Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor

The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.     

黑色素皮质素受体激动剂的高通量筛选模型研究

为了建立黑色素皮质素受体（MC4R）激动剂的高通量筛选方法,将人的MC4R基因质粒（hMC4R/pCDNA3.1）与报告基因质粒（3×CRE/3×MRE/SRE-LUC）按1∶5的比例共转染到HEK293细胞,通过G418筛选,建立了稳定的MC4R激动剂筛选细胞株.利用MC4R内源激动剂α-MSH探索和优化了每孔接种细胞数目、激动剂孵育时间、溶剂DMSO终浓度、荧光素酶底物浓度等筛选条件,建立了可靠的筛选方法.实验表明：当细胞数目为4×10⁴个/孔,激动剂孵育时间为8 h,每孔DMSO终浓度小于1％和α-MSH终浓度为1 μmol/L时,系统Z'-因子接近0.7,能够用于MC4R激动剂的高通量筛选.     

Patients Lacking Sustainable Long-Term Weight Loss after Gastric Bypass Surgery Show Signs of Decreased Inhibitory Control of Prepotent Responses

Background

A considerable number of bariatric patients report poor long-term weight loss after Roux-en-Y gastric bypass (RYGB) surgery. One possibility for an underlying cause is an impairment of cognitive control that impedes this patient group’s dietary efforts.

Objective

To investigate if patients having either poor or good weight loss response, ~12 years after RYGB-surgery, differ in their ability to inhibit prepotent responses when processing food cues during attentional operations—as measure of cognitive control.

Methods

In terms of weight loss following RYGB-surgery, 15 ‘poor responders’ and 15 ‘good responders’, matched for gender, age, education, preoperative body mass index, and years since surgery, were administered two tasks that measure sustained attention and response control: a go/no-go task and a Stroop interference task; both of which are associated with maladaptive eating behaviours.

Results

The poor responders (vs. good responders) needed significantly more time when conducting a go/no-go task (603±134 vs. 519±44 msec, p = 0.03), but the number of errors did not differ between groups. When conducting a Stroop interference task, poor responders read fewer inks than good responders (68±16 vs. 85±10 words, p = 0.002).

Conclusion

Patients lacking sustainable weight loss after RYGB-surgery showed poorer inhibitory control than patients that successfully lost weight. In the authors’ view, these results suggest that cognitive behavioral therapies post-RYGB-surgery may represent a promising behavioral adjuvant to achieve sustainable weight loss in patients undergoing this procedure. Future studies should examine whether these control deficits in poor responders are food-specific or not.     

Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

Nephrotic syndrome, characterized by massive proteinuria, is caused by a large group of diseases including membranous nephropathy (MN) and focal segmental glomerulosclerosis (FSGS). Although the underlying mechanisms are beginning to unravel, therapy is unspecific and far from efficient. It has been suggested that adrenocorticotropic hormone (ACTH) has beneficial effects in patients with MN and possibly in other nephrotic diseases. We have previously reported that ACTH may act directly on podocytes through the melanocortin 1 receptor (MC1R). In the present study, we evaluate the effect of highly specific MC1R agonists in two different nephrotic disease models. Experimental MN: Passive Heymann nephritis (PHN) was induced in rats that were treated for four weeks with MS05, a selective MC1R agonist, or saline. The degree of albuminuria was significantly reduced over time and the effect was sustained one week after treatment withdrawal (p<0.05). Experimental FSGS: Based on a dose-response study, two doses of adriamycin were used for induction of nephropathy in Balb/c mice. Mice were treated with either a synthetic MC1R agonist (BMS-470539), with α-melanocyte stimulating hormone (α-MSH) or with saline. There was no beneficial effect of treatment. In summary, MC1R agonists reduce albuminuria and improve morphology in experimentally induced MN whereas they have no effect in experimental FSGS. The results illustrate the differences in these podocytopathies in terms of signaling mechanisms underlying proteinuria, and progression of disease.     

Hyperfunction of Muscarinic Receptor Maintains Long-Term Memory in 5-HT4 Receptor Knock-Out Mice

