Both 3,5-diiodo-L-thyronine (T2) and T3 modulate glucose-induced insulin secretion.

3,5-diiodo-L-thyronine (T2), a naturally existing iodothyronine, has biological effects on humans, but no information is available on its action on pancreatic b-cells. We evaluated its effect vs triiodothyronine (T3), on glucose-induced insulin secretion in INS-1e cells, a rat insulinoma line, and on human islets. INS-1e were incubated in the presence/absence of T2 or T3 (0.1 nmol/L-10 µmol/L), and glucose (3.3, 7.5, 11.0, and 20 mmol/L). Insulin release and content (at 11.0 and 20 mmol/L glucose) were significantly (p less than 0.01) stimulated by 1-100 nmol/L T2 and 0.1 nmol/L-1.0 µmol/L T3, and inhibited with higher concentrations of both (1–10 µmol/L T2 and 10 µmol/L T3). Human islets were incubated with 3.3 mmol/L glucose in presence/absence of T3 or T2 (0.1 nmol/L, 0.1 µmol/L, and 1 µmol/L). T2 (0.1 nmol/L-0.1 µmol/L) significantly (p less than0.01) stimulated insulin secretion, while higher concentrations (1 µmol/L) inhibited it. A modest increase in insulin secretion was evidenced with 1 µmol/L T3. In conclusion, T2 and T3 have a direct regulatory role in insulin secretion, depending on their concentrations and the glucose level itself. At concentrations near the physiological range, T2 enhances glucose-induced insulin secretion in both rat b-cells and human islets.

3,5-diiodo-L-thyronine increases resting metabolic rate and reduces body weight without undesirable side effects.

Recently, it was demonstrated that 3,5-diiodo-L-thyronine (T2) stimulates the resting metabolic rate (RMR), and reduces body-weight gain of rats receiving a high-fat diet. The aim of this study is to examine the effects of chronic T2 administration on basal metabolic rate and body weight in humans. Two euthyroid subjects volunteered to undergo T2 administration. Body weight, body mass index, blood pressure, heart rate, electrocardiogram, thyroid and liver ultrasonography, glycemia, total cholesterol, triglycerides, free T3 (FT3), free T4 (FT4), T2, thyroid stimulating hormone (TSH) and RMR were evaluated at baseline and at the end of treatment. RMR increased significantly in each subject. After continuing the T2 treatment for a further 3 weeks (at 300 mcg/day), body weight was reduced significantly (p<0.05) (about 4 percent), while the serum levels of FT3, FT4 and TSH, were unchanged. No side effects were observed at the cardiac level in either subject. No significant change was observed in the same subjects taking placebo.

Dietary zinc supplementation of 3xTg-AD mice increases BDNF levels and prevents cognitive deficits as well as mitochondrial dysfunction.

The overall effect of brain zinc (Zn(2+)) in the progression and development of Alzheimer's disease (AD) is still not completely understood. Although an excess of Zn(2+) can exacerbate the pathological features of AD, a deficit of Zn(2+) intake has also been shown to increase the volume of amyloid plaques in AD transgenic mice. In this study, we investigated the effect of dietary Zn(2+) supplementation (30 p.p.m.) in a transgenic mouse model of AD, the 3xTg-AD, that expresses both ß amyloid (Aß)- and tau-dependent pathology. We found that Zn(2+) supplementation greatly delays hippocampal-dependent memory deficits and strongly reduces both Aß and tau pathology in the hippocampus. We also evaluated signs of mitochondrial dysfunction and found that Zn(2+) supplementation prevents the age-dependent respiratory deficits we observed in untreated 3xTg-AD mice. Finally, we found that Zn(2+) supplementation greatly increases the levels of brain-derived neurotrophic factor (BDNF) of treated 3xTg-AD mice. In summary, our data support the idea that controlling the brain Zn(2+) homeostasis may be beneficial in the treatment of AD.

Alterations of brain and cerebellar proteomes linked to Aß and tau pathology in a female triple-transgenic murine model of Alzheimer's disease.

