Regulation of glucose transport in skeletal muscle.

The entry of glucose into muscle cells is achieved primarily via a carrier-mediated system consisting of protein transport molecules. GLUT-1 transporter isoform is normally found in the sarcolemmal (SL) membrane and is thought to be involved in glucose transport under basal conditions. With insulin stimulation, glucose transport is accelerated by translocating GLUT-4 transporters from an intracellular pool out to the T-tubule and SL membranes. Activation of transporters to increase the turnover number may also be involved, but the evidence is far from conclusive. When insulin binds to its receptor, it autophosphorylates tyrosine and serine residues on the beta-subunit of the receptor. The tyrosine residues are thought to activate tyrosine kinases, which in turn phosphorylate/activate as yet unknown second messengers. Insulin receptor antibodies, however, have been reported to increase glucose transport without increasing kinase activity. Insulin resistance in skeletal muscle is a major characteristic of obesity and diabetes mellitus, especially NIDDM. A decrease in the number of insulin receptors and the ability of insulin to activate receptor tyrosine kinase has been documented in muscle from NIDDM patients. Most studies report no change in the intracellular pool of GLUT-4 transporters available for translocation to the SL. Both the quality and quantity of food consumed can regulate insulin sensitivity. A high-fat, refined sugar diet, similar to the typical U.S. diet, causes insulin resistance when compared with a low-fat, complex-carbohydrate diet. On the other hand, exercise increases insulin sensitivity. After an acute bout of exercise, glucose transport in muscle increases to the same level as with maximum insulin stimulation. Although the number of GLUT-4 transporters in the sarcolemma increases with exercise, neither insulin or its receptor is involved. After an initial acute phase, which may involve calcium as the activator, a secondary phase of increased insulin sensitivity can last for up to a day after exercise. The mechanism responsible for the increased insulin sensitivity with exercise is unknown. Regular exercise training also increases insulin sensitivity, which can be documented several days after the final bout of exercise, and again the mechanism is unknown. An increase in the muscle content of GLUT-4 transporters with training has recently been reported. Even though significant progress has been made in the past few years in understanding glucose transport in skeletal muscle, the mechanisms involved in regulating transport are far from being understood.     

Exogenous prolactin delays photo-induced sexual maturity and suppresses ovariectomy-induced luteinizing hormone secretion in the turkey (Meleagris gallopavo)

A direct effect of prolactin (Prl) on gonadotropin secretion has been suggested but not convincingly demonstrated. The secretion of LH in response to photostimulation (phs) and ovariectomy (ovx) was evaluated in adult female turkeys that had received injections of ovine Prl (124 IU/bird/day for 14 days). In experiment 1, oPrl administration initiated on the day of ovx and phs in reproductively quiescent birds suppressed (p less than 0.05) the elevated LH from a peak level of 11.7 +/- 3.5 ng/ml to 5.1 +/- 0.8 ng/ml in ovx hens. The photo-induced LH increase was unaffected by the oPrl treatment in intact birds. In experiment 2, the oPrl treatment was initiated 7 days before ovx and/or phs. This treatment blunted the peak increase in LH level that follows phs in intact (p less than 0.05) and in ovx (p less than 0.05) hens. In both experiments, onset of lay following phs was delayed (p less than 0.05) in the oPrl-treated groups (29.4 +/- 0.9 days vs. 22.3 +/- 0.9 days; 34.8 +/- 0.5 days vs. 25.0 +/- 0.9 days). In experiment 3, administration of oPrl after ovx of laying hens suppressed the LH rise at essentially all sampling times tested. At the end of the experimental period, 6 of the 7 sham-operated, oPrl-treated laying hens, but none of the sham controls, displayed incubation behavior and had Prl levels of 1020 +/- 370 ng/ml compared to 34 +/- 7 ng/ml in vehicle-treated controls. The results suggest a role for Prl in incubation behavior and LH secretion in the turkey.     

Influence of VIP on prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression

Vasoactive intestinal peptide (VIP) is the avian prolactin (PRL)-releasing factor. In the turkey, hypothalamic VIP immunoreactivity and mRNA content, as well as VIP levels in hypophyseal portal blood, are closely related to the state of prolactinemia and the reproductive stage. The present study investigated the role of VIP on prolactinemia in turkey anterior pituitary (AP) cells through PRL gene expression and the role of a cAMP second messenger system on VIP-induced PRL expression. In primary AP cells harvested from hens in different prolactinemic states, steady state promoter activities were positively correlated with secreted PRL levels. VIP increased PRL promoter activities in AP cells from hens with intermediate PRL levels (laying), but not in AP cells from hypoprolactinemic hens (nonphotostimulated reproductively quiescent). However, in AP cells from hyperprolactinemic hens (incubating), PRL promoter activity was down-regulated by VIP. PRL mRNA steady state levels were significantly decreased by the cAMP analogue, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and PRL secretion was down-regulated by the phosphodiesterase blocker, 3-isobutyl-1-methylxanthine (IBMX) in a dose-dependent manner, suggesting that the cAMP second messenger system might be involved in the inhibitory action of dopamine upon VIP-stimulated PRL secretion and gene expression at the pituitary level. In a study of VIP immediate and long-term effects on c-fos expression in relation to PRL expression, VIP dramatically induced c-fos mRNA expression within 5 min, suggesting that VIP-induced c-fos expression might be involved in VIP-stimulated PRL secretion and gene expression. These results provide additional evidence of the functional significance of VIP in PRL gene expression and suggest that changes in PRL promoter activity by VIP may be one of the important inductive mechanisms leading to prolactinemia.     

