Insulin elevates hippocampal GABA levels during ischemia. This is independent of its hypoglycemic effect

There are reports that insulin may protect neurons from the effects of ischemia. The mechanisms for this protection are not fully understood. We studied the extracellular levels of glutamate and GABA in insulin-treated animals exposed to transient forebrain ischemia under normoglycemic and hypoglycemic conditions. In vivo microdialysis technique was used to collect extracellular fluid from the CA1 region of the hippocampus. There was a significant increase in GABA levels in the two insulin-treated sub-groups compared with the controls. GABA levels were < 1 pmol/10 microliters in three 10 min collections prior to ischemia in all the groups. It increased from 11.1 +/- 3.5 pmol/10 microliters in the conrol group to 47 +/- 5 (P < 0.001) in the insulin-treated hypoglycemic group and up to 47.2 +/- 9.3+ (P < 0.005) in the insulin-treated normoglycemic group (two-way ANOVA with repeated measures). Ischemia resulted in an increase in the glutamate levels. The glutamate levels returned to baseline within 30 min of the insult. There were no significant differences in the glutamate levels in three groups. The increase in GABA concentrations in the extracellular space may result in the inhibition of CA1 pyramidal neurons. This may be a possible mechanism of neuronal protection in animals treated with insulin (with or without being hypoglycemic) during ischemia.     

Effect of obesity on the response to insulin therapy in noninsulin-dependent diabetes mellitus

An initial improvement in glycemic control is often followed by gradual deterioration of glycemia during insulin treatment of patients with noninsulin-dependent diabetes mellitus (NIDDM). We examined the causes of such worsening in a 12-month follow-up analysis of 100 insulin-treated NIDDM patients in the Finnish Multicenter Insulin Therapy Study who were treated with either combination therapy with insulin or insulin alone. In the entire study group, glycemic control averaged 9.7 +/- 0.2% at 0 months and 8.0 +/- 0.1%, 8.0 +/- 0.1%, 8.2 +/- 0.1%, and 8.5 +/- 0.2% at 3, 6, 9, and 12 months (P < 0.001 for each time point vs. 0 months). Glycemic control at 12 months was significantly worse than that at 3 (P < 0.001), 6 (P < 0.001), and 9 months (P < 0.02). Baseline body mass index was the most significant predictor of deterioration in glycemic control. During 1 yr, hemoglobin A1c decreased almost 3-fold more (by 1.7 +/- 0.2%; P < 0.001 vs. 0 months) in patients whose baseline weight was below the mean baseline body mass index of 28.1 kg/m2 (nonobese patients) than in those whose weight exceeded 28.1 kg/m2 (obese patients; 0.5 +/- 0.2%; P = NS vs. 0 months; P < 0.01 vs. obese patients). Glycemic control improved similarly over 1 yr in the nonobese subjects and deteriorated similarly in the obese patients regardless of their treatment regimen. Insulin doses, per body weight, were similar in the nonobese and obese patients. The nonobese patients consistently gained less weight during 12 months of combination therapy with insulin (3.5 +/- 0.6 kg at 12 months) than during insulin therapy alone (5.1 +/- 0.6 kg; P < 0.05). The treatment regimen did not influence weight gain in the obese group, who gained 4.4 +/- 1.0 kg during combination therapy with insulin and 4.5 +/- 1.1 kg during insulin therapy alone. We reached the following conclusions: 1) after an initial good response, glycemic control deteriorates more in obese than in nonobese patients with NIDDM; 2) in obese patients, weight gain per se cannot explain the poor glycemic response to combination or insulin therapy, but it may induce a disproportionately large increase in insulin requirements because of greater insulin resistance in the obese than in the nonobese; 3) in nonobese patients, glycemic control improves equally during 1 yr with combination therapy with insulin and insulin alone, but combination therapy with insulin is associated with less weight gain than treatment with insulin alone; 4) weight gain appears harmful, as it is associated with increases in blood pressure and low density lipoprotein cholesterol.     

