A Global Loss of Dio2 Leads to Unexpected Changes in Function and Fiber Types of Slow Skeletal Muscle in Male Mice.

The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.

Mice lacking the thyroid hormone receptor-alpha gene spend more energy in thermogenesis, burn more fat, and are less sensitive to high-fat diet-induced obesity.

Unable to activate brown adipose tissue (BAT) thermogenesis, alphaT3-receptor-deficient mice (Thra-0/0) are cold intolerant. Our objective was to investigate the impact on energy economy and mechanisms of the alternate facultative thermogenesis developed. Energy expenditure (oxygen and food consumption) is elevated in Thra-0/0 mice reared at room temperature. Such difference disappears at thermoneutrality (30 C) and expands as ambient temperature becomes colder (P < 0.001). Despite eating more, Thra-0/0 are leaner than wild-type (WT) mice (P < 0.01), whereas these, whether on chow or high-fat diet, gained more weight (g/d: 0.12 +/- 0.002 vs. 0.08 +/- 0.002 and 0.25 +/- 0.005 vs. 0.17 +/- 0.005, respectively) and adiposity than Thra-0/0 mice (P < 0.001). The respiratory quotient was lower in Thra-0/0 than WT mice (P < 0.001), after feeding or fasted, on chow or high-fat diet, indicating a preference for fat as fuel, which was associated with increased lipoprotein lipase (LPL) expression in skeletal muscle of Thra-0/0 mice but with no differences in gene expression in white adipose tissue. Type-2 deiodinase (D2) was increased in BAT and aerobic muscle of Thra-0/0 mice. This and liver D1 were increased by a high-fat diet in both genotypes, as also were serum T3 and T3/T4 ratio, but more in Thra-0/0 than WT mice (P < 0.001). Remarkably, when studied at thermoneutrality, genotype differences in weight and adiposity gain, respiratory quotient, D2, and LPL disappeared. Thus, disruption of BAT thermogenesis in Thra-0/0 mice activates an alternate facultative thermogenesis that is more energy demanding and associated with reduced fuel efficiency, leanness, increased capacity to oxidize fat, and relative resistance to diet-induced obesity, in all of which muscle LPL and deiodinases play a key role.

Thyroid-adrenergic interactions: physiological and clinical implications.

The sympathoadrenal system, including the sympathetic nervous system and the adrenal medulla, interacts with thyroid hormone (TH) at various levels. Both systems are evolutionary old and regulate independent functions, playing probably independent roles in poikilothermic species. With the advent of homeothermy, TH acquired a new role, which is to stimulate thermogenic mechanisms and synergize with the sympathoadrenal system to produce heat and maintain body temperature. An important part of this new function is mediated through coordinated and, most of the time, synergistic interactions with the sympathoadrenal system. Catecholamines can in turn activate TH in a tissue-specific manner, most notably in brown adipose tissue. Such interactions are of great adaptive value in cold adaptation and in states needing high-energy output. Conversely, in states of emergency where energy demand should be reduced, such as disease and starvation, both systems are turned down. In pathological states, where one of the systems is fixed at a high or a low level, coordination is lost with disruption of the physiology and development of symptoms. Exaggerated responses to catecholamines dominate the manifestations of thyrotoxicosis, while hypothyroidism is characterized by a narrowing of adaptive responses (e.g., thermogenic, cardiovascular, and lipolytic). Finally, emerging results suggest the possibility that disrupted interactions between the two systems contribute to explain metabolic variability, for example, fuel efficiency, energy expenditure, and lipolytic responses.

Temperature homeostasis in transgenic mice lacking thyroid hormone receptor-alpha gene products.

We studied temperature homeostasis in male mice lacking all thyroid hormone receptor-alpha gene products (TRalpha-0/0). As other TRalpha-deficient mice, TRalpha-0/0 mice have lower core body temperature (T(C)) than cognate wild-type controls. We found that obligatory thermogenesis is normal in TRalpha-0/0 and that the lower T(C) at room temperature (RT, 20-22 C) is caused by a down setting of the hypothalamic thermostat. However, TRalpha-0/0 mice are cold intolerant due to impaired facultative thermogenesis. Norepinephrine-induced brown adipose tissue (BAT) thermogenesis is blunted, even though BAT-relevant genes and T(4) deiodinase respond normally to cold stimulation, as do serum T(3), serum glycerol (marker of lipolysis), and heart rate. BAT normally contributes to maintain T(C) at RT, 9 C below thermoneutrality, yet TRalpha-0/0 mice do not show signs of being cold stressed at 20-22 C. Instead, oxygen consumption is greater in TRalpha-0/0 than in wild-type mice at RT, suggesting the recruitment of an alternate, cold-activated form of thermogenesis to compensate for the lack of BAT thermogenesis. These results indicate that TRalpha is necessary for T(3) to modulate the central control of T(C) and for an essential step in norepinephrine activation of BAT thermogenesis but not to sustain obligatory thermogenesis. In addition, the results provide evidence for an alternate form of facultative thermogenesis, which probably originates in skeletal muscle and that is less effective and more energy demanding than BAT thermogenesis.

