Biomedical subjects

S D Clarke

Publications and source records attributed to S D Clarke.

At least 19 recordsLinked to original sources

PubMed1992

Tissue specificity of S14 and fatty acid synthase in vitro transcription.

Hepatic nuclear extracts support in vitro transcription from S14 and fatty acid synthase (FAS) promoters. Renal nuclear extracts support in vitro transcription from S14 promoter constructs at rates comparable to hepatic nuclear extracts, but lack the apparent positive and negative control observed with liver. In contrast, renal nuclear extracts do not support in vitro transcription from the FAS promoter. NF-1 or a related protein in both hepatic and renal extracts contributes to in vitro transcriptional activity from the S14 promoter, but not the FAS promoter. These studies indicate that different mechanisms regulate tissue-specific expression of S14 and FAS.

Animals↗

PubMed1992

Gene expression: nutrient control of pre- and posttranscriptional events.

Regulation of gene expression by specific nutrients has become a major frontier for the next generation of nutrition scientists. The techniques of molecular biology allow us to define nutrient needs, as well as the outcomes of nutrient excesses, in terms of events that govern gene transcription, mRNA processing, mRNA stability, and mRNA translation. Evidence is presented showing that dietary constituents specifically modulate the nuclear events governing gene transcription and transcript processing. For example, fatty acid synthase gene transcription is inhibited by specific polyunsaturated fatty acids; S14 and pyruvate kinase genes contain a specific carbohydrate response element; and editing of apo B-100 to apo B-48 is enhanced by dietary carbohydrate. Nutrients such as iron and glucose are shown to control mRNA stability and translational rates of certain transcripts by regulating the interaction of cytosolic proteins with specific nucleotide sequences. The data of this review clearly demonstrate that nutrients play an active and specific role in governing the expression of select genes.

Animals↗

PubMed1992

Adipocyte fatty acid-binding protein: regulation of gene expression in vivo and in vitro by an insulin-sensitizing agent.

Pioglitazone, a thiazolidinedione, is a novel antidiabetic compound that can lower blood glucose in diabetic rodents by increasing insulin sensitivity in target tissues. We have previously demonstrated that pioglitazone can enhance the insulin- or insulin-like growth factor-1-regulated differentiation of 3T3-L1 cells, a cell line that undergoes morphological and biochemical differentiation to mature adipocytes [Mol. Pharmacol. 41:393-398 (1992)]. In this study, we have examined the effect of pioglitazone on the expression of the adipocyte fatty acid-binding protein (aFABP) in ob/ob mice and 3T3-L1 cells. Administration of the drug to mice was observed to cause a dose-dependent increase in aFABP mRNA expression in epididymal fat, which was correlated with a decrease in blood glucose and insulin levels. Treatment of 3T3-L1 cells with pioglitazone enhanced aFABP expression in a time-dependent fashion. To explore a possible direct effect of pioglitazone on aFABP expression, a chimeric gene was constructed containing the aFABP promoter fused upstream of the bacterial reporter gene for chloramphenicol acetyltransferase. After transfection into 3T3-L1 cells and selection of stable transformants, regulation of the chimeric gene was studied. Pioglitazone, in combination with insulin or insulin-like growth factor-1, was observed to elicit a dose-dependent increase in expression, indicating a role for pioglitazone in regulating transcription of the aFABP gene. Several thiazolidinedione analogs were tested for their ability to induce the expression of the chimeric gene, and it was found that activity in this assay paralleled the structure-activity relationships observed for enhancement of 3T3-L1 cell differentiation. These observations on control of aFABP gene expression by pioglitazone suggest possible mechanisms by which cellular sensitivity to insulin may be regulated.

3T3 Cells↗

PubMed1992

Enhancement of adipocyte differentiation by an insulin-sensitizing agent.

