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S B Smith

Publications and source records attributed to S B Smith.

At least 55 records · Page 3Linked to original sources
PubMed1999

Trapping of megabase-sized DNA molecules during agarose gel electrophoresis.

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated lambda-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.

Bacteriophage lambda↗
PubMed1999

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations.

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.

Animals↗
PubMed1999

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitfvit (vitiligo) mutant mouse.

In order for the retina to function properly, photoreceptor cell outer segments must be in contact with the adjacent retinal pigmented epithelium (RPE). A mouse model homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene manifests disruption of the outer segment/RPE interdigitation and demonstrates progressive loss of the photoreceptor cells. The mouse nevertheless has near normal levels of rhodopsin for many weeks and it is not known whether there is an in vivo loss of adhesion or whether the disruption is visible following tissue processing for histology. To assess this, a mechanical separation experiment was performed in which neural retinas were peeled free from the RPE and examined for the amount of pigment adherent to them. The peeling experiment indicated that control neural retinas retained significant amounts of adherent pigment at all ages examined. Neural retinas of mutant mice at age 2 weeks demonstrated adherent pigment, but older animals retained minimal pigment. Scanning electron microscopy indicated that the RPE cells of control mice were markedly damaged upon peeling and displayed different planes of cleavage, whereas those of mutants showed minimal cellular damage upon peeling, suggestive of decreased adhesion. A recombination experiment revealed that the mutant RPE/eyecup could reappose mutant and control retinas under in vitro conditions, suggesting that RPE fluid transport abilities were intact. The data provide the first direct experimental evidence that the Mitfvit mutant mouse has a naturally occurring retinal detachment and hence support its value as a model for studies of retina/RPE adhesion.

Animals↗
PubMed1999

Delta9 desaturase activity in bovine subcutaneous adipose tissue of different fatty acid composition.

Two experiments were conducted to investigate the relationship between delta9 desaturase (stearoyl-coenzyme A desaturase) activity and fatty acid composition in subcutaneous adipose tissue from cattle of different backgrounds. In Experiment 1, subcutaneous adipose tissue samples were taken from carcasses of pasture-fed cattle and feedlot cattle fed for 100, 200, or 300 d. Adipose tissue from pasture-fed cattle had significantly lower total saturated fatty acids and higher total unsaturated fatty acids than feedlot cattle. Desaturase activity correspondingly was 60-85% higher in pasture-fed cattle than in feedlot cattle. There was no difference in the fatty acid composition or desaturase activity among samples from the 100-, 200-, and 300-d feedlot cattle. In Experiment 2, adipose tissue samples were collected from carcasses of feedlot cattle fed for 180 d with either a standard feedlot ration (control group), or a ration containing rumen-protected cottonseed oil (CSO) for the last 70-80 d. Adipose tissue from the CSO-fed cattle was more saturated than that from the control group, having significantly more 18:0 and less 16:1 and 18:1. Correspondingly, adipose tissue from the CSO group had significantly lower desaturase activity. The elevated 18:2 in adipose tissue from the CSO group confirmed that unsaturated fatty acids (including cyclopropenoid fatty acids) were protected from biohydrogenation. Further studies are needed to determine whether the repression of desaturase activity results from direct inhibition by cyclopropenoic acids or by higher dietary contents of 18:2.

Adipose Tissue↗
PubMed1999

Stable and efficient gene transfer into the mutant retinal pigment epithelial cells of the Mitf(vit) mouse using a lentiviral vector.

PURPOSE: The purpose of the present study was to test whether a lentiviral vector encoding the marker lacZ gene under the control of the human CMV promoter would stably infect a significant number of RPE cells in the vitiligo mouse. This mouse harbors a mutation in the microphthalmia gene in RPE cells that leads to slow progressive photoreceptor cell degeneration. METHODS: Concentrated lentiviral vector HR'CMVlacZ was injected intravitreally into newborn vitiligo mice. Mice were sacrificed at various time points up to two months post-injection and eyes were processed histochemically to detect lacZ expression. RESULTS: The lentiviral vector infected predominantly the RPE and resulted in lacZ expression in numerous RPE cells at all times analyzed. CONCLUSIONS: LacZ expression in vitiligo RPE cells appeared to be stable for a period of at least two months. These results raise the possibility of using a similar lentiviral vector for introduction of a correct copy of the microphthalmia cDNA into the RPE that may ultimately rescue photoreceptor cells in this mutant mouse.