The triple-transgenic Alzheimer (3 × Tg-AD) mouse expresses mutant PS1(M146V), APP(swe), and tau(P301L) transgenes and progressively develops plaques and neurofibrillary tangles with a temporal- and region-specific profile that resembles the neuropathological progression of Alzheimer's disease (AD). In this study, we used proteomic approaches such as two-dimensional gel electrophoresis and mass spectrometry to investigate the alterations in protein expression occurring in the brain and cerebellum of 3 × Tg-AD and presenilin-1 (PS1) knock-in mice (animals that do not develop Aß- or tau-dependent pathology nor cognitive decline and were used as control). Finally, using the Ingenuity Pathway Analysis we evaluated novel networks and molecular pathways involved in this AD model. We identified several differentially expressed spots and analysis of 3 × Tg-AD brains showed a significant downregulation of synaptic proteins that are involved in neurotransmitter synthesis, storage and release, as well as a set of proteins that are associated with cytoskeleton assembly and energy metabolism. Interestingly, in the cerebellum, a structure not affected by AD, we found an upregulation of proteins involved in carbohydrate metabolism and protein catabolism. Our findings help to unravel the pathogenic brain mechanisms set in motion by mutant amyloid precursor protein (APP) and hyperphosphorylated tau. These data also reveal cerebellar pathways that may be important to counteract the pathogenic actions of Aß and tau, and ultimately offer novel targets for therapeutic intervention.

High levels of circulating N-terminal pro-brain natriuretic peptide in patients with hepatitis C.

Many patients chronically infected by hepatitis C virus (HCV) experience symptoms like fatigue, dyspnea and reduced physical activity. However, in many patients, these symptoms are not proportional to the liver involvement and could resemble symptoms of chronic heart failure. To our knowledge, no study evaluated serum levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) in a large series of patients with HCV chronic infection (HCV+). Serum NT-proBNP was assayed in 50 patients HCV+ and in 50 sex- and age-matched controls. HCV+ patients showed significantly higher mean NT-proBNP level than controls (P = 0.001). By defining high NT-proBNP level as a value higher than 125 pg/mL (the single cut-off point for patient under 75 years of age), 34% HCV+ and 6% controls had high NT-proBNP (Fisher exact test; P < 0.001). With a cut-off point of 300 pg/mL (used to rule out chronic heart failure in patients under 75 years of age) 10% HCV+ and 0 controls had high NT-proBNP (Fisher exact test; P = 0.056). With a cut-off point of 900 pg/mL (used for ruling in chronic heart failure in patients with age 50-75) 8% HCV+ patients and 0 controls had high NT-proBNP (Fisher exact test; P = 0.12). The study demonstrates high levels of circulating NT-proBNP in HCV+ patients compared to healthy controls. The increase of NT-proBNP may indicate the presence of a sub-clinical cardiac dysfunction. Further prospective studies quantifying these symptoms in correlation with echocardiography are needed to confirm this association.

Defining the transcriptomic and proteomic profiles of rat ageing skeletal muscle by the use of a cDNA array, 2D- and Blue native-PAGE approach.

We defined the transcriptomic and proteomic profiles of rat ageing skeletal muscle using a combined cDNA array, 2D- and Blue native-PAGE approach. This was allowed to obtain an overview of the interrelated events leading to the transcriptome/proteome/mitoproteome changes likely to underlie the structural/metabolic features of aged skeletal muscle. The main differences were found in genes/proteins related to energy metabolism, mitochondrial pathways, myofibrillar filaments, and detoxification. Concerning the abundance of mitochondrial OXPHOS complexes as well as their supramolecular organization and activity, mitochondria from old rats, when compared with those from young rats, contained significantly lower amounts of complex I (NADH:ubiquinone oxidoreductase), V (FoF1-ATP synthase), and III (ubiquinol:cytochrome c oxidoreductase). The same mitochondria contained a significantly larger amount of complex II (succinate:ubiquinone oxidoreductase), but an unchanged amount of complex IV (cytochrome c oxidase, COX). When comparing the supercomplex profiles between young and old muscle mitochondria, the densitometric analysis revealed that lighter supercomplexes were significantly reduced in older mitochondria, and that in the older group the major supercomplex bands were those representing heavier supercomplexes, likely suggesting a compensatory mechanism that, in ageing muscle, is functionally directed towards substrate channeling and catalytic enhancement advantaging the respirosome.

