Regulation of expression of antioxidant enzymes by vitamin E and curcumin in <Emphasis Type="SmallCaps">l</Emphasis>-thyroxine-induced oxidative stress in rat renal cortex

The present study investigates the antioxidative effects of vitamin E and curcumin against l-thyroxine (T₄)-induced oxidative stress in renal cortex of adult male rats. Rats were made hyperthyroid by administration of l-thyroxine (0.0012%) in their drinking water for 30 days. Vitamin E (200 mg/kg body weight/day) and curcumin (30 mg/kg body weight/day) were supplemented singly or in combination orally for 30 days along with l-thyroxine treatment. The elevated level of oxidative stress parameters (lipid peroxidation and protein carbonylation) and decline level of small antioxidant molecules (reduced glutathione and ascorbic acid) in renal cortex of T₄-treated rats were restored back by supplementation of vitamin E or/and curcumin. Increased superoxide dismutase and catalase activities in kidney cortex of T₄-treated rats were ameliorated in response to vitamin E or/and curcumin treatment. The elevated translated product of Cu/Zn-SOD, Mn-SOD and catalase in T₄-treated rats were differentially reduced by the administration of vitamin E and curcumin independently or in combination. Cu/Zn-SOD expression was ameliorated by both vitamin E and curcumin independently or in combination, whereas Mn-SOD expression was ameliorated by the supplementation of vitamin E or curcumin independently. However, the expression of catalase was alleviated by only supplementation of vitamin E to T₄-treated rats. The results suggest that both vitamin E and curcumin may play an important role in protecting T₄-induced oxidative stress in rat renal cortex by differentially modulating the activities of antioxidant enzymes and oxidative stress parameters.     

Effect of l-triiodothyronine on the mitochondrial α-glycerophosphate dehydrogenase activity,mitochondrial and total protein contents of brain of Singi fish (Heteropneustes fossilis bloch)

A single injection of various doses (0.25, 0.5, 5, 20 and 50 μg/g) of l-triiodothyronine increased the mitochondrial cytochrome-linked α-glycerophosphate dehydrogenase (EC 1.1.99.5) activity and mitochondrial protein content of brain of Singi fish on the 3rd day. l-Triiodothyronine at the dose of 0.1 μg/g did not alter the α-glycerophosphate dehydrogenase activity and mitochondrial protein content of brain. The total protein content of the brain also increased on the 3rd day with 0.5 μg of l-triiodothyronine per g. Increased enzyme activity followed a dose-response relationship of a non-linear fashion. The enhancement of the α-glycerophosphate dehydrogenase activity of fish brain with a dose of 0.5 μg/g was found from the 1st day and it reached to a maximum level from the 3rd to the 5th day. The enzyme activity then sharply declined on the 6th or 7th day. Cycloheximide inhibited the l-triiodothyronine-induced increase in the α-glycerophosphate dehydrogenase activity, mitochondrial and total protein content of fish brain.The present study thus reveals the responsiveness of fish brain to thyroid hormone, and α-glycerophosphate dehydrogenase activity can be taken as a biochemical indices for the expression of thyroid hormone action in fish brain.     

Prevention of ammonia toxicity byl-carnitine: metabolic changes in brain

l-Carnitine when injected in mice 30 min before an LD₁₀₀ of ammonium acetate (12 mmol/kg body weight, intraperitoneal) reduced mortality (100% survival with 16 mmoll-carnitine/kg) and prevented the appearance of symptoms of ammonia toxicity. Brain ammonia decreased in the animals givenl-carnitine. Ammonia decreased the levels of glutamate in brain; they were partially restored byl-carnitine, which also reduced the increase in brain glutamine in animals given only ammonia. The redox state of the brain was altered following ammonia intoxication. The ratio of lactate to pyruvate in the cytosol increased while that of glutamate to -ketoglutarate in the mitochondria decreased. These ratios were partially restored byl-carnitine. The implications of these findings are discussed relative to the mechanism of ammonia toxicity.     

