Shuji Hidaka

ENHANCED EXPRESSION OF UNCOUPLING PROTEIN 2 GENE IN RAT WHITE ADIPOSE TISSUE AND SKELETAL MUSCLE FOLLOWING CHRONIC TREATMENT WITH THYROID HORMONE

Evidence is rapidly emerging which suggests that uncoupling protein 2 (UCP2), by virtue of its ubiquitous expression, may be important for determining basal metabolic rate. To assess the functional modulation of UCP2 gene expression in relation to body weight control, we examined the effects of hyperthyroid state induced by chronic treatment with triiodothyronine (T3) on UCP2 mRNA expression in male rats. Daily subcutaneous injection of T3 (37 pmol/100 g body weight) for 7 days increased UCP2 mRNA expression in brown adipose tissue (BAT), white adipose tissue (WAT) and the soleus muscle 1.6‐, 1.6‐ and 1.7‐fold compared to the controls, respectively, and increased UCP1 mRNA expression in BAT 1.2‐fold. In contrast, the same treatment with T3 decreased both ob mRNA expression in WAT and plasma leptin level 0.5‐fold for each. The present results suggest that T3 may directly increase UCP2 expression independently of leptin action.

Chronic central leptin infusion restores hyperglycemia independent of food intake and insulin level in streptozotocin-induced diabetic rats

We examined the effects of chronic centrally administered leptin on the glucose metabolism of streptozotocin‐induced diabetic (STZ‐D) rats, a model for insulin‐dependent diabetes mellitus. When 3 μg • rat‐1 • day‐1 of leptin was infused into the third ventricle for 6 consecutive days (STZ‐LEP), STZ‐D rats became completely euglycemic. The effect was not seen when the same dosage was administered s.c. Centrally administered leptin did not affect peripheral insulin levels. The feeding volume of STZ‐LEP rats was suppressed to the level of non‐STZ‐D control rats. No improvement of hyperglycemia was noted when STZ‐D rats were pair‐fed to match the feeding volume of STZ‐LEP rats. Thus, the euglycemia of STZ‐LEP rats cannot be due to the decreased feeding volume. In the STZ‐D rat, glucokinase mRNA, a marker of glycolysis, is down‐regulated whereas glucose‐6‐phosphatase mRNA, a marker of gluconeogenesis, and glucose transporter (GLUT) 2, which is implicated in the release of glucose from liver, are up‐regulated. GLUT4, uncoupling protein (UCP) 1, and UCP3 were down‐regulated in brown adipose tissue. These parameters returned to normal upon central infusion of leptin. GLUT4 was not down‐regulated in the skeletal muscle of STZ‐D rats; however, fatty acid binding protein and carnitine palmitoyltransferase I, markers for utilization and β‐oxidation of fatty acids, were up‐regulated and restored when the rats were treated with leptin. The increase and subsequent decrease of fatty acid utilization suggests a decrease of glucose uptake in the skeletal muscle of STZ‐D rats, which was restored upon central leptin administration. We conclude that centrally infused leptin does not control serum glucose by regulating feeding volume or elevating peripheral insulin, but by regulating hepatic glucose production, peripheral glucose uptake, and energy expenditure. The present study indicates the possibility of future development of a new class of anti‐diabetic agents that act centrally and independent of insulin action.—Hidaka, S., Yoshimatsu, H., Kondou, S., Tsuruta, Y., Oka, K., Noguchi, H., Okamoto, K., Sakino, H., Teshima, Y., Okeda, T., Sakata, T. Chronic central leptin infusion restores hyperglycemia independent of food intake and insulin level in streptozotocin‐induced diabetic rats. FASEB J. 16, 509–518 (2002)

Tumor necrosis factor-α regulates in vivo expression of the rat UCP family differentially

Abstract A family of uncoupling proteins (UCPs), free fatty acid anion transporters, plays a crucial role in energy homeostatic thermoregulation. Tumor necrosis factor-α (TNF-α), a member of the cytokine family, is well known as an endogenous pyrogen. To evaluate the interaction of TNF-α with UCPs in thermogenesis, effects of TNF-α on rat UCP gene expression were examined in intrascapular brown adipose tissue (BAT), epididymal white adipose tissue (WAT) and soleus muscle (Muscle). Administration of TNF-α elevated rectal temperature by 0.7°C as well as serum leptin which peaked at 6 h, compared with saline controls. BAT UCP1 mRNA expression was increased by 1.2-fold at 6 h after the TNF-α treatment and decreased by 0.8-fold at 16 h after the treatment. In contrast to UCP1 expression in BAT, UCP2 mRNA expressions in BAT, WAT, and Muscle was increased to reach maximum levels of 1.3-, 1.6- and 1.8-fold, respectively, at 16 h after the treatment. UCP3 mRNA in Muscle, but not in BAT or WAT, was exclusively up-regulated by 1.7-fold at 16 h after the treatment. These results indicate that TNF-α up-regulates UCP gene expression differentially and tissue dependently, and add novel insights into thermogenesis under conditions of malignancy and inflammation.