The thiazolidinediones are a class of novel antidiabetic compounds that enhance the response of target tissues to insulin. Pioglitazone, a thiazolidinedione analog, lowers blood glucose and insulin levels in rodent models of non-insulin-dependent diabetes mellitus. We have studied the effect of pioglitazone on 3T3-L1 cells, a cell line that undergoes differentiation from a preadipocyte fibroblastic morphology to that of an adipocyte. Pioglitazone treatment of preadipocytes enhanced the insulin- or insulin-like growth factor-1 (IGF-I)-regulated differentiation (monitored by the rate of lipogenesis or triglyceride accumulation), whereas treatment of the cells in the absence of insulin or IGF-I resulted in no apparent change in the cellular phenotype. Pioglitazone caused both a leftward shift and enhanced maximum response for the IGF-I-regulated differentiation of the cells, consistent with the idea that the drug enhances the sensitivity of cells to polypeptide hormones. A series of pioglitazone analogs were tested in this system, and variations in activity relative to that of the parent compound were observed. A study of the time required for the drug to exert an effect on differentiation revealed that an increased rate of lipogenesis occurred 16-24 hr after drug treatment in appropriately staged cells. An increased rate of glucose transport and increased activity of lipogenic enzymes were noted in a time frame that correlated with the change in lipogenesis. Analysis of mRNA abundance for Glut-4, lipoprotein lipase, and glucose-6-phosphate dehydrogenase showed that pioglitazone enhanced the insulin induction of these mRNA species. Thus, pioglitazone, in combination with insulin or IGF-I, appears to be exerting effects on the cellular phenotype by eliciting changes in the expression of genes that regulate metabolic pathways leading to the acquisition of the differentiated phenotype.

3T3 Cells↗

PubMed1991

The origin of multiply sigmoid curves of pH-dependence. The partitioning of groups among titration pK values.

An acid, HnA, with n ionizing groups is known to have the same titration curve as an equimolar mixture of n hypothetical monobasic acids, whose dissociation constants are known as the 'titration constants' of the real acid. We show that the pH-dependence of any property of HnA is also represented by the sum of one-site titration curves, characterized by these same titration constants. Since one such property is the degree of dissociation of one of the dissociating groups, a fraction of each group shows each of the various titration pK values, so that the group partitions among them. The n groups therefore share the same n titration pK values but differ in the fractions belonging to each. The one H+ ion per molecule that titrates with each pK is thus made up of the fractions, one from each group, that share this pK value. A group may possess a single pK value, in that it contributes virtually all of this pK and almost nothing to the others, only if either (1) in titrates in a different pH range from the other groups or (2) its affinity for H+ is unaffected by their ionization state.

Acids↗

PubMed1991

Arachidonic acid suppression of fatty acid synthase gene expression in cultured rat hepatocytes.

Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50-500 microM albumin-bound 20:1 (n-9) vs 20:4 (n-6). The induction of fatty acid synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of beta-actin mRNA was not suppressed by 20:4 (n-6). The expression of fatty acid synthase was actually stimulated 2-fold by 20:1 (n-9). It would appear that the in vivo inhibition of fatty acid synthase gene expression by dietary polyunsaturated fatty acids is a specific hepatocelluar event.

Animals↗

PubMed1991

Porcine fatty acid synthase: cloning of a complementary DNA, tissue distribution of its mRNA and suppression of expression by somatotropin and dietary protein.

A cDNA for porcine fatty acid synthase was isolated and used to examine the tissue distribution of fatty acid synthase mRNA within the pig and to determine the impact of recombinant porcine somatotropin (rpSt) and the level of dietary protein on fatty acid synthase mRNA abundance in pig liver and adipose tissue. A 1.5-kb cDNA representing the thioesterase domain of porcine fatty acid synthase was isolated from a lambda gt 11 liver cDNA library. Northern analysis with total RNA extracted from adipose tissue, liver, heart, lung, kidney and intestine revealed a single major fatty acid synthase mRNA species of 8-9 kb. The amount of fatty acid synthase mRNA in hepatic tissue was 25% of the amount in adipose tissue, which suggests that the liver may be a significant site of fatty acid synthesis in the pig. Fatty acid synthase mRNA abundance was significantly reduced in the adipose tissue (P less than 0.01) and the liver (P less than 0.1) by chronic daily administration (60 micrograms/kg) of rpSt. In addition, increasing the amount of dietary protein decreased (P less than 0.1) the abundance of fatty acid synthase mRNA in adipose tissue but had no effect on liver fatty acid synthase expression. In contrast, the abundance of adipose fatty acid binding protein mRNA was unaffected by rpSt or dietary protein. These data indicate that the reduction in the level of fatty acid synthase mRNA is a factor in the pSt-mediated suppression of fatty acid synthesis in porcine adipose tissue.

Adipose Tissue↗

PubMed1990

Induction of adipose fatty acid binding protein (a-FABP) by insulin-like growth factor-1 (IGF-1) in 3T3-L1 preadipocytes.