Evidence for a compensated thermogenic defect in transgenic mice lacking the mitochondrial glycerol-3-phosphate dehydrogenase gene.

A role for mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) in thermogenesis was investigated in transgenic mice lacking the mGPD gene (mGPD-/-). Reared and studied at 22 C, these mice have a small, but significant, reduction (7-10%) in energy expenditure, as evidenced by oxygen consumption (QO2) and food intake, and show signs of increased brown adipose tissue (BAT) stimulation, higher plasma T4 and T3 concentrations, as well as increased uncoupling protein 3 (UCP3) expression in muscle. When acclimated at thermoneutrality temperature (32 C), QO2 decreased in both genotypes, but the difference between them widened to 16%, whereas BAT underwent atrophy, and plasma T4 and T3 levels and UCP3 mRNA decreased, yet T3 and UCP3 persisted at significantly higher levels in mGPD-/- mice. Such differences disappeared when the mice were rendered hypothyroid. A compensatory role for the observed changes in BAT, thyroid hormone levels, and UCP3 was investigated with a 2-h cold challenge of 12 C in euthyroid and hypothyroid mice. No hypothermia ensued if the mice had been acclimated at 22 C, but when acclimated at 32 C, euthyroid mGPD-/- mice became significantly more hypothermic than the wild-type controls. When rendered hypothyroid, this difference was accentuated, and the mGPD-/- mice developed profound hypothermia ( approximately 28 vs. 34 C in wild-type mice; P < 0.001). Thus, mGPD-deficient mice have, despite increased plasma T4 and T3, a small, but distinct, reduction in obligatory thermogenesis, which is compensated by increased BAT facultative thermogenesis and by thyroid hormone-dependent mechanisms using other proteins, possibly UCP3. The results support a role for mGPD in thyroid hormone thermogenesis.

Opposing effects of glucocorticoids on beta(3)-adrenergic receptor expression in HIB-1B brown adipocytes.

Glucocorticoids (GC) have been reported to promptly repress beta(3)-adrenergic receptor (beta(3)-AR) gene transcription in a white adipose tissue cell line. However, the effect of these hormones on beta(3)-AR expression in brown adipose tissue in vivo suggests a more complex mechanism of action. To avoid potential in vivo confounding variables, we investigated the effect of GC on the beta(3)-AR of HIB-1B brown adipocytes. While beta(3)-AR mRNA had same rapid turnover as in white fat cells, 1.5-2 h, the time course of its descent following dexamethasone was complex. A rapid initial descent beta(3)-AR mRNA with t(1/2) approximately 1.6 h was consistent with a prompt, complete inhibition of transcription. Such rapid initial phase was followed approximately 2 h later by a plateau or even an increase of beta(3)-AR mRNA, to descend thereafter following a slower single exponential (t(1/2) approximately 10 h). The change in the time course was abrogated by cycloheximide, and was not due to dexamethasone degradation or stabilization of beta(3)-AR mRNA at later times after dexamethasone. In vivo, a sufficiently large dose of dexamethasone was associated with a transient approximately 70% reduction of brown adipose tissue beta(3)-AR mRNA by 4 h and full recovery by 24 h. These findings suggest that GC have two opposing effects on beta(3)-AR gene expression: they rapidly and directly inhibit transcription but also induce a rapidly turned-over protein (C/EBPbeta?) that stimulates gene transcription. The relative magnitude of these two effects may vary explaining apparently discrepant observations.

