Malic Enzyme mRNA Levels Research Articles

Effects of Clostridium butyricum and Enterococcus faecium on growth performance, lipid metabolism, and cecal microbiota of broiler chickens

To investigate the effects of Clostridium butyricum and Enterococcus faecium on the growth performance, lipid metabolism, and cecal microbiota of broilers, 264 one-day-old male Ross 308 broiler chicks were randomly allocated into four treatments with six replicates in a 2 × 2 factorial arrangement and fed four diets with two levels of C. butyricum (0 or 1 × 10⁹ cfu/kg) and two levels of E. faecium (0 or 2 × 10⁹ cfu/kg) for 42 days. There was no significant interaction between C. butyricum and E. faecium on the growth performance, lipid metabolism, and cecal microbiota of broilers. However, broilers supplemented with E. faecium had lower (P = 0.022) serum leptin level at day 21 and higher (P < 0.001) fatty acid synthase (FAS), malic enzyme (ME), and acetyl-CoA carboxylase (ACC) mRNA levels in the liver at day 42. Supplementation of C. butyricum improved (P < 0.05) the average daily feed intake and average daily gain, increased (P = 0.016) the serum insulin level at 21 days of age, enhanced (P < 0.05) the content of intramuscular fat, activities of FAS in the liver and lipoprotein lipase (LPL) in the breast muscle, mRNA expression of FAS, ME, and ACC in the liver and LPL in the breast muscle at 42 days of age, but reduced (P = 0.030) cecal Bacteroidetes relative abundance at 21 days of age. The results of this study indicate that the increased intramuscular fat content of broilers fed C. butyricum as observed may be the result of enhanced lipogenesis.

AMP-activated protein kinase and carbohydrate response element binding protein: A study of two potential regulatory factors in the hepatic lipogenic program of broiler chickens

This study investigated the effects of fasting and refeeding on AMP-activated protein kinase (AMPK) and carbohydrate response element binding protein (ChREBP) mRNA, protein and activity levels; as well as the expression of lipogenic genes involved in regulating lipid synthesis in broiler chicken (Gallus gallus) liver. Fasting for 24 or 48 h produced significant declines in plasma glucose (at 24 h), insulin and thyroid hormone (T3) levels that were accompanied by changes in mRNA expression levels of hepatic lipogenic genes. The mRNA levels of malic enzyme (ME), ATP-citrate lyase (ACL), acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1) and thyroid hormone responsive Spot 14 (Spot 14) declined in response to fasting. Refeeding for 24 h increased mRNA levels for each of these genes, characterized by a significant increase ('overshoot') above fed control values. No change in mRNA expression of the two AMPK alpha subunit genes was observed in response to fasting or refeeding. In contrast, ChREBP and sterol regulatory element binding protein-1 (SREBP-1) mRNA levels decreased during fasting and increased with refeeding. Phosphorylation of AMPK alpha subunits increased modestly after a 48 h fast. However, there was no corresponding change in the phosphorylation of ACC, a major downstream target of AMPK. Protein level and DNA-binding activity of ChREBP increased during fasting and declined upon refeeding as measured in whole liver tissue extracts. In general, evidence was found for coordinate transcriptional regulation of lipogenic program genes in broiler chicken liver, but specific regulatory roles for AMPK and ChREBP in that process remain to be further characterized.

Evidence that triiodothyronine decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue

Conflicting results have been reported regarding the effect of triiodothyronine (T 3) on serum leptin and adipose tissue leptin gene expression in human and animals. The aim of the present study was to evaluate the effect of administration of increasing doses of T 3 on serum leptin concentration and on leptin mRNA abundance in white adipose tissue of rats. The results presented in this paper indicate that administration of single different doses of T 3 to euthyroid rats resulted dose dependent increases of serum total T 3 concentrations which are associated with a decrease in white adipose tissue leptin mRNA level. The leptin mRNA level in white adipose tissue was negatively correlated with serum total T 3 concentration ( r = − 0.8, p < 0.001). Like white adipose tissue leptin mRNA level, serum leptin concentration decreased after T 3 administration, and was also negatively correlated with the serum T 3 concentration ( r = − 0.8, p < 0.001). In contrast, administration of T 3 to the same rats led to a significant increase in white adipose tissue expression of the malic enzyme gene (malic enzyme activity and malic enzyme mRNA level), a known target gene for T 3. The results indicate that T 3 exerts a selective inhibitory effect on white adipose tissue leptin gene expression in vivo. A conclusion is that T 3 decreases rat serum leptin concentration by down-regulation of leptin gene expression in white adipose tissue.