Melatonin Modulates Thyroid Hormones and Splenocytes Proliferation Through Mediation of Its MT1 and MT2 Receptors in Hyperthyroid Mice

Hyperthyroidism is characterized by an increased metabolic rate with the alteration of immune activity. The pineal hormone melatonin regulates various physiological activities through sensitization of MT1 and MT2 membrane receptors in mammals. In the present study we have evaluated the involvement of MT1 and MT2 receptors in melatonin mediated modulation of thyroid hormones and splenocyte proliferation in experimentally induced hyperthyroidic mice. The l-thyroxine treatment induced the hyperthyroidism in mice evidenced with hypersecretion of T3 and T4 hormones from thyroid gland. Hyperthyroidic state increased the TSH hormone level which might be inducing hyper activity in thyroid gland. Exogenous melatonin suppressed the thyroid hormones level as well as TSH level in circulation. The l-thyroxine treatment increased the splenocyte proliferation and showed synergic effects along with melatonin. l-thyroxine treated mice alone or along with melatonin treatment showed differential expression pattern of MT1 and MT2 receptors protein in thyroid and spleen tissues. It seems that melatonin regulates thyroid hormones and splenocyte proliferation through activation of MT1 and MT2 receptors.     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

Purification and characterization of two allelic forms of l-glycerol 3-phosphate dehydrogenase from inbred strains of mice

l-Glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) was purified from the muscle of BALB/cJ and C57BL/6J mice. The half-lives of the enzyme at 50 C were 6 and 33 min, respectively, for the BALB/cJ and C57BL/6J strains. Enzyme preparations from the two strains of mice were compared with respect to the following properties and found to be essentially indistinguishable: K _m values for dihydroxyacetone phosphate, NADH, l--glycerophosphate, and NAD⁺; maximum velocity; competitive inhibition by inorganic phosphate; pH optimum; energy of activation; electrophoretic mobility; molecular weight and subunit molecular weight. From these data, it is concluded that the kinetic properties of the purified enzyme are not the factors responsible for the differences in activity found in crude homogenates of mouse tissues.This work was supported by NIH Research Grant HD 06712 from the National Institute of Child Health and Human Development and by an allocation from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Enzymatic synthesis of l-β-chloroalanine using amino acid dehydrogenase

l--Chloroalanine is a useful intermediate for the synthesis of several l-amino acids. Conditions for synthesizing optically pure l--chloroalanine from 3-chloropyruvate using alanine dehydrogenase (AlaDH), leucine dehydrogenase and phenylalanine dehydrogenase with a regeneration of NADH by formate dehydrogenase (FDH) were investigated. The enzymatic reaction was carried out at neutral pH because of a chemical instability of 3-chloropyruvate on the alkaline side. Commercially available AlaDH from Bacillus stearothermophilus IFO 12550 showed the highest activity for the production of l--chloroalanine at pH 7.5. The K _m and V _max values for 3-chloropyruvate of AlaDH were calculated to be 300 units/mg and 62.5 mm, respectively. Although 3-chloropyruvate had no inhibitory effect on AlaDH, it acted as a non-competitive inhibitor with FDH. 3-Chloropyruvate was added into the reaction mixture in a stepwise manner to avoid the inhibition. l--Chloroalanine was produced with high chemical (>90%) and optical yields (100% enantiometric excess) and at a high concentration (43 g/l).     

Feedback inhibition of homoserine dehydrogenase and threonine deaminase in the genusBifidobacterium

Feedback inhibition of crude and purified extracts of homoserine dehydrogenase and threonine deaminase activities in the genusBifidobacterium was studied. Homoserine dehydrogenase was partially or completely inhibited byl-threonine, and a marked inhibitory effect byl-isoleucine on threonine deaminase was observed. In the speciesBifidobacterium cuniculi high levels ofl-valine reversed the inhibitory effect ofl-isoleucine. The -aminobutyric acid-resistant mutant Ru 326/106 of the speciesB. ruminale, overproducer ofl-isoleucine, had a derepressed homoserine dehydrogenase and a lesser feedback inhibition byl-threonine. Homoserine dehydrogenase appeared to be in bifids specifically NAD dependent. The regulatory mechanisms of aspartate family amino acid biosynthesis in bifidobacteria was discussed.     