Tumor necrosis factor-alpha in sera of obese patients: fall with weight loss

In view of the recent demonstration that obesity in animals and humans is associated with an increase in tumor necrosis factor-alpha (TNFalpha) expression, that this expression falls with weight loss, and that TNFalpha may specifically inhibit insulin action, the possibility that TNFalpha may be a mediator of insulin resistance has been raised. We have undertaken this study to investigate whether serum TNFalpha concentrations are elevated in obese subjects, whether they fall after weight loss, and whether this fall parallels the fall in insulin release after glucose challenge. Obese patients (age range: 25-54, weight mean +/- SD: 96.4 +/- 13.8 kg, body mass index: 35.7 +/- 5.6 kg/m2) were started on a diet program. The mean weight fell to 84.5 +/- 11.3 (P < 0.0001) and body mass index to 31.3 +/- 4.9 (P < 0.0001). Plasma TNFalpha concentrations were markedly elevated in the obese (3.45 +/- 0.16 pg/mL), when compared with controls (0.72 +/- 0.28 pg/mL), and fell significantly (2.63 +/- 1.40 pg/mL) after weight loss (P < 0.02). The magnitude of insulin release after glucose (75 g) challenge (area under the curve) also fell significantly (P < 0.01) after weight loss. The magnitude of weight loss and fall in TNFalpha were related to basal body weight (r = 0.57, P < 0.001) and basal TNFalpha (r = 0.55, P < 0.001) concentrations, respectively, but not to each other or to the glucose-induced insulin release (area under the curve). We conclude that obesity is associated with increased plasma TNFalpha concentrations, which fall with weight loss. Because circulating TNFalpha may mediate insulin resistance in the obese, a fall in TNFalpha concentrations may contribute to the restoration of insulin resistance after weight loss, Thus, TNFalpha may be an important circulating cytokine, which may provide a potentially reversible mechanism for mediating insulin resistance.     

Disparate effects of insulin on isolated rabbit afferent and efferent arterioles

Despite evidence that insulin per se may be an important regulator of glomerular hemodynamics, little is known about its direct action on the glomerular afferent arterioles (Af-Art) and efferent arterioles (Ef-Art), the crucial vascular segments that control glomerular hemodynamics. In the present study, we examined the direct effect of physiological concentrations of insulin on isolated microperfused rabbit Af- and Ef-Arts. After cannulation, vessels were equilibrated in insulin-free medium for 30 min. To determine whether insulin causes vasodilation or constriction, increasing doses (5, 20, and 200 microU/ml) were added to the bath and lumen of arterioles that were either preconstricted to 50% of control diameter with norepinephrine or left nonpreconstricted. Insulin caused no vasoconstriction in either Af- or Ef-Arts, but it reversed norepinephrine-induced constriction in Ef-Arts but not Af-Arts (suggesting a vasodilator action selective to the Ef-Art): at 200 microU/ml, insulin increased Ef-Art luminal diameter by 75.8 +/- 7.0% from the preconstricted level (n = 6; P < 0.008). The vasorelaxant effect of insulin on Ef-Arts was not affected by blockade of either endothelium-derived relaxing factor/nitric oxide or prostaglandin synthesis. Despite the lack of effect of insulin on Af-Art when added after the equilibration period, when Af-Arts were equilibrated in the presence of either 20 or 200 microU/ml insulin, their basal diameter was significantly reduced (11.7 +/- 0.9 microns; P < 0.025, n = 6, and 12.0 +/- 0.9 microns; P < 0.025, n = 7, respectively) compared with nontreated Af-Arts (16.2 +/- 1.3 microns; n = 7). In conclusion, this study demonstrates that at physiological concentrations, insulin dilates NE-constricted Ef-Arts, while insulin pretreatment enhances Af-Art tone. The disparate actions of insulin on the Af- vs the Ef-Art may contribute to its beneficial effect on glomerular hypertension.     