Prevention and reversal of renal injury by leptin in a new mouse model of diabetic nephropathy

Diabetic nephropathy is the leading cause of end‐stage renal disease, for which effective therapy to prevent the progression at advanced stages remains to be established. There is also a long debate whether diabetic glomerular injury is reversible or not. Lipoatrophic diabetes, a syndrome caused by paucity of adipose tissue, is characterized by severe insulin resistance, dyslipidemia, and fatty liver. Here, we show that a genetic model of lipoatrophic diabetes (A‐ZIP/F‐1 mice) manifests a typical renal injury observed in human diabetic nephropathy that is associated with glomerular hypertrophy, diffuse and pronounced mesangial widening, accumulation of extracellular matrix proteins, podocyte damage, and overt proteinuria. By crossing A‐ZIP/F‐1 mice with transgenic mice overexpressing an adipocyte‐derived hormone leptin, we also reveal that leptin completely prevents the development of hyperglycemia and nephropathy in A‐ZIP/F‐1 mice. Furthermore, continuous leptin administration to A‐ZIP/F‐1 mice by minipump beginning at 40 weeks of age significantly alleviates the glomerular injury and proteinuria. These findings demonstrate the therapeutic usefulness of leptin at least for a certain type of diabetic nephropathy. The model presented here will serve as a novel tool to analyze the molecular mechanism underlying not only the progression but also the regression of diabetic nephropathy.

Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins.

Chronic administration of leptin has been shown to reduce adiposity through energy intake and expenditure. The present study alms to examine how acute central infusion of leptin regulates peripheral lipid metabolism, as assessed by markers indicative of their mobilization and utilization. A bolus infusion of 1 μg/rat leptin into the third cerebroventricle increased the expression of mRNA for hormone-sensitive lipase (HSL), an indicator of lipolysis, in white adipose tissue (WAT). This was accompanied by elevation of plasma levels of glycerol, but not of free fatty acids, as compared to the saline control (P < 0.03). The same treatment with leptin decreased plasma insulin levels but did not affect the plasma glucose level (P < 0.05 for insulin). Among the major regulators of the transportation or utilization of energy substrates, leptin treatment increased expression of mRNA for uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), UCP2 in WAT, and UCP3 in quadriceps skeletal muscle, but not those for fatty acid–binding protein in WAT, carnitine phosphate transferase-1, a marker for β oxidation of fatty acids in muscle, nor glucose transporter 4 in WAT and muscle (P < 0.01 for HSL, P < 0.05 for UCP1, and P < 0.005 for UCP2 and UCP3). These results indicate that, even in a single bolus, leptin may regulate the mobilization and/or utilization of energy substrates such as fatty acids by affecting lipolytic activity in WAT and by increasing the expression of UCPs in BAT, WAT, and muscle.

MOLECULAR CLONING OF RAT UNCOUPLING PROTEIN 2 CDNA AND ITS EXPRESSION IN GENETICALLY OBESE ZUCKER FATTY (FA/FA) RATS

We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.

Hypoleptinemia, but not hypoinsulinemia, induces hyperphagia in streptozotocin-induced diabetic rats

To assess the dominance between hypoinsulinemia and hypoleptinemia as factors in the development of hyperphagia in streptozotocin (STZ)‐induced diabetes mellitus (STZ‐DM) rodents with respect to hormone–neuropeptide interactions, changes in gene expression of agouti gene‐related protein (AGRP) in the arcuate nucleus of the hypothalamus were investigated using STZ‐DM rats, fasting Zucker fa/fa rats and STZ‐DM agouti (STZ‐DM Ay/a) mice. AGRP mRNA and neuropeptide Y mRNA were both significantly up‐regulated in STZ‐DM rats, which are associated with body weight loss, hyperglycemia, hypoinsulinemia and hypoleptinemia. We proceeded to analyze whether insulin or leptin played the greater role in the regulation of AGRP using Zucker fa/fa rats. The AGRP mRNA did not differ significantly between fasted fa/fa rats, which have both leptin‐insensitivity and hypoinsulinemia, and fed Zuckers, which have leptin‐insensitivity and hyperinsulinemia. We further found that up‐regulation of AGRP expression was normalized by infusion of leptin into the third cerebroventricle (i3vt), but not by i3vt infusion of insulin, although up‐regulation of AGRP was partially corrected by systemic insulin infusion. The latter finding supports hypoleptinemia as a key‐modulator of STZ‐DM‐induced hyperphagia because systemic insulin infusion, at least partially, restored hypoleptinemia through its acceleration of fat deposition, as demonstrated by the partial recovery of lost body weight. After STZ‐DM induction, Ay/a mice whose melanocortin‐4 receptor (MC4‐R) was blocked by ectopic expression of agouti protein additionally accelerated hyperphagia and up‐regulated AGRP mRNA, implying that the mechanism is triggered by a leptin deficit rather than by the main action of the message through MC4‐R. Hypoleptinemia, but not hypoinsulinemia per se, thus develops hyperphagia in STZ‐DM rodents. These results are very much in line with evidence that hypothalamic neuropeptides are potently regulated by leptin as downstream targets of its actions.