Murine 3T3-L1 cells were cultured in the presence of fetal bovine serum (FBS) washed with an anion exchange resin and charcoal. Using the abundance of a-FABP and fatty acid synthase (FAS) as criteria of differentiation, IGF-1 was found to be 10-fold more potent than insulin as an inducer of preadipocyte differentiation. As little as 0.5 nM IGF-1 induced expression of FAS and a-FABP mRNAs whereas a minimum of 5 nM insulin was required. The data indicate IGF-1 specifically induces the expression of a-FABP in 3T3-L1 preadipocytes whereas the effect of insulin is likely via insulin's binding to the IGF-1 receptor.

Adipose Tissue↗

PubMed1990

The purification and characterization of a fatty acid binding protein specific to pig (Sus domesticus) adipose tissue.

Western-blot analysis using antiserum to 3T3-L1-cell fatty acid binding protein (FABP) revealed that pig adipose tissue contains a 15 kDa protein immunologically similar to the murine protein. This 15 kDa protein was purified from pig adipose tissue by sequential application of Sephadex G-50 gel filtration, cation exchange and covalent chromatography on Thiol-Sepharose-4B. The purity of the pig protein was established by two-dimensional polyacrylamide-gel electrophoresis. Isoelectric focusing indicated that the pig adipose FABP (a-FABP) exists with two charge isoforms (pI 5.1 and 5.2), both of which persist after delipidation. The N-terminus of the purified pig a-FABP was blocked; however, cleavage with CNBr allowed recovery of a 12-amino-acid peptide which was identical with the murine a-FABP sequence (residues 36-48) at 10 of 12 positions. The pig a-FABP bound 12-(9-anthroyloxy)oleic acid saturably and stoichiometrically, with an apparent dissociation constant of 1.0 microM. Northern-blot analysis using the cDNA for the murine 3T3-L1 FABP revealed that the pig a-FABP was expressed exclusively in adipose tissue.

Adipose Tissue↗

PubMed1990

Suppression of rat hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fat.

The objective of this research was to determine whether dietary polyunsaturated fatty acids suppress hepatic fatty acid synthase (FAS) mRNA levels by altering FAS gene transcription. Male Sprague-Dawley rats were meal-fed for 10 d a high glucose diet supplemented with 20% digestible energy as menhaden oil or tripalmitin. The transcription rate for FAS was determined by nuclear run-on analysis in hepatic nuclei isolated from rats 2 h postmeal. The values for transcription rates of FAS and S14 (a putative lipogenic protein) in rats fed menhaden oil were only 6 and 21%, respectively, of the rates in rats fed the tripalmitin diet (p less than 0.02). Gene transcription for beta-actin and phosphoenolpyruvate carboxykinase did not differ between treatments. The reduction in hepatic FAS mRNA levels caused by dietary polyunsaturated fats appears to be caused primarily by an inhibition of FAS transcription. The control of transcription by polyunsaturated fats appears not to be mediated by cAMP because the transcription rate for phosphoenolpyruvate carboxykinase (whose gene is very sensitive to cAMP stimulation) was unaffected by the source of dietary fat.

Animals↗

PubMed1990

Nutritional control of rat liver fatty acid synthase and S14 mRNA abundance.

The objective of this research was to evaluate the change in abundance of S14 and fatty acid synthase (FAS) mRNAs under a variety of nutritional conditions to evaluate the hypothesis that the regulation of the S14 gene is similar to that of other proteins involved in lipid metabolism and that changes in S14 expression are comparable to those that occur in FAS expression. Livers from rats fed a high carbohydrate diet were found to contain 350- and 100-fold more S14 and FAS mRNA than livers from rats fasted for 48 h. Although feeding a high fat diet increased S14 and FAS mRNA above fasting (P less than 0.05), the level of S14 and FAS mRNAs was only 5% and 4%, respectively, of the amount in the high carbohydrate group. Both S14 and FAS mRNAs accumulated quickly upon intubation of fasted rats with a solution of sucrose. The earliest rise in these mRNAs occurred within 60 min; by 240 min after gavage, each mRNA had increased 30-fold. The rapid induction of FAS and S14 mRNAs was also observed during ingestion of a high glucose meal. Hepatic FAS and S14 mRNA decreased 80-90% and 60%, respectively, during the 21-h interval between meals. This degree of mRNA loss was estimated to require a half-life for FAS and S14 mRNA of less than 8 h and less than 12 h, respectively. Regression analysis of the three dietary studies revealed a correlation coefficient for the relationship between S14 and FAS mRNA abundance ranging between 0.88 and 0.96.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1990

Dietary polyunsaturated fats uniquely suppress rat liver fatty acid synthase and S14 mRNA content.

