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S D Clarke

Publications and source records attributed to S D Clarke.

At least 55 records · Page 3Linked to original sources
PubMed1994

Dietary polyunsaturated fatty acid regulation of gene transcription.

We have known for nearly 30 years that dietary polyenoic (n-6) and (n-3) fatty acids potentially inhibit hepatic fatty acid biosynthesis. The teleological explanation for this unique action of PUFAs resides in their ability to suppress the synthesis of (n-9) fatty acids. By inhibiting fatty acid biosynthesis, dietary PUFAs reduce the availability of substrate for delta 9 desaturase (7, 22, 34, 36) and in turn reduce the availability of (n-9) fatty acids for incorporation into plasma membranes. In this way, essential biological processes dependent on essential fatty acids (e.g. reproduction and trans-dermal water loss) continue to operate normally. Therefore, if essential fatty acid intake did not regulate (n-9) fatty acid synthesis, the survival of the organism would be threatened. During the past 20 years, we have gradually elucidated the cellular and molecular mechanisms by which dietary PUFAs modulate fatty acid biosynthesis and (n-9) fatty acid availability. Central to this mechanism has been our ability to determine that dietary PUFAs regulate the transcription of genes coding for lipogenic enzymes (12, 40). The potential mechanisms by which PUFAs govern gene transcription are numerous, and it is unlikely that any one mechanism can fully elucidate the nuclear actions of PUFA. The difficulty in providing a unifying hypothesis at this time stems from: (a) the many metabolic routes taken by PUFAs upon entering the hepatocyte (Figure 1); and (b) the lack of identity of a specific PUFA-regulated trans-acting factor. However, the studies described above indicate that macronutrients, like PUFA, are not only utilized as fuel and structural components of cells, but also serve as important mediators of gene expression (12, 14, 40). As regulators of gene expression, PUFAs (or metabolites) are thought to affect the activity of transcription factors, which in turn target key cis-linked elements associated with specific genes. Whether this targeting involves DNA-protein interaction or the interaction of PUFA-regulated factors is unclear. A better understanding of the nuclear actions of PUFA will clarify the role of these compounds in lipid metabolism and lead to a better understanding of the role of PUFAs in disease processes such as insulin-resistant diabetes and certain forms of cancer.

Animals↗
PubMed1994

Coordinate regulation of glycolytic and lipogenic gene expression by polyunsaturated fatty acids.

Using a combination of in vivo and in vitro studies, we have investigated the impact of polyunsaturated fatty acids (PUFA) on the expression of several genes encoding proteins involved in hepatic glycolysis and lipogenesis. Meal-training rats to a high glucose diet containing 10% triolein led to a significant induction of hepatic mRNAs encoding glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), malic enzyme (ME), and the S14 protein (S14), but had no effect on thyroid hormone receptor-beta 1 (TR beta 1) and c/EBP alpha gene expression. Replacing triolein with menhaden oil attenuated (by 50-90%) the induction of mRNA encoding GK, ME, PK, FAS, and S14. This effect was rapid (within hours) and for FAS and S14, directed at the transcriptional level. The mRNAs encoding TR beta 1, c/EBP alpha and beta-actin were unaffected by menhaden oil. Studies with cultured primary hepatocytes showed that C18:3,omega 3 (n-3), C18:3,omega 6 (n-6), C20:4, omega 6 (n-6), and C20:5,omega 3 (n-3) were all equally effective at suppressing the level of mRNAs encoding FAS, S14, and PK. This effect was specific for glycolytic and lipogenic enzymes, as expression of beta-actin was not affected by these fatty acids. Moreover, the fatty acids had only marginal effects on cell viability as judged by lactate dehydrogenase release. These data indicate that polyunsaturated fatty acids coordinately regulate the expression of several enzymes involved in carbohydrate and lipid metabolism. The mechanism of control does not require extrahepatic factors or fatty acid metabolism.

Animals↗
PubMed1993

Polyunsaturated fatty acids inhibit S14 gene transcription in rat liver and cultured hepatocytes.