Importance of peripheral insulin levels for insulin-induced suppression of glucose production in depancreatized dogs

It is generally believed that glucose production (GP) cannot be adequately suppressed in insulin-treated diabetes because the portal-peripheral insulin gradient is absent. To determine whether suppression of GP in diabetes depends on portal insulin levels, we performed 3-h glucose and specific activity clamps in moderately hyperglycemic (10 mM) depancreatized dogs, using three protocols: (a) 54 pmol.kg-1 bolus + 5.4 pmol.kg-1.min-1 portal insulin infusion (n = 7; peripheral insulin = 170 +/- 51 pM); (b) an equimolar peripheral infusion (n = 7; peripheral insulin = 294 +/- 28 pM, P < 0.001); and (c) a half-dose peripheral infusion (n = 7), which gave comparable (157 +/- 13 pM) insulinemia to that seen in protocol 1. Glucose production, use (GU) and cycling (GC) were measured using HPLC-purified 6-[3H]- and 2-[3H]glucose. Consistent with the higher peripheral insulinemia, peripheral infusion was more effective than equimolar portal infusion in increasing GU. Unexpectedly, it was also more potent in suppressing GP (73 +/- 7 vs. 55 +/- 7% suppression between 120 and 180 min, P < 0.001). At matched peripheral insulinemia (protocols 2 and 3), not only stimulation of GU, but also suppression of GP was the same (55 +/- 7 vs. 63 +/- 4%). In the diabetic dogs at 10 mM glucose, GC was threefold higher than normal but failed to decrease with insulin infusion by either route. Glycerol, alanine, FFA, and glucagon levels decreased proportionally to peripheral insulinemia. However, the decrease in glucagon was not significantly greater in protocol 2 than in 1 or 3. When we combined all protocols, we found a correlation between the decrements in glycerol and FFAs and the decrease in GP (r = 0.6, P < 0.01). In conclusion, when suprabasal insulin levels in the physiological postprandial range are provided to moderately hyperglycemic depancreatized dogs, suppression of GP appears to be more dependent on peripheral than portal insulin concentrations and may be mainly mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and by the suppressive effect of insulin on glucagon secretion. These results suggest that a portal-peripheral insulin gradient might not be necessary to effectively suppress postprandial GP in insulin-treated diabetics.     

Down-regulation of hepatic and renal 11 beta-hydroxysteroid dehydrogenase in rats with liver cirrhosis

Twenty-four crossbred primiparous sows were used to investigate the influence of insulin administration after weaning on the intrafollicular insulin-like growth factor i (IGF-I) system. Sows received 0.4 i.u. insulin kg-1 bodyweight or an equivalent volume of saline for 3 days (n = 5 insulin; n = 4 saline) or 5 days (n = 5 insulin; n = 6 saline) after weaning or served as untreated controls on day 1 (n = 4). The number and diameters of ovarian follicles were recorded, and fluid was aspirated from the 20 largest follicles for determination of oestradiol and IGF-I by radioimmunoassay and of insulin-like growth factor-binding proteins (IGFBPs) by western ligand blotting. The walls of the follicles were collected for mRNA analysis by RNase protection assay or granulosa cells were collected for estimation of apoptosis by flow cytometry. Insulin treatment resulted in smaller diameters of all follicles (P < 0.05) and tended (P < 0.07) to increase the number of follicles available on day 5 compared with saline-treated animals (19.8 versus 17.8). The concentration of oestradiol in follicular fluid from large (7-10 mm) follicles on days 3 and 5 was reduced (treatment by size class interaction; P < 0.05) by insulin treatment. Insulin also reduced intrafollicular concentrations of IGF-I at days 3 and 5 after weaning (treatment by day interaction; P < 0.02) while the amounts of IGFBP-3 and IGFBPs of molecular mass 30 and 22 kDa decreased from day 3 to day 5 in saline-treated animals only (treatment by day interaction; P < 0.05). Gene expression for IGF-I increased in saline-treated animals but decreased fourfold in insulin-treated sows from day 3 to day 5 (treatment by day interaction; P < 0.002). Gene expression for IGFBP-d decreased (P < 0.04) from day 3 to day 5, while expression of IGFBP-2 was unaffected by treatment or day. Overall, insulin influenced the IGF-I system in a manner consistent with slowing follicular growth and possibly allowed more follicles to become available for ovulation.     

