Signaling through Tyr985 of Leptin Receptor as an Age/Diet-Dependent Switch in the Regulation of Energy Balance

Leptin regulates energy homeostasis through central activation of multiple signaling pathways mediated by Ob-Rb, the long form of leptin receptor. Leptin resistance underlies the pathogenic development of obesity, which is closely associated with environmental factors. To further understand the physiological function of leptin signaling mechanisms, we generated a knock-in line of mice (Y985F) expressing a mutant Ob-Rb with a phenylalanine substitution for Tyr⁹⁸⁵, one of the three intracellular tyrosines that mediate leptin''s signaling actions. Surprisingly, whereas young homozygous Y985F animals were slightly leaner, they exhibit adult-onset or diet-induced obesity. Importantly, both age-dependent and diet-induced deterioration of energy balance was paralleled with pronounced leptin resistance, which was largely attributable to attenuation of leptin-responsive hypothalamic STAT3 activation as well as prominently elevated expression of hypothalamic SOCS3, a key negative regulator of leptin signaling. Thus, these results unmask distinct binary roles for Try⁹⁸⁵-mediated signaling in energy metabolism, acting as an age/diet-dependent regulatory switch to counteract age-associated or diet-induced obesity.As a component of the metabolic syndrome, obesity is closely associated with increased risk for the development of type 2 diabetes and cardiovascular disorders (16). Arising from a chronic imbalance between energy intake and expenditure, the pathogenic progression of obesity is attributable to the complex interactions between genetic factors and environmental influences. In mammals, energy balance is maintained through multiple homeostatic mechanisms that operate coordinately in response to hormonal and nutritional cues. Leptin is an adipose-secreted hormone (43) that plays a pivotal role in the regulation of energy metabolism. Acting through its active-form receptor Ob-Rb in distinct classes of leptin-responsive neurons (11, 14, 34), leptin activates multiple signaling pathways in the hypothalamus to regulate food intake and energy expenditure. Mice with deficiency in leptin (ob/ob) or its functional receptor (db/db) develop morbid obesity, hyperphagia, and diabetes (20). Impaired leptin responsiveness, i.e., leptin resistance (33), is a key characteristic of the metabolic defects that are responsible for disrupted energy control, presumably underlying the pathogenic development of human obesity (29). Although diminished leptin signaling has been found to occur in association with aging (21, 39) or feeding of a high-fat diet (HFD) (17, 18), the exact physiological mechanisms linking the environmental factors to the impairment in leptin-mediated regulation of energy metabolism remain largely elusive.Leptin binds to Ob-Rb and elicits an array of subsequent intracellular signaling cascades (7, 22) via Jak2 phosphorylation. The mouse Ob-Rb comprises three cytoplasmic tyrosine residues, Tyr⁹⁸⁵, Tyr¹⁰⁷⁷, and Tyr¹¹³⁸, which are known to be phosphorylated and mediate leptin''s physiological functions (22, 26). The phosphorylated Tyr¹¹³⁸ is thought to recruit STAT3 (1), thereby activating the JAK2-STAT3 pathway, which has been shown to play an important role in the control of energy balance (2), whereas our recent investigation has demonstrated crucial actions in vivo for Ob-Rb tyrosine-dependent as well as tyrosine-independent mechanisms in the regulation of energy and glucose homeostasis (26). Our earlier studies in vitro as well as observations from other laboratories have also documented that phosphorylation at Tyr⁹⁸⁵ leads to recruitment of SH2-containing protein tyrosine phosphatase 2 (SHP2) (28, 41) and activation of extracellular signal-regulated kinase (ERK) (9). On the other hand, phosphorylated Tyr⁹⁸⁵ has been postulated to serve as a docking site for SOCS3, thereby exerting an antagonizing effect on Tyr¹¹³⁸-mediated STAT3 activation (8). Consistent with this, a leptin-activated autoinhibitory action in vivo has recently been suggested for Tyr⁹⁸⁵ in the l/l mice expressing a mutant leptin receptor where Tyr⁹⁸⁵ was replaced with leucine (10). However, whereas elevated hypothalamic expression of SOCS3 has been reported to occur in aged rodents (36) or in mice with diet-induced obesity (18, 42), it has yet to be understood whether Ob-Rb Tyr⁹⁸⁵-mediated mechanisms are physiologically connected to altered SOCS3 expression, particularly in the face of aging or high dietary fat intake. Moreover, direct in vivo evidence also has been lacking with respect to whether there exist potential interplays between Ob-Rb Tyr⁹⁸⁵ signaling and other Ob-Rb tyrosine-dependent mechanisms, which act to influence the homeostatic control of energy balance.To gain further insight into the roles of Ob-Rb intracellular tyrosine phosphorylation in mediating leptin''s physiological functions in vivo, we previously generated two lines of mice expressing mutant leptin receptors with phenylalanine substitution for all three tyrosines or for Tyr¹¹³⁸ alone, revealing the metabolic contribution of both tyrosine-dependent and -independent actions in energy homeostasis (26). Here we investigated the physiological consequences of abrogation of signaling through Ob-Rb Tyr⁹⁸⁵ via characterization of the knock-in mice generated by introducing a phenylalanine substitution mutation at this site. We examined the impact of deficiency in Tyr⁹⁸⁵-mediated signaling upon the susceptibility of mice to age-associated and diet-induced energy imbalance, attempting to explore the potential mechanistic links between leptin resistance and environmental influences such as aging and overnutrition.     

