Impairments in availability of insulin to liver in vivo and in binding of insulin to purified hepatic plasma membrane during aging

The concentration of insulin in portal vein blood decreases 50–60% in fed or fasted rats as they age from 12- to 24-months. The binding capacity of purified hepatic plasma membrane for insulin decreases approximately 60% as rats age from 2- to 24-months, whereas the dissociation constant for insulin is not altered. It is proposed that these factors may contribute significantly to previously documented examples of age-dependent modifications in liver enzyme regulation.     

Relationships between insulin and glucose metabolism and pituitary-ovarian functions in fasted heifers

The effects of fasting between Days 8 and 16 of the estrous cycle on plasma concentrations of luteinizing hormone (LH), progesterone, cortisol, glucose and insulin were determined in 4 fasted and 4 control heifers during an estrous cycle of fasting and in the subsequent cycle after fasting. Cortisol levels were unaffected by fasting. Concentrations of insulin and glucose, however, were decreased (p less than 0.05) by 12 and 36 h, respectively, after fasting was begun and did not return to control values until 12 h (insulin) and 4 to 7 days (glucose) after fasting ended. Concentrations of progesterone were greater (p less than 0.05) in fasted than in control heifers from Day 10 to 15 of the estrous cycle during fasting, while LH levels were lower (p less than 0.01) in fasted than in control heifers during the last 24 h of fasting. Concentrations of LH increased (p less than 0.01) abruptly in fasted heifers in the first 4 h after they were refed on Day 16 of the fasted cycle. Concentrations (means +/- SEM) of LH also were greater (p less than 0.05) in fasted (11.2 +/- 2.6 ng/ml) than in control (4.7 +/- 1.2 ng/ml) heifers during estrus of the cycle after fasting; this elevated LH was preceded by a rebound response in insulin levels in the fasted-refed heifers, with insulin increasing from 176 +/- 35 pg/ml to 1302 +/- 280 pg/ml between refeeding and estrus of the cycle after fasting. Concentrations of LH, glucose and insulin were similar in both groups after Day 2 of the postfasting cycle. Concentrations of progesterone in two fasted heifers and controls were similar during the cycle after fasting, whereas concentrations in the other fasted heifers were less than 1 ng/ml until Day 10, indicating delayed ovulation and (or) reduced luteal function. Thus, aberrant pituitary and luteal functions in fasted heifers were associated with concurrent fasting-induced changes in insulin and glucose metabolism.     

Metabolic and mitochondrial disturbances in streptozotocin-treated Sprague-Dawley and Sherman rats

This study provides explanation for conflicting evidence in the literature relating to changes in mitochondrial function and metabolic parameters during chemically induced diabetes. Diabetes of 3 days' duration (early ketosis) did not alter heart, kidney, or liver mitochondrial respiratory rates with glutamate or succinate even though serum glucose and triglycerides were elevated. Diabetes of 5 weeks' duration did not alter kidney or liver mitochondrial function in the fed adult rat although weight gain was depressed. The amount of kidney mitochondrial protein isolated per gram of tissue was increased by 30% in the diabetic. This increase was reversed by insulin treatment as were the other biochemical modalities measured. Superimposition of a 24-hr fast resulted in enhanced gluconeogenesis as measured by an animal weight loss of 17% within 24 hr (liver weight loss, 21%) and an elevation of serum urea nitrogen by 180% compared to fasted control. Respiratory rates of diabetic kidney mitochondria with glutamate were unaffected in the fasted animal whereas diabetic liver mitochondrial respiratory rates during succinate oxidation were reduced by 43%. Respiratory control was unchanged in the fasted diabetic rat. All the observed changes were reversed by insulin. Variation in the serum and liver metabolic indices (urea nitrogen, creatinine, glycerol, free fatty acids, free amino acids, triglycerides, and glucose) and liver mitochondrial responses to 7 weeks of chemically induced diabetes was affected by the rat strain, Sprague-Dawley versus Sherman, and rat weight, 72 g versus 222 g. Liver mitochondrial respirations in fed Sherman rats were not depressed by diabetes. Both rat strains had elevated liver free fatty acids and glutamate dehydrogenase activity in the diabetic state. Serum leucine, isoleucine, and valine were more elevated and serum lysine and arginine were more depressed in the diabetic Sprague-Dawley rat than in the Sherman rat. Conjectures on these results are presented in the text.     

