Responses of chickens subjected to thyroid hormone depletion-repletion

The purpose of this experiment was to determine the relationship between lipid metabolism and the expression of specific genes in chickens fed methimazole to produce hypothyroidism. Male, broiler chickens growing from 14 to 28 days of age were fed diets containing 18% crude protein and either 0 or 1 g methimazole per kg of diet. At 28 days, these two groups were further subdivided into groups receiving 18% crude protein diets containing either 0 or 1 mg triiodothyronine (T(3)) per kg. Birds were sampled at intervals from 0 to 120 h. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), isocitrate dehydrogenase (ICD-NADP), aspartate aminotransferase (AAT) activities and the expression of the genes for ME, fatty acid synthase (FAS), NADP-ICD, AAT and acetyl coenzyme carboxylase (ACC). Gene expression was estimated with real time RT-PCR assays. Expression rates were noted as C(t)'s. Dietary methimazole decreased IVL and ME at 28 days of age. T(3) and supplementation for 1 day restored both IVL and ME. Paradoxically, continuing T(3) replenishment for a longer period decreased IVL without affecting ME activity. Although methimazole decreased ME gene expression, there was only a transitory relationship between enzyme activity and gene expression when plasma T(3) was replenished with exogenous T(3). These data explain the apparent dichotomies in lipid metabolism elicited by changes in the thyroid state of animals. Most metabolic changes in response to feeding T(3) occurred within a short period of time, suggesting that changes in intermediary metabolism preceded morphological changes. Furthermore, the thyroid state of the animal will determine responses to exogenous T(3).     

Further studies on short-term adaptations in the expression of lipogenic genes in broilers

This experiment was conducted to determine possible relationships between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens (Gallus gallus) growing from 7 to 28days of age were fed diets containing 12 or 30% protein ad libitum. Both groups were then switched to the diets containing the opposite level of protein. Birds were sampled at 0, 6, 9, 12, 18 and 24h following the switch in protein levels. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), aspartate aminotransferase (AAT) and isocitrate dehydrogenase (NADP) (ICD) activities. In addition, ME, AAT, ICD, fatty acid synthase (FAS), and acetyl coenzyme carboxylase (ACC) gene expression rates were determined. IVL and ME activities were inversely related to dietary protein levels (12 to 30%) and to acute changes from 12 to 30%. In contrast, expression of ME, FAS and ACC genes was decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out; however, that metabolic regulation at the gene level only occurs when feeding very high or very low levels of dietary protein.     

Expression of lipogenic enzymes in chickens

Hubbard x Hubbard chickens (Gallus gallus) growing from 7 to 28 days of age were fed 12 or 30% protein diets and then switched to the diets containing the opposite level of protein. Birds were killed on days 28, 29, 30 and 31. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), isocitrate dehydrogenase (ICD) and aspartate aminotransferase (AAT) activities and the expression of the genes for ME, fatty acid synthase (FAS) and acetyl coenzyme carboxylase (ACC). Gene expression was determined with a combined RT-PCR using SYBR green as a fluorescent probe monitored in a real time mode. IVL and ME activity were inversely related to dietary protein levels (12 to 30%) and to acute changes in either level. In contrast, both ICD and AAT activities were increased by any increase in dietary protein. Lipogenic gene expression was inversely related to protein level, whether fed on an acute or chronic basis. It appears that real time RT-PCR is an acceptable method of estimating gene expression in birds. In addition, further work will focus on primer sizes that might further optimize RT-PCR as an instrument for studying the regulation of avian lipid metabolism. Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. However, it should be pointed out that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.     