蓝花侧金盏化学成分的研究

从藏药蓝花侧金盏的乙醇溶液中分离得到8个化合物,通过波谱学方法分别鉴定为三十一烷醇(1)、对甲酰基肉桂酸(2)、芹菜素(3)、木犀草素(4)、荭草苷(5)、木犀草素-7-O-β-D-葡萄糖苷(6)、异荭草苷(7)及侧金盏醇(8)。化合物1、2、4、6、7均首次从该种植物中分离得到。     

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

Background  

The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).     

Reversal of aging‐associated hippocampal synaptic plasticity deficits by reductants via regulation of thiol redox and NMDA receptor function

Deficits in learning and memory accompanied by age‐related neurodegenerative diseases are closely related to the impairment of synaptic plasticity. In this study, we investigated the role of thiol redox status in the modulation of the N‐methyl‐d ‐aspartate receptor (NMDAR)‐dependent long‐term potentiation (LTP) in CA1 areas of hippocampal slices. Our results demonstrated that the impaired LTP induced by aging could be reversed by acute administration of reductants that can regulate thiol redox status directly, such as dithiothreitol or β‐mercaptoethanol, but not by classical anti‐oxidants such as vitamin C or trolox. This repair was mediated by the recruitment of aging‐related deficits in NMDAR function induced by these reductants and was mimicked by glutathione, which can restore the age‐associated alterations in endogenous thiol redox status. Moreover, antioxidant prevented but failed to reverse H₂O₂‐induced impairment of NMDAR‐mediated synaptic plasticity. These results indicate that the restoring of thiol redox status may be a more effective strategy than the scavenging of oxidants in the treatment of pre‐existing oxidative injury in learning and memory.     

Effects of genistein on β‐catenin signaling and subcellular distribution of actin‐binding proteins in human umbilical CD105‐positive stromal cells