Animals↗
PubMed1999

Conjugated linoleic acid inhibits proliferation but stimulates lipid filling of murine 3T3-L1 preadipocytes.

This study documented the effects of conjugated linoleic acid (CLA) on the proliferation and differentiation of 3T3-L1 preadipocytes. During proliferation, preadipocytes were cultured in Dulbecco's modified Eagle's medium (DMEM), 100 g/L fetal bovine serum (FBS), 0. 584 g/L L-glutamine and 0 (control), 0.5, 1.0, 5.0 or 10.0 mg/L CLA. Proliferation of 3T3-L1 preadipocytes was measured directly by cell counting and indirectly by radiolabeled thymidine incorporation into DNA at 96 h postinoculation. Conjugated linoleic acid was not cytotoxic during proliferation or differentiation. The 0.5, 1.0, 5.0 or 10.0 mg/L CLA treatments inhibited proliferation by 8, 12, 31 and 36%, respectively (all P < 0.05). Treatment with 10 mg/L CLA or 10 mg/L linoleic acid (cis-9,12) reduced the incorporation of 3H-thymidine into DNA by 56 and 35%, respectively, suggesting that some portion of the effect of CLA on preadipocyte proliferation was nonspecific. After the initiation of differentiation, preadipocytes were cultured in DMEM, 100 g/L FBS, 0.584 g/L L-glutamine, 1.7 micromol/L insulin and 0 (control), 0.5, 1.0, 5.0 or 10.0 mg/L CLA. Radiolabeled glucose incorporation into cellular lipids was increased from 7.4 to 11.1, 11.1, 17.4 and 22.5 nmol/(h.10(6 )cells) (all P < 0.05) by 0.5, 1.0, 5.0 and 10.0 mg/L CLA, respectively. A media concentration of 10 mg/L CLA increased total cellular CLA (from 0 to 0.16 +/- 0.01 micromol/10(6 )cells), palmitic acid (from 0.47 to 1.10 +/- 0.03 micromol/10(6 )cells) and palmitoleic acid (from 0.24 to 0.81 +/- 0.03 micromol/10(6 )cells) (means +/- pooled SEM; all P < 0.05). Conjugated linoleic acid had no effect on arachidonic acid content, but decreased its proportion (g arachidonic acid/100 g total fatty acids) by >50% (P < 0.05). These data indicate that CLA inhibited proliferation and promoted de novo lipogenesis and lipid filling in 3T3-L1 preadipocytes, suggesting that CLA may reduce overall fat accumulation in growing animals by inhibiting stromal vascular preadipocyte hyperplasia.

3T3 Cells↗
PubMed1999

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.

Animals↗
PubMed1999

Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development.

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.

3T3 Cells↗
PubMed1999

Metabolism and morphology of brown adipose tissue from Brahman and Angus newborn calves.

The objective of this study was to compare adipocyte morphology and lipogenesis between breed types (Angus vs Brahman) in brown adipose tissue (BAT) and white adipose tissue (WAT) from newborn calves. The Brahman calves (n = 7) were born during the fall season, whereas the Angus calves were born in fall (n = 6) or the following spring (n = 4). At parturition, Brahman cows were lighter than fall Angus cows, but were heavier than spring Angus cows (P < .05). Birth weights and perirenal BAT weights were greater in spring-born, but not in fall-born Angus calves, than in Brahman calves (P < .05). Fall-born Angus BAT contained 63% more (P < .05) adipocytes/100 mg tissue and contained a greater proportion (P < .05) of adipocytes with mean diameters of 40 to 50 microm, and fewer adipocytes with diameters of 60 microm or greater, than Brahman BAT. Brahman BAT contained two-to-three times as many beta-receptors as Angus BAT (P < .05), although the dissociation constant (Kd) was not different between breed types. Mitochondria in Brahman BAT were primarily spherical, whereas Angus BAT mitochondria were elongated, and mitochondrial cross-sectional area tended (P = .08) to be greater in Brahman BAT than in Angus BAT. The mitochondrial uncoupling protein (UCP) mRNA concentration (per 10(6) cells) was greater in Brahman BAT than in BAT from fall-born Angus calves. Lipogenesis from acetate was greater in Angus BAT than in Brahman BAT (P < .05), and glucose and palmitate contributed a greater proportion of carbon to lipogenesis in Brahman BAT than in Angus BAT. These differences in lipogenesis between breed types were not observed in s.c. WAT. The WAT from both breed types contained adipocytes with distinct brown adipocyte morphology, suggesting an involution of BAT to WAT in utero. We conclude that differences in UCP gene expression cannot cause the greater peak thermogenesis of Angus calves; however, differences between breed types in lipid metabolism and(or) mitochondrial morphology may contribute to this phenomenon.

Adipose Tissue↗
PubMed1999

Postnatal development of stearoyl coenzyme A desaturase gene expression and adiposity in bovine subcutaneous adipose tissue.

This investigation addressed the hypothesis that stearoyl coenzyme A desaturase (SCD) gene expression would serve as a postnatal marker of adipocyte differentiation in bovine s.c. adipose tissue. Samples of tailhead s.c. adipose tissue were obtained by biopsy from preweaning steer calves 2.5 wk, 5 mo, and 7.5 mo of age and from yearling steers 12 mo of age. Samples also were obtained at slaughter when the steers were 18 mo of age. The steers sampled as yearlings were fed native pasture from weaning until 12 mo of age, and the steers sampled at slaughter were fed a high-concentrate diet from 12 to 18 mo of age. Major peak adipocyte volumes for the 2.5-wk-, 5-mo-, and 7.5-mo-old steers were 14, 270, and 700 pL, respectively (P < .001). The steers did not gain weight during pasture feeding, and at 12 mo of age peak adipocyte volume had decreased (P = .009) to 270 pL. At this time, a second, smaller population of adipocytes had appeared with a peak volume of 115 pL. At slaughter, adjusted fat thickness of the steers was 1.60 +/- .13 cm, the USDA yield grade of the carcasses was 3.51 +/- .31, and peak adipocyte volume had increased (P = .01) to over 2,500 pL. The number of adipocytes per 100 mg of adipose tissue doubled (P = .006) between 2.5 wk and 5 mo of age, concurrent with the nearly 20-fold increase in peak adipocyte volume, indicating that this was a period of apparent adipocyte hyperplasia. Uncoupling protein mRNA was undetectable at all stages of postnatal growth, indicating that differentiating tailhead s.c. adipocytes do not acquire brown adipocyte characteristics postnatally. Lipogenesis expressed on a cellular basis was low in all preweaning samples and increased significantly above preweaning values only in the 18-mo-old steers. Stearoyl coenzyme A desaturase mRNA concentration also was low in all preweaning samples, but it peaked (P = .07) at 12 mo of age. Because the peak in SCD mRNA concentration preceded a significant rise in lipogenesis and lipid filling, we conclude that the level SCD gene expression may be indicative of the extent of terminal differentiation in bovine tailhead s.c. adipose tissue.

Acetates↗
PubMed1999

Stearoyl-coenzyme A desaturase gene expression during growth in adipose tissue from obese and crossbred pigs.

This investigation addressed the hypothesis that, as a marker of adipocyte differentiation, stearoyl-coenzyme A desaturase (SCD) gene expression would be greater during growth in obese pigs than in crossbred, contemporary pigs. Suckled pigs from a single litter were removed from the sow for sampling at 0, 3, 10, and 17 d. The number of litters at 0, 3, 10, and 17 d of age was zero, two, three, and three (obese sows) and four, two, three, and three (crossbred sows), respectively. Postweaning pigs were removed from the sow at 14 d of age. One set of postweaning pigs was fed a high-fat, milk-based diet from d 28 to 49; pigs were killed on d 28 and 49 for sampling. The grain-fed pigs were switched to a pelleted, grain-based grower diet at d 28, and samples were obtained at 31, 35, or 49 d of age. Adipose tissue from all pigs in a litter for preweaning and postweaning pigs was pooled for the measurement of cellularity and SCD mRNA. There were significant genetic and age effects for adipocyte diameter and volume; overall, adipocytes from obese pigs were larger than those from crossbred pigs. Stearoyl-coenzyme A desaturase mRNA was barely detectable at 0 d of age and increased (P < .01) by 20-fold by 49 d of age. There was a significant genetic x age interaction (P = .026); there was more SCD mRNA in adipose tissue from obese pigs than in that from crossbred pigs during the suckling period, whereas crossbred pigs exhibited greater SCD gene expression than obese pigs during the postweaning period. The lesser SCD gene expression in postweaning obese pigs was caused by a strong depression in SCD gene expression in the grain-fed obese pigs. The data indicate that SCD gene expression provides a marker for terminal differentiation, especially in preweaning pigs. Furthermore, these results provide additional evidence that SCD gene expression is up-regulated by diets high in saturated fatty acids.

