Liver Malic Enzyme Research Articles

Digestible protein levels and metabolic responses in juvenile piapara (Megaleporinus obtusidens)

The piapara (Megaleporinus obtusidens) is an omnivorous fish with great market potential. However, little is known about its dietary requirements. This study aimed to determine the effects of dietary protein on growth and physiological indicators in piapara. Three hundred piapara juveniles (24.0 ± 1.3 g) were distributed in 20 tanks of 130-L (15 fish/tank) provided with continuous aeration and water. Five isoenergetic diets (14.0 MJ/kg) were formulated to contain increasing levels of digestible protein (21, 24, 27, 30, and 33% DP). After 77 days, weight gain and ammonia concentration in water were found to increase linearly with increasing digestible protein (DP) level (p<0.05). Protein productive value (PPV) reached a maximum at 28.4% DP. No relationship was found between DP level and serum ammonia, liver alanine aminotransferase (ALT) activity, hepatosomatic index (HSI), liver glycogen (GL) or liver lipid (LL) content (p>0.05). Mesenteric fat index (MFI) and liver malic enzyme (ME) activity followed a quadratic pattern (p<0.05) and reached a maximum at 25.8% and 27.1% DP, respectively. Muscle lipid (ML) content decreased linearly with increasing DP level (p<0.05), whereas liver glucose-6-phosphate dehydrogenase (G6PD) activity decreased linearly as DP level increased (p<0.05). Results showed that a diet containing 28.4% PD provides better crude protein retention efficiency and reduces amino acid deamination in M. obtusidens. Low protein diets increased energy retention efficiency, whereas fish fed high protein diets had higher ammonia excretion.

Supplemental L-arginine improves feed conversion and modulates lipid metabolism in male and female broilers from 29 to 42 days of age

The excessive accumulation of abdominal fat in broilers is an economic waste. Supplemental dietary L-arginine has been shown to reduce lipogenesis in broilers, but studies on this subject are still scarce. Two experiments were conducted in a 5×2 factorial design, with five L-arginine concentrations in diets (0, 3, 6, 9 and 12 g/kg) provided to male and female broilers, from 29 to 42 days of age, reared in boxes or cages, with six replicates of 23 broilers per box and six replicates of three broilers per cage, totalling 1560 broilers. Data on performance, carcass and cuts yield, abdominal fat deposition, chemical composition of the breast, lipid profile and liver enzyme activity were evaluated in experiment 1. In experiment 2, the balance and retention of nitrogen, metabolizability coefficients and metabolizable energy of feed were determined and the treatments were evaluated in six replicates of three broilers per cage, totalling 180 broilers. In both experiments, there was no interaction (P > 0.05) between L-arginine concentration in the diet and sex of the broilers for all parameters evaluated. Male broilers showed higher (P < 0.05) weight gain, noble cuts yield, levels of CP and mineral matter in the breast, better feed conversion (P < 0.05) and lower (P < 0.05) malic enzyme activity in the liver and abdominal fat deposition. On the other hand, regardless of sex, the increase in L-arginine concentration in the diet improved (P < 0.05) the feed conversion in addition to reducing (P < 0.05) serum levels of total cholesterol and low-density lipoprotein cholesterol, the malic enzyme activity in the liver and abdominal fat deposition. In conclusion, male broilers had better productive results than females. However, supplementing the diet of male or female broilers from 29 to 42 days of age with L-arginine at a concentration of 6.87 g/kg represents a nutritional strategy to improve feed conversion and reduce circulating triacylglycerol and cholesterol levels, NADPH synthesis by liver malic enzyme and abdominal fat deposition, without negatively affecting the carcass and noble cuts yield, the amount of nitrogen excreted by the broilers and the energy value of the feed.

Dietary glutamine, glutamate, and aspartate supplementation improves hepatic lipid metabolism in post-weaning piglets.

A previous study has demonstrated that early weaning significantly suppressed hepatic glucose metabolism in piglets. Glutamate (Glu), aspartate (Asp) and glutamine (Gln) are major metabolic fuels for the small intestine and can alleviate weaning stress, and therefore might improve hepatic energy metabolism. The objective of this study was to investigate the effects of administration of Glu, Asp and Gln on the expression of hepatic genes and proteins involved in lipid metabolism in post-weaning piglets. Thirty-six weaned piglets were assigned to the following treatments: control diet (Control; basal diet + 15.90 g/kg alanine); Asp, Gln and Glu-supplemented diet (Control + AA; basal diet + 1.00 g/kg Asp + 5.00 g/kg Glu + 10.00 g/kg Gln); and the energy-restricted diet supplemented with Asp, Gln and Glu (Energy− + AA; energy deficient diet + 1.00 g/kg Asp + 5.00 g/kg Glu + 10.00 g/kg Gln). Liver samples were obtained on d 5 and 21 post-weaning. Piglets fed Energy− + AA diet had higher liver mRNA abundances of acyl-CoA oxidase 1 (ACOX1), succinate dehydrogenase (SDH), mitochondrial transcription factor A (TFAM) and sirtuin 1 (SIRT1), as well as higher protein expression of serine/threonine protein kinase 11 (LKB1), phosphor-acetyl-CoA carboxylase (P-ACC) and SIRT1 compared with piglets fed control diet (P < 0.05) on d 5 post-weaning. Control + AA diet increased liver malic enzyme 1 (ME1) and SIRT1 mRNA levels, as well as protein expression of LKB1 and P-ACC on d 5 post-weaning (P < 0.05). On d 21 post-weaning, compared to control group, Glu, Gln and Asp supplementation up-regulated the mRNA levels of ACOX1, ME1 and SIRT1 (P < 0.05). These findings indicated that dietary Glu, Gln and Asp supplementation could improve hepatic lipid metabolism to some extent, which may provide nutritional intervention for the insufficient energy intake after weaning in piglets.

Atmospheric ammonia alters lipid metabolism-related genes in the livers of broilers (Gallus gallus).

Atmospheric ammonia in animal housing is reported to have adverse effects on livestock performance and animal health. Previous experiments have found that 75ppm ammonia reduced the production performance and altered body fat distribution quality of broilers. In this study, we examined the body fat distribution, serum metabolites and lipid metabolism gene expression of broiler exposed to ammonia. A total of 400 chickens were randomly allocated to four groups with four replicates and received ammonia treatments at 0, 25, 50 and 75ppm, respectively, for 3weeks. The average daily feed intake and weight gain were decreased when broiler was exposed to ammonia concentration exceeding 50ppm (p<.05). The increased abdominal fat and reduced thickness of subcutaneous adipose were found in broilers of 75ppm group (p<.05). When ammonia exceeded 50ppm, the content of fat in breast muscle of broiler was increased, and when ammonia was higher than 25ppm, the fat in liver was increased (p<.05). It showed that the fat content in liver was a sensitive index for broilers exposed to ammonia. Furthermore, ammonia exposure had no significant effect on total cholesterol and triglyceride in serum, but significantly increased the relative mRNA expression of acetyl-CoA carboxylase (p=.046) and malic enzyme in liver (p=.038), which indicated that ammonia exposure may increase the de novo fat synthesis in liver. In addition, ammonia increased the high-density lipoprotein cholesterol (p=.02) and activity of hepatic lipase in serum (p<.001), which indicated that ammonia exposure may improve the transportation of cholesterol to liver. To conclude, our results indicated that ammonia exposure might increase the de novo fat synthesis in liver and increased the transportation of cholesterol to liver. In addition, the concentration of ammonia in poultry house should be limited lower than 25ppm based on the variation of hepatic fat content.

Nutritional and metabolic responses in common dentex (Dentex dentex) fed on different types and levels of carbohydrates

The present study was aimed to evaluate the capacity of common dentex (Dentex dentex) to efficiently use dietary carbohydrates. So, the effects of different type and levels of carbohydrates on growth performance, feed utilization, fish composition, plasma metabolites and key metabolic pathways in liver and white muscle of common dentex are presented. Nine isonitrogenous (43%) and isoenergetic (22MJkg−1) diets were formulated combining three types, pregelatinized starch (PS), dextrin (Dx) and maltodextrin (Mx), and three levels (12, 18 and 24%) of carbohydrates. Growth performance was not significantly influenced by treatments. The best feed utilization was observed in 18% Mx group. Higher hepatic lipid content was found in fish fed lower dietary carbohydrate levels. PS induced higher liver and white muscle hexokinase and pyruvate kinase activities compared to the lower values observed for Mx. Malic enzyme and glucose 6-phosphate dehydrogenase in liver and white muscle were lower in Mx group. The influence of dietary carbohydrates source was more noticeable than those induced by the carbohydrate level, when glycolysis and lipogenesis pathways were considered. Common dentex is able to use properly dietary carbohydrates, although optimal dietary inclusion levels are below 24%. The greater protein-sparing effect was promoted by the less complex carbohydrate (maltodextrin) and the best feed utilization indices were obtained at intermediate levels of inclusion (18%).