This study was performed to define the roles of actin‐binding proteins in the regulation of actin filament assembly associated with cellular signal transduction pathways in stromal cell proliferation. Genistein, a tyrosine protein kinase inhibitor, decreased the intracellular Ca²⁺ and attenuated cell proliferation and DNA synthesis through the β‐catenin and cyclin D1 pathway in human umbilical CD105‐positive cells. Immunoprecipitation studies using anti‐β‐actin antibody revealed that several actin‐binding proteins implicated in cells include formin‐2 (FMN‐2), caldesmon (CaD), tropomyosin (Tm), and profilin. Protein levels of these proteins in whole cell lysates were not significantly changed by genistein. Three Tm isoforms, Tm‐1, Tm‐2, and Tm‐4, were found to be present in cells. Genistein caused a reduction in levels of mRNAs coding for Tm‐1 and Tm‐4, but had no significant effect on Tm‐2 mRNA levels. Immunofluorescence confocal scanning microscopy indicated that changes in the subcellular distribution of Tm and CaD, in which the diffuse cytosolic staining was shifted to show colocalization with actin stress fibers. In contrast, genistein‐induced accumulation of FMN‐2 and profilin in the peri‐nuclear area. Silencing of FMN‐2 by small interfering RNA resulted in increases of intracellular Ca²⁺ and rendered genistein resistance in decreasing intracellular Ca²⁺ in cells. These results provide the novel findings that genistein acts by modulating the cellular distribution of actin‐binding proteins in association with alterations of cellular signal transduction pathways in human stromal cell proliferation. J. Cell. Physiol. 223: 423–434, 2010. © 2010 Wiley‐Liss, Inc.     

Dopamine D1 receptor activation of adenylyl cyclase, not phospholipase C, in the nucleus accumbens promotes maternal behavior onset in rats

A body of evidence supports the idea that the mesolimbic dopamine (DA) system modulates the natural increase in responsiveness female rats show toward offspring (biological or foster) at birth. In the absence of the full hormonal changes associated with pregnancy and birth, female rats do not show immediate responsiveness toward foster offspring. Activation of the mesolimbic DA system can produce an immediate onset of maternal behavior in these females. For example, female rats that are hysterectomized and ovariectomized on day 15 of pregnancy (15HO) and presented with pups 48 hours later normally show maternal behavior after 2-3 days of pup exposure, but will show maternal behavior on day 0 of testing after microinjection of the DA D₁ receptor agonist, SKF 38393, into the nucleus accumbens (NA) at the time of pup presentation. DA D₁ receptor stimulation is known to activate cAMP intracellular signaling cascades via its stimulation of adenylyl cyclase (AC). However, some DA D₁ receptors are also linked to phospholipase C (PLC) and are capable of activating phosphatidylinositol signaling cascades. SKF 38393 stimulates both types of D₁ receptors. Here we provide evidence that the facilitatory effects of DA D₁ receptor stimulation in the NA on maternal behavior are mediated by AC-linked DA D₁ receptors. By examining the effects of intra-NA application of SKF 83822, a drug which selectively binds DA D₁-AC receptors, or SKF 83959, a drug which selectively activates D₁-PLC-linked receptors, we find that only SKF 83822 facilitates maternal behavior onset.     

Construction of Small-Caliber,Polydiaxanone Cyclohexanone Vascular Stents

Objective of this article is to construct and characterize a three-layered small-caliber, artificial vascular stent. The outer layer of the stent consisted of small intestine submucosa (SIS), the middle layer was the polydioxanone (PDS) vascular stents, and the inner layer. The SIS and PDS were attached with 8-0 PDS thread. Crosslinking with a 10% collagen/chondroitin sulfate solution and 0.020% glutaraldehyde secured the structure. The stent was implanted into the muscles on both sides of the canines’ spine at 2, 4, 12, and 24 weeks. A MTT assay using L-929 cells measured the cytotoxicity of the implant. Histological and microscopy analyses were employed to examine the degradation characteristics of the PDS stent. Biomechanical properties of the stent were tested and compared to those of normal physiological blood vessels. The PDS stent burst pressure (43.5 ± 8.3) kPa, rupture intensity (19.1 ± 1.56) N, strain ratio (42.88 ± 3.16)%, and radial compliance (5.96 ± 0.87)%/100 mmHg were similar to that of physiological vessels. The cytotoxicity test showed that the PDS stent complied with specifications for biological materials for medical applications, with a cell toxicity ranging from 0 to 1. After 12 weeks, SIS and collagen sponge were completely replaced by fibrous connective tissue. Although there was some degradation of PDS, inflammatory cell infiltration subsided. After 24 weeks, the scaffold material began to absorb the new fibers and became filled with inflammatory cells and macrophages. This artificial vascular stent met the requirements of transplant experiments and should be further investigated for future clinical applications.     

