Sodium-calcium exchanger and R-type Ca(2+) channels mediate spontaneous [Ca(2+)]i oscillations in magnocellular neurones of the rat supraoptic nucleus.

Isolated supraoptic neurones generate spontaneous [Ca(2+)]i oscillations in isolated conditions. Here we report in depth analysis of the contribution of plasmalemmal ion channels (Ca(2+), Na(+)), Na(+)/Ca(2+) exchanger (NCX), intracellular Ca(2+) release channels (InsP3Rs and RyRs), Ca(2+) storage organelles, plasma membrane Ca(2+) pump and intracellular signal transduction cascades into spontaneous Ca(2+) activity. While removal of extracellular Ca(2+) or incubation with non-specific voltage-gated Ca(2+) channel (VGCC) blocker Cd(2+) suppressed the oscillations, neither Ni(2+) nor TTA-P2, the T-type VGCC blockers, had an effect. Inhibitors of VGCC nicardipine, ω-conotoxin GVIA, ω-conotoxin MVIIC, ω-agatoxin IVA (for L-, N-, P and P/Q-type channels, respectively) did not affect [Ca(2+)]i oscillations. In contrast, a specific R-type VGCC blocker SNX-482 attenuated [Ca(2+)]i oscillations. Incubation with TTX had no effect, whereas removal of the extracellular Na(+) or application of an inhibitor of the reverse operation mode of Na(+)/Ca(2+) exchanger KB-R7943 blocked the oscillations. The mitochondrial uncoupler CCCP irreversibly blocked spontaneous [Ca(2+)]i activity. Exposure of neurones to Ca(2+) mobilisers (thapsigargin, cyclopiazonic acid, caffeine and ryanodine); 4-aminopyridine (A-type K(+) current blocker); phospholipase C and adenylyl cyclase pathways blockers U-73122, Rp-cAMP, SQ-22536 and H-89 had no effect. Oscillations were blocked by GABA, but not by glutamate, apamin or dynorphin. In conclusion, spontaneous oscillations in magnocellular neurones are mediated by a concerted action of R-type Ca(2+) channels and the NCX fluctuating between forward and reverse modes.

Physiology of spontaneous [Ca(2+)]i oscillations in the isolated vasopressin and oxytocin neurones of the rat supraoptic nucleus.

The magnocellular vasopressin (AVP) and oxytocin (OT) neurones exhibit specific electrophysiological behaviour, synthesise AVP and OT peptides and secrete them into the neurohypophysial system in response to various physiological stimulations. The activity of these neurones is regulated by the very same peptides released either somato-dendritically or when applied to supraoptic nucleus (SON) preparations in vitro. The AVP and OT, secreted somato-dendritically (i.e. in the SON proper) act through specific autoreceptors, induce distinct Ca(2+) signals and regulate cellular events. Here, we demonstrate that about 70% of freshly isolated individual SON neurones from the adult non-transgenic or transgenic rats bearing AVP (AVP-eGFP) or OT (OT-mRFP1) markers, produce distinct spontaneous [Ca(2+)]i oscillations. In the neurones identified (through specific fluorescence), about 80% of AVP neurones and about 60% of OT neurones exhibited these oscillations. Exposure to AVP triggered [Ca(2+)]i oscillations in silent AVP neurones, or modified the oscillatory pattern in spontaneously active cells. Hyper- and hypo-osmotic stimuli (325 or 275 mOsmol/l) respectively intensified or inhibited spontaneous [Ca(2+)]i dynamics. In rats dehydrated for 3 or 5days almost 90% of neurones displayed spontaneous [Ca(2+)]i oscillations. More than 80% of OT-mRFP1 neurones from 3 to 6-day-lactating rats were oscillatory vs. about 44% (OT-mRFP1 neurones) in virgins. Together, these results unveil for the first time that both AVP and OT neurones maintain, via Ca(2+) signals, their remarkable intrinsic in vivo physiological properties in an isolated condition.

Phthalate is associated with insulin resistance in adipose tissue of male rat: role of antioxidant vitamins.

Diethyl hexyl phthalate (DEHP) is a plasticizer, commonly used in a variety of products, including lubricants, perfumes, hairsprays and cosmetics, construction materials, wood finishers, adhesives, floorings and paints. DEHP is an endocrine disruptor and it has a continuum of influence on various organ systems in human beings and experimental animals. However, specific effects of DEHP on insulin signaling in adipose tissue are not known. Adult male albino rats of Wistar strain were divided into four groups. Control, DEHP treated (dissolved in olive oil at a dose of 10, and 100 mg/kg body weight, respectively, once daily through gastric intubations for 30 days) and DEHP + vitamin E (50 mg/kg body weight) and C (100 mg/kg body weight) dissolved in olive oil and distilled water, respectively, once daily through gastric intubations for 30 days. After the completion of treatment, adipose tissue was dissected out to assess various parameters. DEHP treatment escalated H(2)O(2) and hydroxyl radical levels as well as lipid peroxidation in the adipose tissue. DEHP impaired the expression of insulin signaling molecules and their phosphorelay pathways leading to diminish plasma membrane GLUT4 level and thus decreased glucose uptake and oxidation. Blood glucose level was elevated as a result of these changes. Supplementation of vitamins (C & E) prevented the DEHP-induced changes. It is concluded that DEHP-induced ROS and lipid peroxidation disrupts the insulin signal transduction in adipose tissue and favors glucose intolerance. Antioxidant vitamins have a protective role against the adverse effect of DEHP.

Protective role of lycopene against Aroclor 1254-induced changes on GLUT4 in the skeletal muscles of adult male rat.

Aroclor 1254 is the commercial mixture of highly toxic environmental pollutant, polychlorinated biphenyls (PCBs). Being immensely durable, it is extensively used and widely distributed. Studies show that Aroclor 1254 causes a variety of adverse health effects through free radical generation. The present investigation was designed to check the effect of Aroclor 1254 on the glucose transporter protein, GLUT4, which plays a key role in glucose homeostasis. The protective role of lycopene against the adverse effect of Aroclor 1254 was also tested. Group 1 rats received corn oil as vehicle and served as control. Groups 2, 3, and 4 were administered with Aroclor 1254 [2 mg kg(-1) body weight (b.w.) day(-1)] intraperitoneally for 30 days. Groups 3 and 4 received lycopene (2 and 4 mg kg(-1) b.w. day(-1), respectively) orally in addition to Aroclor 1254. After 30 days, animals were euthanized and the skeletal muscles were dissected to determine the following parameters: GLUT4 messenger RNA (mRNA), GLUT4 protein (both plasma membrane and cytosolic fractions), and (14)C-2-deoxyglucose uptake. Though there was no change in GLUT4 mRNA and fasting plasma glucose levels, Aroclor 1254 significantly decreased the GLUT4 protein level in both the subcellular fractions of the gracilis and triceps muscles. Most important, (14)C-2-deoxyglucose uptake showed a significant decrease in Aroclor 1254 alone treated rats, and Aroclor 1254 plus 4 mg lycopene supplementation treatment maintained the same at par with control. Thus, Aroclor 1254 has adverse effects on GLUT4 translocation and (14)C-2-deoxyglucose uptake, and lycopene administered along with Aroclor 1254 has a protective role over it.

Diethyl hexyl phthalate-induced changes in insulin signaling molecules and the protective role of antioxidant vitamins in gastrocnemius muscle of adult male rat.