Thyroid hormone receptor-α gene knockout mice are protected from diet-induced hepatic insulin resistance.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in the United States and is strongly associated with hepatic insulin resistance. We examined whether the thyroid hormone receptor-α (Thra) would be a potential therapeutic target to prevent diet-induced NAFLD and insulin resistance. For that purpose, we assessed insulin action in high-fat diet-fed Thra gene knockout (Thra-0/0) and wild-type mice using hyperinsulinemic-euglycemic clamps combined with (3)H/(14)C-labeled glucose to assess basal and insulin-stimulated rates of glucose and fat metabolism. Body composition was assessed by (1)H magnetic resonance spectroscopy and energy expenditure by indirect calorimetry. Relative rates of hepatic glucose and fat oxidation were assessed in vivo using a novel proton-observed carbon-edited nuclear magnetic resonance technique. Thra-0/0 were lighter, leaner, and manifested greater whole-body insulin sensitivity than wild-type mice during the clamp, which could be attributed to increased insulin sensitivity both in liver and peripheral tissues. Increased hepatic insulin sensitivity could be attributed to decreased hepatic diacylglycerol content, resulting in decreased activation of protein kinase Cε and increased insulin signaling. In conclusion, loss of Thra protects mice from high-fat diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. Therefore, thyroid receptor-α inhibition represents a novel pharmacologic target for the treatment of NAFLD, obesity, and type 2 diabetes.

Physiological importance and control of non-shivering facultative thermogenesis.

This review examines general and evolutionary aspects of temperature homeostasis, focusing on mammalian facultative or adaptive thermogenesis and its control by the sympathetic nervous system and hormones. Thyroid hormone acquired a new role with the advent of homeothermy enhancing facultative thermogenesis by interacting synergistically with the sympathetic nervous system, and directly increasing basal metabolic rate (obligatory thermogenesis). Facultative thermogenesis is triggered by cold. The major site of facultative thermogenesis in mammals is brown adipose tissue, endowed with abundant mitochondria rich in a protein called uncoupling protein-1. This protein can uncouple phosphorylation in a controlled manner, releasing the energy of the proton-motive force as heat. Its synthesis and function are regulated synergistically by the sympathetic nervous system and thyroid hormone and modulated by other hormones directly, or indirectly, modulating sympathetic activity as well as thyroid hormone secretion and activation in brown adipose tissue. Alternate, evolutionary older forms of facultative thermogenesis activated in transgenic mice with disabled brown adipose tissue thermogenesis reveal this latter as the culmination of energy-efficient facultative thermogenesis.

Postpartum granulomatous hypophysitis with sphenoid sinus involvement: a case study.

OBJECTIVE: To report an unusual case of granulomatous hypophysitis with sphenoid sinus involvement in a woman presenting with headaches and visual field deficits approximately 2 weeks after a normal delivery. METHODS: We present the history, physical findings, hormonal assay results, pituitary imaging, surgical findings, and pathology findings at presentation and then follow-up data at several times encompassing 1 year of observation. We also performed a literature review on granulomatous hypophysitis. RESULTS: A 29-year-old woman presented with headache and visual disturbances 11 days after childbirth. Magnetic resonance imaging revealed a sellar mass with suprasellar extension, compression of the optic chiasm, possible invasion of the cavernous sinuses, and sinus mucosal thickening. A subtotal resection was performed through the transsphenoidal route. Histologic examination demonstrated extensive nonvasculitic granulomatous tissue in pituitary and sphenoid mucosal samples. Investigation for known causes of granulomatous hypophysitis was negative. She required desmopressin and levothyroxine replacement postoperatively. Sequential follow-up revealed spontaneous resolution of the residual mass in 5 months. CONCLUSION: Unique features of this case include the concomitant presence of granulomatous lesions in the pituitary gland and the sphenoid sinus, its manifestation in the early postpartum period, and the spontaneous resolution of the residual granulomatous lesions in both the sphenoid sinus and the sella turcica.

Type 2 iodothyronine deiodinase levels are higher in slow-twitch than fast-twitch mouse skeletal muscle and are increased in hypothyroidism.

Because of its large mass, relatively high metabolic activity and responsiveness to thyroid hormone, skeletal muscle contributes significantly to energy expenditure. Despite the presence of mRNA encoding the type 2 iodothyronine-deiodinase (D2), an enzyme that activates T(4) to T3, very low or undetectable activity has been reported in muscle homogenates of adult humans and mice. With a modified D2 assay, using microsomal protein, overnight incubation and protein from D2 knockout mouse muscle as a tissue-specific blank, we examined slow- and fast-twitch mouse skeletal muscles for D2 activity and its response to physiological stimuli. D2 activity was detectable in all hind limb muscles of 8- to 12-wk old C57/BL6 mice. Interestingly, it was higher in the slow-twitch soleus than in fast-twitch muscles (0.40 ± 0.06 vs. 0.076 ± 0.01 fmol/min · mg microsomal protein, respectively, P < 0.001). These levels are greater than those previously reported. Hypothyroidism caused a 40% (P < 0.01) and 300% (P < 0.001) increase in D2 activity after 4 and 8 wk treatment with antithyroid drugs, respectively, with no changes in D2 mRNA. Neither D2 mRNA nor activity increased after an overnight 4 C exposure despite a 10-fold increase in D2 activity in brown adipose tissue in the same mice. The magnitude of the activity, the fiber specificity, and the robust posttranslational response to hypothyroidism argue for a more important role for D2-generated T(3) in skeletal muscle physiology than previously assumed.

