The mushroom body D1 dopamine receptor controls innate courtship drive.

Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, we investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. Here, we report that the D1 dopamine receptor dDA1 regulates courtship drive in naïve males. The wild-type naïve males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg-less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/ß and γ neurons but not α/ß or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience-dependent courtship or other learning and memory processes. We also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short-term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, our study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.

Emergent Synapse Organizers: LAR-RPTPs and Their Companions.

Leukocyte common antigen-related receptor tyrosine phosphatases (LAR-RPTPs) have emerged as key players that organize various aspects of neuronal development, including axon guidance, neurite extension, and synapse formation and function. Recent research has highlighted the roles of LAR-RPTPs at neuronal synapses in mediating distinct synaptic adhesion pathways through interactions with a host of extracellular ligands and in governing a variety of intracellular signaling cascades through binding to various scaffolds and signaling proteins. In this chapter, we review and update current research progress on the extracellular ligands of LAR-RPTPs, regulation of their extracellular interactions by alternative splicing and heparan sulfates, and their intracellular signaling machineries. In particular, we review structural insights on complexes of LAR-RPTPs with their various ligands. These studies lend support to general molecular mechanisms underlying LAR-RPTP-mediated synaptic adhesion and signaling pathways.

Efficacy and safety of teneligliptin, a novel dipeptidyl peptidase-4 inhibitor, in Korean patients with type 2 diabetes mellitus: a 24-week multicentre, randomized, double-blind, placebo-controlled phase III trial.

We assessed the 24-week efficacy and safety of teneligliptin, a novel dipeptidyl peptidase-4 inhibitor, in Korean patients with type 2 diabetes mellitus (T2DM) that was inadequately controlled with diet and exercise. The present study was designed as a multicentre, randomized, double-blind, placebo-controlled, parallel-group, phase III study. Patients (n = 142) were randomized 2 : 1 into two different treatment groups as follows: 99 received teneligliptin (20 mg) and 43 received placebo. The primary endpoint was change in glycated haemoglobin (HbA1c) level from baseline to week 24. Teneligliptin significantly reduced the HbA1c level from baseline compared with placebo after 24 weeks. At week 24, the differences between changes in HbA1c and fasting plasma glucose (FBG) in the teneligliptin and placebo groups were -0.94% [least-squares (LS) mean -1.22, -0.65] and -1.21 mmol/l (-1.72, -0.70), respectively (all p < 0.001). The incidence of hypoglycaemia and adverse events were not significantly different between the two groups. This phase III, randomized, placebo-controlled study provides evidence of the safety and efficacy of 24 weeks of treatment with teneligliptin as a monotherapy in Korean patients with T2DM.

Efficacy and safety of teneligliptin, a dipeptidyl peptidase-4 inhibitor, combined with metformin in Korean patients with type 2 diabetes mellitus: a 16-week, randomized, double-blind, placebo-controlled phase III trial.

The aim of the present study was to assess the efficacy and safety of teneligliptin in combination with metformin in Korean patients with type 2 diabetes mellitus who were inadequately controlled with metformin monotherapy. Patients [glycated haemoglobin (HbA1c) 7.0-10.0%, on stable metformin ≥1000 mg/day] were randomized 2 : 1 to receive 20 mg teneligliptin plus metformin (n = 136) or placebo plus metformin (n = 68). The primary endpoint was the change in HbA1c levels from baseline to week 16. The mean baseline HbA1c was 7.9% in the teneligliptin group and 7.8% in the placebo group. The differences between the teneligliptin and placebo groups regarding changes in HbA1c and fasting plasma glucose levels were -0.78 % and -1.24 mmol/l (22.42 mg/dl), respectively, at week 16. The incidence of adverse events was similar between the groups. The addition of teneligliptin once daily to metformin was effective and generally well tolerated in Korean patients with type 2 diabetes.

A cross-sectional evaluation of seasonality as a determinant of endothelial function.

Endothelium-dependent vasodilation is impaired in obese versus lean humans. We set out to evaluate whether agonist-mediated endothelium-dependent vasodilation varies by season in a cross-sectional dataset of lean and obese humans, and whether this effect differed by obesity status. All vascular studies performed in our laboratory over a 12 year period from 1997 to 2009 were evaluated. Endothelium-dependent vasodilation was measured invasively using thermodilution in the leg as the response to intra-arterially infused methacholine chloride. Resting blood pressure was measured concurrently by cuff and intra-arterially. The association of endothelium-dependent vasodilation and blood pressure measurements with season was evaluated, comparing responses in lean and obese subjects. Endothelium-dependent vasodilation differed between lean and obese subjects, and varied across seasons (p=0.02), but without an interaction between season effect and obesity status. The proportion of obese versus lean subjects differed significantly across seasons in our dataset, and after adjusting for this factor the apparent seasonal variation in endothelium-dependent vasodilation was lost (p=0.12), and was not present in either lean or obese subjects separately. Systolic arterial blood pressure varied across seasons, and this effect remained significant after adjusting obesity status (p=0.019 and p=0.043 for blood pressures measured intra-arterially and by cuff respectively). In our cross-sectional dataset, seasonal variations in blood pressure were seen but we did not observe an association between season and endothelium-dependent vasodilation in lean or obese subjects.