Changes in glucose-stimulated insulin secretion after long-term treatment of C57BL mice with glibenclamide

An insulin response to increased glucose concentrations could not be found in vivo and in vitro after long-term treatment of C57BL/KsJ and C57BL/6J mice with Glibenclamide. This missing stimulation of insulin secretion was not the result of an exhaustion of the islets or a disturbed (pro)insulin biosynthesis as demonstrated by measurements of insulin content of the islets and by in vitro (pro)insulin biosynthesis experiments. In the presence of glucose (15 mmol/l) theophylline increased the insulin secretion of isolated islets of Glibenclamide-treated mice to values similar to control islets. The insulin response to an i.p. glucose loading was found to be normal in comparison with control mice 1-2 weeks after the Glibenclamide treatment had been finished.     

Effect of leptin on insulin, glugacon and somatostatin secretion in the perfused rat pancreas.

We have investigated the effect of rat leptin as well as the 22-56 fragment of this molecule on pancreatic hormone secretion in the perfused rat pancreas. In pancreases from fed rats, leptin failed to alter the insulin secretion elicited by glucose, arginine or tolbutamide, but inhibited the insulin response to both CCK-8 and carbachol, secretagogues known to act on the B-cell by increasing phospholipid turnover. This insulinostatic effect was also observed with the 22-56 leptin fragment. In pancreases obtained from 24-hour fasted rats, no effect of leptin on carbachol-induced insulin output was found, perhaps as a consequence of depressed B-cell phospholipid metabolism. Leptin did not influence glucagon or somatostatin release. Our results do not support the concept of leptin as a major regulator of B-cell function. Leptin inhibition of carbachol-induced insulin output might reflect a restraining effect of this peptide on the cholinergic stimulation of insulin release.     

Effect of fasting and insulin on the glucagon-induced orthophosphate incorporation to the isolated perfused rat liver.

Isolated perfused fed rat livers spontaneously liberated glucose and orthophosphate to the medium; 24-hr fasted rat livers did not exhibit these phenomena. In perfused fed rat livers, glucagon (2 mug) increased glucose output and promoted orthophosphate incorporation. In perfused fed rat livers, insulin (250 or 500 mU) inhibited the spontaneous liberation of glucose and orthophosphate. Comparable doses of insulin significantly reduced the glucagon (2 mug)-induced increase in glucose output from perfused fed rat liver, but did not affect orthophosphate uptake by the organ.     

Endogenous triacylglycerol utilization by the isolated rat atria

The isolated atria from 24 h fasted rats, either in the presence of glucose or in a substrate-free medium containing 2-deoxyglucose, mobilized the endogenous triacylglycerol (TG) to a greater extent than those from fed rats. The TG of the fasted atria had almost disappeared at the end of the 90 min incubation in the substrate-free plus 2-deoxyglucose medium, whereas in those from fed rats a mobilization-resistant portion of about 40% of the TG pool remained. This finding coincided with a lower decay of the contractile and pacemaker activities in the atria from fasted rats. Insulin abolished the TG mobilization in the atria from fed rats in the presence of glucose, but it was ineffective in the fasted atria. These data suggest that the endogenous-TG and glucose share in supporting the atrial functions, that insulin is involved in the control of TG consumption only in the fed state and that the greater TG mobilization in the fasted atria, at least partly, meets the energy requirements of the tissue.     

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

The stimulation of lipogenesis in white adipose tissue from fed rats by corticosterone

The direct effects of a physiological concentration of corticosterone (50 ng ml-1) in presence of insulin (200 microU ml-1) on lipid synthesis and CO2 production from glucose and glycerol release were evaluated in vitro in white adipose tissue after pre-incubation with the hormones. Lipid synthesis was 27% higher after 24 h and 66% higher after 48h pre-incubation with corticosterone and insulin compared with insulin alone. Basal and adrenaline-stimulated glycerol release and CO2 production were unchanged after pre-incubation with both hormones compared with insulin alone. We propose that corticosterone acts as a pro-lipogenic hormone on adipose tissue in the fed rat, in contrast to its glucose sparing effects in the fasted animal.     

Insulin receptor substrate 3 is not essential for growth or glucose homeostasis.

The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulin-stimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes from IRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.     