Effects of exercise on energy-regulating hormones and appetite in men and women.

When previously sedentary men and women follow exercise training programs with ad libitum feeding, men lose body fat, but women do not. The purpose of this study was to evaluate whether this observation could be related to sex differences in the way energy-regulating hormones and appetite perception respond to exercise. Eighteen (9 men, 9 women) overweight/obese individuals completed four bouts of exercise with energy added to the baseline diet to maintain energy balance (BAL), and four bouts without energy added to induce energy deficit (DEF). Concentrations of acylated ghrelin, insulin, and leptin, as well as appetite ratings were measured in response to a meal after a no-exercise baseline and both exercise conditions. In men, acylated ghrelin area under the curve (AUC) was not different between conditions. In women, acylated ghrelin AUC was higher after DEF (+32%) and BAL (+25%), and the change from baseline was higher than men (P < 0.05). In men, insulin AUC was reduced (-17%) after DEF (P < 0.05), but not BAL. In women, insulin AUC was lower (P < 0.05) after DEF (-28%) and BAL (-15%). Leptin concentrations were not different across conditions in either sex. In men, but not in women, appetite was inhibited after BAL relative to DEF. The results indicate that, in women, exercise altered energy-regulating hormones in a direction expected to stimulate energy intake, regardless of energy status. In men, the response to exercise was abolished when energy balance was maintained. The data are consistent with the paradigm that mechanisms to maintain body fat are more effective in women.

Thermogenic mechanisms and their hormonal regulation.

Increased heat generation from biological processes is inherent to homeothermy. Homeothermic species produce more heat from sustaining a more active metabolism as well as from reducing fuel efficiency. This article reviews the mechanisms used by homeothermic species to generate more heat and their regulation largely by thyroid hormone (TH) and the sympathetic nervous system (SNS). Thermogenic mechanisms antecede homeothermy, but in homeothermic species they are activated and regulated. Some of these mechanisms increase ATP utilization (same amount of heat per ATP), whereas others increase the heat resulting from aerobic ATP synthesis (more heat per ATP). Among the former, ATP utilization in the maintenance of ionic gradient through membranes seems quantitatively more important, particularly in birds. Regulated reduction of the proton-motive force to produce heat, originally believed specific to brown adipose tissue, is indeed an ancient thermogenic mechanism. A regulated proton leak has been described in the mitochondria of several tissues, but its precise mechanism remains undefined. This leak is more active in homeothermic species and is regulated by TH, explaining a significant fraction of its thermogenic effect. Homeothermic species generate additional heat, in a facultative manner, when obligatory thermogenesis and heat-saving mechanisms become limiting. Facultative thermogenesis is activated by the SNS but is modulated by TH. The type II iodothyronine deiodinase plays a critical role in modulating the amount of the active TH, T(3), in BAT, thereby modulating the responses to SNS. Other hormones affect thermogenesis in an indirect or permissive manner, providing fuel and modulating thermogenesis depending on food availability, but they do not seem to have a primary role in temperature homeostasis. Thermogenesis has a very high energy cost. Cold adaptation and food availability may have been conflicting selection pressures accounting for the variability of thermogenesis in humans.

Regulation of uncoupling protein-2 mRNA in L6 myotubules: I: Thiazolidinediones stimulate uncoupling protein-2 gene expression by a mechanism requiring ongoing protein synthesis and an active mitogen-activated protein kinase.

