Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.     

褐色脂肪组织代谢与肥胖

肥胖是由于机体能量储存与消耗的失衡而产生的.褐色脂肪组织通过产热的形式,能够将体内过多的能量释放出来,以减少能量积累,避免造成肥胖.现从褐色脂肪组织的结构、分布、功能以及调控机制等方面,对褐色脂肪组织与肥胖症的关系作一综述,旨在为防治肥胖症及相关疾病寻找理论基础和实验依据.     

Developmental role of tryptophan hydroxylase in the nervous system

The serotonin 5-hydroxytryptamine (5-HT) neurotransmitter system contributes to various physiological and pathological conditions. 5-HT is the first neurotransmitter for which a developmental role was suspected. Tryptophan hydroxylase (TPH) catalyzes the rate-limiting reaction in the biosynthesis of 5-HT. Both TPH1 and TPH2 have tryptophan hydroxylating activity. TPH2 is abundant in the brain, whereas TPH1 is mainly expressed in the pineal gland and the periphery. However, TPH1 was found to be expressed predominantly during the late developmental stage in the brain. Recent advances have shed light on the kinetic properties of each TPH isoform. TPH1 showed greater affinity for tryptophan and stronger enzymic activity than TPH2 under conditions reflecting those in the developing brain stem. Transient alterations in 5-HT homeostasis during development modify the fine wiring of brain connections and cause permanent changes to adult behavior. An increasing body of evidence suggests the involvement of developmental brain disturbances in psychiatric disorders. These findings have revived a long-standing interest in the developmental role of 5-HT-related molecules. This article summarizes our understanding of the kinetics and possible neuronal functions of each TPH during development and in the adult.     

Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization

Tryptophan hydroxylase (TPH) catalyses the first and rate limiting step in the biosynthesis of the neurotransmitter serotonin. There are two TPH isoenzymes in humans, encoded by two different genes: TPH1 and the recently described TPH2. We have expressed both human enzymes and various deletion mutants of TPH2 (DeltaN44, DeltaC17, DeltaC19, DeltaC51) in COS7 cells. TPH1 and 2 displayed different kinetic properties with a lower K(m) value of TPH1. Removal of 44 amino acids from the N-terminus of TPH2 resulted in a 3-4-fold increased V(max), which indicates a strong inhibitory function of this part on the enzymes activity. TPH1 and 2 were able to form homooligomers and also heterooligomers with each other. The different deletion mutants (DeltaC17, DeltaC19 and DeltaC51), which lack the putative C-terminal leucine zipper tetramerization domain, existed as monomeric enzymes. While short deletions (DeltaC17 and DeltaC19) hardly changed V(max) values, the DeltaC51 mutant lost 99% of TPH activity. These data identify a region between the C-terminal oligomerization domain and the catalytic domain, which is indispensable for TPH2 activity.     

脂肪组织功能失调在动脉血栓相关疾病中的作用

肥胖与代谢综合征是传统心血管疾病的危险因素.多项临床研究表明,肥胖也会增加患血栓性疾病(如急性心肌梗死和脑卒中)的风险.脂肪组织与血小板反应性增加和高凝状态形成以及纤溶功能降低等存在着重要联系.脂肪组织还是一个高度活跃的内分泌器官,其表达和分泌具有重要功能的脂肪因子和脂质代谢物参与调控全身代谢.深入地了解脂肪组织的内分...     

Decreased brown adipose tissue thermogenic activity following a reduction in brain serotonin by intraventricular p-chlorophenylalanine

The effects of reducing brain serotonin (5-HT) levels by means of intracerebral-ventricular injections of the tryptophan antagonist p-chlorophenylalanine (PCPA) were investigated in male rats. Six days after the operation, PCPA-treated rats, either fedad libitum or pair-fed to the food intake of control rats, showed decreased thermogenic activity and capacity in their interscapular brown adipose tissue (BAT) and also increased fat storage in their white adipose tissue (WAT). These results indicate that serotonergic synapses might play a regulatory role in the sympathetic control of BAT thermogenesis and in the rate of WAT deposition (by an as yet unidentified mechanism), in addition to their well established role in controlling food intake.     

肥胖及2型糖尿病患者内脏脂肪基因差异表达研究

目的：筛查在正常人、单纯性肥胖患者及肥胖伴2型糖尿病患者内脏脂肪组织中差异表达的基因。方法：利用自制的高密度cDNA芯片,比较正常人、单纯性肥胖患者及肥胖伴2型糖尿病患者内脏脂肪组织中差异表达的基因,以寻找脂肪组织特异的与肥胖及糖尿病发生有关的基因。结果：和正常人相比,在肥胖患者及肥胖伴2型糖尿病患者中上调的基因分别有119个和257个,下调的基因分别有46和58个。这些基因中有77个在两组中均上调,其中包括与代谢有关的基因,如丙酮酸脱氢酶激酶4（PDK4）以及窖蛋白、金属硫因蛋白等;8个基因在两组中均下调,其中包括脂肪合成途径中的关键酶,如3-羟基-3-甲基戊二酸单酰辅酶A（MGA）合成酶、脂肪酸合成酶及硬脂酰辅酶A脱氢酶。另外,酪氨酸-3单加氧酶-色氨酸-5单加氧酶活化蛋白θ（YWHAZ）仅在肥胖伴2型糖尿病患者中上调,而在单纯性肥胖患者中不变,该基因所编码的蛋白在胰岛素信号转导途径中起着负调控的作用。结论：脂肪组织中脂肪生成下降、脂肪酸氧化增加可能是肥胖及2型糖尿病中胰岛素抵抗发生的共同原因,其它基因功能的改变也可能参与了肥胖及2型糖尿病的发生,而胰岛素信号转导受阻可能是肥胖向糖尿病转化的促进因素。对这些基因的进一步研究将有助于更好地了解肥胖及糖尿病的发生机制。     

Endogenous adipocyte apolipoprotein E is colocalized with caveolin at the adipocyte plasma membrane

Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism.     

Leptin-induced adipose apoptosis: Implications for body weight regulation

Great strides have been made in understanding the genetics of body weight regulation, in part due to the study of rodent models of obesity that are characterized by mutations affecting leptin or its receptors. Leptin, produced in adipose tissue, acts both centrally and peripherally to orchestrate complex metabolic and behavioral changes that increase loss of adipose tissue, including suppressing food intake and increasing thermogenesis. In addition, recent evidence indicates that leptin acts centrally to trigger an apoptotic process resulting in adipocyte deletion. Loss of adipocytes by apoptosis may provide an explanation for the unexpected delay in return to initial energy status following leptin treatments. This review summarizes the major aspects of leptin-induced adipose tissue apoptosis, including some of the newest findings about possible mechanisms of action.