Combined administration of sodium chloro-2-propionate and insulin on the secretion of glucagon by the pancreas in the diabetic rat

This work was designed to study the effects of sodium 2-chloropropionate (2CP) alone or combined with insulin, in vitro, on glucagon secretion from pancreas isolated from rats, made diabetic by streptozotocin (66 mg/kg i.p.). The pancreata were perfused with a physiological solution containing 2.8 mM glucose (0.5 g/l) and glucagon secretion was stimulated by an arginine infusion (5 mM) for 30 min. When 2CP (1 mM) and/or insulin (4 IU/l) were applied, they were infused from the start of the organ perfusion. In the presence of glucose alone, a marked decrease in glucagon output was observed in diabetic rat pancreas. The arginine perfusion induced a biphasic glucagon secretion both in normal and diabetic rat pancreas; this response was however clearly reduced in diabetic rat pancreas. In diabetic rat pancreas, the infusion of either 2CP or insulin had no effect on glucagon output in presence of glucose alone, nor did it modify the response to arginine. In contrast, the combined infusion of insulin and 2CP induced different effects depending on the conditions: whereas in presence of glucose alone it restored a glucagon output close to that recorded in normal rat pancreas, it did not modify the response to arginine.     

Induction of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes. Requirement for insulin and dexamethasone

To determine the relative contributions of glucose, insulin, dexamethasone, and triiodothyronine to the induction of hepatic glucose-6-phosphate dehydrogenase, hepatocytes isolated from normal or adrenalectomized rats, either fasted or fed, were examined in culture. Addition of insulin (42 milliunits/ml, 0.9 microM) and dexamethasone (1 microM) to hepatocytes obtained from 3-day-fasted rats and cultured for 48 h in serum-free Dulbecco's medium resulted in a 7- to 11-fold increase in Glc-6-P dehydrogenase specific activity compared with a 2- to 3-fold increase in activity in control cultures incubated without added hormones. The effects of insulin and dexamethasone were independent of DNA synthesis, dose-dependent, and additive; each contributing about one-half of the total response. Medium glucose was neither sufficient nor necessary for the insulin- or dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Addition of triiodothyronine (10 microM) preferentially blocked the dexamethasone-stimulated increase in Glc-6-P dehydrogenase specific activity. Insulin failed to stimulate the induction of Glc-6-P dehydrogenase in hepatocytes obtained from normal fed rats or from fasted and fed adrenalectomized rats. However, insulin caused a significant increase in the Glc-6-P dehydrogenase specific activity of these cells when dexamethasone was concurrently added to the culture medium.     

Fetal rat islet insulin deficiency following maternal administration of streptozotocin

Pancreatic islets were isolated from the fetuses of normal rats and rats made diabetic by the iv administration of streptozotocin (STZ) on either Day 3 or 5 of pregnancy. Of the rats made diabetic on Day 3, one group also received insulin injections at the appearance of glucosuria. Maternal blood glucose on Day 20 of gestation was significantly different in the diabetic rats (405 +/- 27 mg/dl) from the normal (97 +/- 1 mg/dl) and insulin-treated diabetic rats (69 +/- 9 mg/dl). While fetal weight was significantly decreased in the STZ-treated rats (2.64 +/- 0.13 g vs 3.52 +/- 0.05 g for the control group, P less than 0.005), fetal glucose was significantly higher in the STZ-treated than in normal pups (342 +/- 11 vs 35 +/- 1 mg/dl, P less than 0.005). Both fetal weight and glucose were normalized by insulin treatment: 3.16 +/- 0.18 g and 31 +/- 7 mg/dl, respectively. Insulin release from fetal islets of diabetic dams was blunted after a week in culture both in basal and stimulated conditions. After 2 weeks in culture, there was partial recovery in the insulin response to glucose but it did not equal to that measured in fetal islets from the normal and insulin-treated diabetic rats. These data suggest maternal hyperglycemia severely impairs fetal weight and insulin release from fetal rat islets in vitro, and correction of the hyperglycemia by insulin treatment not only improves fetal weight and glucose concentrations, but it also normalizes insulin release from fetal rat islets in vitro.     