Polyunsaturated fatty acids (PUFAs) have been shown to have significant effects on hepatic lipogenic gene expression. The S14 gene has been used as a model to examine the effects of PUFAs on hepatic lipogenic gene expression. In vivo studies showed that feeding rats a high carbohydrate diet containing menhaden oil rapidly (within hours) and significantly (> or = 50%) attenuates hepatic S14 gene transcription and S14 mRNA abundance. The suppressive effect of menhaden oil was both gene and tissue specific. The effect of PUFAs on expression of the S14 mRNA and a transfected S14 fusion gene (i.e., S14CAT4.3) was examined in cultured hepatocytes in the presence of triiodothyronine (T3), insulin, dexamethasone, and albumin under serum-free conditions. Whereas T3 stimulated both S14 mRNA (> 40-fold) and S14CAT4.3 (> 100-fold), eicosapentaenoic acid (C20:5 omega 3) significantly attenuated (> or = 80%) both S14 mRNA and S14CAT activity in a dose-dependent fashion. The effects of C20:5 on hepatocyte gene expression were both gene and fatty acid specific. Deletion analysis of transfected S14CAT fusion genes indicated that the S14 thyroid hormone response element (at -2.5 to -2.9 kb) was not sensitive to C20:5 control. The cis-linked PUFA response elements were localized to a region within the S14 proximal promoter (at -80 to -220 bp). This region also contains cis-acting elements that potentiate T3 activation of S14 gene transcription. These studies suggest that C20:5 (or its metabolites) regulates factors within the S14 proximal promoter region that are important for T3 activation of S14 gene transcription.

Adipose Tissue↗
PubMed1993

Calcium measurements with a new high-affinity n.m.r. indicator in the isolated perfused heart.

A new n.m.r. indicator, 1,2-bis-(2-[1-(hydroxycarbony)ethyl- (hydroxycarbonylmethyl)]amino-5-fluorophenoxy)ethane (DiMe-5FBAPTA), with a higher affinity for calcium (apparent Kd 46 nM, pH 7.2, 30 degrees C) than the parent 5FBAPTA chelator (Kd 537 nM, pH 7.1, 30 degrees C) has been used to measure the cardiac intracellular free Ca2+ ([Ca2+]i). DiMe-5FBAPTA was loaded into Langendorff-perfused ferret hearts maintained at 30 degrees C using the acetoxymethyl ester (AM) derivative. The intracellular concentration required to achieve an adequate signal-to-noise (S/N) ratio (> 10:1) for the n.m.r. spectra caused a similar reduction in developed pressure to that obtained using 5FBAPTA-AM. The DiMe-5FBAPTA was used to estimate [Ca2+]i in diastole, through the calcium transient and at rest in the presence of the slow calcium channel blocker diltiazem. At a pacing frequency of 1.0 Hz, end-diastolic [Ca2+]i was 198 +/- 30 nM (n = 9), and reducing the pacing frequency to 0.2 Hz lowered [Ca2+]i to 89 +/- 13 nM (n = 5). Perfusion with diltiazem (100 microM) for 60 min lowered [Ca2+]i to 10 +/- 1 nM (n = 4) in unpaced hearts and to 94 +/- 24 nM (n = 4) in hearts paced at 1.0 Hz. The [Ca2+]i transient measured with DiMe-5FBAPTA was sharper and delayed compared with the transient measured previously with 5FBAPTA. Co-loading the two indicators provided evidence that the indicator with the higher Kd had a dominant effect on the end-diastolic [Ca2+]i. The lower values for end-diastolic [Ca2+]i obtained with DiMe-5FBAPTA are consistent with fluorescent indicator measurements. These observations suggest that perturbations of [Ca2+]i caused by the new indicator are less than those induced by 5FBAPTA. DiMe-5FBAPTA therefore represents a useful step in the development of 19F-n.m.r. calcium indicators.

Animals↗
PubMed1993

Thermogenesis secondary to transdermal water loss causes growth retardation in essential fatty acid-deficient rats.

Manifestations of essential fatty acid (EFA) deficiency in rats include growth retardation and increased transdermal water loss. The extra metabolic energy requirement could be caused by greater evaporative water loss from the skin surface. To test this hypothesis, 38 weanling rats were randomly assigned to one of two environments, control (CE) at 20 degrees C and 40% humidity or warm/humid (WHE) at 30 degrees C and 90% humidity. Half of the 20 CE rats were fed an EFA-adequate diet and the other 10 an EFA-deficient diet; the 18 WHE rats were also equally partitioned to the two diets. CE and WHE animals were independently group-fed to maintain equal energy intakes within each environment. Weight gain at 90 days was lower for CE EFA-deficient rats than for CE EFA-adequate rats (P < .0001). Growth rates in the WHE to 140 days did not differ. Mean weights at 90 days were as follows: CE EFA-adequate, 196 g; CE EFA-deficient, 148 g; WHE EFA-adequate, 148 g; WHE EFA-deficient, 135 g. In both CE and WHE animals, the triene/tetraene ratio of both serum and liver phospholipids (PL) was 100-fold greater for EFA-deficient versus EFA-adequate diets. PL fatty acids of liver in CE and WHE EFA-deficient rats contained 2.09 and 1.92 micrograms of 20:3 omega 9 per micrograms phosphorus (Pi), respectively, compared with 0.03 and 0.02 microgram 20:3 omega 9/micrograms Pi in CE and WHE EFA-adequate rats. These results indicate equivalent degrees of EFA deficiency in the two environments.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Treatment of secretory pituitary adenoma with radiation therapy.