Contributions of dysglycaemia, obesity, and insulin resistance to impaired endothelium-dependent vasodilation in humans.

BACKGROUND: Individual effects of hyperglycaemia and obesity to impair vascular health are recognized. However, the relative contributions of dysglycaemia versus other obesity-related traits to vascular dysfunction have not been systematically evaluated. METHODS: We undertook a cross-sectional evaluation of factors contributing to vascular function in 271 consecutive subjects, categorized as non-obese normal glucose tolerant (n = 115), non-obese dysglycaemic (n = 32), obese normal glucose tolerant (n = 57), obese dysglycaemic (n = 38), or type 2 diabetic (n = 29). Vascular function was measured invasively as leg blood flow responses to methacholine chloride, an endothelium-dependent vasodilator. Categorical and continuous analyses were carried out to assess the contributions of hyperglycaemia to vascular dysfunction. RESULTS: Even among normoglycaemic subjects, obese subjects had impaired vascular function compared to non-obese subjects (p = 0.004). Vascular function was also impaired in non-obese dysglycaemic subjects (p = 0.04 versus non-obese normoglycaemic subjects), to a level comparable to normoglycaemic obese subjects. Within obese subject groups, gradations of dysglycaemia including the presence of diabetes were not associated with further worsening of these vascular responses beyond the effect of obesity alone (p = not significant comparing all obese groups, p < 0.001 versus lean normoglycaemic subjects). After univariate and multivariable modelling analyses we found that effects of glycaemia were less powerful than effects of insulin resistance and obesity on vascular dysfunction. CONCLUSIONS: Dysglycaemia contributes to impaired vascular function in non-obese subjects, but obesity and insulin resistance are more important determinants of vascular function in obese and diabetic subjects.

Resistance exercise did not alter intramuscular adipose tissue but reduced retinol-binding protein-4 concentration in individuals with type 2 diabetes mellitus.

Lipid accumulation in muscle is associated with diminished insulin sensitivity. It was hypothesized that resistance exercise decreases muscular adipose tissue and reduces the level of retinol-binding protein-4 (RBP4), which is linked to adipose tissue and insulin sensitivity in diabetics. Forty-four women with type 2 diabetes were randomly assigned to three groups for a period of 12 weeks: control (asked to maintain a sedentary lifestyle); resistance exercise (elastic band exercise at moderate intensity five times per week); and aerobic exercise (walking for 60 min at moderate intensity five times per week). Subcutaneous (SCAT), subfascial (SFAT) and intramuscular (IMAT) adipose tissues at mid-thigh level were assessed using computed tomography, and RBP4 level and insulin sensitivity (fractional disappearance rate of insulin, k(ITT)) were assessed before and after intervention. Changes in SCAT, SFAT, IMAT, RBP4 and k(ITT) were similar among the three groups. Within-group analysis revealed that body mass index and waist circumference decreased significantly in both exercise groups, but RBP4 decreased significantly only with resistance exercise. Resistance exercise did not alter muscular adipose tissue or improve insulin sensitivity.

The effects of total energy expenditure from all levels of physical activity vs. physical activity energy expenditure from moderate-to-vigorous activity on visceral fat and insulin sensitivity in obese Type 2 diabetic women.

AIMS: We examined the effects of physical activity with or without dietary restriction for 3 months on regional fat and insulin sensitivity and compared the effect of total energy expenditure from all levels of physical activity with that of physical activity energy expenditure from moderate-to-vigorous exercise in obese women with Type 2 diabetes. METHODS: In this randomized, controlled trial, we assessed change of body weight, abdominal visceral fat area, subcutaneous fat area and insulin sensitivity, expressed as K(ITT), and monitored total energy expenditure and physical activity energy expenditure using an accelerometer during a 12-week intervention in four groups: control, diet, exercise and diet plus exercise. RESULTS: The mean body mass index was 28.0 +/- 2.7 kg/m(2) and the mean duration of diabetes was 8 +/- 6 years. Both the diet and diet plus exercise groups showed significant body weight loss compared with the control group (P < 0.05). However, the visceral fat area was reduced only in the diet and exercise group (P = 0.017) and the subcutaneous fat area was reduced only in the diet group (P = 0.009). Mean energy intake was an independent determinant of the change in subcutaneous fat area (P = 0.020) and mean total anergy expenditure was an independent determinant of visceral fat area (P = 0.002). Insulin sensitivity K(ITT) was associated with physical activity energy expenditure (P = 0.006), energy intake (P = 0.047) and the change in fructosamine level (P = 0.016) but not with changes in body weight, subcutaneous fat area, visceral fat area or adipokine level. CONCLUSIONS: Exercise had an additive effect to dietary restriction on visceral fat reduction. Visceral fat area was associated with total energy expenditure, but insulin sensitivity was associated with physical activity energy expenditure.