Short sequence paperMolecular cloning of rat uncoupling protein 2 cDNA and its expression in genetically obese Zucker fatty (fa/fa) rats1

We isolated rat UCP2 cDNA, which has been proposed to play an important role in mammalian thermogenesis and body weight regulation. The nucleotide sequence of the cDNA revealed that the rat UCP2 protein is composed of 309 amino acid residues, and is 99% and 95% identical to the mouse and human proteins, respectively. The molecular weight of rat UCP2, calculated from the predicted amino acid sequence, was 33,369, and the UCP2 protein of this size was detected when the cDNA was expressed in vitro. Northern blot analysis revealed that the corresponding mRNA is approximately 1.7 kb in size, and is expressed in a variety of rat organs, with predominant expression in the heart, lung and spleen. UCP2 mRNA levels in the heart, liver, muscle and epididymal adipose tissue of Zucker fatty (fa/fa) rats were comparable to those in the lean littermates, while ob mRNA level markedly increased in the epididymal adipose tissue of Zucker (fa/fa) rats.

Alteration of heart uncoupling protein-2 mRNA regulated by sympathetic nerve and triiodothyronine during postnatal period in rats

To provide tissue-specific and developmental characteristics of gene expression of rat heart uncoupling protein-2 (UCP2), we investigated developmental alterations of UCPs mRNA expression in the heart and brown adipose tissue (BAT), and examined possible up-regulators of heart UCP2 expression using in vitro studies. Heart UCP2 mRNA expression was low during the early postnatal days followed by a rapid and significant increase in the 2nd postnatal week. Heart UCP3 mRNA remained undetectable until the 2nd postnatal week when the expression reached a small but significant peak. BAT UCP1 mRNA was abundantly expressed in the neonate, but the expression rapidly decreased to the adult level. The studies using cultured cardiomyocytes demonstrated that both 10(-8) M triiodothyronine and 10(-7) M isoproterenol, but not phenylephrine, increased UCP2 mRNA expression. These results indicate that the sympathetic nervous system and/or thyroid hormones may be involved in the up-regulation of heart UCP2 gene expression during postnatal development. The increase in postnatal heart UCP2 may provide a key link between the postnatal energy shift and adaptation of rat pups to their novel environment.

Molecular cloning of rat brain mitochondrial carrier protein-1 cDNA and its up-regulation during postnatal development.

Brain mitochondrial carrier protein-1 (BMCP1), a new member of the mitochondrial uncoupling carrier, has been shown to be expressed predominantly in the brain of the mice and humans. We cloned rat BMCP1 cDNA and investigated its mRNA level during postnatal development and under various metabolic conditions. The nucleotide sequence of the cDNA revealed that rat BMCP1 protein was composed of 322 amino acid residues, and was 99 and 96% identical to the mouse and human proteins and 29, 33 and 35% identical to rat uncoupling protein (UCP) 1, UCP2 and UCP3, respectively. The molecular weight was predicted to be 36017 Da and the protein of this size was detectable when the cDNA was expressed in vitro. Using Northern blot analysis, the corresponding mRNA, approximately 1.8-kb in size, was found expressed predominantly in the cerebrum, cerebellum and hypothalamus. A unique developmental pattern was identified in the brain, where BMCP1 expression was low in their fetal life, but significantly elevated in the first postnatal week. Thereafter BMCP1 mRNA was maintained to be gradually increased. In 48-h fasted or insulin-induced hypoglycemic rats, BMCP1 mRNA expression in the hypothalamus slightly, but significantly, decreased compared with that in their appropriate controls. The present results indicate that BMCP1 may be involved in pathogenesis of mitochondrial dysfunction in neurons induced by aging or neurodegenerative disorders, and perhaps in energy balance in the brain.