The long-term effect of glucagon on pyruvate kinase activity in primary cultures of hepatocytes

Glucagon caused a marked decrease in the total L-pyruvate kinase activity of control hepatocytes maintained in monolayer culture (t1/2 = 54 h), while the addition of insulin to hepatocytes isolated from a fasted rat caused a four- to fivefold increase in the total enzyme activity. Maintenance of L-pyruvate kinase in control cultures of hepatocytes was shown to require insulin. However, when 1 microM glucagon was present in the medium, the total L-pyruvate kinase activity was not maintained even in the presence of 1 microM insulin, but rather the total L-pyruvate kinase activity of the cells steadily declined from 12.1 to 5.7 units/mg DNA by the 6th day in culture. The increase in the total L-pyruvate kinase activity of fasted hepatocytes cultured in the presence of insulin was shown to result from an increase in protein synthesis, since actinomycin D and cycloheximide blocked the insulin-induced increase in the enzyme activity. The addition of 1 microM glucagon to cultures of fasted hepatocytes also blocked the insulin-induced increase in total L-pyruvate kinase activity. Since glucagon decreased the total L-pyruvate kinase activity in control hepatocytes and blocked the increase in L-pyruvate kinase activity in fasted hepatocytes, it is suggested that, in addition to the phosphorylation of L-pyruvate kinase by a cAMP-dependent protein kinase, glucagon also acts to decrease the synthesis of L-pyruvate kinase in vitro.     

Secretory and electrophysiological characteristics of insulin cells from gastrectomized mice: evidence for the existence of insulinotropic agents in the stomach

Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for 1 h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca(2+) current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca(2+) influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocytosis and glucose-stimulated insulin release may reflect the lack of a gastric agent that serves to maintain an appropriate insulin response to glucose and an appropriate exocytotic response to depolarisation by raising cAMP in a special glucose-sensitive compartment possibly regulated by a soluble adenylate cyclase.     

Dietary fat type alters glucose metabolism in isolated rat hepatocytes

Dietary fat type can influence the regulation of carbohydrate metabolism in multiple tissue types. The influence of feeding high-fat (40% of kilocalories) diets containing either menhaden oil (MO) or coconut oil (CO) on hepatic glycogenolytic and gluconeogenic capacities was studied in isolated rat hepatocytes. Estimates of both glycogenolytic and gluconeogenic capacities were performed on hepatocytes isolated from fed and fasted animals, respectively. In MO-fed animals, both basal and hormone-stimulated rates of glucose production were significantly greater than those in CO-fed animals. However, both groups displayed a similar maximal increase in glucose production above basal for glucagon and epinephrine (2.3- and 1.9-fold, respectively). Basal rates of adenosine 3′,5′-cyclic phosphate (cAMP) production were not different between groups whereas glucagon-stimulated cAMP production was increased twofold in the MO-fed group. In both MO and CO groups, the addition of 10 nM insulin reduced glucose production in fed animals to similar absolute rates. In animals fasted for 24 hours, gluconeogenic capacity was estimated using 10 mM pyruvate, lactate, or glycerol. Glucose production from all substrates was significantly greater in CO-fed animals. In addition to increased gluconeogenic rates, maximal phosphoenolpyruvate carboxykinase (PEPCK) activity was increased in the CO-fed group. Insulin reduced glucose production in both dietary groups, but the absolute rate of glucose production was 28% greater in the CO-fed group relative to the MO-fed group. In summary, dietary fat type can markedly influence the regulation of hepatic glucose metabolism in multiple metabolic pathways. MO feeding promoted glycogenolysis and sensitivity to insulin whereas CO feeding favored gluconeogenesis and reduced insulin sensitivity.     

Long-term treatment with atrial natriuretic peptide inhibits ATP production and insulin secretion in rat pancreatic islets

Atrial natriuretic peptide (ANP) levels correlate with hyperglycemia in diabetes mellitus, but ANP effects on pancreatic islet β-cell insulin secretion are controversial. ANP was investigated for short- and long-term effects on insulin secretion and mechanisms regulating secretion in isolated rat pancreatic islets. A 3-h incubation with ANP did not affect basal or glucose-stimulated islet insulin secretion. However, 7-day culture of islets with 5.5 mM glucose and ANP (1 nM - 1 μM) markedly inhibited subsequent glucose (11 mM)-stimulated insulin secretion; total islet insulin content was not affected. Following ANP removal for 24 h, the islet insulin-secretory response to glucose was restored. The insulin-secretory response to other insulin secretagogues, including α-ketoisocaproic acid, forskolin, potassium chloride, and ionomycin were also markedly inhibited by chronic exposure to ANP. However, the combination of potassium chloride and α-ketoisocaproic acid was sufficient to overcome the inhibitory effects of ANP on insulin secretion. The glucose-stimulated increases in islet ATP levels and the ATP/ADP ratio were completely inhibited in ANP 7-day-treated islets vs. control; removal of ANP for 24 h partially restored the glucose response. ANP did not affect islet glycolysis. ANP significantly increased levels of islet activated hormone-sensitive lipase and the expression of uncoupling protein-2 and peroxisome proliferator-activated receptor-δ and -α. Although islet ANP-binding natriuretic peptide receptor-A levels were reduced to 60% of control after 7-day culture with ANP, the ANP-stimulated cGMP levels remained similar to control islet levels. Thus, long-term exposure to ANP inhibits glucose-stimulated insulin secretion and ATP generation in isolated islets.     