The objective of these studies was to demonstrate that dietary polyunsaturated, but not saturated, fatty acids decrease mRNA abundance for fatty acid synthase (FAS) and S14. A series of experiments involving adult and weanling rats was designed to examine the ability of saturated (tripalmitin), (n-9) monounsaturated (triolein), (n-6) di-unsaturated (safflower oil), and (n-3) polyenic (fish oil) fatty acids to suppress the gene expression of FAS and S14. Dietary polyunsaturated fats reduced by 75-90% the hepatic abundance of FAS and S14 mRNA. Fish oil, rich in 20- and 22-carbon polyenic fatty acids, was more effective than safflower oil, whereas tripalmitin and triolein were without effect. Polyunsaturated fats were also very effective at preventing the rise in FAS and S14 mRNA associated with weaning. The inhibitory action of polyunsaturated fat was rapidly (less than 3 h) removed by deleting the fat from the meal. Regression analysis revealed a high correlation (0.81-0.94) between FAS and S14 expression among the various dietary studies. These data support the hypothesis that dietary polyunsaturated fats uniquely regulate the gene expression of lipogenic enzymes and that the mediator is likely a specific entity derived from the long-chain polyenic fatty acids. Moreover, the high correlation between FAS and S14 expression supports the hypothesis that S14 is a member of the lipogenic protein family and has potential as a model gene for the study of FAS expression.

Animals↗

PubMed1990

Potency of polyunsaturated and saturated fats as short-term inhibitors of hepatic lipogenesis in rats.

Three dietary studies using male Sprague-Dawley rats conditioned to meal-eat a high glucose, fat-free diet and one in vitro study with isolated rat hepatocytes were designed to examine the hypothesis that polyunsaturated fats (i.e., safflower oil or linoleate) are more potent acute inhibitors of liver fatty acid synthesis than are saturated fats (i.e., beef tallow or palmitate). Fat in the first in vivo study was administered via intubation (1500 mg/rat) whereas in the second and third in vivo studies fat was added to the meal in amounts of 50, 100, 250 or 500 mg/g fat-free diet. When the rats were in a postprandial condition, significant suppression of hepatic lipogenesis required the meal to contain 38% of its energy as fat (i.e., 250 mg/g fat-free diet). At this level of fat, safflower oil was more inhibitory than beef tallow (p less than 0.05). The inhibition constant (Ki) for palmitate inhibition of fatty acid synthesis by isolated hepatocytes was fourfold greater than linoleate's Ki (fatty acid/albumin ratio of 1.4/1). When fat constituted 50% of the ingested energy, beef tallow was equivalent to safflower oil as an inhibitor of lipogenesis. Although a single meal containing 50 mg safflower oil/g fat-free diet did not decrease fatty acid synthesis, it effectively delayed the induction of lipogenesis during the first 30 min of the adaptive decrease in lipogenic enzymes attributed to polyunsaturated fats extends to short-term regulatory mechanisms.

Animals↗

PubMed1990

Inhibition of triiodothyronine's induction of rat liver lipogenic enzymes by dietary fat.

The objective of these studies was to demonstrate that the reduction in lipogenic enzymes caused by ingestion of dietary polyunsaturated fat can in part be attributed to an inhibition of triiodothyronine's induction of hepatic lipogenic enzymes. A 3 x 4 factorial design was employed to examine the effect of diets containing no fat, beef tallow or safflower oil on the triiodothyronine-mediated (0, 2, 4 or 8 micrograms/100 g injected daily intraperitoneally) induction of rat liver lipogenic enzymes. Triiodothyronine (T3) administration induced (p less than 0.05) the activity of malic enzyme, fatty acid synthase and glucose-6-phosphate dehydrogenase in a dose-dependent manner. Malic enzyme activity was increased five- to sevenfold by 8 micrograms T3/100 g daily. Fatty acid synthase activity at the 8 micrograms dose had increased two- to threefold whereas glucose-6-phosphate dehydrogenase activity was elevated only 15-35%. Beef tallow and safflower oil supplementation of the high glucose, fat-free diet significantly reduced the T3 induction of all the enzymes. Safflower oil was more effective than tallow as a repressor of T3 action. The effect of dietary fat, particularly safflower oil, was to increase the amount of T3 required to induce the activity of lipogenic enzymes. In a second study, with a 2 x 2 factorial design, daily injection of 15 micrograms T3/100 g was found to overcome the safflower oil inhibition of lipogenic enzymes. These data support the hypothesis that polyunsaturated fats uniquely suppress the gene expression of lipogenic enzymes by functioning as competitive inhibitors of T3 action, possibly at the nuclear receptor level.