A retrospective review of 44 patients with secretory pituitary adenoma treated with radiation therapy (median total dose of 4,500 cGy with a median fraction size of 225 cGy) was performed to analyze response rates and possible variables associated with failure to respond and with complications. The treatment technique used for 75% of the patients was a combination field; an opposed-lateral fields technique was used for the remainder. Median follow-up was 78.5 months, with 59% followed up for more than 60 months and 34% for more than 120 months. Overall survival was 90%, and disease-free survival was 62%. Response rates were 86% for the group with prolactinoma, 67% for the group with acromegaly, and 50% for the group with Cushing disease; the overall response rate was 71%. Findings of suprasellar extension and those from treatment with opposed-lateral fields correlated significantly with failure to respond. A higher percentage of patients with invasive macroadenomas also failed to respond. More complications were found in patients treated with opposed-lateral fields, but the numbers were too small to reach significance. Radiation therapy remains an important adjunct for the treatment of many patients with secretory pituitary adenoma.

Acromegaly↗
PubMed1993

Effect of in vivo somatotropin treatment of growing pigs on adipose tissue lipogenesis.

The present study was undertaken to determine the effects of porcine somatotropin (pST) on glucose flux rates, lipogenic enzyme activities, and the abundance of fatty acid synthase mRNA in pig adipose tissue. Barrows were injected daily with 120 micrograms of pST/kg BW (n = 6) or excipient (n = 6). On d 11 of treatment, pigs were slaughtered (empty BW = 77 +/- 2 kg) and subcutaneous adipose tissue was collected. Basal incorporation of [14C]glucose into total lipid decreased by 86% with pST treatment, whereas glucose oxidation to CO2 decreased by 79%. Insulin (10 ng/mL) stimulated both glucose oxidation and incorporation into lipid by a small increment of similar magnitude for both treatment groups. Rates of lipogenesis determined in vitro were highly correlated with similar measurements made in vivo on the same set of animals (r = .76). The reduction in basal rates of lipogenesis corresponded to a 79% decrease in total (activated) acetyl-coenzyme A carboxylase activity and a 67% decrease in fatty acid synthase activity. Reduced nicotinamide adenine dinucleotide phosphate-generating enzymes were decreased to a lesser extent. Northern blot analysis of RNA from the same animals reveal a 90% decrease in mRNA for fatty acid synthase in the pST-treated group. The correlation between mRNA abundance and enzyme activity for fatty acid synthase was .90. These data indicate that the pST-induced reduction in adipose tissue lipid accretion in growing barrows is largely a result of diminished rates of lipogenesis that are manifestations of the decreased activities of the fatty acid-synthesizing enzymes. These changes seem to result from suppression of genes that encode for the lipogenic enzymes.

Acetyl-CoA Carboxylase↗
PubMed1993

Regulation of fatty acid synthase gene expression: an approach for reducing fat accumulation.

Fatty acid synthase (FAS) catalyzes the last step in the fatty acid biosynthetic pathway. The tissue concentration of FAS, which is affected by a number of hormonal and dietary factors, is a key determinant for the maximal capacity of a tissue to synthesize fatty acids by the de novo pathway. The complete nucleotide sequence of the avian and rat FAS transcripts has been cloned. In addition, a 1.5-kb cDNA that represents the thioesterase domain of the pig FAS protein plus the entire 3'-untranslated region of the transcript was isolated from a porcine liver cDNA library. Using these FAS cDNA tools, FAS mRNA transcripts have been found in most tissues, including adipose, liver, lung, brain, kidney, and small intestine. Moreover, the abundance of FAS mRNA in a tissue determines the rate of FAS protein synthesis, and ultimately the tissue content of FAS protein. In the liver, the rate of FAS gene transcription dictates the level of FAS mRNA, whereas the FAS mRNA content of adipose tissue seems to be determined by factors that affect gene transcription and mRNA stability. Adaptive changes in the abundance of FAS mRNA seem to occur primarily in hepatic and adipose tissues, whereas FAS expression in other tissue types is resistant to nutritional and hormonal manipulations. This review presents the concept that the tissue-specific adaptation in FAS gene expression can be exploited to develop a tissue-specific inhibitor of FAS gene expression and, hence, reduce the tissue capacity for fat accretion through the de novo biosynthetic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
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↗