3,5-Diiodo-L-thyronine rapidly enhances mitochondrial fatty acid oxidation rate and thermogenesis in rat skeletal muscle: AMP-activated protein kinase involvement.

Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-l-thyronine (T(2)) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T(2) to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 h after its injection into hypothyroid rats. Administration of T(2) induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoylcarnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T(2) was able to 1) activate the AMPK-ACC-malonyl-CoA metabolic signaling pathway known to direct lipid partitioning toward oxidation and 2) increase the importing of fatty acids into the mitochondrion. These results suggest that T(2) stimulates mitochondrial fatty acid oxidation by activating several metabolic pathways, such as the fatty acid import/beta-oxidation cycle/FADH(2)-linked respiratory pathways, where fatty acids are imported. T(2) also enhanced skeletal muscle mitochondrial thermogenesis by activating pathways involved in the dissipation of the proton-motive force not associated with ATP synthesis ("proton leak"), the effect being dependent on the presence of free fatty acids inside mitochondria. We conclude that skeletal muscle is a target for T(2), and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis, T(2) could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.

Thyroid hormones as molecular determinants of thermogenesis.

Thyroid hormones (TH) are major modulators of energy metabolism and thermogenesis. It is generally believed that 3,5,3'-triiodo-l-thyronine (T3) is the only active form of TH, and that most of its effects are mediated by nuclear T3 receptors, which chiefly affect the transcription of target genes. Some of these genes encode for the proteins involved in energy metabolism. However, a growing volume of evidence now indicates that other iodothyronines may be biologically active. Several mechanisms have been proposed to explain the calorigenic effect of TH, but none has received universal acceptance. Cold acclimation/exposure and altered nutritional status are physiological conditions in which a modulation of energy expenditure is particularly important. TH seem to be deeply involved in this modulation, and this article will review some aspects of their possible influence in these conditions.

Biological effects of 3,5-diiodothyronine (T(2)).

This article is principally intended to describe the facts concerning the actions of 3,5-diiodothyronine (T(2)). Until recent years, T(2), because of its very low affinity for thyroid hormone receptors (THR), was considered an inactive metabolite of thyroid hormones (TH) (thyroxine (T(4)) and triiodo-L-thyronine (T(3))). Several observations, however, led to a reconsideration of this idea. Early studies dealing with the biological activities of this iodothyronine revealed its ability to stimulate cellular/mitochondrial respiration by a nuclear-independent pathway. Mitochondria and bioenergetic mechanisms seem to be major targets of T(2), although outside the mitochondria T(2) also has effects on carriers, ion-exchangers, and enzymes. Recent studies suggest that T(2) may also affect the transcription of some genes, but again the underlying mechanisms seem to be different from those actuated by T(3). The accumulated evidence permits the conclusion that the actions of T(2) do not simply mimic those of T(3) but instead are specific actions exerted through mechanisms that are independent of those actuated by T(3) and do not involve THR.

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria.

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.

Thyroid hormone and uncoupling proteins.

Thyroid hormone (TH/T3) exerts many of its effects on energy metabolism by affecting gene transcription. However, although this is an important target for T3, only a limited number of T3-responsive genes have been identified and studied. Among these, the genes for uncoupling proteins (UCPs) have attracted the interest of scientists. Although the role of UCP1 seems quite well established, uncertainty surrounds the physiological function of the recently discovered UCP1 analogs, UCP2 and UCP3. The literature suggests that T3 affects both the expression and the activity of each of these UCPs but further studies are needed to establish whether the mechanisms activated by the hormone are the same. Recently, because of their larger range of expression, much attention has been devoted to UCP2 and UCP3. Most detailed studies on the involvement of these proteins as mediators of the effects of T3 on metabolism have focused on UCP3 because of its expression in skeletal muscle. T3 seems to be unique in having the ability to stimulate the expression and activity of UCP3 and this may be related to the capacity of T3 to activate the integrated biochemical processes linked to UCP activity, such as those related to fatty acids, coenzyme Q and free radicals.