Patients suffering from dementia of Alzheimer''s type express less serotonin 4 receptors (5-HTR₄), but whether an absence of these receptors modifies learning and memory is unexplored. In the spatial version of the Morris water maze, we show that 5-HTR₄ knock-out (KO) and wild-type (WT) mice performed similarly for spatial learning, short- and long-term retention. Since 5-HTR₄ control mnesic abilities, we tested whether cholinergic system had circumvented the absence of 5-HTR₄. Inactivating muscarinic receptor with scopolamine, at an ineffective dose (0.8 mg/kg) to alter memory in WT mice, decreased long-term but not short-term memory of 5-HTR₄ KO mice. Other changes included decreases in the activity of choline acetyltransferase (ChAT), the required enzyme for acetylcholine synthesis, in the septum and the dorsal hippocampus in 5-HTR₄ KO under baseline conditions. Training- and scopolamine-induced increase and decrease, respectively in ChAT activity in the septum in WT mice were not detected in the 5-HTR₄ KO animals. Findings suggest that adaptive changes in cholinergic systems may circumvent the absence of 5-HTR₄ to maintain long-term memory under baseline conditions. In contrast, despite adaptive mechanisms, the absence of 5-HTR₄ aggravates scopolamine-induced memory impairments. The mechanisms whereby 5-HTR₄ mediate a tonic influence on ChAT activity and muscarinic receptors remain to be determined.     

Sequence Variants of Toll Like Receptor 4 and Late-Onset Alzheimer's Disease

Background

Toll like receptor 4 (TLR4) has been related to inflammation and beta-amyloid deposition in Alzheimer''s disease (AD) brain. No study has explored the association between haplotype-tagging single nucleotide polymorphisms (htSNPs) of TLR4 and AD risk previously and ApoE e4 status alone showed low sensitivity in identifying late-onset AD (LOAD) patients.

Methods

A total of 269 LOAD patients were recruited from three hospitals in northern Taiwan (2007–2010). Controls (n = 449) were recruited from elderly health checkup and volunteers of the hospital during the same period of time. Five common (frequency≥5%) TLR4 htSNPs were selected to assess the association between TLR4 polymorphisms and the risk of LOAD in the Chinese ethnic population.

Results

Homozygosity of TLR4 rs1927907 was significantly associated with an increased risk of LOAD [TT vs. CC: adjusted odds ratio (AOR) = 2.45, 95% confidence interval (CI) = 1.30–4.64]. After stratification, the association increased further in ApoE e4 non-carriers (AOR = 3.07) and in hypertensive patients (AOR = 3.60). Haplotype GACGG was associated with a decreased risk of LOAD (1 vs. 0 copies: AOR = 0.59, 95% CI = 0.36–0.96; 2 vs. 0 copies: AOR = 0.31, 95% CI = 0.14–0.67) in ApoE e4 non-carriers. ApoE e4 status significantly modified this association (p _interaction = 0.01). These associations remained significant after correction for multiple tests.

Conclusions

Sequence variants of TLR4 were associated with an increased risk of LOAD, especially in ApoE e4 non-carriers and in hypertensive patients. The combination of TLR4 rs1927907 and ApoE e4 significantly increased the screening sensitivity in identifying LOAD patients from 0.4 to 0.7.     

Hormone Substitution after Gastric Bypass Surgery in Patients with Hypopituitarism Secondary to Craniopharyngioma

Objective: Craniopharyngiomas (CPs) are benign brain tumors presenting frequently in childhood and are treated by surgery with or without radiotherapy. About 50% of cured patients suffer from eating disorders and obesity due to hypothalamic damage, as well as hypopituitarism, necessitating subsequent hormone substitution therapy. Gastric bypass surgery has been reported to be an efficient treatment strategy for morbid hypothalamic obesity. However, so far it is unknown whether oral hormone substitution is affected by impaired intestinal drug absorption, potentially leading to severe hypopituitarism or pituitary crisis.Methods: Four morbidly obese CP patients with panhypopituitarism treated by gastric bypass surgery were included in this retrospective analysis. Dosages of hormone substitution therapy, blood concentrations of hormones, potential complications of impaired drug absorption, and anthropometric characteristics were investigated pre- and postoperatively after 6 to 14 months and 13 to 65 months.Results: In all CP patients (3 female/1 male; baseline body mass index, 49 ± 7 kg/m²), gastric bypass resulted in distinct weight loss (-35 ± 27 kg). In follow-up examinations, mean daily dosage of thyroid hormone (levothyroxine_baseline 156 ± 44 μg/day versus levothyroxine_follow-up 150 ± 30 μg/day), hydrocortisone (hydrocortisone_baseline 29 ± 12 mg/day versus hydrocortisone_follow-up 26 ± 2 mg/day), growth-hormone (somatotropin_baseline 0.9 ± 0.5 mg/day versus somatotropin_follow-up 1.0 ± 0.4 mg/day), and desmopressin (desmopressin_baseline 222 ± 96 μg/day versus desmopressin_follow-up 222 ± 96 μg/day) substitution was unchanged. No patient developed adrenal insufficiency. Oral thyroid/hydrocortisone absorption testing performed in 1 patient indicated sufficient gastrointestinal drug absorption after bariatric surgery.Conclusion: Our preliminary results suggest that oral hormone substitution therapy is not impaired following gastric bypass operation in CP patients with morbid obesity, indicating that it might be a safe and effective treatment strategy.Abbreviations:BMI = body mass indexCP = craniopharyngiomafT4 = free thyroxineGH = growth hormoneIGF-1 = insulin-like growth factor 1     