Adipose Tissue↗
PubMed1999

Expression of folate receptor alpha in the mammalian retinol pigmented epithelium and retina.

PURPOSE: Folic acid is essential for DNA, RNA, and protein synthesis, and deficiencies in folate can lead to nutritional amblyopia and optic neuropathy. The transport of folate from the choroidal blood supply to the retina is only now beginning to be understood. The reduced-folate transporter was reported recently to be present in cultured human retinal pigment epithelial (RPE) cells and is thought to be localized to the apical region of these cells. The authors hypothesize that the RPE plays a role in the vectorial transport of folate from the choroidal blood to the neural retina and uses not only the reduced-folate transporter but also the folate receptor alpha in mediating this transport. The purpose of the present study was to determine whether the folate receptor alpha was present in the RPE and, if so, whether it was distributed along the basolateral membrane of the RPE, supporting a role for the protein in the initial steps of folate transport into the RPE. METHODS: The expression of the folate receptor alpha in mouse RPE was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), functional assays, in situ hybridization, immunohistochemistry, and laser scanning confocal microscopy. RESULTS: RT-PCR analysis, cloning of the RT-PCR product, and subsequent sequencing established that folate receptor alpha mRNA transcripts are expressed in the mouse RPE/choroid and are expressed also in the neural retina. A heterologous functional expression assay using MTX(R)-ZR-75-1 cells showed that the folate receptor alpha cDNA obtained by RT-PCR from the RPE/choroid complex and the neural retina was functional as assessed by the binding of folic acid and by the uptake of N5-methyltetrahydrofolate. In situ hybridization localized the folate receptor alpha mRNA to the mouse RPE cells and to cells of the neural retina. The folate receptor alpha was detected immunohistochemically in the mouse and rat RPE and in several layers of the neural retina. Laser scanning confocal microscopy revealed the distribution of the folate receptor alpha along the basolateral region of the RPE and not the apical region. CONCLUSIONS: The present work represents the first analysis of the folate receptor alpha expression in intact mammalian retina. The receptor is present and functional in mouse RPE. It is distributed specifically along the basolateral surface of the RPE and is proposed to work in a coordinated manner with the reduced-folate transporter in the vectorial transport of folate from the choroidal blood to the neural retina.

Animals↗
PubMed1998

Transient overexpression of the Microphthalmia gene in the eyes of Microphthalmia vitiligo mutant mice.

The murine microphthalmia gene (Mitf) encodes a basic helix-loop-helix transcription factor thought to regulate transcription of genes encoding proteins of the pigmentation pathway. It may promote pigment cell survival and development. The protein encoded by Mitf appears to be critical for eye development, because mutant alleles demonstrate varying degrees of ocular malformation. One of the mildest of these is the Mitf vitiligo (Mitfvit) mutant allele, which exhibits uneven pigmentation of the retinal pigment epithelium (RPE) and slow, progressive photoreceptor cell loss, eventually leading to blindness. In the present study, the expression of Mitf during early eye development in the Mitfvit mutant was compared with that of pigmented wild type mice. Mitf expression quantified by reverse transcriptase-polymerase chain reaction amplification demonstrated a transient elevation of Mitf between embryonic day 10.5 (E10.5) and E13.5 in the Mitfvit mutant compared with wild type mice. In situ hybridization analysis confirmed this elevation and localized Mitf expression to the neuroepithelium during onset of optic vesicle formation (E9.0-E9.5) and, subsequently, to the RPE during optic cup formation (E10-E11.5) in both mutant and wild type eyes. This is the first report of transient elevation of Mitf in any of the Mitf mutants, and the elevation may be relevant to altered levels of pigmentation proteins as well as to the RPE abnormalities observed in the Mitfvit mutant.