Uncoupling proteins-2 and 3 influence obesity and inflammation in transgenic mice.

OBJECTIVE: To test the hypothesis that either uncoupling protein-2 UCP2 or UCP3 or both together influence obesity and inflammation in transgenic mice. DESIGN: We generated 12 lines of transgenic mice for both human UCP2 and 3 using native promoters from a human bacterial artificial chromosome (BAC) clone. The BAC expresses no genes other than UCP2 and 3. Mice used for experiments are N4 or higher of backcross to C57BL/6J (B6). Each experiment used transgenic mice and their nontransgenic littermates. RESULTS: Northern blots confirmed expression on human UCP2 in adipose and spleen, while human UCP3 expression was detectable in gastrocnemius muscle. Western blots demonstrated a four-fold increase of UCP2 protein in spleens of Line 32 transgenic animals. Heterozygous mice of four lines showing expression of human UCP2 in spleen were examined for obesity phenotypes. There were no significant differences between Lines 1 and 32, but female transgenics of both lines had significantly smaller femoral fat depots than the control (littermate) mice (P=0.015 and 0.005, respectively). In addition, total fat of transgenic females was significantly less in Line 1 (P=0.05) and almost significantly different in Line 32 (P=0.06). Male Line 1 mice were leaner (P=0.04) while male Line 32 mice were almost significantly leaner (P=0.06). Heterozygous mice of Lines 35 and 44 showed no significant differences from the nontransgenic littermate controls. Effects of the UCP2/UCP3 transgene on obesity in Line 32 mice were confirmed by crossing transgenic mice with the B6.Cg-Ay agouti obese mice. B6.Cg-Ay carrying the UCP2/UCP3 transgene from Line 32 were significantly leaner than nontransgenic B6.Cg-Ay mice. Line 32 UCP2/UCP3 transgenics showed increased hypothalamic Neuropeptide (NPY) levels and food intake, with reduced spontaneous physical activity. Transgenic baseline interleukin4 (IL-4) and interleukin6 (IL-6) levels were low with lower or later increases after endotoxin injection compared to wild-type littermates. Endotoxin-induced fever was also diminished in transgenic male animals. Low-density lipoprotein (LDL) cholesterol levels were significantly higher in both Line 1 and 32 transgenics (P=0.05 and 0.001, respectively) after they had been placed on a moderate fat-defined diet containing 32% of calories from fat for 5 weeks. CONCLUSION: Moderate overexpression of UCP2 and 3 reduced fat mass and increased LDL cholesterol in two independent lines of transgenic mice. Thus, the reduced fat mass cannot be due to insertional mutagenesis since virtually identical fat pad weights and masses were observed with the two independent lines. Line 32 mice also have altered inflammation and mitochondrial function. We conclude that UCP2 and/or 3 have small but significant effects on obesity in mice, and that their mechanism of action may include alterations of metabolic rate.

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase.

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.

Uncoupling protein 2 mRNA expression and respiratory parameters in Kupffer cells isolated from euthyroid and hyperthyroid rat livers.

OBJECTIVE: The levels of uncoupling protein 2 (UCP2) mRNA and determinants of respiration (ATP synthesis, proton leak and non-mitochondrial respiration) were evaluated in Kupffer cells isolated from the livers of normal euthyroid, acute hyperthyroid and chronic hyperthyroid rats. METHODS: After liver perfusion, Kupffer cells were purified by density-gradient centrifugation followed by counterflow centrifugal elutriation. UCP2 mRNA levels were measured by Northern blot and respiratory parameters by polarographic method. RESULTS: In cells isolated from hyperthyroid (tri-iodothyronine (T(3))-treated) rats, the effect of T(3) treatment on the UCP2 mRNA level varied: it was more than doubled (P<0.05) in acutely T(3)-treated rats but, after chronic (3-week) T(3) treatment, it was only 30% (not statistically significant) above the control (euthyroid) level. In Kupffer cells from the livers of chronic hyperthyroid rats, we observed an increase in total respiration rate, with an increase in the percentage attributable to the proton leak and a corresponding decrease in the percentage attributable to ATP synthesis (no alteration was observed in the percentage attributable to non-mitochondrial respiration). In the acute hyperthyroid rats, no significant differences were observed in any of the respiratory parameters, although they all tended to increase. CONCLUSION: These data are indicative of a possible uncoupling effect of UCP2 in Kupffer cells. T(3), by enhancing the expression of UCP2, could play a role in the energy homeostasis of these cells.

Uncoupling protein-3 is a molecular determinant for the regulation of resting metabolic rate by thyroid hormone.

Thyroid hormones increase energy expenditure, partly by reducing metabolic efficiency. The control of specific genes at the transcriptional level is thought to be the major molecular mechanism. However, both the number and the identity of the thyroid hormone-controlled genes remain unknown, as do their relative contributions. Uncoupling protein-3, a recently identified member of the mitochondrial transporter superfamily and one that is predominantly expressed in skeletal muscle, has the potential to be a molecular determinant for thyroid thermogenesis. However, changes in mitochondrial proton conductance and resting metabolic rate after physiologically mediated changes in uncoupling protein-3 levels have not been described. Here, in a study on hypothyroid rats given a single injection of T(3), we describe a strict correlation in terms of time course between the induced increase in uncoupling protein-3 expression (at mRNA and protein levels) and decrease in mitochondrial respiratory efficiency, on the one hand, and the increase in resting metabolic rate, on the other. First, we describe our finding that uncoupling protein-3 is present and regulated by T(3) only in metabolically relevant tissues (such as skeletal muscle and heart). Second, we follow the time course (at 0, 6, 12, 24, 48, 65, 96, and 144 h) of both uncoupling protein-3 mRNA levels and mitochondrial uncoupling protein-3 density in gastrocnemius muscle and heart. In both tissues, the maximal (12-fold) increase in uncoupling protein-3 density was reached at 65 h. The resting metabolic rate [lO(2)(kg(0.75))(-1)h(-1)] showed the same time course, and at 65 h the increase vs. time zero was 45% (1.316 +/- 0.026 vs. 0.940 +/- 0.007; P < 0.001). At the same time point, gastrocnemius muscle mitochondria showed a significantly higher nonphosphorylating respiration rate (nanoatoms of oxygen per min/mg protein; increase vs. time zero, 40%; 118 +/- 4 vs. 85 +/- 9; P < 0.05), whereas the membrane potential decreased by 8% (168 +/- 2 vs. 182 +/- 4; P < 0.05). These data are diagnostic of mitochondrial uncoupling. The results reported here provide the first direct in vivo evidence that uncoupling protein-3 has the potential to act as a molecular determinant in the regulation of resting metabolic rate by T(3).

Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats.

Fibrates are hypolipidemic drugs that activate the peroxisome proliferator-activated receptors. Since fibrates may also increase energy expenditure, we investigated whether fenofibrate (FF) had this effect in diet-induced obese rats. A 2-month administration of a high-fat palatable diet to adult rats increased body weight by 25% and white adipose mass by 163% compared with a standard diet. These effects were prevented by FF, both when administered for the 2 months of high-fat feeding and when given for only the second month. Consequently, FF-treated rats had a final body weight and white adipose tissue mass similar to untreated animals on the standard diet. FF also increased resting metabolic rate, hepatic peroxisomal and mitochondrial palmitoyl-dependent oxygen uptake and mRNA levels of acyl-CoA oxidase and lipoprotein lipase. Finally, FF lowered mRNA levels of uncoupling protein-2 and did not affect mitochondrial respiration in skeletal muscle. Therefore, FF seems to act as a weight-stabilizer mainly through its effect on liver metabolism.

Control of energy metabolism by iodothyronines.