Alterations in regulation of insulin biosynthesis in pregnancy and starvation studied in isolated rat islets of Langerhans

1. Insulin biosynthesis in isolated rat islets of Langerhans was determined by the incorporation of [(3)H]leucine into newly synthesized islet proteins. Anti-insulin serum covalently coupled to a solid phase (CNBr-activated Sepharose 4B) was used to separate the immunoreactive proinsulin and insulin from other islet proteins. This method was applied to a study of the regulation of insulin biosynthesis in isolated rat islets of Langerhans during pregnancy, and immediately after a period of food deprivation. 2. Islets isolated from pregnant rats showed an increased basal rate of synthesis compared with the non-pregnant controls. In addition, they showed a significant increase in biosynthesis of proinsulin and insulin in comparison with the normal islets over a range of glucose concentrations of 2-20mm. 3. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine significantly increased the insulin-synthetic response of normal islets over the glucose range 5-20mm, so that their glucose response approached that of islets from pregnant rats. 4. Normal female rates were injected with a long-acting progesterone derivative (hydroxyprogesterone hexanoate), to investigate the role of progesterone on the increased insulin biosynthesis observed in islets in pregnancy. There appeared to be no marked difference in insulin biosynthesis between the islets from the progesterone-injected and control rats in the presence of 2mm- or 6mm-glucose alone. However, in the presence of 4mm- or 6mm-glucose and 3-isobutyl-1-methylxanthine there was a significant increase in insulin biosynthesis in the progesterone-treated animals. 5. Total islet protein biosynthesis was determined by the incorporation of [(3)H]leucine into trichloroacetic acid-precipitable islet proteins. Islets isolated from normal rats showed a 1.6-fold increase in incorporation over the glucose concentration range 2-20mm, and this value remained unchanged during starvation; however, rates of incorporation were significantly raised in islets isolated from pregnant rats in the presence of 20mm-glucose. 6. Islets from starved and fed control rats were incubated in the presence of increasing concentrations of glucose or glucose+3-isobutyl-1-methylxanthine. The islets isolated from the starved animals showed a diminished insulin-synthetic response to glucose as compared with the controls; this response was partially restored to normal values by elevation of cyclic AMP concentrations by using 3-isobutyl-1-methylxanthine. 7. It is suggested that the alterations in glucose-stimulated insulin biosynthesis observed in islets during pregnancy and after a period of starvation could be attributable, at least in part, to a long-term alteration of the cyclic AMP system, and in pregnancy to a direct or indirect effect of progesterone on beta-cell function.     

Islet-cell metabolism during insulin release. Effects of glucose, citrate, octanoate, tolbutamide, glucagon and theophylline

1. Concentrations of glucose 6-phosphate and 6-phosphogluconate were studied in islets of Langerhans isolated from rat pancreas and incubated in the presence of various agents that induce insulin release. 2. In response to rising concentrations of extracellular glucose (2-10mm) there is a linear increase in the intracellular concentration of glucose 6-phosphate, though this is not the case for 6-phosphogluconate, the intracellular concentration of which only increases when the external glucose concentration exceeds 5mm. 3. Tolbutamide, octanoate and citrate, all of which promote insulin secretion from isolated islets, increase the intracellular concentrations of glucose 6-phosphate and 6-phosphogluconate. The results obtained in the presence of octanoate and citrate are compatible with an inhibitory effect of citrate on islet-cell phosphofructokinase. 4. Theophylline and glucagon when incubated with islets in vitro promote insulin release and cause a rise in 6-phosphogluconate concentration and not in that of glucose 6-phosphate. 5. It is suggested that the further metabolism of glucose 6-phosphate through a pathway other than glycolysis is essential for insulin release. One such pathway involves its oxidation to 6-phosphogluconate, which seems to be a necessary accompaniment of insulin secretion due to glucose. The possibility that agents other than glucose promote insulin release by enhancing the oxidation of glucose 6-phosphate through this pathway is discussed.