Pharmacological Doses of Triiodothyronine Upregulate Lipogenic Enzyme Gene Expression in Rat White Adipose Tissue

Triiodothyronine (T (3)) is known to increase liver lipogenic enzyme gene expression both in vivo and in tissue culture. Conflicting results have been reported on the effect of T (3) on lipogenic enzyme gene expression in white adipose tissue. The results presented in this paper indicate that administration of pharmacological doses of T (3) in rats leads to increased fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), ATP-citrate lyase (ACL) and malic enzyme (ME) activity in white adipose tissue. The increase in lipogenic enzyme activity was associated with increased FAS, ACC, ACL and ME mRNA levels. The response was dose-dependent. Activity of lipogenic enzyme and the lipogenic enzyme mRNA levels were positively correlated to serum T (3) concentration. The in vivo effect of T (3) on lipogenic enzyme gene expression could be reproduced in primary white rat adipocyte culture. In conclusion, the results presented in this paper indicate that T (3) exerts a stimulatory effect on lipogenic enzyme gene expression in white adipose tissue both in vivo and in tissue culture. Significant effects of T (3) on lipogenic enzyme gene expression were only observed in the presence of relatively high (pharmacological) concentrations of the hormone.

A Peroxisome Proliferator-Activated Receptor α/γ Dual Agonist with a Unique in Vitro Profile and Potent Glucose and Lipid Effects in Rodent Models of Type 2 Diabetes and Dyslipidemia

LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)alpha/gamma dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPARgamma partial agonist with relatively less but significant PPARalpha agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPARgamma full agonist with no PPARalpha activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPARgamma full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPARalpha/gamma dual agonist.

Epigallocatechin gallate attenuates diet-induced obesity in mice by decreasing energy absorption and increasing fat oxidation

To examine the antiobesity effect of epigallocatechin gallate (EGCG), a green tea bioactive polyphenol in a mouse model of diet-induced obesity. Obesity was induced in male New Zealand black mice by feeding of a high-fat diet. EGCG purified from green tea (TEAVIGO) was supplemented in the diet (0.5 and 1%). Body composition (quantitative magnetic resonance), food intake, and food digestibility were recorded over a 4-week period. Animals were killed and mRNA levels of uncoupling proteins (UCP1-3), leptin, malic enzyme (ME), stearoyl-CoA desaturase-1 (SCD1), glucokinase (GK), and pyruvate kinase (PK) were analysed in different tissues. Also investigated were acute effects of orally administered EGCG (500 mg/kg) on body temperature, activity (transponders), and energy expenditure (indirect calorimetry). Dietary supplementation of EGCG resulted in a dose-dependent attenuation of body fat accumulation. Food intake was not affected but faeces energy content was slightly increased by EGCG, indicating a reduced food digestibility and thus reduced long-term energy absorption. Leptin and SCD1 gene expression in white fat was reduced but SCD1 and UCP1 expression in brown fat was not changed. In liver, gene expression of SCD1, ME, and GK was reduced and that of UCP2 increased. Acute oral administration of EGCG over 3 days had no effect on body temperature, activity, and energy expenditure, whereas respiratory quotient during night (activity phase) was decreased, supportive of a decreased lipogenesis and increased fat oxidation. Dietary EGCG attenuated diet-induced body fat accretion in mice. EGCG apparently promoted fat oxidation, but its fat-reducing effect could be entirely explained by its effect in reducing diet digestibility.

Altered expression of nuclear hormone receptors and coactivators in mouse heart during the acute-phase response.