One of the most widely recognized effects of thyroid hormones (TH) in adult mammals is their influence over energy metabolism. In the past, this has received much attention but, possibly because of the complex mode of action of thyroid hormones, no universally accepted mechanism to explain this effect has been put forward so far. Significant advances in our understanding of the biochemical processes involved in the actions of TH have been made in the last three decades and now it seems clear that TH can act through both nuclear-mediated and extranuclear-mediated pathways. TH increase energy expenditure, partly by reducing metabolic efficiency, with control of specific genes at the transcriptional level, being is thought to be the major molecular mechanism. However, both the number and the identity of the thyroid-hormone-controlled genes remain unknown, as do their relative contributions. The recent discovery of uncoupling proteins (UCPs) (in addition to UCP1 in brown adipose tissue) in almost all tissues in animals, including humans, has opened new perspectives on the understanding of the mechanisms involved in the regulation of energy metabolism by thyroid hormones. Other approaches have included the various attempts made to attribute changes in respiratory activity to a direct influence of thyroid hormones over the mitochondrial energy-transduction apparatus. In addition, an increasing number of studies has revealed that TH active in the regulation of energy metabolism include not only T3, but also other iodothyronines present in the biological fluids, such as 3,5-diiodothyronine (3,5-T2). This, in turn, may make it possible to explain some of the effects exerted by TH on energy metabolism that cannot easily be attributed to T3.

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis.

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using 'top-down' elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.

3,5-diiodo-L-thyronine regulates glucose-6-phosphate dehydrogenase activity in the rat.

Thyroid hormones influence the activity of lipogenic enzymes such as malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PD). The effect of T3 on ME is exerted at the transcriptional level, but it is unclear if its effect on G6PD is also nuclear mediated. Furthermore, other iodothyronines that have been shown to possess biological activity (such as diiodothyronines) could contribute to this enzyme's regulation. In this study the effects of 3,5-diiodothyronine (T2) on the aforementioned enzymes were examined and compared with those of T3. Rats made hypothyroid by propylthiouracil and iopanoic acid treatment were used throughout. Enzyme activities were determined spectrophotometrically, and G6PD messenger RNA (mRNA) expression was analyzed by Northern blotting using a human G6PD complementary DNA probe. Injections of T2 to hypothyroid animals significantly enhanced the activity of both enzymes. The effect of T2 on ME was nuclear mediated and mimicked the effect of T3. The effects of T2 and T3 on G6PD differed. Injection of T3 into hypothyroid rats induced an increase in both enzyme activity and G6PD mRNA expression, indicating a nuclear-mediated effect. The effect of T2 on G6PD activity, on the other hand, was not nuclear mediated. The injection of T2 into hypothyroid animals did not change G6PD mRNA expression, and the strong increase in the enzyme's activity (from +70% to +300%) was unaffected by simultaneous injection of protein synthesis inhibitors. As the lowest dose of 1 microg T2/100 g BW affects G6PD activity 3-5 times more than the same dose of T3, these data provide the first evidence that T2 is a factor capable of regulating G6PD activity.

Brain uncoupling protein 2: uncoupled neuronal mitochondria predict thermal synapses in homeostatic centers.

Distinct brain peptidergic circuits govern peripheral energy homeostasis and related behavior. Here we report that mitochondrial uncoupling protein 2 (UCP2) is expressed discretely in neurons involved in homeostatic regulation. UCP2 protein was associated with the mitochondria of neurons, predominantly in axons and axon terminals. UCP2-producing neurons were found to be the targets of peripheral hormones, including leptin and gonadal steroids, and the presence of UCP2 protein in axonal processes predicted increased local brain mitochondrial uncoupling activity and heat production. In the hypothalamus, perikarya producing corticotropin-releasing factor, vasopressin, oxytocin, and neuropeptide Y also expressed UCP2. Furthermore, axon terminals containing UCP2 innervated diverse hypothalamic neuronal populations. These cells included those producing orexin, melanin-concentrating hormone, and luteinizing hormone-releasing hormone. When c-fos-expressing cells were analyzed in the basal brain after either fasting or cold exposure, it was found that all activated neurons received a robust UCP2 input on their perikarya and proximal dendrites. Thus, our data suggest the novel concept that heat produced by axonal UCP2 modulates neurotransmission in homeostatic centers, thereby coordinating the activity of those brain circuits that regulate daily energy balance and related autonomic and endocrine processes.