenergic receptor was the same in ghosts from pigs fed the three diets. The number of receptors per cell was less in ghosts from pigs fed the low-fat diet compared with pigs fed either high-fat diet: receptor number per unit surface area was not different, implying that receptor number was related to adipocyte size. Activity of the membrane-bound enzyme, 5′ -nucleotidase was the same in ghosts from pigs fed the three diets. Isoproterenol-stimulated adipocyte lipolytic activity tended (P