Photophysical Probes of the Amorphous Solid State of Proteins

The properties of amorphous solid proteins influence the texture and stability of low-moisture foods, the shelf-life of pharmaceuticals, and the viability of seeds and spores. We have investigated the relationship between molecular mobility and oxygen permeability in dry food protein films—bovine α-lactalbumin (α-La), bovine β-lactoblobulin (β-Lg), bovine serum albumin (BSA), soy 11S globulin, and porcine gelatin—using phosphorescence from the triplet probe erythrosin B. Measurements of the phosphorescence decay in the absence (nitrogen) and presence (air) of oxygen versus temperature provide estimates of the non-radiative decay rate for matrix-induced quenching (k _TS0) and oxygen quenching (k _Q[O₂]) of the triplet state. Since the oxygen quenching constant is the product of the oxygen solubility ([O₂]) and a term (k _Q) proportional to the oxygen diffusion coefficient, it is a measure of the oxygen permeability through the films. For all proteins except gelatin, Arrhenius plots of k _TS0 reveal a gradual increase of apparent activation energy across a broad temperature range starting at ∼50 °C; this suggests that there is a steady increase in the available modes of molecular motion with increasing temperature within the protein matrix. Arrhenius plots for k _Q[O₂] were linear for all proteins with activation energies ranging from 24 to 29 kJ/mol. The magnitude of the oxygen quenching constants varied in the different proteins; the rates were approximately 10-fold higher in α-La, β-Lg, and BSA than in 11S glycinin and gelatin. Although the rate of oxygen permeability was not directly affected by the increased mobility of the protein matrix, plots of k _Q[O₂] versus k _TS0 were linear over nearly three orders of magnitude in the protein films, suggesting that the matrix mobility plays a specific role in modulating oxygen permeability. This effect may reflect differences in matrix-free volume that directly influence both mobility and oxygen solubility.     

Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss

In this report, we investigated the frequency and spectrum of mitochondrial 12S rRNA variants in a large cohort of 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mutational analysis of 12S rRNA gene in these subjects identified 68 (54 known and 14 novel) variants. The frequencies of known 1555A>G and 1494C>T mutations were 3.96% and 0.18%, respectively, in this cohort with nonsyndromic and aminoglycoside-induced hearing loss. Prevalence of other putative deafness-associated mutation at positions 1095 and 961 were 0.61% and 1.7% in this cohort, respectively. Furthermore, the 745A>G, 792C>T, 801A>G, 839A>G, 856A>G, 1027A>G, 1192C>T, 1192C>A, 1310C>T, 1331A>G, 1374A>G and 1452T>C variants conferred increased sensitivity to ototoxic drugs or nonsyndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this rRNA. However, other variants appeared to be polymorphisms. Moreover, 65 Chinese subjects carrying the 1555A>G mutation exhibited bilateral and sensorineural hearing loss. A wide range of severity, age-of-onset and audiometric configuration was observed among these subjects. In particular, the sloping and flat-shaped patterns were the common audiograms in individuals carrying the 1555A>G mutation. The phenotypic variability in subjects carrying these 12S rRNA mutations indicated the involvement of nuclear modifier genes, mitochondrial haplotypes, epigenetic and environmental factors in the phenotypic manifestation of these mutations. Therefore, our data demonstrated that mitochondrial 12S rRNA is the hot spot for mutations associated with aminoglycoside ototoxicity.     

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor,is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

Background  

The downstream of tyrosine kinase/docking protein (Dok) adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk) receptor family, which has three members (TrkA, TrkB and TrkC), are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction.