Galanin-stimulated high-affinity GTPase activity in plasma membranes from RINm5F cells.

GTPase activity was studied in plasma membranes purified from the clonal beta-cell line RINm5F. GTPase activities were identified as two broad classes with high or low affinity for GTP. The low-affinity GTPase activity had a Km > 60 microM. In contrast, the high-affinity activity had a Km of 225 nM. Only the high-affinity activity was stimulated by galanin. The stimulated activity had a higher Km (448 nM) and Vmax (75 pmol P(i).min-1.mg-1 protein) compared with the basal. This does not necessarily reflect a complex mechanism of stimulation. Rather, it may reflect that basal activity most likely results from multiple GTPases, whereas the stimulated activity probably reflects one or two specific GTPases. Galanin stimulated the high-affinity GTPase, over the concentration range in which it inhibits stimulated insulin secretion, to a maximal rate 80% greater than the basal rate. The EC50 was 5 nM. Murine and porcine galanin had similar potencies and intrinsic activities on the GTPase. Treatment of the RINm5F cells with PTX before making membranes completely eliminated the stimulatory effect of galanin. Thus, galanin stimulates PTX-sensitive GTPase activity in RINm5F cell membranes in a manner consistent with receptor activation.

Dehydroepiandrosterone sulfate and beta-cell function: enhanced glucose-induced insulin secretion and altered gene expression in rodent pancreatic beta-cells.

Administration of dehydroepiandrosterone (DHEA), or its sulfated form (DHEAS), controls hyperglycemia in diabetic rodents without directly altering insulin sensitivity. We show that DHEAS enhanced glucose-stimulated insulin secretion when administered in vivo to rats or in vitro to beta-cell lines, without changing cellular insulin content. Insulin secretion increased from 3 days of steroid exposure in vitro, suggesting that DHEAS did not directly activate the secretory processes. DHEAS selectively increased the beta-cell mRNA expression of acyl CoA synthetase-2 and peroxisomal acyl CoA oxidase in a time-dependent manner. Although DHEAS is a peroxisomal proliferator, it did not alter the mRNA expression of peroxisomal proliferator-activated receptor (PPAR) alpha or beta, or enhance the activity of transfected PPAR alpha, beta, or gamma in vitro. Thus, DHEAS directly affected the beta-cell to enhance glucose-stimulated insulin secretion and increased the mRNA expression of specific beta-cell mitochondrial and peroxisomal lipid metabolic enzymes. This effect of DHEAS on insulin secretion may contribute to the amelioration of hyperglycemia seen in various rodent models of diabetes.

A critical period in the development of the insulin secretory response to glucose in fetal rat pancreas.

Insulin secretion by fetal rat pancreas was studied at 19.5 and 20.5 days of gestation. Over this 24-hour period, the response to glucose changed rapidly from one that is insensitive to the calcium channel antagonist nitrendipine but markedly enhanced by the presence of the inhibitor of fatty acid oxidation 2-bromostearate, to one that is larger, sensitive to nitrendipine but now not enhanced by 2-bromostearate. The 19.5-day pancreas that is not affected by nitrendipine when responding to glucose alone, is inhibited by nitrendipine when the response to glucose is enhanced by 2-bromostearate. The data suggest a possible metabolic change in the developing B-cell in which fatty acid oxidation is decreased, glucose oxidation increased, and a change in stimulus-secretion coupling from a (KATP) channel-independent mechanism alone, to a combination of that system with the (KATP) channel-dependent system. This could be achieved by a simple increase in the strength of the signal from glucose metabolism.

Glucose-induced time-dependent potentiation and "run down" of insulin secretion in islets of young rats.

A "run down" phenomenon in isolated islets from young rats has been detected, in which the amount of insulin released in response to glucose decreases with time. This has been studied in conjunction with glucose-induced time-dependent potentiation (TDP) to see if the two are related. Islets, isolated from 7-day-old rats, exposed to 16.7 mM glucose for the first time after 30, 75 and 120 minutes of perifusion in Krebs-Ringer bicarbonate buffer (KRB) and 2.8 mM glucose, showed a progressive loss of responsiveness, and less insulin was released at the later times. This run down of the responses was prevented by successive challenges with 16.7 mM glucose and marked TDP was observed. The earlier the islets were exposed to glucose the more effectively was the run down prevented. Also, and perhaps causally related to the prevention of run down, the earlier the exposure to glucose the greater the TDP of a subsequent response. The results could possibly be explained in terms of a shift in metabolism in the isolated 7-day islets, with resultant diminution of the signals for the stimulation of insulin release and TDP. Early, and repeated exposure to high glucose would appear to minimize and delay the metabolic shift.

Glucose-induced insulin release in islets of young rats: time-dependent potentiation and effects of 2-bromostearate.

The development of glucose-stimulated insulin release and time-dependent potentiation (TDP) has been studied in isolated islets from 7-, 14-, and 21-day-old and 3-mo-old rats. Responses were small at 7 days and changed little at 14 days. At 21 days the amount of insulin released in response to glucose was two times that at 14 days but was still less than one-half that released by 3-mo islets. Glucose-induced TDP was absent at 7 days but was present at 21 days. The second phase response to glucose decreased with perifusion time in 7-, 14-, and 21-day islets. In 7- and 21-day islets, high glucose in the presence of 2-bromostearate, an inhibitor of fatty acid oxidation, prevented the time-dependent decrease in responses; in addition, it induced TDP and enhanced TDP in the 7-day and 21-day islets, respectively. The data suggest that, in the young islet, glucose metabolism fails to inhibit fatty acid oxidation as it does in the mature islet and that this leads to a diminished signal for stimulus-secretion coupling.

Maternal diet and insulin secretion in islets of lactating, nonlactating, and young rats.

The effects of dietary phosphorus (P) on insulin secretion by pancreatic islets of rats at peak lactation (14 days after parturition) and by islets of nonlactating previously pregnant rats have been determined. In addition, the effects of changed dietary P in the maternal diet on insulin secretion by islets of the 1- and 14-day-old offspring were studied. Lactating rats had increased food intake relative to nonlactating rats and correspondingly increased P intake on the 0.4 and 0.7% P diets. Serum P levels were elevated in lactating rats consuming the 0.7% P feed relative to those on the 0.4% P feed and also relative to the nonlactating rats on the 0.7% P feed. Lactating rats had decreased bone calcium compared with the nonlactating rats. Islets from lactating rats on the 0.7% P feed secreted more insulin in response to glucose or to 3-isobutyl-1-methylxanthine (IBMX) than lactating rats on the 0.4% P feed. Additionally, lactating rats on the 0.7% P feed secreted more insulin in response to glucose than nonlactating rats on the same 0.7% P feed. The maternal diet had no influence on the endocrine pancreatic responses of the 1- and 14-day-old offspring in response to glucose or IBMX.

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase.

Studies on the mode of action of galanin to inhibit insulin release in RINm5F cells have shown that basal and glyceraldehyde-stimulated release were both inhibited. Galanin was inhibitory at concentrations in the low nanomolar range. Binding studies with 125I-labeled galanin indicated that the RINm5F cells exhibit a single set of sites estimated to be of the order of 30,000 sites/cell. Displacement of 125I-galanin by galanin from the receptor sites occurred over a similar concentration range to that which inhibited insulin release. Half-displacement was achieved with 2 nM galanin. Measurements of bis-(1,3-diethylthiobarbiturate) trimethineoxonol (bis-oxonol) fluorescence showed that galanin hyperpolarized the RINm5F cell plasma membrane. Measurements of intracellular free calcium, [Ca2+]i by means of the fluorescent indicator fura-2 showed that galanin decreased [Ca2+]i. As galanin did not inhibit either basal or glyceraldehyde-stimulated insulin release in the presence of the Ca2+ channel blocker nitrendipine, the hyperpolarization and reduction of Ca2+ entry appear to be a possible explanation for the galanin effects. However, quantitatively, the effects on membrane potential and [Ca2+]i appear to be insufficient to account for the potent inhibition of insulin release. Furthermore, evidence for an additional mechanism of action was obtained from experiments with 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester which stimulates insulin secretion by at least two mechanisms, one Ca2+ dependent and one Ca2+ independent. TPA-stimulated insulin release was inhibited by galanin over the same concentration range as for the inhibition of glyceraldehyde-stimulated release. Galanin inhibited TPA-stimulated release in the presence of maximally effective concentrations of nitrendipine and in the absence of extracellular Ca2+. These effects cannot be explained by hyperpolarization of the plasma membrane and consequent reduction of Ca2+ entry via the voltage-dependent Ca2+ channels. One suggested mechanism for the action of galanin is inhibition of adenylate cyclase. However, it was found that galanin inhibits insulin release even in the presence of 8-Br-cAMP, an agent which effectively bypasses adenylate cyclase. Therefore, an additional mechanism for the inhibitory effect of galanin must be present. All of the effects of galanin were sensitive to pertussis toxin. These data suggest two G-protein-dependent actions of galanin, one to hyperpolarize the plasma membrane and one at a distal point in stimulus-secretion coupling, close to the exocytotic event.