Rate Limiting Factors in Melanocortin 1 Receptor Signalling Throughthe cAMP Pathway

The melanotropic actions of α‐melanocyte‐stimulating hormone (α‐MSH) and other melanocortins are mediated by activation of the melanocortin 1 receptor (MC1R). This G protein‐coupled receptor is positively coupled to Gs and triggers the cyclic adenosine mono‐phosphate (cAMP) pathway. Mutations of the MC1R gene are associated with skin type and pigmentation phenotypes, and with increased risk of skin cancers. Genetic studies have demonstrated an heterozygote carrier effect for these associations, suggesting the importance of variant allele dosage. This could be accounted for, at least partially, if the number of MC1R molecules, rather than the Gs protein or the effector enzyme, adenylyl cyclase, is limiting for the activation of the signalling pathway. However, the nature of the limiting factor(s) in MC1R signalling has not been investigated. We addressed this question by comparing the cAMP output of clones of human melanoma cell lines enriched in MC1R by stable transfection. We also analysed heterologous cell systems widely used for functional studies of MC1R. We show that cAMP production in clones of Chinese hamster ovary cells stably expressing the MC1R is a linear function of receptor number up to high, supraphysiological levels of approximately 50 000 α‐MSH binding sites per cell. Enrichment of human melanoma cell lines with MC1R also results in increased cAMP levels, with a small leftward shift of the agonist dose–response curves. Therefore, at physiological expression levels second‐messenger generation is dependent on receptor density. Within melanoma cells and also likely in normal melanocytes, MC1R appears the limiting factor controlling the output of the cAMP signalling pathway.     

Melanocortin-4 Receptor Gene Polymorphisms in Obese Patients

Obesity is a complex disease caused by both genetics and environmental factors. Melanocortin-4 receptor (MC4R) (MIM 155541) gene polymorphisms were reported to be the cause of monogenic obesity in humans. We studied three polymorphisms (Val50Met, Val103Ile, and Ser58Cys) and a mutation (Asn274Ser) of the MC4R gene in 203 obese patients and in 110 healthy subjects in the Turkish population. A high incidence of Val103Ile and Val50Met polymorphisms as well as the Asn274Ser mutation was found in the obese patients, whereas no significant correlation was found regarding the Ser58Cys polymorphism. We conclude that there is a concordance between the polymorphisms (Val103Ile, Val50Met, Ser58Cys) that were first studied in the Turkish population with obesity.     

Upregulated Expression of C-X-C Chemokine Receptor 4 Is an Independent Prognostic Predictor for Patients with Gastric Cancer

Aberrant chemokine (C-X-C motif) receptor CXCR4 expressions in malignant tissues have been reported, but its role in gastric cancer prognosis remains unknown. Our studies were designed to investigate the expression and prognostic significance of CXCR4 in patients with gastric cancer. CXCR4 expression was retrospectively analyzed by immunohistochemistry in 97 patients with gastric adenocarcinoma from China. Results were assessed for association with clinical features and overall survival by using Kaplan-Meier analysis. Prognostic values of CXCR4 expression and clinical outcomes were evaluated by Cox regression analysis. A molecular prognostic stratification scheme incorporating CXCR4 expression was determined by using receiver operating characteristic (ROC) analysis. The results show that CXCR4 predominantly localized in the cell membranes and cytoplasm. The protein level of CXCR4 was upregulation in gastric cancer tissues and upregulated expression of CXCR4 was only significantly associated with Lauren classification (P<0.001). Increased CXCR4 expression in gastric cancer tissues was positively correlated with poor overall survival of gastric cancer patients (P<0.001). Further multivariate Cox regression analysis suggested that intratumoral CXCR4 expression was an independent prognostic indicator for the disease. Applying the prognostic value of intratumoral CXCR4 density to TNM stage system showed a better prognostic value in patients with gastric cancer. In conclusion, intratumoral CXCR4 expression was recognized as an independent prognostic marker for the overall survival of patients with gastric cancer. On the basis of TNM stage, detection of CXCR4 expression will be helpful for predicting prognosis for patients with gastric cancer.     