Severe sepsis results in the decreased uptake and oxidation of fatty acids in the heart and cardiac failure. Some of the key proteins required for fatty acid uptake and oxidation in the heart have been shown to be downregulated after endotoxin (LPS) administration. The nuclear hormone receptors, peroxisome proliferator-activated receptor (PPAR) and thyroid receptor (TR), which heterodimerize with the retinoid X receptor (RXR), are important regulators of fatty acid metabolism and decrease in the liver after LPS administration. In the present study, we demonstrate that LPS treatment produces a rapid and marked decrease in the mRNA levels of all three RXR isoforms, PPARalpha and PPARdelta, and TRalpha and TRbeta in the heart. Moreover, LPS administration also decreased the expression of the coactivators CREB-binding protein (CBP)/p300, steroid receptor coactivator (SRC)-1, SRC-3, TR-associated protein (TRAP)220, and PPARgamma coactivator (PGC)-1, all of which are required for the transcriptional activity of RXR-PPAR and RXR-TR. In addition, the mRNA levels of the target genes malic enzyme, Spot 14, sarcoplasmic reticulum Ca2+-ATPase, or SERCA2, the VLDL receptor, fatty acyl-CoA synthetase, fatty acid transporter/CD36, carnitine palmitoyltransferase Ibeta, and lipoprotein lipase decrease in the heart after LPS treatment. The decrease in expression of RXRalpha, -beta, and -gamma, PPARalpha and -delta, and TRalpha and -beta, and of the coactivators CBP/p300, SRC-1, SRC-3, TRAP220, and PGC-1 and the genes they regulate, induced by LPS in the heart, could account for the decreased expression of key proteins required for fatty acid oxidation and thereby play an important role in cardiac contractility. These alterations could contribute to the myocardial dysfunction that occurs during sepsis.

Peroxisome Proliferator-activated Receptor α-mediated Pathways Are Altered in Hepatocyte-specific Retinoid X Receptor α-deficient Mice

Retinoid x receptor alpha (RXRalpha) serves as an active partner of peroxisome proliferator-activated receptor (PPARalpha). In order to dissect the functional role of RXRalpha and PPARalpha in PPARalpha-mediated pathways, the hepatocyte RXRalpha-deficient mice have been challenged with physiological and pharmacological stresses, fasting and Wy14,643, respectively. The data demonstrate that RXRalpha and PPARalpha deficiency are different in several aspects. At the basal untreated level, RXRalpha deficiency resulted in marked induction of apolipoprotein A-I and C-III (apoA-I and apoC-III) mRNA levels and serum cholesterol and triglyceride levels, which was not found in PPARalpha-null mice. Fasting-induced PPARalpha activation was drastically prevented in the absence of hepatocyte RXRalpha. Wy14,643-mediated pleiotropic effects were also altered due to the absence of hepatocyte RXRalpha. Hepatocyte RXRalpha deficiency did not change the basal acyl-CoA oxidase, medium chain acyl-CoA dehydrogenase, and malic enzyme mRNA levels. However, the inducibility of those genes by Wy14,643 was markedly reduced in the mutant mouse livers. In contrast, the basal cytochrome P450 4A1, liver fatty acid-binding protein, and apoA-I and apoC-III mRNA levels were significantly altered in the mutant mouse livers, but the regulatory effect of Wy14,643 on expression of those genes remained the same. Wy14,643-induced hepatomegaly was partially inhibited in hepatocyte RXRalpha-deficient mice. Wy14,643-induced hepatocyte peroxisome proliferation was preserved in the absence of hepatocyte RXRalpha. These data suggested that in comparison to PPARalpha, hepatocyte RXRalpha has its unique role in lipid homeostasis and that the effect of RXRalpha, -beta, and -gamma is redundant in certain aspects.

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in α-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary α-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.

Effect of clofibrate on malic enzyme and leptin mRNAs level in rat brown and white adipose tissue.

Two previous studies have reported contradictory results regarding the effect of fibrates treatment on obese (ob) gene expression in rodents. The purpose of the present study was to reinvestigate this issue. We examined the effect of clofibrate (fibrate derivative) administration for 14 days to rats on malic enzyme (as an adequate control of fibrates action) and leptin mRNAs level in the white and brown adipose tissues (WAT and BAT, respectively). The malic enzyme activity and malic enzyme mRNA level in white adipose tissue increased significantly after clofibrate feeding. In brown adipose tissue, the drug treatment resulted in depression of malic enzyme activity and malic enzyme mRNA level. Under the same conditions, leptin mRNA level did not change in these tissues. The results presented in this paper provide further evidence that the clofibrate (activator of peroxisome proliferator activated receptor alpha), feeding is without effect on ob gene expression in rat white and brown adipose tissue. Furthermore, the present study demonstrates that clofibrate causes opposite effects on malic enzyme gene expression in WAT (up-regulation) and BAT (down-regulation).