Studies on doses of methimazole (MMI) and its administration regimen on broiler metabolism

We designed three experiments to determine both the optimal dose of and time on experiment for methimazole (MMI; 1-methyl-2-mercaptimidazole). Our goals were to determine if chicken growth was related to thyroid hormone levels and if intermediary metabolism changed along with changes in thyroid hormone levels. Initiating MMI at one week of age decreased (P<0.01) plasma thyroid levels and growth in four-week old birds. In contrast, initiating MMI at two and three weeks of age decreased (P<0.05) hormone levels without affecting growth as severely. Although initiating MMI at two weeks of age depressed (P<0.05) plasma thyroid hormones at four weeks, there was little change in vitro lipogenesis at four weeks. Again, initiating MMI at one week of age decreased body weight, plasma thyroid hormones and in vitro lipogenesis at four weeks of age. In addition, this treatment also decreased (P<0.05) malic enzyme activity at this same age period. The second experiment showed that MMI, initiated at 14 days, had no significant effect on 28-day body weight and again decreased both plasma T(3) and T(4) but T(3) replacement increased plasma T(3) in both 14-28-day treatment groups. All body weights were similar at 30 days, however. Lastly, diets containing graded levels of MMI decreased thyroid hormones and body weight (0>0.25>0.5>1 g MMI/kg). In contrast, only the two higher levels (0.5 and 1 g MMI/kg) decreased in vitro lipogenesis. Growth depression, caused by MMI feeding, can occur without changes in lipid metabolism. The length of MMI administration may be as important as dose level in obtaining effects (growth, thyroid hormone depression and inhibition of lipogenesis).     

Chicken hepatic metabolism in vitro. Protein and energy relations in the broiler chicken--VI. Effect of dietary protein and energy restrictions on in vitro carbohydrate and lipid metabolism and metabolic hormone profiles

1. Ross male broiler chicks growing from 14 to 28 days of age were fed 14 and 20% protein diets (4 kcal day-1/body wt0.66) or 20 and 28% protein diets (2.8 kcal day-1/body wt0.66) in a 2 x 2 factorial arrangement to determine the effects of protein and energy intakes on in vitro lipogenesis (IVL) and net glucose production (NGP). Plasma concentrations of insulin, glucagon, thyroid hormones (T3 and T4) and somatomedin-C (Sm-C) were estimated by radioimmunoassay. 2. There was a significant (P less than 0.05) decrease in IVL in the chicks given the higher daily protein intake. 3. The higher protein intake increased (P less than 0.05) NGP while the lower energy intake decreased (P less than 0.05) NGP. 4. Insulin, both thyroid hormones and Sm-C were affected by dietary energy and protein intakes.     

Short term changes in the expression of lipogenic genes in broilers (Gallus gallus)

The purpose of these experiments were to determine possible relationships between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12 or 30% protein ad libitum. Both groups were then switched on day 28 to the diets containing the opposite level of protein. Birds were killed on day 28 (basal values prior to the switch) and at 12, 18 and 24 h post switch. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12 to 30%) and to acute changes from 12 to 30%. Malic enzyme, fatty acid synthase and acetyl coenzyme A carboxylase genes were constant over a dietary protein range of 12 to 21% as in previous experiments, but decreased by feeding a 30% protein diet in the present experiments (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. Metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.     

Dietary protein regulates in vitro lipogenesis and lipogenic gene expression in broilers

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed graded levels of dietary crude protein. Male, broiler chickens growing from 7 to 28 days of age were fed diets containing 12, 21 or 30% protein ad libitum. In addition, another group of birds was fed on a regimen consisting of a daily change in the dietary protein level (12 or 30%). This latter group was further subdivided such that one-half of the birds received each level of protein on alternating days. Birds were sampled from 28 to 30 days of age. Measurements taken included in vitro lipogenesis, malic enzyme activity the expression of the genes for malic enzyme, fatty acid synthase and acetyl coenzyme carboxylase. In vitro lipogenesis and malic enzyme activity were inversely related to dietary protein levels (12-30%) and to acute changes from 12 to 30%. In contrast, expression of malic enzyme, fatty acid synthase and acetyl CoA carboxylase genes were constant over a dietary protein range of 12-21%, but decreased by feeding a 30% protein diet (acute or chronic feeding). Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out, however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.     

Thyroid status,but not insulin status,affects expression of avian uncoupling protein mRNA in chicken

The aim of this study was to investigate the hormonal regulation of the avian homolog of mammalian uncoupling protein (avUCP) by studying the impact of thyroid hormones and insulin on avUCP mRNA expression in chickens (Gallus gallus). For 3 wk, chicks received either a standard diet (control group), or a standard diet supplemented with triiodothyronine (T(3); T3 group) or with the thyroid gland inhibitor methimazole (MMI group). A fourth group received injections of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th wk of age, all animals received two daily injections of either human insulin or saline solution. The results indicate a twofold overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a clear downregulation (-74%) in MMI chickens compared with control chickens. Insulin injections had no significant effect on avUCP mRNA expression in chickens. This study describes for the first time induction of avUCP mRNA expression by the thermogenic hormone T(3) in chickens and supports a possible involvement of avUCP in avian thermogenesis.     

Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major increase in P450 reductase protein or activity was detectable over a 3-day period. Together, these studies establish that thyroid hormone regulates P450 reductase expression by pretranslational mechanisms. They also suggest that other regulatory mechanisms, which may involve changes in P450 reductase protein stability and/or changes in the translational efficiency of its mRNA, are likely to occur.     

Dietary propylthiouracil and 17 beta-estradiol benzoate implant effects on liver carbohydrate and lipid metabolism in chicks

1. Forty-eight chicks (21 days old) were implanted (+/- 50 mg 17-beta estradiol; EBI) and fed diets containing +/- 0.1% propylthiouracil (PTU) for 7 days to determine the role of gonadal hormones in the regulation of energy metabolism in the hypothyroid chick. 2. In vitro lipogenesis (IVL) and glucose production (NGP) were measured in liver explants. 3. Liver glycogen (GLY) metabolism was studied in particulate fractions (50,000 x g) of liver. 4. Glycogen synthetase activity (GLYSYN) was assayed in the presence of glucose-6-phosphate (G-6P; 0, 0.15 and 10 mM) to determine activation states. 5. PTU and EBI increased (P less than 0.05) both GLY concentration and GLYSYN activity in chicks but, did not increase G-6P activity ratios or the in vitro activation of GLYSYN. 6. Both PTU and EBI increased (P less than 0.05) IVL and NGP. 7. Estradiol magnifies the effects of PTU in chicks suggesting an interaction between thyroid status and gonadal function.     

The effects of selenium depletion and repletion on the metabolism of thyroid hormones in the rat

Rats were fed selenium-deficient (less than 0.005 mg selenium/kg) or selenium-supplemented diets (0.1 mg selenium/kg, as Na2SeO2) for up to five wks from weaning to assess the effects of developing selenium deficiency on the metabolism of thyroid hormones. Within two wks 3:5,3'-triiodothyronine (T3) production from thyroxine (T4) in liver homogenates from selenium-deficient rats was significantly lower compared with the activity in liver homogenates from selenium-supplemented rats. This decreased activity was probably responsible, in part, for the higher T4 and lower T3 concentrations in plasma from the selenium-deficient rats after 3, 4, and 5 weeks of experiment. Repletion of selenium-deficient rats with single intra-peritoneal injections of 200 micrograms selenium/kg body wt. (as Na2SeO3) 5 days before sampling reversed the effects of the deficiency on thyroid hormone metabolism and significantly increased liver and plasma glutathione peroxidase activities. However a dose of 10 micrograms selenium/kg body wt given to rats of similar low selenium status had no effect on thyroid hormone metabolism or glutathione peroxidase activity but did reverse the increase in hepatic glutathione S-transferase activity characteristic of severe selenium deficiency. Imbalances in thyroid hormone metabolism are an early consequence of selenium deficiency and are probably not related to changes in hepatic xenobiotic metabolizing enzymes associated with severe deficiency.     

AdipoR1 and AdipoR2 gene expression are regulated by thyroid hormones in adipose tissue

The aim of this study was to examine whether the relative gene expression of AdipoR1 and AdipoR2 in rat adipose tissue is altered by thyroid hormones, and whether this might relate to their circulating thyroid hormones and adiponectin levels. Hyper- and hypothyroidism were induced by daily oral administration of levothyroxine and methimazole in rats, respectively, over a 42 days period. Real-time PCR analysis was performed to evaluate the changes in AdipoR1 and AdipoR2 mRNA levels in the adipose tissue on days 15, 28, 42, and also 2 weeks after the cessation of treatment. In response to treatment with methimazole, mRNA levels of AdipoR1 and AdipoR2 decreased in the white adipose tissue compared to the euthyroid rats (p < 0.05). This decline was reversible 2 weeks after treatment cessation. The mRNA levels of AdipoR1 and AdipoR2 were increased in the hyperthyroid group of animals compared to euthyroid control (p < 0.05), and its changes were reversible 2 weeks after treatment cessation (P < 0.05). Adiponectin receptors gene expression levels in the adipose tissue of treated animals have positive correlations with thyroid hormones concentrations. Our results suggest that AdipoR1 and AdipoR2 gene expression is regulated by thyroid hormones in hypo- and hyperthyroidism.     

Brain biogenic amines and altered thyroid function.

Evidence has been presented that alterations in thyroidal status produce marked changes in the metabolism of several biogenic amines in developing brain. Neonatal hypothyroidism induced either by ¹³¹I or by an anti-thyroid agent, methimazole, markedly decreased the concentrations of norepinephrine, dopamine and 5-hydroxytryptamine and the activity of their rate-limiting enzymes, tyrosine hydroxylase and tryptophan hydroxylase. However, the levels of 5-hydroxyindoleacetic acid, the chief metabolite of 5-hydroxytryptamine were elevated in several regions of the brain. Whereas thyroid deficiency in early life produced no appreciable change in whole brain monoamine oxidase activity, it was increased in mid brain and decreased in the hypothalamus. Brain acetylcholine levels were significantly elevated and the activity of acetylcholinesterase remained unchanged in rats made hypothyroid at 1 day of age. Delaying thyroidectomy for 20 days after birth produced less appreciable changes in norepinephrine and 5-hydroxytryptamine metabolism. Thyroid deficiency suppressed the ontogenesis of behavioural arousal and spontaneous locomotor activity. The administration of L-triiodothyronine to hypothyroid animals in early life restored the metabolism of various neurohumors virtually to the normal limits. However, when the replacement therapy was postponed until adulthood, L-triiodothyronine failed to produce any restorative effects, suggesting that a critical period exists in early life during which thyroid hormone must be present to permit normal developmental pattern of central amines. Data also have been obtained demonstrating that neonatal hyperthyroidism induced by daily administration of L-triiodothyronine results in an increased turnover of norepinephrine and 5-hydroxytryptamine. These amine changes were accompanied by a marked rise in the spontaneous locomotor activity in hyperthyroid rats. Finally, chronic treatment with lithium, an antimanic drug, also known to suppress thyroid hormone production, significantly decreased not only the spontaneous locomotor activity, but also changes in the turnover of 5-hydroxytryptamine and norepinephrine in neonatally hyperthyroid rats.     

Thyroid hormone regulates the obesity gene tub

Thyroid hormone T3/T4 is a major regulator of energy metabolism in vertebrates, and defects in thyroid status are frequently associated with changes in body weight. It is demonstrated here that thyroid hormone regulates in vivo and in vitro the tub gene, which when mutated in tubby mice causes obesity, insulin resistance and sensory deficits. Hypothyroidism in rats altered tub mRNA and protein in discrete brain areas. These changes could be attributed to thyroid hormone deficiency since T3/T4 treatment restored normal tub expression. T3 also upregulated tub mRNA within 4–6 h in neuronal cells in culture, suggesting that T3 is a positive regulator of tub gene expression. Thus, these results establish a novel pathway of T3 action and provide an important molecular link between thyroid status and the tubby-associated syndrome.     

Thyroidal involvement in the expression of avian muscular dystrophy

We showed previously that propylthiouracil (PTU), a thyroid inhibitor, could alleviate several major signs of hereditary muscular dystrophy in chickens. The goals of the present investigation were to: (1) determine whether a nearly athyroid condition (achieved within two days after hatching by surgical thyroidectomy plus PTU) during an 11-day period beneficially affects the dystrophic condition when followed by triiodothyronine (T3) replacement to 33 days of age; (2) determine the beneficial effects on the expression of avian dystrophy when the thyroidectomized-PTU-treated chickens received a wide range of moderate to low T3 replacement doses beginning by two days after thyroidectomy; and (3) examine the thyroid hormone receptor system in dystrophic muscle for a possible abnormality. Thyroid deprivation increased muscle function (righting ability) and reduced plasma creatine kinase activity in dystrophic chickens. The major thyroid-related abnormality in dystrophic pectoralis muscles was an increased maximum binding capacity of solubilized nuclear T3 receptors.     

Effects of thyroid hormone and adrenalectomy on [Na+ + K+]ATPase in the ob/ob mouse

The absence of the thyroid stimulation of hepatic [Na+ + K+]ATPase in obese (ob/ob) mice has been investigated. A wide range of tri-iodothyronine (T3) concentrations failed to enhance the hepatic [Na+ + K+]ATPase of ob/ob mice made hypothyroid by methimazole treatment but glycerophosphate dehydrogenase activity was maximally stimulated at low doses of T3. Adrenalectomy increased the basal activity of [Na+ + K+]ATPase to levels found in lean control mice and restored the response of this enzyme to T3. Body weight gain was unaffected by the induction of hypothyroidism in either lean or ob/ob mice. It is concluded that the adrenal steroids play an important role in the expression of [Na+ + K+]ATPase activity in the ob/ob mouse.     

Tissue-specific effect of refeeding after short- and long-term caloric restriction on malic enzyme gene expression in rat tissues

Restricting food intake to a level below that consumed voluntarily (85%, 70% and 50% of the ad libitum energy intake for 3 or 30 days) and re-feeding ad libitum for 48 h results in an increase of malic enzyme (ME) gene expression in rat white adipose tissue. The increase of ME gene expression was much more pronounced in rats maintained on restricted diet for 30 days than for 3 days. The changes in ME gene expression resembled the changes in the content of SREBP-1 in white adipose tissue. A similar increase of serum insulin concentration was observed in all groups at different degrees of caloric restriction and refed ad libitum for 48 h. Caloric restriction and refeeding caused on increase of ME activity also in brown adipose tissue (BAT) and liver. However, in liver a significant increase of ME activity was found only in rats maintained on the restricted diet for 30 days. No significant changes after caloric restriction and refeeding were found in heart, skeletal muscle, kidney cortex, and brain. These data indicate that the increase of ME gene expression after caloric restriction/refeeding occurs only in lipogenic tissues. Thus, one can conclude that caloric restriction/refeeding increases the enzymatic capacity for fatty acid biosynthesis.     

Localization and role of leptin in the thyroid gland of the lizard Podarcis sicula (Reptilia, Lacertidae)

Leptin, the product of the ob gene, is a hormone secreted by adipocytes that regulates food intake and energy expenditure. The hypothalamus-pituitary-thyroid axis is markedly influenced by the metabolism status, being suppressed during food deprivation. The present study was designed to ascertain whether (1) lizard thyroid gland expresses the long form of leptin receptor (Ob-Rb) and (2) the leptin administration affects the thyroid gland activity in this species (and to verify whether leptin plays a similar role in reptiles as observed in the other vertebrates). The presence of leptin receptor in the thyroid gland of Podarcis sicula was demonstrated by immunohistochemical technique (avidin-biotin-peroxidase complex--ABC method). The role of leptin in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Leptin (0.1 mg/100 g body wt)/day increased T4 and T3 release for 3 days but decreased the plasma concentration of TSH; using LM clear signs of stimulation in the thyroid gland were observed. These findings suggest that systemic administration of leptin stimulates the morphophysiology of the thyroid gland in the lizard through a direct mechanism involving Ob-Rb.