The SNPs of Melanocortin 4 Receptor (MC4R) Associated with Body Weight in Beagle Dogs

Melanocortin 4 receptor (MC4R), which is associated with inherited human obesity, is involoved in food intake and body weight of mammals. To study the relationships between MC4R gene polymorphism and body weight in Beagle dogs, we detected and compared the nucleotide sequence of the whole coding region and 3′- and 5′- flanking regions of the dog MC4R gene (1214 bp). In 120 Beagle dogs, two SNPs (A420C, C895T) were identified and their relation with body weight was analyzed with RFLP-PCR method. The results showed that the SNP at A420C was significantly associated with canine body weight trait when it changed amino acid 101 of the MC4R protein from asparagine to threonine,while canine body weight variations were significant in female dogs when MC4R nonsense mutation at C895T. It suggested that the two SNPs might affect the MC4R gene’s function which was relative to body weight in Beagle dogs. Therefore, MC4R was a candidate gene for selecting different size dogs with the MC4R SNPs (A420C, C895T) being potentially valuable as a genetic marker.     

Dynamics of Change in Total and Regional Body Composition After Gastric Bypass in Obese Patients

Little is known on patterns of change over time in body composition, especially lean body mass (LBM), during massive weight loss after Roux‐en‐Y gastric bypass (RYGB) in obese patients. We performed sequential measurements of total and regional body composition in patients after RYGB, and we compared a subsample of patients after surgery to a nonsurgical control group of similar age and body fatness. We used dual‐energy X‐ray absorptiometry (DXA) before and at 3, 6, and 12 months after RYGB in 42 obese women (before surgery: age 39.5 ± 11.6 years; BMI 44.6 ± 6.1 kg/m²; mean ± s.d.) and in 48 control obese women referred for nonsurgical weight management, before weight loss. During 1‐year follow‐up after RYGB, there was a continuous decrease in body weight (?36.0 ± 12.5 kg at 1 year), total fat mass (FM) (?26.0 ± 9.1 kg), as well as in trunk and appendicular FM. In contrast, the decrease in total LBM (?9.8 ± 4.8 kg at 1 year), as well as in trunk and appendicular LBM, plateaued after 3–6 months. Rates of loss in weight, FM, and LBM were highest during the first 3‐month period after RYGB (6.4 ± 1.8, 4.1 ± 1.7, and 2.3 ± 1.2 kg/month, respectively), then decreased continuously for FM but plateaued for LBM. There was no evidence of a decrease in total, trunk, or appendicular LBM in weight‐reduced subjects compared to the control group. In conclusion, follow‐up of these obese women revealed a differential pattern of change in FM and LBM after RYGB. Despite an important loss in LBM, especially during the 3–6 months of initial period, LBM appears to be spared thereafter.     

Health Outcomes of Gastric Bypass Patients Compared to Nonsurgical,Nonintervened Severely Obese

Favorable health outcomes at 2 years postbariatric surgery have been reported. With exception of the Swedish Obesity Subjects (SOS) study, these studies have been surgical case series, comparison of surgery types, or surgery patients compared to subjects enrolled in planned nonsurgical intervention. This study measured gastric bypass effectiveness when compared to two separate severely obese groups not participating in designed weight‐loss intervention. Three groups of severely obese subjects (N = 1,156, BMI ≥ 35 kg/m²) were studied: gastric bypass subjects (n = 420), subjects seeking gastric bypass but did not have surgery (n = 415), and population‐based subjects not seeking surgery (n = 321). Participants were studied at baseline and 2 years. Quantitative outcome measures as well as prevalence, incidence, and resolution rates of categorical health outcome variables were determined. All quantitative variables (BMI, blood pressure, lipids, diabetes‐related variables, resting metabolic rate (RMR), sleep apnea, and health‐related quality of life) improved significantly in the gastric bypass group compared with each comparative group (all P < 0.0001, except for diastolic blood pressure and the short form (SF‐36) health survey mental component score at P < 0.01). Diabetes, dyslipidemia, and hypertension resolved much more frequently in the gastric bypass group than in the comparative groups (all P < 0.001). In the surgical group, beneficial changes of almost all quantitative variables correlated significantly with the decrease in BMI. We conclude that Roux‐en‐Y gastric bypass surgery when compared to severely obese groups not enrolled in planned weight‐loss intervention was highly effective for weight loss, improved health‐related quality of life, and resolution of major obesity‐associated complications measured at 2 years.     

黑素皮质素受体对动物采食量和能量稳态的调控

黑素皮质素受体是G-蛋白耦联受体超家族成员。5个黑素皮质素受体基因已经被克隆和鉴定,并有不同的组织分布和生物学功能。本文综述了黑素皮质素受体3和受体4基因调控采食量和能量稳态的研究进展。 Abstract:The melanocortin receptors are members of the super-family of G-protein coupled receptors.To date,five melanocortin receptor genes (MC1R-MC5R) have been cloned and characterized.These receptorsdiffer in their tissue distributions and physiological roles.This review focuses on the roles of MC3R and MC4R in regulation of food intake and energy homeostasis.     

Postprandial Hypoglycemia in Patients after Gastric Bypass Surgery Is Mediated by Glucose-Induced IL-1β

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