Intermittent feeding programme and addition of &amp;lt;i&amp;gt;Bacillus subtilis&amp;lt;/i&amp;gt; based probiotics to the diet of growing broiler chickens: Influence on growth, hepatic enzymes and serum lipid metabolites profile

Abstract. This study was conducted to examine the effects of Intermittent feeding programme (IFP) and Bacillus subtilis-based probiotic (BSP) addition in diet on liver malic enzyme and isocitrate dehydrogenase activity, lipid metabolism and performance in broiler chickens. Five hundred and four one-day old male broiler chicks were randomly allocated in four experimental treatments (T1, T2, T3 and T4). Groups T1 (control diet) and T3 (Bacillus subtilis-based probiotic diet) were fed ad libitum, whereas groups T2 (control diet) and T4 (Bacillus subtilis-based probiotic diet) served as IFP from day 8 to the end of the experiment. The data on initial body weight, weekly feed consumption and body weight gain were recorded up to six weeks of age. Carcass composition, blood parameters and hepatic enzyme activity were measured at the end of the experiment. Although body weight gain was not significantly different among any of the treatments, the birds raised under IFP consumed significantly (P&lt;0.05) lower feed (207 g) and utilized their feed more efficiently (1.78) than those of the control group fed ad libitum (1.84). Carcass weight as a percentage of live weight was not affected by probiotic supplementation on the diet, but IFP significantly reduced (P&lt;0.05) broiler carcass weight. However, the liver malic and isocitrate dehydrogenase enzyme activity was not significantly different between IFP and BSP groups. All serum lipid metabolites concentration decreased (P&lt;0.05) with probiotic treatment. It may be concluded that dietary supplementation with BSP may influence the pathway of fat metabolism through promotion and/or suppression of serum lipid metabolites.

White adipose tissue re-growth after partial lipectomy in high fat diet induced obese Wistar rats

The effects of partial removal of epididymal (EPI) and retroperitoneal (RET) adipose tissues (partial lipectomy) on the triacylglycerol deposition of high fat diet induced obese rats were analyzed, aiming to challenge the hypothesized body fat regulatory system. Male 28-day-old wistar rats received a diet enriched with peanuts, milk chocolate and sweet biscuits during the experimental period. At the 90th day of life, rats were submitted to either lipectomy (L) or sham surgery. After 7 or 30 days, RET, EPI, liver, brown adipose tissue (BAT), blood and carcass were obtained and analyzed. Seven days following surgery, liver lipogenesis rate and EPI relative weight were increased in L. After 30 days, L, RET and EPI presented increased lipogenesis, lipolysis and percentage of small area adipocytes. L rats also presented increased liver malic enzyme activity, BAT lipogenesis, and triacylglycerol and corticosterone serum levels. The partial removal of visceral fat pads affected the metabolism of high fat diet obese rats, which leads to excised tissue re-growth and possibly compensatory growth of non-excised depots at a later time.

A STUDY OF ADLAY ON LOWERING SERUM AND LIVER LIPIDS IN HAMSTERS

ABSTRACT The functional component of adlay responsible for lowering blood lipids was studied using an animal model. Forty male hamsters were randomly divided into four dietary groups, including control, whole grain adlay (WGA), oil‐free adlay (OFA) and adlay oil (AO) groups. All groups were fed a high fat diet containing 11.8% oil and 0.2% cholesterol for 8 weeks. The results showed that the WGA, OFA and AO diets could decrease triacylglycerol (TAG), total cholesterol (TC) and low‐density lipoprotein cholesterol (LDL‐C) concentrations in serum (P &lt; 0.05). Specifically, all lipid levels were significantly lowered in the WGA and OFA groups as compared to the AO group (P &lt; 0.05). Ingestion of WGA or OFA also reduced liver TAG and TC concentrations (P &lt; 0.05). The data demonstrated that WGA, OFA and AO prolonged the lag phase of LDL oxidation (P &lt; 0.05). Moreover, hamsters fed with WGA or OFA had lower activity of malic enzyme in liver than those fed with control or AO diet (P &lt; 0.05). WGA, OFA and AO diets were also found to enhance the activity of glucose‐6‐phosphate dehydrogenase. Thus, the beneficial components of adlay were mainly in the OFA portion.PRACTICAL APPLICATIONSAdlay is one of the most common components used in Chinese herbal medicine for hypolipidemic effect and immunoregulation. Numerous studies had demonstrated that adlay can decrease serum total cholesterol, triacylglycerol and low‐density lipoprotein cholesterol concentrations in animal models and human subjects. However, the shelf‐life of processed adlay products is relatively short due to the easy oxidation of its oil. According to the results of this study, it seems reasonable to remove the oil component of adlay grains to prevent its peroxidation and the development of off‐flavor in products while retaining most of the hypolipidemic effects of adlay.

Polychlorinated Biphenyls 105 and 118 Form Thyroid Hormone Receptor Agonists after Cytochrome P4501A1 Activation in Rat Pituitary GH3 Cells

BackgroundPolychlorinated biphenyls (PCBs) may interfere with thyroid hormone (TH) signaling by reducing TH levels in blood, by exerting direct effects on TH receptors (TRs), or both.ObjectiveOur objective was to identify individual PCBs that directly affect TH signaling by acting on the TR.MethodsWe administered a mixture of six PCB congeners based on their ortho substitution pattern, including PCBs 77 and 126 (non-ortho), PCBs 105 and 118 (mono-ortho), and PCBs 138 and 153 (di-ortho), to pregnant Sprague-Dawley rats from gestational days (G) 6 to 16. This mixture, or various combinations of the components, was also evaluated in a transient transfection system using GH3 cells.ResultsThe mixture reduced serum TH levels in pregnant rats on G16 but simultaneously up-regulated the expression of malic enzyme in liver. It also functioned as a TR agonist in vitro; however, none of the individual PCB congeners comprising this mixture were active in this system. Using the aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone, and the cytochrome P450 (CYP)1A1 antagonist ellipticine, we show that the effect of the mixture on the thyroid hormone response element required AhR and CYP1A1.ConclusionsWe propose that PCB 126 induces CYP1A1 through the AhR in GH3 cells, and that CYP1A1 activates PCB 105 and/or 118 to a form a compound that acts as a TR agonist. These data suggest that some tissues may be especially vulnerable to PCBs interfering directly with TH signaling due to their capacity to express CYP1A1 in response to coplanar PCBs (or other dioxin-like molecules) if sufficient mono-ortho PCBs are present.

Effects of fructose and sucrose intake on lipid metabolism in 9‐month‐old rats feeding on different levels

This study was designed to evaluate effects of fructose(F) and sucrose(S) intake on lipid metabolism in 9-month-old rats. A total of 100 Spraque-Dawley male rats were raised 10 rats in each group, for one month, in 10 different groups. One group was raised of the control diet that contain cornstarch of source of carbohydrate(100% of CHO). Three groups were raised of varying levels of S of 25%, 50% and 100% of CHO, labeled here as 25S, 50S and 100S, respectively. Three groups were raised of varying levels of F of 25%, 50% and 100% of CHO, labeled here as 25F, 50F and 100F, respectively. The last three groups were raised of varying levels of F of 14%, 28% and 57% of carbohydrate, labeled here as 14F, 28F and 57F, respectively. Note that the last three groups have a similar level of sweetness by 25S, 50S and 100S, respectively. Total lipid, triglyceride(TG) and total cholesterol in plasma and liver, HDL-cholesterol concentration and free fatty acid(FFA) contents in plasma were measured. Plasma malic enzyme(ME) and glucose-6-phosphate dehydrogenase(G-6-PDH) activity were also determined. Total lipid, TG concentrations in plasma were not significantly different among all the groups. Total and HDL-cholesterol concentrations of 25F, 50F, 100F tended to be higher than those of the other groups. Plasma FFA content of S intake groups was higher than that of F intake groups. Liver ME and G-6-PDH activities of all non-control diet groups were significantly higher than those of the control group. Both S and F intakes about equally increased the levels of enzyme activities. In conclusion, S and F intakes had little effect on lipid metabolism. This research was supported by grants from Daesang Co. Ltd.

Characterization of the interactions between Asp141 and Phe236 in the Mn2+-l-malate binding of pigeon liver malic enzyme.

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.

Increased rate of cholesterologenesis--a possible cause of hypercholesterolemia in experimental chronic renal failure in rats.

Hypercholesterolemia plays an important role in the lipid abnormalities in chronic renal failure (CRF). It is thought to contribute to both a progression of renal failure and atherosclerosis. Despite intensive research, the etiopathogenesis of hypercholesterolemia in CRF patients is still obscure. The present study was designed to evaluate the possible role of cholesterol overproduction in the development of hypercholesterolemia associated with experimental CRF. We found that plasma total cholesterol and cholesterol distributed in VLDL, LDL and HDL concentrations were significantly enhanced in CRF rats. Simultaneously, the rate of liver cholesterol biosynthesis in vivo (measured by determining the incorporation of tritium from tritiated water intraperitoneally injected into cholesterol ), liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and liver HMG-CoA reductase mRNA presence were elevated. Significant increases in activity of liver malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, NADPH-producing enzyme (required for cholesterol synthesis) have also been observed in CRF rats. In conclusion, the increased rate of liver cholesterol biosynthesis due to increase of HMG-CoA reductase and NADPH-producing enzyme gene expression could be one of the possible causes of hypercholesterolemia in CRF animals.

Steroid receptor coactivator-1 deficiency causes variable alterations in the modulation of T(3)-regulated transcription of genes in vivo.

Thyroid hormone exerts its biological effect by binding to a TR. Both liganded and unliganded TRs regulate the transcription of T(3)-responsive genes. Cofactors with activating or repressing function modulate the transcriptional regulation by TRs. We showed that steroid receptor coactivator 1 (SRC-1)-deficient mice (SRC-1(-/-)) exhibit partial resistance to thyroid hormone at the level of the pituitary thyrotrophs. To determine whether SRC-1 deficiency affects globally T(3)-dependent transcriptional regulation, we studied the effects of thyroid hormone deprivation and replacement on the expression of several genes in different tissues of SRC-1(-/-) and wild-type mice (SRC-1(+/+)). Thyroid hormone deficiency was induced by a low iodine diet (LoI) supplemented with propylthiouracil (PTU) for 2 wk. L-T(3) was injected ip for the last 4 d in one group (PTU+T(3) group), and another group (PTU group) received only vehicle. Levels of mRNAs for T(3)-responsive genes were determined by Northern blotting: GH and TSH beta in pituitary; type 1 iodothyronine 5'-deiodinase, spot 14 (S14), and malic enzyme in liver; and sarcoplasmic reticulum calcium adenosine triphosphatase 2 and myosin heavy chain alpha and beta in heart. Serum parameters, TSH, total cholesterol, creatine kinase, and alkaline phosphatase (AP), were also measured. Hypothyroidism produced a comparable increase in TSH beta mRNA in both genotypes, but its suppression by L-T(3) was attenuated in SRC-1(-/-) mice. In contrast, hypothyroidism failed to reduce S14 mRNA levels in SRC-1(-/-) mice. As a consequence, the response to L-T(3) was not observed in these mice. SRC-1 deficiency had no effect on the expression of the rest of the T(3)-responsive genes examined. Of the four serum parameters, the T(3)-mediated decrease in TSH and changes in AP were attenuated in SRC-1(-/-) mice. We conclude that SRC-1 deficiency altered the expression of only some of the T(3)-responsive genes. SRC-1 appears to be involved not only in transcriptional activation by liganded TRs, but also in the suppression by liganded or unliganded TRs. Some of the effects of SRC-1 may be TR isoform specific.