Magnesium responsiveness to insulin and insulin-like growth factor I in erythrocytes from normotensive and hypertensive subjects

Depletion of intracellular free magnesium (Mg(i)) is a characteristic feature of insulin resistance in essential hypertension, but it is not clear to what extent low Mg(i) levels contribute to insulin resistance, result from it, or both. As insulin-like growth factor I (IGF-I) may improve insulin resistance, we investigated whether this peptide could similarly improve Mg(i) responsiveness to insulin in hypertension, and whether this effect was related to any direct IGF-I effect on Mg(i). 31P-Nuclear magnetic resonance spectroscopy was used to measure Mg(i) in erythrocytes from 13 fasting normotensive and 10 essential hypertensive subjects before and 30, 60, and 120 min after incubation with a physiologically maximal dose of insulin (200 microU/mL) and with different doses of recombinant human IGF-I (0.1-100 nmol/L). In normotensive subjects, IGF-I elevated Mg(i) (P < 0.05) in a dose- and time-dependent fashion, as did insulin (P < 0.05). However, in hypertensive subjects, maximal Mg(i) responses to insulin, but not to IGF-I, were blunted [insulin, 163+/-11 to 177+/-10 micromol/L (P=NS); IGF-I, 164+/-6 to 190+/-11.7 micromol/L (P < 0.05)]. Furthermore, for insulin, but not for IGF-I, cellular Mg(i) responsiveness was closely and directly related to basal Mg(i) levels (insulin: r=0.72; P < 0.01; IGF-I: r=0.18; P=NS). Lastly, blunted Mg(i) responses to insulin could be reversed by preincubation of hypertensive cells with IGF-I. We conclude that 1) both IGF-I and insulin stimulate erythrocyte Mg(i) levels; 2) cellular Mg(i) responses to insulin, but not to IGF-I, depend on basal Mg(i) levels, i.e. the higher the Mg(i) the greater the sensitivity to insulin; and 3) IGF-I potentiates insulin-induced stimulation of Mg(i) at doses that themselves do not raise Mg(i). These effects of IGF-I may underlie at least in part its ability to improve insulin sensitivity clinically. Together, these data support a role for IGF-I in cellular magnesium metabolism and emphasize the importance of magnesium as a determinant of insulin action.     

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

Short-term hypocaloric nutrition but not bed rest decrease insulin sensitivity and IGF-I bioavailability in healthy subjects: the importance of glucagon

Hyperinsulinemic, normoglycemic clamps were performed before and after 24 h of either hypocaloric nutrition or bed rest in healthy subjects. Decreased insulin sensitivity and insulin-like growth factor-I (IGF-I) bioavalibility, as measured by the serum IGF-I/insulin-like growth factor binding protein-1 (IGFBP-1) ratio, was found after fasting, whereas no metabolic changes were found after bed rest. Glucagon seems to be a key regulator of IGFBP-1 after brief hypocaloric nutrition. Hypocaloric nutrition and immobilization may add to the catabolic response to surgery and other trauma. Presently, six healthy subjects were studied before and after a 24-h period of hypocaloric nutrition (200 kcal/24 h, fast) or immobilization (bed rest) using the hyperinsulinemic (0.8 mU.kg-1.min-1), normoglycemic (4.5 mmol/L) clamp, indirect calorimetry, and circulating levels of substrates and hormones. After fast, body weight decreased (P < 0.05), and nitrogen balance was negative (-10 +/- 1 g urea nitrogen/24 h). Basal levels of free fatty acids, glucagon, and IGFBP-1 increased (P < 0.05), whereas c-peptide levels and the IGF-I/IGFBP-1 ratio decreased (P < 0.05). However, no change was found in basal levels of IGF-1 or substrate oxidation. Furthermore, changes (%) in basal levels of glucagon after fast correlated to IGFBP-1 (r = 1.0, P < 0.05), whereas the suppressibility of IGFBP-1 by insulin was maintained at normal levels. During clamps, glucose infusion rates (GIR) decreased after fast (-43 +/- 13%, mean +/- SEM, P < 0.001). Although not significantly, clamp levels of fat oxidation tended to increase and glucose oxidation tended to decrease. Levels of IGFBP-1 during clamps were higher as compared with the control clamp (P < 0.05). No adverse metabolic changes were seen after bed rest, and no change in GIR during clamps were seen as compared with the control measurement (0 +/- 14%). After brief hypocaloric nutrition, insulin sensitivity is reduced, whereas IGF-I bioavalibility is reduced by an increase in levels of IGFBP-1. Glucagon seems to contribute to the increase in IGFBP-1 during these conditions.     