Alleles↗
PubMed1998

Expression of tyrosinase and the tyrosinase related proteins in the Mitfvit (vitiligo) mouse eye: implications for the function of the microphthalmia transcription factor.

Mitf (Microphthalmia transcription factor), a basic-helix-loop-helix zipper protein, encoded at the microphthalmia (Mitf) locus, regulates the transcription of the gene encoding tyrosinase, the rate-limiting enzyme in melanin biosynthesis, by binding the DNA sequence CATGTG. This binding site is present also in the genes encoding two tyrosinase related proteins, TRP-1 and TRP-2. To gain insight into the function of Mitf in vivo, we determined whether there was a difference in the levels of these proteins in the RPE/choroid of the vitiligo (Mitfvit) mouse, in which there is a mutation of the Mitf gene. This mouse has alteration of RPE pigmentation and function that presumably leads to slow progressive loss of photoreceptor cells. The RPE/choroid was dissected from eyes of vitiligo and C57BL/6 wild-type mice at postnatal ages 2, 4, 7, 10, 14, 21 and 42 days. Extracts of pooled tissues were subjected to electrophoresis and immunoblotting. The levels of tyrosinase, TRP-1 and TRP-2 were determined densitometrically following immunodetection with rabbit antipeptide antisera. In addition, the tyrosine hydroxylase activity of tyrosinase as assayed radiometrically. Levels of TRP-1 were 3-7 fold greater in control RPE/choroid compared with mutants. This marked difference in protein level was observed at the earliest age examined (P2) and persisted throughout the first two weeks. Tyrosinase levels in mutants were similar to controls at P2 and P4, but were reduced at P10 and beyond. Tyrosinase activity was diminished also in mutants by P10. Levels of TRP-2 were similar between mutants and controls, although the typical decrease seen in controls after P14 was attenuated in the mutant mice. There is a significant reduction in the level of TRP-1 in the RPE/choroid of the Mitfvit mouse. The data suggests that transcription of the gene encoding TRP-1 is extremely dependent upon functional Mitf. It provides in vivo evidence that Mitf regulates the transcription of the gene encoding TRP-1 as well as tyrosinase.

Aging↗
PubMed1998

Complete unfolding of the titin molecule under external force.

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z- and M-lines of the vertebrate muscle sarcomere. Several earlier studies have implicated titin as playing a fundamental role in maintaining sarcomeric structural integrity and generating the passive force of muscle. The elastic properties of titin were characterized in recent single-molecule mechanical works that described the molecule as an entropic spring in which partial unfolding may take place at high forces during stretch and refolding at low forces during release. In the present work titin molecules were stretched using a laser tweezer with forces above 400 pN. The high external forces resulted in complete mechanical unfolding of the molecule, characterized by the disappearance of force hysteresis at high forces. Titin refolded following complete denaturation, as the hysteresis at low forces reappeared in subsequent stretch-release cycles. The broad force range throughout which unfolding occurred indicates that the various globular domains in titin require different unfolding forces due to differences in the activation energies for their unfolding.

Antibodies↗
PubMed1998

Positional analysis of triacylglycerols from bovine adipose tissue lipids varying in degree of unsaturation.

The objective of this study was to demonstrate that changing the fatty acid composition of bovine adipose tissue concurrently changed (i) proportions of triacylglycerol species, (ii) fatty acid composition of triacylglycerol species, and (iii) positional distribution of the component fatty acids of the triacylglycerol species. To achieve this, we took advantage of adipose tissue lipids, from cattle fed in Australia and Japan, that varied widely in fatty acid composition and melting points. Treatment groups produced in Australia were cattle fed: a cornbased diet (MUFA1); a grain-based diet containing whole cottonseed (SFA); a grain-based diet containing protected cottonseed oil (PUFA); and a grain-based diet that resulted in high contents of trans fatty acids (TFA). Treatment groups produced in Japan (MUFA2 and MUFA3) were diets of unknown composition fed for over 300 d. The MUFA1, MUFA2, and MUFA3 samples all were rich in monounsaturated fatty acids, varying only in the proportions of the individual monounsaturates. The SFA, PUFA, and TFA samples had relatively high concentrations of stearic acid (18:0), PUFA, and TFA, respectively. Slip points (indicative of melting points) were 45.1, 41.5, 38.5, 30.7, 28.4, and 22.8 degrees C, for the SFA, TFA, PUFA, MUFA1, MUFA2, and MUFA3 groups, respectively (P < 0.05). Triacylglycerols were separated by high-performance liquid chromatography on a silver nitrate-impregnated column into sn-1,2,3-saturated fatty acid triacylglycerol (SSS); [triacylglycerols containing two saturated acids and one trans-monounsaturated fatty acid (SMMt sn-positions unknown)]; sn-1-saturated, 2-monounsaturated, 3-saturated triacylglycerol (SMS); sn-1-saturated, 2-monounsaturated 3-trans-monounsaturated triacylglycerol (SMMt); sn-1-saturated, 2,3-monounsaturated fatty acid triacylglycerol (SMM); sn-1-saturated, 2-polyunsaturated, 3-trans-monounsaturated triacylglycerol; sn-1,2,3-monounsaturated fatty acid triacylglycerol (MMM); and sn-1-saturated, 2-polyunsaturated, 3-monounsaturated triacylglycerol. Fatty acid methyl esters of each triacylglycerol species also were determined, and further analysis indicated sn-2, and sn-1/3 positions. As the percentage oleic acid increased in the total lipid extract, the proportions of SMM and MMM increased (e.g., from 31.4 and 2.4% in the SFA group to 55.4 and 17.8% in the MUFA3 group). The elevated 18:0 in the SFA group (26%) was reflected in increased percentages of SSS and SSM, and caused an increase in the proportion of 18:0 in all triacylglycerol species relative to the other treatment groups. The percentage of 18:0 in the sn-1/3 positions was elevated markedly in the SMS fraction of the SFA group (to 44%); this would account for the high melting point of the fat of these animals. We conclude that long-term feeding of cattle is sufficient to produce significant alterations in fatty acid composition in bovine adipose tissue. Alterations in the fatty acid composition of bovine adipose tissue changed both the distribution and the composition of the triacylglycerol species, which, in turn, accounted for marked differences in melting points among treatment groups.

Adipose Tissue↗
PubMed1998

Starvation depresses acylglycerol biosynthesis in bovine subcutaneous but not intramuscular adipose tissue homogenates.

The primary objective of this study was to document the effects of starvation on acylglycerol biosynthesis in homogenates of intramuscular and subcutaneous adipose tissues. Adipose tissue samples were obtained from 8th-13th thoracic rib sections from 12 Angus cattle (six steers plus six heifers). Three steers and three heifers were starved for 72 h prior to slaughter while the remainder were slaughtered 4 h after food was withheld. Fat-free 700 x g centrifugal fractions were used to measure the esterification of radiolabeled sn-glycerol 3-phosphate (G-3-P) into acylglycerols at 1.0 mM palmitic or stearic acid, or 0.2 mM oleic, linoleic, or alpha-linolenic acid. There were significant tissue x fatty acid interactions for rates of incorporation into diacylglycerols and triacylglycerols; in subcutaneous, but not intramuscular homogenates, palmitic > stearic = oleic = linoleic = alpha-linolenic acid. Subcutaneous homogenates incorporated a greater percentage of G-3-P into triacylglycerols, and a lesser percentage into phospholipid, than intramuscular homogenates (P < 0.05). In intramuscular homogenates, the primary product of G-3-P esterification to saturated fatty acids was phospholipids. When unsaturated fatty acid served as substrates, triacylglycerols and phospholipids were produced in equal proportions in intramuscular homogenates, and triacylglycerols were the predominant product in subcutaneous homogenates. Intramuscular adipose tissue homogenates exhibited no response to starvation, whereas triacylglycerol and diacylglycerol synthesis was depressed by approximately 50% in subcutaneous adipose tissue homogenates. Similarly, phosphatidic phosphohydrolase activity, initially greater in subcutaneous than in intramuscular adipose tissue, was decreased by approximately 50% by starvation in subcutaneous adipose tissue, but not in intramuscular adipose tissue. We conclude that differences in rates of diacylglycerol and triacylglycerol biosynthesis, and response to starvation, between intramuscular and subcutaneous adipose tissues were due to dissimilarities in the activity of phosphatidic phosphohydrolase.

Adipose Tissue↗