Steroid Receptor Coactivator-1 Deficiency Causes Variable Alterations in the Modulation of T3-Regulated Transcription of Genes in Vivo

Thyroid hormone exerts its biological effect by binding to a TR. Both liganded and unliganded TRs regulate the transcription of T3-responsive genes. Cofactors with activating or repressing function modulate the transcriptional regulation by TRs. We showed that steroid receptor coactivator 1 (SRC-1)-deficient mice (SRC-1−/−) exhibit partial resistance to thyroid hormone at the level of the pituitary thyrotrophs. To determine whether SRC-1 deficiency affects globally T3-dependent transcriptional regulation, we studied the effects of thyroid hormone deprivation and replacement on the expression of several genes in different tissues of SRC-1−/− and wild-type mice (SRC-1+/+). Thyroid hormone deficiency was induced by a low iodine diet (LoI) supplemented with propylthiouracil (PTU) for 2 wk. l-T3 was injected ip for the last 4 d in one group (PTU+T3 group), and another group (PTU group) received only vehicle. Levels of mRNAs for T3-responsive genes were determined by Northern blotting: GH and TSHβ in pituitary; type 1 iodothyronine 5′-deiodinase, spot 14 (S14), and malic enzyme in liver; and sarcoplasmic reticulum calcium adenosine triphosphatase 2 and myosin heavy chain α and β in heart. Serum parameters, TSH, total cholesterol, creatine kinase, and alkaline phosphatase (AP), were also measured. Hypothyroidism produced a comparable increase in TSHβ mRNA in both genotypes, but its suppression by l-T3 was attenuated in SRC-1−/− mice. In contrast, hypothyroidism failed to reduce S14 mRNA levels in SRC-1−/− mice. As a consequence, the response to l-T3 was not observed in these mice. SRC-1 deficiency had no effect on the expression of the rest of the T3-responsive genes examined. Of the four serum parameters, the T3-mediated decrease in TSH and changes in AP were attenuated in SRC-1−/− mice. We conclude that SRC-1 deficiency altered the expression of only some of the T3-responsive genes. SRC-1 appears to be involved not only in transcriptional activation by liganded TRs, but also in the suppression by liganded or unliganded TRs. Some of the effects of SRC-1 may be TR isoform specific.

Effect of Metal Binding on the Structural Stability of Pigeon Liver Malic Enzyme

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.

3,5,3'-Triiodothyronine (T3) and 3',5'-Diiodothyrone (T2) Have Short-Term Effects on Lipid Metabolism in a Teleost Anabas testudineus (Bloch): Evidence From Enzyme Activities

As thyroid hormones are known to have long-term and short-term effects on metabolism and only the long term effects been examined in detail, in the present investigation a comparative study has been mad to elucidate the short-term effects of T3 and T2 on enzymes of lipid metabolism in a fish Anabas testudineus. The time dependent studies involved assays of specific, activities of lipogenic enzymes and membrane ATPase- Anabas responded to T3 treatment with a significant increase in the liver malic enzyme activity (after 1 hr) and the activity pattern was reversed after 6 hrs in low dose (0.25µg T3) treated group. T2 treatment also increased the malic enzyme activity within one hour after administration. T2 caused an Increase, in the activity of glucose-6-phosphate dehydrogenase under both time courses, HMG-CoA Reductase activity was reduced upon T3 and T2 administration except for T2 at one hour. T3 treatment significantly enhanced Na+K+-ATPase activity while T2 decreased it except by low dose at one hour. Both T3 and T2 treatment influenced the activities of enzymes of lipid synthesis in a dose and time dependent manner emphasising a short-term effect of these thyroid hormones in Anabas.

Characterization of the functional role of asp 141, asp 194, and asp464 residues in the Mn2+‐l‐malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Thyroid Hormone Action on Liver, Heart, and Energy Expenditure in Thyroid Hormone Receptor -Deficient Mice