Valsartan increases circulating adiponectin levels without changing HOMA-IR in patients with type 2 diabetes mellitus and hypertension.

Evaluating increasing circulating adiponectin levels is becoming an important strategy in the prevention of diabetes mellitus and cardiovascular events. This study was designed to investigate the effect of the angiotensin II receptor blocker valsartan on blood adiponectin levels and insulin sensitivity in patients with type 2 diabetes and mild-to-moderate hypertension. A total of 91 Korean patients were treated with 80 mg/day valsartan for 4 weeks followed by 160 mg/day for a further 8 weeks. Blood pressure, adiponectin levels and metabolic parameters were measured before and after treatment. The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as an insulin sensitivity index. Valsartan significantly decreased mean blood pressure and increased circulating adiponectin levels. There were no differences in metabolic parameters, including HOMA-IR, glycosylated haemoglobin and lipid levels before and after treatment. These results indicated that valsartan increases circulating adiponectin levels, but does not change insulin sensitivity in patients with type 2 diabetes and mild-to-moderate hypertension.

Classical reward conditioning in Drosophila melanogaster.

Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.

A novel octopamine receptor with preferential expression in Drosophila mushroom bodies.

Octopamine is a neuromodulator that mediates diverse physiological processes in invertebrates. In some insects, such as honeybees and fruit flies, octopamine has been shown to be a major stimulator of adenylyl cyclase and to function in associative learning. To identify an octopamine receptor mediating this function in Drosophila, putative biogenic amine receptors were cloned by a novel procedure using PCR and single-strand conformation polymorphism. One new receptor, octopamine receptor in mushroom bodies (OAMB), was identified as an octopamine receptor because human and Drosophila cell lines expressing OAMB showed increased cAMP and intracellular Ca2+ levels after octopamine application. Immunohistochemical analysis using an antibody made to the receptor revealed highly enriched expression in the mushroom body neuropil and the ellipsoid body of central complex, brain areas known to be crucial for olfactory learning and motor control, respectively. The preferential expression of OAMB in mushroom bodies and its capacity to produce cAMP accumulation suggest an important role in synaptic modulation underlying behavioral plasticity.

Tripartite mushroom body architecture revealed by antigenic markers.

We have explored the organization of the axonal lobes in Drosophila mushroom bodies by using a panel of immunohistochemical markers. These markers consist of antibodies to eight proteins expressed preferentially in the mushroom bodies: DAMB, DCO, DRK, FASII, LEO, OAMB, PKA RII, and RUT. Previous to this work, four axonal lobes, two projecting dorsally (alpha and alpha') and two medially (beta and gamma), had been described in Drosophila mushroom bodies. However, our analysis of immunohistochemically stained frontal and sagittal sections of the brain revealed three medially projecting lobes. The newly distinguished lobe, which we term beta', lies along the dorsal surface of beta, just posterior to gamma. In addition to resolving a fifth lobe, our studies revealed that there are specific lobe sets defined by equivalent marker expression levels. These sets are (1) the alpha and beta lobes, (2) the alpha' and beta' lobes, and (3) the gamma lobe and heel (a lateral projection formed by a hairpin turn of some of the peduncle fibers). All of the markers we have examined are consistent with these three sets. Previous Golgi studies demonstrate that each mushroom body cell projects one axon that branches into a dorsal lobe and a medial lobe, or one unbranched axon that projects medially. Taken together with the lobe sets listed above, we propose that there are three major projection configurations of mushroom body cell axons: (1) one branch in the alpha and one in the beta lobe, (2) one branch in the alpha' and one in the beta' lobe, and (3) one unbranched axon projecting to the heel and the gamma lobe. The fact that these neuron types exhibit differential expression levels of a number of mushroom body genes suggests that they may have corresponding functional differences. These functions may be conserved in the larvae, as several of these genes were expressed in larval and embryonic mushroom bodies as well. The basic mushroom body structure, including the denritic calyx, peduncle, and lobes, was already visible by the late stages of embryogenesis. With new insights into mushroom body organization, and the characterization of markers for developing mushroom bodies, we are beginning to understand how these structures form and function.