Hexachlorobenzene, a dioxin-type compound, increases malic enzyme gene transcription through a mechanism involving the thyroid hormone response element.

Hexachlorobenzene (HCB) is a dioxin-type chemical that acts mainly through the aryl hydrocarbon receptor. Chronic exposure of rats to HCB increases the activity of malic enzyme (ME). In this report, we show that this increase is correlated with an induction of ME messenger RNA (mRNA) levels, with the maximal HCB effect achieved after 9 days of intoxication. This effect is specific for ME, as other liver enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase, and mitochondrial alpha-glycerol-3-phosphate dehydrogenase, are not affected by HCB. The induction of ME mRNA levels is accompanied by an increase in ME promoter activity, as demonstrated by transient transfection experiments performed in rat hepatoma H35 cells. In an attempt to identify the cis-regulatory elements responsible for the HCB effect, different promoter deletions and mutations were used. The results obtained localize the responsive region between positions -315 and -177. This region does not contain either consensus xenobiotic response or activating protein-1 elements, the two main mediators of dioxin compounds described to date. In contrast, a thyroid hormone response element (TRE) is located between -281 to -261. Deletions and mutations of the TRE element do not respond to HCB, demonstrating that this element mediates the response of this dioxin-type compound. As ME gene expression is regulated mainly by thyroid hormones, we next investigated the role of T3 receptor (T3R) in the ME gene transcriptional induction mediated by HCB. Using Scatchard analysis, we show that neither T3R binding features for its ligand nor alpha1 or beta1T3R mRNA levels are changed with the toxic. In gel shift assays, however, we observed that protein/DNA complexes formed on TRE from the ME promoter were induced by HCB. Using an oligonucleotide with a mutation that eliminates the TRE function, we demonstrate a loss of the induced protein/DNA complexes. Together, these data suggest that the dioxin-type compound HCB increases ME gene transcription by modulating the levels of still unidentified nuclear proteins that bind to the TRE element of the ME promoter.

Tissue-specific effect of clofibrate on rat lipogenic enzyme gene expression

Fibrate derivatives are commonly used to treat hyperlipidaemia; however, the mechanism of the antilipidaemic action of these drugs is still unknown. The effect of clofibrate (fibrate derivative) administration for 14 days on lipogenesis and on malic enzyme (EC 1.1.1.40) and fatty acid synthase (EC 2.3.1.85) gene expression in brown and white adipose tissues and in the liver was examined in rats. The rate of brown adipose tissue lipogenesis in the clofibrate-treated animals was significantly lower than that of the control rats. The rate of liver and white adipose tissue lipogenesis was not affected significantly by clofibrate. In brown adipose tissue, the drug treatment resulted in a depression of fatty acid synthase and malic enzyme mRNA levels. The fatty acid synthase mRNA level did not change significantly in the liver, whereas the malic enzyme mRNA level increased approximately 6-fold in this organ after clofibrate treatment. The malic enzyme mRNA level in white adipose tissue increased about 2-fold, while the fatty acid synthase mRNA level was unchanged after clofibrate feeding. The results presented in this paper provide further evidence that the hypolipidaemia caused by treatment of rats with clofibrate cannot be related to the inhibition of fatty acid synthesis in the liver and white adipose tissue. These data also indicate that clofibrate exhibits tissue specificity.

Insulin regulation of malic enzyme gene expression in rat liver: evidence for nuclear proteins that bind to two putative insulin response elements.