Neural network subtyping of depression

OBJECTIVE: To examine the mechanisms by which weight loss improves glycemic control in overweight subjects with NIDDM, particularly the relationships between energy restriction, improvement in insulin sensitivity, and regional and overall adipose tissue loss. RESEARCH DESIGN AND METHODS: Hyperinsulinemic glucose clamps were performed in 20 subjects (BMI = 32.0 +/- 0.5 [SEM] kg/m2, age = 48.4 +/- 2.7 years) with normal glucose tolerance (NGT) (n = 10) or mild NIDDM (n = 10) before and on the 4th (d4) and 28th (d28) days of a reduced-energy (1,100 +/- 250 [SD] kcal/day) formula diet. Body composition changes were assessed by dual energy x-ray absorptiometry and insulin secretory changes were measured by insulin response to intravenous glucose before and after weight loss. RESULTS: In both groups, energy restriction (d4) reduced fasting plasma glucose (FPG) (delta FPG: NGT = -0.4 +/- 0.2 mmol/l and NIDDM = -1.1 +/- 0.03 mmol/l, P = 0.002), which was independently related to reduced carbohydrate intake (partial r = 0.64, P = 0.003). There was a marked d4 increase in percent of insulin suppression of hepatic glucose output (HGO) in both groups (delta HGO suppression: NGT = 28 +/- 15% and NIDDM = 32 +/- 8%, P = 0.002). By d28, with 6.3 +/- 0.4 kg weight loss, FPG was further reduced (d4 vs. d28) in NIDDM only (P = 0.05), and insulin sensitivity increased in both groups (P = 0.02). Only loss of abdominal fat related to improvements in FPG (r = 0.51, P = 0.03) and insulin sensitivity after weight loss (r = 0.48, P = 0.05). In contrast to insulin action, there were only small changes in insulin secretion. CONCLUSIONS: Both energy restriction and weight loss have beneficial effects on insulin action and glycemic control in obesity and mild NIDDM. The effect of energy restriction is related to changes in individual macronutrients, whereas weight loss effects relate to changes in abdominal fat.     

Markers of bone turnover in hyperthyroidism and the effects of treatment

Serum osteocalcin (OC) and bone-specific alkaline phosphatase (B-ALP), reflecting bone formation, and urinary pyridinoline cross-link (Pyr) excretion, reflecting bone resorption, have been measured in 27 patients with hyperthyroidism and 30 age-matched controls using direct and novel immunoassays. Hyperthyroid patients had higher (P < 0.001) levels of all 3 markers compared with control values: Pyr, 246 +/- 181 nmol/mmol creatinine vs. 40 +/- 12 (+515%); OC, 55 +/- 23 vs. 23 +/- 7.4 micrograms/L (+139%); and B-ALP, 22 +/- 17 vs. 10.0 +/- 5.0 micrograms/L (+120%). OC and Pyr levels were elevated above the normal range in most patients and were significantly correlated with serum free T3 concentrations (r = 0.53; P < 0.01 and r = 0.76; P < 0.001; for OC and Pyr, respectively). B-ALP levels were elevated in 11 of the 27 patients and did not correlate with serum thyroid hormone concentrations. After therapy for hyperthyroidism, Pyr and OC levels returned to normal within 1 month, whereas B-ALP transiently increased after 1 month before falling to baseline levels. The relapse of hyperthyroidism observed in 1 patient was associated with a steep increase in bone markers. These results indicate that Pyr, measured using a new and convenient immunoassay, is a highly sensitive marker for altered bone metabolism in hyperthyroidism. The increases in OC and B-ALP were less impressive, suggesting an imbalance between resorption and formation with subsequent rapid bone loss in untreated hyperthyroidism. OC and B-ALP also appear to reflect different aspects of osteoblast metabolism during the treatment of hyperthyroid patients.     