Enhanced muscle insulin receptor autophosphorylation with short-term aerobic exercise training

Exercise training improves insulin action in skeletal muscle, but the mechanisms of this effect are not completely understood. In particular, the role of the insulin receptor (IR) is unclear. We examined the IR and an enzyme indicative of oxidative capacity in muscle in relation to improved insulin action in 20 previously sedentary individuals before and after a 7-day program of moderate-intensity cycle ergometry. After training, insulin sensitivity increased 33% (6.20 +/- 0.91 vs. 8.22 +/- 1.12 min. microU(-1). ml(-1) mean +/- SE, pre- vs. posttraining, respectively, P < 0.05). The mitochondrial marker enzyme cytochrome c oxidase (COX) increased in vastus lateralis biopsies by 21% (P < 0.05). After training, IR autophosphorylation, determined by ELISA, was significantly increased by approximately 40% at insulin concentrations from 1 to 100 nM (P < 0.05). The training-induced improvements in IR autophosphorylation were significantly correlated with changes in muscle COX content (r = 0.65, P < 0.05). These studies indicate that, in this model of increased physical activity, improvements in IR function are an early adaptation to exercise in humans, are correlated with increases in muscle oxidative capacity, and likely contribute to the beneficial effects of exercise training on insulin action.     

Structural prediction and comparative docking studies of psychrophilic ��- Galactosidase with lactose, ONPG and PNPG against its counter parts of mesophilic and thermophilic enzymes

Enzymes from psychrophiles catalyze the reactions at low temperatures with higher specific activity. Among all the psychrophilic enzymes produced, cold active β-galactosidase from marine psychrophiles revalorizes a new arena in numerous areas at industrial level. The hydrolysis of lactose in to glucose and galactose by cold active β-galactosidase offers a new promising approach in removal of lactose from milk to overcome the problem of lactose intolerance. Herein we propose, a 3D structure of cold active β-galactosidase enzyme sourced from Pseudoalteromonas haloplanktis by using Modeler 9v8 and best model was developed having 88% of favourable region in ramachandran plot. Modelling was followed by docking studies with the help of Auto dock 4.0 against the three substrates lactose, ONPG and PNPG. In addition, comparative docking studies were also performed for the 3D model of psychrophilic β-galactosidase with mesophilic and thermophilic enzymes. Docking studies revealed that binding affinity of enzyme towards the three different substrates is more for psychrophilic enzyme when compared with mesophilic and thermophilic enzymes. It indicates that the enzyme has high specific activity at low temperature when compared with mesophilic and thermophilic enzymes.     

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle

Background

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.

Methodology/Principal Findings

To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and ¹H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H₂O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.

Conclusions

This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.     

Expression of vasoactive intestinal peptide receptor messenger RNA in the hypothalamus and pituitary throughout the turkey reproductive cycle

Vasoactive intestinal peptide (VIP) has been implicated in the regulation of avian reproductive activity and appears to act at the level of the hypothalamus and pituitary. This in situ hybridization histochemistry study describes the distribution of VIP receptor mRNA expression in the hypothalamus and the pituitary of reproductively active (laying) and quiescent (nonphotostimulated, incubating, and photorefractory) female turkeys and characterizes the differences observed in VIP receptor gene expression. VIP receptor mRNA, while expressed throughout the hypothalamus, was specifically expressed in areas known to contain GnRH-I neurons in the chicken, i.e., the lateral septum, medial preoptic area, anterior hypothalamus, and paraventricular nucleus. Significant differences in VIP receptor mRNA expression between different reproductive states was observed only within the infundibular nuclear complex. VIP receptor mRNA was markedly less in nonphotostimulated and photorefractory hens as compared with laying and incubating hens. The most dense VIP receptor mRNA was found in the anterior pituitary, where it was 2.4- and 3.0-fold greater in laying and incubating hens, respectively, as compared with that in nonphotostimulated ones. Hens that stopped incubating and became photorefractory displayed pituitary VIP receptor mRNA levels similar to those of nonphotostimulated birds. The changes in pituitary VIP receptor mRNA expression were positively correlated with known changes in pituitary prolactin (PRL) mRNA expression and PRL content and release. These findings indicate that the variations in PRL secretion observed across the turkey reproductive cycle are, in part, regulated by changes in VIP receptors at the pituitary level.     

The segregation of Escherichia coli minichromosomes constructed in vivo by recombineering

Circularized regions of the chromosome containing the origin of replication, oriC, can be maintained as autonomous minichromosomes, oriC plasmids. We show that oriC plasmids containing precise, pre-determined segments of the chromosome can be generated by a simple in vivo recombineering technique. We generated two such plasmids carrying fluorescent markers. These were transferred to a recipient strain with a different fluorescent marker near the chromosomal copy of oriC. Thus the fates of the oriC plasmid and chromosomal origins could be followed independently in living cells by fluorescence microscopy. In contrast to a previous report, we show that there is a strong tendency of oriC plasmid copies to accumulate at the cell center as a single or double focus at the plane of cell division. This is not simply due to exclusion from the nucleoid space but rather appears to be a specific recognition and retention of the plasmid by some central-located cell site.