Animals↗

PubMed1990

Limits of medium-chain and long-chain triacylglycerol utilization by neonatal piglets.

Fifteen unsuckled neonatal piglets from three litters were fasted for 2 h after birth then allotted within litter to a 2 x 3 factorial arrangement of treatments and given by stomach tube 2.36, 4.91 or 11.32 g of either (1-14C)-triolein or (1-14C)-trioctanoin/BW (kg).75. The hourly 14CO2 production was measured for 12 h for estimating the oxidation rate of dosed triacylglycerols. The contents of each segment of the digestive tract were collected for 14C and lipid analyses for calculating absorption of the dosed triacylglycerols. Grams of trioctanoin absorbed (P less than .01) and oxidized (P less than .001) by piglets were higher than grams of triolein. The maximum amounts of trioctanoin and triolein absorbed in 12 h were 2.77 +/- .29 and 1.83 +/- .36 g/BW (kg).75, respectively. The maximum amounts of trioctanoin and triolein oxidized in 12 h were 1.60 +/- .14 and .33 +/- .17 g/BW (kg).75, respectively. The maximum amounts of trioctanoin and triolein oxidization during 1-h periods were .231 +/- .017 and .049 +/- .021 g/BW (kg).75, respectively. The calculated maximum percentage of piglets' daily maintenance energy needs supplied by trioctanoin oxidation was 42.0%, compared with 10.6% from triolein. The maximum utilization of trioctanoin occurred at the intermediate dose. Results indicated that trioctanoin was used more effectively than triolein; it may be useful as a source of supplemental energy to improve the survival of piglets. A single dose should provide about 6 g/body wt (kg).75.

Animals↗

PubMed1990

Focal brain lesions on computed tomography in patients with acquired immune deficiency syndrome.

Neurologic involvement is common in patients with acquired immune deficiency syndrome (AIDS). To assess the computed tomographic findings in AIDS, the authors reviewed 12 consecutive patients found to have focal brain lesions representing AIDS complications on computed tomography and having histologic or clinical proof of the diagnosis. Five patients had toxoplasmosis, six had lymphoma and one had both conditions sequentially. The finding of multiple lesions and mass effects with edema was more common with toxoplasmosis. In six patients who had toxoplasmosis 33 nodules were observed compared with only 8 in seven patients who had lymphoma. Disease extending linearly along the ventricular wall was typically seen in patients who had lymphoma.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Digestion and absorption of fish oil by neonatal piglets.

In order to establish the digestibility and absorption of fish oil, 18 unsuckled, newborn piglets were tube-fed an amount of fish oil calculated to provide the piglets' maintenance energy needs for 12 h. After fat administration the piglets were either intubated with 25 ml of fat-free milk replacer per h, or fasted. The digesta of the stomach, small intestine, and large intestine (digesta plus feces) were collected 12 h after fish oil intubation. Disappearance of total extractable fat was 95 +/- 1 and 94 +/- 1% for fed and fasted piglets, respectively. True absorption of the lipid was calculated from the disappearance of eicosapentaenoic acid and was 99% for both fed and fasted piglets. Nearly 50% of the stomach lipid was as diglycerides and free fatty acids, suggesting that hydrolysis of the fish oil was initiated in the stomach. Small intestinal lipid was 63-65% free fatty acids, indicating that the piglet pancreatic lipase was of sufficient activity to hydrolyze fish oil triglyceride. Neonatal piglets appeared to have ample capacity to digest and absorb fish oil, which supports the concept that lipid supplementation could improve the energy status of the newborn piglet.

Absorption↗

PubMed1989

Lack of specificity of polyunsaturated fats in the inhibition of rat liver glucose-6-phosphate dehydrogenase.

The mechanism of the effect of polyunsaturated fatty acids (PUFA) on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G6PDH) was studied in young, male Wistar rats. Starvation-refeeding increased G6PDH level above that seen in ad libitum-fed animals (enzyme overshoot). A second episode of starvation-refeeding produced even higher levels of G6PDH activity (induction increment). Interposing a high fat diet (containing PUFA) between starvation and feeding the inducer diet abolished one-half to two-thirds of the overshoot. Feeding a high fat diet between the two starvations abolished the induction increment. Inhibitors of arachidonic acid metabolism were not able to reverse the PUFA effect. In another set of experiments it was shown that both linoleic and linolenic acid are equally effective in either reducing the overshoot or abolishing the induction increment. The evidence was interpreted as supporting a hypothesis that the PUFA effect does not require the formation of a specific end product of arachidonic metabolism in a direct way.

Animals↗