Diabetes in rats is characterized by insulin deficiency accompanied by a decrease in lipogenic enzymes. The malic enzyme (ME) gene, which encodes an important lipogenic enzyme, was used to investigate insulin regulation of gene expression. ME mRNA levels were reduced by more than 90% in the liver of diabetic rats. The administration of insulin (3 U/15 days) to either control or diabetic rats increased ME mRNA by 2- to 10-fold, respectively. Since diabetes reduces circulating T3 and the levels of nuclear T3-receptors, the potential role of thyroid hormone on insulin regulation of ME gene expression was also evaluated in thyroidectomized-diabetic rats. In these animals the levels of ME mRNA were undetectable but were increased by insulin even in the absence of thyroid hormones. These in vivo effects of insulin and T3 were not additive. The transcription rate of the gene was also reduced in the diabetic liver and recovered after insulin therapy. By computer analyses we have identified two different putative insulin response elements (IREs) in the ME gene promoter, hereafter referred to as IRE-I (-683 to -692), which is similar to the phosphoenol pyruvate carboxy kinase promoter IRE and IRE-II (-161 to -170), which is similar to the glyceraldehyde phosphate dehydrogenase gene promoter IRE-A. Results from gel retardation assays suggest that a single nuclear protein binds to IRE-I whereas two different nuclear proteins bind to IRE-II. The protein/IRE-I complex increased in liver nuclear extracts from diabetic rats and decreased after insulin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor-Induced Alterations in Hepatic Malic Enzyme and Carnitine Palmitoyltransferase Activity

To better understand the role of the liver in the hypertriglyceridemia observed in a tumor-bearing state, we have examined tumor-induced alterations in hepatic lipogenesis and fatty acid oxidation. The effects of differing tumor burden as well as tumor excision on the activity and mRNA levels of malic enzyme and carnitine palmitoyltransferase were studied in Fisher 344 rats bearing a methylcholanthrene-induced sarcoma. Serum triacylglycerols and plasma nonesterified fatty acids (NEFA) levels were both elevated with increasing tumor burden ( P < 0.05 vs control). The elevation disappeared with tumor removal. Malic enzyme activity of tumor bearers, compared with control rats, declined with an increase in tumor burden. These two variables were negatively correlated ( r = -0.90, P < 0.01). The changes in activity were accompanied by positively correlated changes in mRNA levels ( r = 0.73, P < 0.01). Carnitine palmitoyltransferase activity was not altered, even in the presence of a large tumor burden. Hepatic lipogenesis, reflected by malic enzyme activity, was tumor-dependent and was significantly reduced during the period of circulating hypertriglyceridemia. Fatty acid oxidation, reflected by carnitine palmitoyltransferase activity, was not enhanced in spite of an ample supply of NEFAs to the liver from the peripheral tissues. The data suggest that neither hepatic lipogenesis nor fatty acid oxidation contribute to hypertriglyceridemia in the tumor-bearing state.

Regulation of malic-enzyme-gene expression by cAMP and retinoic acid in differentiating brown adipocytes.

Brown adipose tissue (BAT) is composed of highly specialized cells, whose main function is to produce heat under adrenergic stimulation, uncoupling oxidative phosphorylation. For this function, lipogenesis must be accurately regulated. Malic enzyme has a central role in lipogenesis and is strongly expressed in brown adipocytes. In this work, we study the modulation by adrenergic stimuli, cAMP effectors and retinoic acid on the induction produced by insulin and 3,5,3'-triiodothyronine on malic-enzyme-gene expression. Primary cultures of differentiating brown adipocytes have been used. The results obtained demonstrate that physiological doses of norepinephrine do not modify malic-enzyme mRNA levels when acting alone, but considerably reduce the induction produced by insulin, 3,5,3'-triiodothyronine or both together. Other cAMP inducers such as glucagon, forskolin or 8-bromo-cAMP, greatly inhibit both, basal and 3,5,3'-triiodothyronine-induced malic-enzyme-gene gene expression. Retinoic acid abolishes basal and also inhibits 3,5,3'-triiodothyronine-induced malic-enzyme-gene expression.