Purification,characterization and regulation of a monomeric l-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min^-1 mg^-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPT hexulose-6-phosphate isomerase - FPLC fast protein liquid chromatography     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

The convulsant drugs 3-mercaptopropionate and methionine sulfoximine inhibitl-glutamate andl-aspartate binding to a hydrophobic protein fraction from rat cerebral cortex

The action of the convulsant drugs, methionine sulfoximine (MSO), 3-mercaptopropionate (3-MP), megimide (MG), and allylglycine on the binding ofl-[¹⁴C]aspartate,l-[¹⁴C]glutamate and [¹⁴C]GABA to a hydrophobic protein fraction isolated from rat cerebral cortex was studied. Using the convulsant at 10^–4 M concentration and the radioactive ligands at 10⁶ M the binding ofl-[¹⁴C]glutamate was inhibited 60% by 3-MP and 40% by MSO, while MG and allylglycine had no effect. The binding ofl-[¹⁴C]aspartate was inhibited 55%, and 10–20% by 3-MP and MSO, respectively, while MG and allylglycine had not effect. None of the drugs mentioned, except for a minimal inhibition by MG, altered the binding of [¹⁴C]GABA. Neither MSO nor 3-MP affected the high-affinity sites forl-[¹⁴C]glutamate orl-[¹⁴C]aspartate, but they had a strong inhibitory action on the medium affinity site. These results are discussed in relation to the possible mechanism of action of these drugs onl-glutamate andl-aspartate receptors.     

Comparative analysis of thyroxine distribution in ascidian larvae

The ascidian endostyle is a mucus-secreting pharyngeal organ, it has iodine-concentrating activity and the biosynthesis of thyroid hormones has been well documented. According to our recent findings, ascidians possess thyroid hormones, which are localized in mesenchymal cells. We have studied the presence and localization of l-thyroxine (T₄) in Ascidia malaca (Traustedt), Ascidiella aspersa (Müller), Phallusia mamillata (Cuvier) and Ciona intestinalis (Linnaeus) larvae and its involvement in metamorphosis. In vivo treatment of swimming larvae with exogenous T₄ and thiourea (a thyroid hormone synthesis inhibitor), demonstrate the presence of T4 during larval development. These results were confirmed by in vitro experiments utilizing dot blotting, radioimmunoassay and immunoperoxidase staining. The hormone was localized in mesenchymal cells of all four ascidians, spread out in the body cavity, under the adhesive papillae and around the intestine. The presence of TH in mesenchymal cells could be related to blood cells, musculature and heart tissue differentiation. The results suggest that this hormone could be involved in the control of metamorphosis.     

Regulation of biosynthesis of aspartate family amino acids in the photosynthetic bacterium Rhodopseudomonas palustris

The four amino acids of the aspartate family (l-lysine, l-methionine, l-threonine, and l-isoleucine) are produced in bacteria by a branched biosynthetic pathway. Regulation of synthesis of early common intermediates and of carbon flow through distal branches of the pathway requires operation of a number of subtle feedback controls, which are integrated so as to ensure balanced synthesis of the several end products. Earlier studies with nonsulfur purple photosynthetic bacteria were instrumental in revealing the existence of alternative regulatory schemes, and in this communication we report on the control pattern of a representative of this physiological group not previously investigated, Rhodopseudomonas palustris. The results obtained from study of the properties of four key regulatory enzymes of the aspartate family pathway (-aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine deaminase) and of the effects of exogenous amino acids (i. e., the end products) on growth of the bacterium indicate that the control schema in Rps. palustris differs substantially from the schemes described for other Rhodopseudomonas species, but resembles the regulatory pattern observed in Rhodospirillum rubrum.Abbreviations A absorbancy - AK -aspartokinase - ASA aspartate -semialdehyde - DTT dithiothreitol - HS l-homoserine - HSDH homoserine dehydrogenase - HSK homoserine kinase - I l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-isoleucine - KU Klett-Summerson photometer units - L l-lysine - M l-methionine - ME -mercaptoethanol - PABA p-aminobenzoic acid - T l-threonine - TD threonine deaminase - RCV synthetic growth medium (see text) - YP agar medium containing 0.3% yeast extract, 0.3% peptone, and 1.5% agar - Y2T synthetic growth medium (see text)     

A possible localization of α-glycerophosphate dehydrogenase to the inner boundary membrane of mitochondria in livers from rats fed with ethanol

Summary The morphology and respiration of liver mitochondria were studied in rats fed with ethanol for eight months. Morphometric analysis shows an increase of the volume fraction of mitochondria, a decrease of the density of crista membranes but the density of surface area remained unaltered in the ethanol treated rats as compared to the controls. The ethanol treatment caused a reduced capacity of the mitochondria to oxidize succinate. The capacity to oxidize -glycerophosphate remained unchanged.As it is known that succinate dehydrogenase as well as -glycerophosphate dehydrogenase are bound to the inner mitochondrial membrane, different localization is proposed of the two enzymes in this membrane. Thus, there is a good correlation between the reduction of inner mitochondrial membrane and succinate oxidation. As the activity of -glycerophosphate dehydrogenase remained unchanged, this enzyme should be localized to that part of the inner mitochondrial membrane, which is called inner boundary membrane, and which is not altered by the ethanol treatment.The work is a part of investigations made possible by financial support from the Swedish Medical Research Council, Project No. B71-12Y-2364-04.     

Simultaneous and selective production of levan and poly(γ-glutamic acid) by <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis(natto) Takahashi, used to prepare the fermented soybean product natto, was grown in a basal medium containing 5% (w/w) sucrose and 1.5% (w/w) l-glutamate and produced 58% (w/w) poly(-glutamic acid) and 42% (w/w) levan simultaneously. After 21 h, 40–50 mg levan ml^-1had been produced in medium containing 20% (w/w) sucrose but without l-glutamate. In medium containing l-glutamic acid but without sucrose, mainly poly(-glutamic acid) was produced. Revisions requested 28 August 2004/14 October 2004; Revisions received 11 October 2004/22 November 2004     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Protective effect of Premna tomentosa extract (L. verbanacae) on acetaminophen-induced mitochondrial dysfunction in rats

Allurement of herbs as health beneficial foods (physiologically functional foods) and as a source material for the development of new drugs, has led to greater furtherance in the study of herbal medicines during recent years. Plant extracts are being utilized to treat a wide variety of diseases like hepatotoxicity. Premna tomentosa is one such medicinal plant used widely in Indian ayurvedic medicine for the treatment of liver disorders. This study appraised the effectiveness of P. tomentosa leaf extract in protecting the liver against mitochondrial damage induced by acetaminophen, since mitochondrial injury has been investigated as a potential initiator of hepatotoxicity. Normal Wistar strain rats were pre-treated with P. tomentosa extract (750 mg/kg, orally) for 15 days and then intoxicated with acetaminophen (640 mg/kg, orally). Mitochondria were isolated from liver of experimental animals and assessed for the levels of lipid peroxide products, GSH and mitochondrial enzymes (isocitrate dehydrogenase, -keto glutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase and cytochrome-C-oxidase). The levels of Lipid peroxidation products were increased and the levels of the other assessed parameters were significantly decreased in hepatotoxicity induced animals. Whereas, the levels were brought back to normal in P. tomentosa pre-treated rats, which shows the protective effect of the extract against mitochondrial damage. Presence of anti-oxidant compound d-limonene (58%) in P. tomentosa leaves, which is known to enhance conjugation of toxic metabolites by maintaining liver GSH concentrations may explain the hepatoprotective property of the extract.     

Normal thyroid thermogenesis but reduced viability and adiposity in mice lacking the mitochondrial glycerol phosphate dehydrogenase

The mitochondrial glycerol phosphate dehydrogenase (mGPD) is important for metabolism of glycerol phosphate for gluconeogenesis or energy production and has been implicated in thermogenesis induced by cold and thyroid hormone treatment. mGPD in combination with the cytosolic glycerol phosphate dehydrogenase (cGPD) is proposed to form the glycerol phosphate shuttle, catalyzing the interconversion of dihydroxyacetone phosphate and glycerol phosphate with net oxidation of cytosolic NADH. We made a targeted deletion in Gdm1 and produced mice lacking mGPD. On a C57BL/6J background these mice showed a 50% reduction in viability compared with wild-type littermates. Uncoupling protein-1 mRNA levels in brown adipose tissue did not differ between mGPD knockout and control pups, suggesting normal thermogenesis. Pups lacking mGPD had decreased liver ATP and slightly increased liver glycerol phosphate. In contrast, liver and muscle metabolites were normal in adult animals. Adult mGPD knockout animals had a normal cold tolerance, normal circadian rhythm in body temperature, and demonstrated a normal temperature increase in response to thyroid hormone. However, they were found to have a lower body mass index, a 40% reduction in the weight of white adipose tissue, and a slightly lower fasting blood glucose than controls. The phenotype may be secondary to consequences of the obligatory production of cytosolic NADH from glycerol metabolism in the mGPD knockout animal. We conclude that, although mGPD is not essential for thyroid thermogenesis, variations in its function affect viability and adiposity in mice.