Chronic exposure of rat fat cells to insulin enhances lipolysis and activation of partially purified hormone-sensitive lipase

The activity of adipose tissue hormone-sensitive lipase in animals with hyperinsulinemia has been reported to be increased compared with that in control animals. We examined whether this results from a direct effect of insulin on the tissue and whether it is accompanied by alteration in the regulation of lipolysis. When rat epididymal fat pads are incubated in culture medium with bovine serum albumin for 2-4 h with 2 ng/ml or 50 microU/ml of insulin, hormone-sensitive lipase activity in the postmicrosomal supernatant fraction after acid precipitation and activation with ATP-Mg2+ increases significantly compared with preparations from tissues incubated with the vehicle. The specific activities of hormone-sensitive lipase in sonicates of adipocytes after primary culture with insulin at concentrations from 10 to 4000 ng/ml (250 microU to 100 mU/ml) increase in an insulin-dose-related manner. Lipolysis in response to 10(-7) M isoproterenol also increases in an insulin-dose-dependent manner. Enhancement of isoproterenol-mediated lipolysis is not attributable to a difference in the triglyceride content of the cells. Lipolysis caused by the beta-agonist could be completely blocked by the simultaneous presence of insulin in both control and insulin-treated cells reflecting normal responsiveness of both types of cells to the acute effect of insulin. Although an increase in lipolysis is seen with norepinephrine and growth hormone after insulin treatment, other lipolytic agents such as ACTH, thyrotropin, and glucagon evoke similar responses in insulin-treated and control cells. The simultaneous presence of growth hormone and insulin during the 16-h culture results in additive effects on the subsequent response of the cells to 10(-7) M isoproterenol compared with the responses of the cells cultured with each hormone alone. beta-Agonist-mediated cAMP accumulation in the presence of Ro-20.1724, a specific phosphodiesterase inhibitor, is significantly higher in cells cultured in the presence of insulin than in control cells. Forskolin (1-25 microM) increases the lipolytic responses of insulin-treated cells compared with control cells, but the maximal response of the insulin-treated cells to forskolin is lower than that to isoproterenol. We conclude that changes produced by chronic insulin treatment involve more than one site along the lipolytic cascade.     

Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal beta-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, O3, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7.5 microM, compared with a scrambled (MSO3) control oligo (n = 3, P < 0.01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1.78 +/- 0.13 (MSO3) vs 0.19 +/- 0.14 (O3) pmol/10(6) cells per 240 min, n = 7, P < 0.01). A significant increase in insulin mRNA (100 +/- 10% (MSO3) vs 124 +/- 8% (O3), n = 3, P < 0.05) and insulin content (13.0 +/- 0.9 (MSO3) vs 17.4 +/- 1.4 (O3) pmol/10(6) cells, n = 7, P = 0.028) was observed following treatment with O3 at 7.5 microM. O8, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than O3, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated beta-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.     

The three-dimensional bone interface of an osseointegrated implant. I: A morphometric evaluation in initial healing