Lipogenic Enzyme Gene Expression in Rat Liver during Developnment after Birth1

The time courses and the regulation of lipogenic enzyme gene expression during development after birth have been investigated. The mRNA concentrations and activities of liver lipogenic enzymes (acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase) were very low in all the suckling rats, regardless of dietary fat of the mothers. After weaning to the same diet as the mothers, the mRNA and enzyme levels were greatly increased by the fat-free or hydrogenated fat diet but not so greatly increased by the corn or fish oil diet. The mRNA concentrations of all the groups reached maximum at 4-6 weeks old and then decreased, usually to 40-60% of the maximal levels. It appeared that the gene expression after weaning is subject to strong nutritional regulation, as well as developmental regulation. The plasma levels of triiodothyronine and insulin were low during suckling. Malic enzyme mRNA level was increased by triiodothyronine treatment even during suckling, but the absolute increase was much less than after weaning. Thus, the gene expression of lipogenic enzymes during suckling appeared to be suppressed by nutritional and hormonal regulation, or may not be sufficiently developed. On the other hand, the hepatic triacylglycerol levels were increased slightly at 2 weeks old and greatly at 3 weeks. As the gene expression of lipogenic enzymes was still low at that time, the major triacylglycerols appear to be obtained from milk and accumulated in preparation for weaning.

Gene expression of lipid storage-related enzymes in adipose tissue of the genetically obese Zucker rat. Co-ordinated increase in transcriptional activity and potentiation by hyperinsulinaemia.

The genetically obese Zucker rat displays excessive fat storage capacity which is due to a tissue-specific increase in the activities of a number of lipid storage-related enzymes in adipose tissue. The aim of this study was to investigate the molecular mechanism responsible for this phenomenon. Lean (Fa/fa) and obese (fa/fa) Zucker rats were studied during the early stages of adipose tissue overdevelopment, both before (at 16 days of age) and after (at 30 days of age) the emergence of hyperinsulinaemia, in order to delineate the effects of the fatty genotype independently of those of hyperinsulinaemia. Lipoprotein lipase (LPL), glycerophosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malic enzyme (ME) mRNA levels in the adipose tissue of lean and obese rats were assessed by Northern blot analysis, and the relative transcription rates of the corresponding genes were compared in the two genotypes by a nuclear run-on assay. In normoinsulinaemic 16-day-old pre-obese rats, mRNA levels were increased over control values (LPL, 5-fold; ME, 2-fold; GAPDH, 3-fold), in close correlation with genotype-mediated differences in enzyme activities. Stimulation of the transcription rates of the ME and GAPDH genes was observed in obese rats, which could fully account for differences in steady-state mRNA levels. At this age, GPDH activity, mRNA level and transcription rate were similar in the two genotypes. In hyperinsulinaemic 30-day-old obese rats, a 6-7-fold increase in both mRNA and the transcription rate of GPDH emerged, together with an amplification of the genotype-mediated differences observed in younger animals (GAPDH, 6-fold; ME, 7.9-fold; LPL, 10-fold). These results demonstrate that the obese genotype exerts a co-ordinated control on the expression of these genes in adipose tissue, mainly at the transcriptional level. This genotype effect is greatly amplified by the development of hyperinsulinaemia.

Nutritional regulation and tissue-specific expression of the malic enzyme gene in the chicken. Transcriptional control and chromatin structure.

Refeeding starved chicks causes a 25- to 50-fold increase in the level of malic enzyme mRNA in liver. To define the regulated steps, we measured transcriptional activity of the malic enzyme gene using the nuclear run-on assay and a variety of DNA probes specific to the malic enzyme gene. Refeeding starved chicks stimulated transcription of the malic enzyme gene in liver by 40- to 50-fold. An increased transcription rate was detectable at 1.5 h, was maximal at 3 h, and remained high at 24 h of refeeding. The level of nuclear precursor RNA for malic enzyme assessed by hybridization with intron-specific probes was high in liver of refed birds, and barely detectable in that of starved birds. These results indicate that nutritional regulation of the level of malic enzyme mRNA is transcriptional. Low levels of malic enzyme mRNA in brain, kidney, and heart correlated well with low rates of transcription of the malic enzyme gene in these tissues. In contrast to liver, neither the rate of transcription nor the steady-state level of malic enzyme mRNA was affected by refeeding starved birds. A series of DNase I-hypersensitive sites were located within 4000 base pairs upstream of the transcription start site of the malic enzyme gene in liver. The DNase I-hypersensitive region extending from the start of transcription to 400 base pairs upstream was much more pronounced in the refed state than in the starved state. This change in DNase I hypersensitivity followed the same time course as increased transcription of the malic enzyme gene. This DNase I-hypersensitive region also was present at low intensity in kidney and heart independently of nutritional state. The three constitutive DNase I-hypersensitive sites further upstream were present in liver but not in kidney or heart.