DAMB, a novel dopamine receptor expressed specifically in Drosophila mushroom bodies.

The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning.

Neuroanatomy: mushrooming mushroom bodies.

A revolution is spreading in the study of mushroom bodies, structures within the insect brain that mediate learning and memory processes and pheromonal discrimination of the opposite sex.

7,12-Dimethylbenz[a]anthracene-induced mouse keratinocyte malignant transformation independent of Harvey ras activation.

Independent clones of mouse keratinocytes initiated in vitro gave rise to tumor phenotypes typical of mouse skin multistage carcinogenesis and histologically similar to human tumors of the skin, and head and neck. High-molecular-weight genomic DNAs isolated from two 7,12-dimethylbenz[a]anthracene (DMBA)-initiated murine epithelial carcinoma cell lines and one papilloma cell line were examined for transforming activity by transfection into NIH3T3 cells. DNAs from each of these cell lines resulted in the formation of foci morphologically unlike foci containing an activated c-Ha-ras oncogene. Following polymerase chain reaction amplification of the c-Ha-ras gene, Xba I restriction analysis and oligonucleotide differential hybridization did not detect 61st, 12th, or 13th codon mutations. Southern and Northern analysis confirmed that the normal c-Ha-ras gene was not activated by amplification or overexpression. These results provide evidence that 7,12-dimethylbenz[a]anthracene-induced malignant transformation of murine keratinocytes occurred independent of point mutations associated with c-Ha-ras activation. The absence of an activated c-Ha-ras oncogene in these cell lines distinguishes our model from other mouse models of carcinogenesis and may provide a model for functional genetic changes during initiation and progression of human epithelial cancers.

Alternatively spliced p53 RNA in transformed and normal cells of different tissue types.

The alternatively spliced RNA species of tumor suppressor gene p53, containing an additional 96 bases derived from intron 10, is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal and carcinoma cells. The presence of this alternatively spliced RNA in 10T1/2 fibroblast cells, mouse liver and testis suggests that this alternative splicing may be universal. The level of alternatively spliced p53 RNA was increased coordinately with that of regularly spliced p53 in 10T1/2 cells in response to epidermal growth factor. Immunoprecipitation analysis of epidermal cells using monoclonal antibodies which recognize different epitopes of p53 suggested that distinct p53 proteins may be translated from both RNA species. Considering previous observations on the potential importance of carboxyl terminal sequences in p53 function, knowledge of the ubiquitous presence of alternatively spliced p53 is important for future studies of p53 function in normal cells and in oncogenesis.

Altered expression of wild-type p53 tumor suppressor gene during murine epithelial cell transformation.

An epidermal cell model in which initiated, benign tumor-producing and carcinoma stages were derived from a cloned parental cell strain was used to examine p53 expression during multistage epithelial carcinogenesis. Increased steady-state levels of p53 RNA were detected in squamous cell carcinomas compared to papilloma and normal epidermal cells. Nontumorigenic initiated cell precursors of the carcinomas exhibited normal p53 expression, localizing altered p53 regulation to the malignant conversion stage. Immunoprecipitation and Western immunoblot analyses demonstrated elevated levels of p53 protein in the moderately differentiated carcinoma compared to normal cells, and negligible levels of p53 in the poorly differentiated carcinoma cells. Sequence analysis of p53 complementary DNA from normal and carcinoma cells revealed no mutations in the coding or 5'- and 3'-untranslated regions, suggesting a novel mechanism of p53 inactivation.

Altered levels of endogenous retrovirus-like sequence (VL30) RNA during mouse epidermal cell carcinogenesis.

Gene expression during mouse keratinocyte carcinogenesis was examined in a clonal cell model. Tumor cells from three separate initiated cell lineages were compared with their nontumorigenic precursors and with the progenitor cell strain prior to treatment with 7,12-dimethylbenz[a]anthracene (DMBA). The steady-state levels of VL30 RNA in normal and papilloma cells were regulated by extracellular Ca2+ (which controls proliferation and differentiation in normal epidermal keratinocytes) and culture density. In contrast, steady-state levels of VL30 RNA were not regulated by these factors in the squamous cell carcinoma or the anaplastic carcinoma cells. VL30 expression was Ca2+ dependent in the initiated cell precursors within each tumor cell lineage, suggesting that the loss of response to extracellular Ca2+ was associated with the malignant conversion stage of carcinogenesis. No differences between normal and tumor cells were found in the cellular RNA levels of five additional proto-oncogenes. The mouse epidermal cell model should provide a means for direct assessment of a potential functional role of VL30 sequences in cancer development.