The mitochondrial uncoupling protein-2 (UCP2) can uncouple phosphorylation to subserve several functions. It has been reported that the insulin sensitizers, thiazolidinediones (TZDs), increase UCP2 mRNA levels and, more recently, that TZDs stimulate UCP2 reporter genes but that the sequences involved do not bind peroxisome proliferator-activated receptor gamma (PPARgamma). We report here that TZDs stimulated UCP2 gene (ucp2) transcription in L6 myotubules involving an indirect mechanism. L6 cells contained comparatively small amounts of PPARgamma mRNA but clearly detectable amounts of PPARgamma2 protein. UCP2 mRNA levels were increased in a time- and concentration-dependent manner by TZDs. UCP2 mRNA had slow turnover (t 1/2 approximately 38 h), and this was not affected by TZDs. Bisphenol A diglycidyl ether, a PPARy antagonist, concentration dependently inhibited the TZD-induced increase in UCP2 mRNA. Blockade of protein synthesis with cycloheximide as well as abrogation of mitogen-activated protein kinase (MAPK) activity with PD98059 or U0126 also prevented the TZD-induced increase in UCP2 mRNA. As with autologous UCP2 mRNA, TZDs stimulated reporter gene expression directed by ucp2 sequences in transiently transfected L6 cells. The effect was enhanced by cotransfection of PPARgamma + retinoid X receptor gamma and prevented by MEK blockade. TZDs, however, did not increase the activation of MAPK, nor did its activation by other means (change of medium, insulin-like growth factor-1, insulin) increase UCP2 mRNA, indicating that phosphorylation is not limiting. These results suggest that TZDs indirectly stimulate ucp2 transcription by inducing-via PPARgamma-limiting amounts of a protein, which must be phosphorylated by MAPK to stimulate the gene.

Regulation of uncoupling protein-2 mRNA in L6 myotubules: II: Thyroid hormone amplifies stimulation of uncoupling protein-2 gene by thiazolidinediones and other peroxisome proliferator-activated receptor ligands in L6 myotubules: evidence for a priming effect.

The stimulation of the uncoupling protein-2 gene (ucp2) by thyroid hormone (triiodothyronine [T3]) in vivo is variable, suggesting complex interactions and even the possibility of indirect effects. We investigated the effect of T3 on ucp2 expression in L6 myotubules. Alone, T3 did not significantly stimulate ucp2 expression in L6 cells, but it amplified the stimulation by thiazolidinediones (TZDs). L6 cells expressed both alpha1 and beta1 thyroid hormone receptors and the data were consistent with the effect being mediated by these receptors. T3 also enhanced the stimulation of ucp2 by the nonselective peroxisome proliferator-activated receptor (PPAR) ligands bezafibrate and carbacyclin, but not that by oleic acid or norepinephrine. L6 cells expressed PPARbeta and PPARgamma, but not PPARalpha. As short as a 1-h preexposure of L6 cells to T3 was sufficient to amplify the effect of PPAR ligands. Neither transcription nor translation was needed for this effect of T3. T3 did not affect the t1/2 of UCP2 mRNA. The histone deacetylases inhibitor trichostatin A (TSA) stimulated the expression of ucp2 but did not add to the effect of T3 nor did this hormone enhance the effect of TSA. These results suggest that T3 selectively enhances the transcriptional stimulation of ucp2 by TZDs and nonselective PPAR ligands by priming the gene to a transactivating signal(s) generated by such ligands.

Mice with deletion of the mitochondrial glycerol-3-phosphate dehydrogenase gene exhibit a thrifty phenotype: effect of gender.

To define the role of mitochondrial glycerol-3-phosphate dehydrogenase (mGPD; EC 1.1.99.5) in energy balance and intermediary metabolism, we studied transgenic mice not expressing mGPD (mGPD-/-). These mice had approximately 14% lower blood glucose; approximately 50% higher serum glycerol; approximately 80% higher serum triglycerides; and at thermoneutrality, their energy expenditure (Qo(2)) was 15% lower than in wild-type (WT) mice. Glycerol-3-phosphate levels and lactate-to-pyruvate ratios were threefold elevated in muscle, but not in liver, of mGPD-/- mice. WT and mGPD-/- mice were then challenged with a high-fat diet, fasting, or food restriction. The high-fat diet caused more weight gain and adiposity in mGPD-/- than in WT female mice, without the genotype differentially affecting Qo(2) or energy intake. After a 30-h fast, WT female lost 60% more weight than mGPD-/- mice but these latter became more hypothermic. When energy intake was restricted to 50-70% of the ad libitum intake for 10 days, mGPD-/- female mice lost less weight than WT controls, but they had lower Qo(2) and body temperature. WT and mGPD-/- male mice did not differ significantly in their responses to these challenges. These results show that the lack of mGPD causes significant alterations of intermediary metabolism, which are more pronounced in muscle than liver and lead to a thrifty phenotype that is more marked in females than males. Lower T(4)-to-T(3) conversion in mGPD-/- females and a greater reliance of normal females on mGPD to respond to high-fat diets make the lack of the enzyme more consequential in the female gender.