Effect of Hydrocortisone on the Ability of Thyrotropin to Increase Deoxyribonucleic Acid Synthesis and Iodide Uptake in FRTL-5 Rat Thyroid Cells: Opposite Regulation of Adenosine 3\5'-Monophosphate Signal Action

In FRTL-5 rat thyroid cells, hydrocortisone alters two TSH-increased cAMP-mediated activities in an opposite manner. Thus, in a concentration-dependent fashion, hydrocortisone synergistically enhances TSH-increased thymidine incorporation into DNA, whereas it inhibits TSH-induced iodide uptake. The effect of hydrocortisone on TSH-increased thymidine incorporation is specific, in that it has only a minimal ability by itself to increase thymidine incorporation into DNA and slightly inhibits the activity of insulin or insulin-like growth factor-I. The effect of hydrocortisone on TSH-induced iodide uptake does not result from altered iodide efflux, but, rather, from a decrease in the maximal velocity, not the Km, of iodide influx, i.e. from a decrease in the effective number of iodide porters. The action of a cAMP analog to increase thymidine incorporation into DNA or iodide uptake was also increased or decreased, respectively, by hydrocortisone, whereas hydrocortisone did not diminish the ability of TSH to increase cAMP levels in the cells. The different effect of hydrocortisone on these two cAMP-mediated activities reflects, therefore, regulation of cAMP signal action rather than the ability of TSH to generate a cAMP signal. Twenty-four hours after TSH, actinomycin-D superinduces iodide uptake and abolishes the action of hydrocortisone to inhibit iodide uptake. It has been suggested that actinomycin-D superinduces iodide uptake in FRTL-5 cells by a posttranscriptional action, inhibition of mRNA degradation, rather than by its known transcriptional actions linked to DNA synthesis. More recent studies of the effect of actinomycin-D, given under identical circumstances, on TSH-stimulated malic enzyme mRNA levels directly validate this hypothesis. We, thus, suggest that the opposite action of hydrocortisone on the two cAMP-mediated activities may reflect positive and negative regulation of the action of cAMP at different steps in the transduction process, one being transcriptional (DNA synthesis) and the other posttranscriptional (induction of iodide porter activity).

Thyrotropin Regulation of Malic Enzyme in FRTL-5 Rat Thyroid Cells

TSH-induced increases in malic enzyme mRNA levels in FRTL-5 rat thyroid cells are paralleled by increases in malic enzyme activity and are mimicked by 8-bromo-cAMP. Apparent approximately 4 h after TSH challenge and maximal after 16 h, they decline by 24 h and are at basal levels by 48 h. The increase occurs in the absence of a measurable effect of TSH on DNA synthesis related to cell growth, since [3H] thymidine incorporation into DNA is still at basal levels 24 h after TSH challenge and is maximal only at 48 h. A protein(s) whose formation is inhibited by cycloheximide appears to be critical to the ability of TSH to increase malic enzyme mRNA levels. Thus, cycloheximide given 30 min before TSH prevents the hormone-induced increase in malic enzyme mRNA; also, when given 24 h after TSH, cycloheximide accelerates the loss of the TSH-induced increase in malic enzyme mRNA. In neither case does cycloheximide affect beta-actin mRNA levels. A second factor(s) whose formation is prevented by actinomycin-D appears to be important for the decrease in malic enzyme mRNA levels seen 24 and 48 h after TSH challenge. Thus, in experiments in which it is given 24 h after TSH, actinomycin-D preserves the hormone-induced increase in malic enzyme mRNA levels rather than accelerating the decrease, as does cycloheximide. In the same experiment, beta-actin mRNA levels decrease to less than 10-20% of control values over the same period; this factor also, therefore, appears to exhibit some degree of specificity.(ABSTRACT TRUNCATED AT 250 WORDS)