Thyroid hormone (TH) responsive genes can be both positively and negatively regulated by TH through receptors (TR) α and β expressed in most body tissues. However, their relative roles in the regulation of specific gene expression remain unknown. The TRβ knockout mouse, which lacks both TRβ1 and TRβ2 isoforms, provides a model to examine the role of these receptors in mediating TH action. TRβ deficient (TRβ−/−) mice that show no compensatory increase in TRα, and wild-type (TRβ+/+) mice of the same strain were deprived of TH by feeding them a low iodine diet containing propylthiouracil, and were then treated with supraphysiological doses of L-T3 (0.5, 5.5, and 25 μg/day/mouse). TH deprivation alone increased the serum cholesterol concentration by 25% in TRβ+/+ mice and reduced it paradoxically by 23% in TRβ−/− mice. TH deprivation reduced the serum alkaline phosphatase (AP) concentration by 31% in TRβ+/+ mice but showed no change in the TRβ−/− mice. Treatment with L-T3 (0.5 to 25μ g/mouse/day) caused a 57% decrease in serum cholesterol and a 231% increase in serum AP in the TRβ+/+ mice. The TRβ−/− mice were resistant to the L-T3 induced changes in serum cholesterol and showed increase in AP only with the highest L-T3 dose. Basal heart rate (HR) in TRβ−/− mice was higher than that of TRβ+/+ mice by 11%. HR and energy expenditure (EE) in both TRβ+/+ and TRβ−/− mice showed similar decreases (49 and 46%)and increases (49 and 41%) in response to TH deprivation and L-T3 treatment, respectively. The effect of TH on the accumulation of messenger RNA (mRNA) of TH regulated liver genes was also examined. TH deprivation down regulated spot 14 (S14) mRNA and showed no change in malic enzyme (ME) mRNA in both TRβ+/+ and TRβ−/− mice. In contrast treatment with L-T3 produced an increase in S14 and ME but no change in TRβ−/− mice. From these results, it can be concluded that regulation of HR and EE are independent of TRβ. With the exception of serum cholesterol concentration and liver ME mRNA accumulation, all other markers of TH action examined during TH deprivation exhibited the expected responses in the absence of TRβ. Thus, as previously shown for serum TSH, TRβ is not absolutely necessary for some changes typical of hypothyroidism to occur. In contrast, except for HR and EE, the full manifestation of TH-mediated action required the presence of TRβ.

Thyroid hormone action on liver, heart, and energy expenditure in thyroid hormone receptor beta-deficient mice.

Thyroid hormone (TH) responsive genes can be both positively and negatively regulated by TH through receptors (TR) alpha and beta expressed in most body tissues. However, their relative roles in the regulation of specific gene expression remain unknown. The TR beta knockout mouse, which lacks both TR beta1 and TR beta2 isoforms, provides a model to examine the role of these receptors in mediating TH action. TR beta deficient (TR beta-/-) mice that show no compensatory increase in TR alpha, and wild-type (TR beta+/+) mice of the same strain were deprived of TH by feeding them a low iodine diet containing propylthiouracil, and were then treated with supraphysiological doses of L-T3 (0.5, 5.5, and 25 microg/day/mouse). TH deprivation alone increased the serum cholesterol concentration by 25% in TR beta+/+ mice and reduced it paradoxically by 23% in TR beta-/- mice. TH deprivation reduced the serum alkaline phosphatase (AP) concentration by 31% in TR beta+/+ mice but showed no change in the TR beta-/- mice. Treatment with L-T3 (0.5 to 25 microg/mouse/day) caused a 57% decrease in serum cholesterol and a 231% increase in serum AP in the TR beta+/+ mice. The TR beta-/- mice were resistant to the L-T3 induced changes in serum cholesterol and showed increase in AP only with the highest L-T3 dose. Basal heart rate (HR) in TR beta-/- mice was higher than that of TR beta+/+ mice by 11%. HR and energy expenditure (EE) in both TR beta+/+ and TR beta-/- mice showed similar decreases (49 and 46%) and increases (49 and 41%) in response to TH deprivation and L-T3 treatment, respectively. The effect of TH on the accumulation of messenger RNA (mRNA) of TH regulated liver genes was also examined. TH deprivation down regulated spot 14 (S14) mRNA and showed no change in malic enzyme (ME) mRNA in both TR beta+/+ and TR beta-/- mice. In contrast treatment with L-T3 produced an increase in S14 and ME but no change in TR beta-/- mice. From these results, it can be concluded that regulation of HR and EE are independent of TR beta. With the exception of serum cholesterol concentration and liver ME mRNA accumulation, all other markers of TH action examined during TH deprivation exhibited the expected responses in the absence of TR beta. Thus, as previously shown for serum TSH, TR beta is not absolutely necessary for some changes typical of hypothyroidism to occur. In contrast, except for HR and EE, the full manifestation of TH-mediated action required the presence of TR beta.

Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes.

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.