Diethyl hexyl phthalate (DEHP) is an endocrine disruptor, it influences various organ systems in human beings and experimental animals. DEHP reduced the serum testosterone and increased the blood glucose, estradiol, T(3) and T(4) in rats. However, the effect of DEHP on insulin signaling and glucose oxidation in skeletal muscle is not known. Adult male albino rats were divided into four groups: Group I: Control; Groups II and III: DEHP treated (dissolved in olive oil at a dose of 10 and 100mg/kg body weight, respectively, once daily through gastric intubation for 30 days); and Group IV: DEHP (100mg/kg body weight) plus vitamins E (50mg/kg body weight) and C (100mg/kg body weight) dissolved in olive oil and distilled water, respectively, once daily through gastric intubation for 30 days. On completion of treatment, animals were euthanized and perfused (whole body); gastrocnemius muscle was dissected out and subjected to assessment of various parameters. DEHP treatment increased the H(2)O(2), hydroxyl radical levels and lipid peroxidation which disrupt the membrane integrity and insulin receptor. DEHP impaired the insulin signal transduction, glucose uptake and oxidation through decreased expression of plasma membrane GLUT4, which may partly be responsible for the elevation of fasting blood glucose level. The present study suggests that DEHP exposure affects glucose oxidation in skeletal muscle and is mediated through enhanced lipid peroxidation, impaired insulin signaling and GLUT4 expression in plasma membrane. Antioxidant vitamins (C and E) have a protective role against the adverse effect of DEHP.

Sex steroids influence glucose oxidation through modulation of insulin receptor expression and IRS-1 serine phosphorylation in target tissues of adult male rat.

Skeletal muscle, liver, and adipose tissue are major insulin responsive target organs that also express androgen receptor. Testosterone (T) plays a role in maintaining normal insulin sensitivity in men but its effects on insulin target tissues are not fully understood. Our previous study showed that orchidectomy impairs glucose oxidation through decreased insulin receptor (IR) mRNA expression in skeletal muscles, liver, and adipose tissue of male rat. Furthermore, T replacement restored IR mRNA expression in skeletal muscles and liver, but did not have any effect in adipose tissue. In the present study, orchidectomy decreased IR mRNA and protein levels in muscle, liver, and adipose tissue. Treatment with a combination of T plus estradiol (E) was necessary to restore the IR mRNA and protein to control levels in adipose tissue. T or E treatment alone had no effect on IR mRNA levels in adipose tissue. T alone also had no effect on the IR protein, whereas E alone had a stimulatory effect. In comparison, in muscle and liver, T or T plus E restored the IR mRNA and protein to control levels. In muscle and liver, E alone had no effect on IR mRNA expression but restored the IR protein. In addition, orchidectomy was seen to have a stimulatory effect on IRS-1 Serine(636/639) phosphorylation in the three tissues studied. Following T, E or combined supplementation to castrated rats, the pattern of IRS-1 serine phosphorylation was restored to normal control levels. Furthermore, orchidectomy decreased serum insulin and glucose oxidation in all three tissues, and this was restored by T and its combination with E replacement, whereas E alone had no effect. It is concluded from the present study that sex steroid deficiency induces impaired glucose oxidation in insulin responsive tissues, which is mediated through reduced IR expression, and increased IRS-1 serine phosphorylation.

Impact of excess aldosterone on glucose homeostasis in adult male rat.

BACKGROUND: Clinical and experimental data demonstrate that excess aldosterone and insulin interact at target tissues. It has been shown that increased levels of aldosterone contribute to development of insulin resistance and thus act as a risk factor for the development of type-II diabetes mellitus. However, the molecular mechanisms involved are yet to be identified. The present study was designed to assess the impact of excess aldosterone on GLUT2 and GLUT4 gene expression and glucose uptake in liver and skeletal muscles (gracilis and quadriceps) of adult male rat. METHODS: Healthy adult male albino rats of Wistar strain (Rattus norvegicus), weighing 180-210 g were used in the present study. The rats were divided into two groups (control and aldosterone treated) each consisting of five animals. Rats were treated with aldosterone at a dose of 10 microg/kg body weight, subcutaneously, twice daily at 8 AM and 6 PM for 15 days. RESULTS: Excess aldosterone impaired the rate of glucose uptake through defective expression of GLUT2 and GLUT4 genes and also decreased translocation of GLUT4 to the plasma membrane. CONCLUSION: Excess aldosterone has an adverse effect on glucose uptake in liver and skeletal muscle and the defective expression of GLUT2 and GLUT4 appears to be responsible for such changes.