This study was designed to determine the effect of a potent cholecystokinin antagonist, L-364,718, on canine pancreatic endocrine function following partial pancreatectomy. Plasma glucose, insulin, and glucagon were determined over a 2-hr interval following an intravenous bolus of 0.5 g/kg glucose in a 50% solution. The following groups were established: normal animals (group A, n = 5), normal animals pretreated with 20 nmole/kg L-364,178 (group B, n = 5), partially pancreatectomized animals (group C, n = 5), and partially pancreatectomized animals pretreated with 20 nmole/kg L-364,178 (group D, n = 5). In contrast to animals with an intact pancreas, pretreatment with L-364,718 following partial pancreatectomy resulted in a significant decrease in peak insulin (group C = 132.8 +/- 13.0 microU/ml vs Group D = 90.4 +/- 16.1 microU/ml, P < 0.05) and the basal-to-peak insulin difference (group C = 111.9 +/- 11.5 microU/ml vs group D = 77.5 +/- 16.6 microU/ml, P < 0.05). Despite this, the rate of glucose utilization (K value) was significantly increased in the partially pancreatectomized animals given the antagonist (group C = -1.22 +/- 0.22%/min vs group D = -2.79 +/- 0.427%/min) and there were no significant differences in basal or peak glucose when comparing the groups given L-364,718 with the groups given placebo (group A vs B and group C vs D). Thus, the CCK antagonist L-364,718 significantly decreases peak insulin in partially pancreatectomized animals but not in nonoperative control animals. There is a paradoxical increase in the rate of glucose utilization but no effect on glucose homeostasis. The effect of this antagonist in other models of reduced islet cell reserve (i.e., pancreas transplantation) remains to be determined.     

Serum beta-2 microglobulin is a marker of high bone remodelling in elderly women

Beta-2 microglobulin (beta2m), the water soluble extrinsic light chain of class I MHC, has been recently isolated from the adult bone culture medium. Serum beta2m plays a role as a bone-derived growth factor regulating both osteoblast and osteoclast cell activity. Serum beta2m has been proposed as a bone remodeling biological marker in high bone turnover conditions. The purpose of our study was to determine the relationship between beta2m and vitamin D status in post-menopausal women. We have studied 44 healthy women from 20 to 80 years with normal hepatic and renal function, without diabetes mellitus and/or inflammatory, tumoral or infectious diseases. We measured the serum levels of calcium, phosphorus, parathyroid hormone (PTH), vitamin D binding protein (DBP), 25-OHD3 (calcidiol), 1,25(OH)2D3 (calcitriol) and beta2m. Serum beta2m levels increased with age (r = 0.54, P < 0.001). Post-menopausal women had higher serum levels than pre-menopausal women of beta2m (1.76 +/- 0.22 mg/l vs. 1.35 +/- 0.2 mg/l, P < 0.01); PTH (61.5 +/- 7.5 ng/ml vs. 39 +/- 6 ng/ml, P < 0.001) and lower serum levels of 25-OHD3 (7.5 +/- 2.3 ng/ml vs. 18.2 +/- 2.5 ng/ml, P < 0.001). Moreover, serum levels of beta2m were negatively correlated with 25-OHD3 (r = -0.34, P < 0.05) and with ionized calcium (r = -0.45, P < 0.01) and positively with PTH (r = 0.48, P < 0.01). These results support the role of beta2m as a regulator of bone metabolism and its potential use as a marker of high bone turnover in post-menopausal women, specially in elderly women with vitamin D deficiency and secondary hyperparathyroidism.     

Time-action characteristics of regular and NPH insulin in insulin-treated diabetics

The time course of action of regular and NPH insulins injected sc was studied in 15 insulin-treated diabetics over a 24-h period during which they received a constant infusion of glucose. The blood glucose began to decline in 1.2 +/- 0.1 h (range, 0.5--2) and reached its nadir in 5.7 +/- 0.3 h (range, 4--8) after the sc injection of regular insulin. The peak effect of regular insulin usually persisted for several hours, and the total duration of action was 16.2 +/- 1.1 h (range, 9--24). Both the time of peak effect and the total duration of action were considerably prolonged compared to data provided in standard textbooks. Free insulin increased to a peak in 2.7 +/- 0.3 h (range, 1--4) after regular insulin injection and then returned to baseline by 8.8 +/- 0.96 h. Subcutaneous injection of NPH insulin decreased the blood glucose by 2.4 +/- 0.5 h (range, 1--7), with a maximal effect at 11.0 +/- 1.4 h (range, 5--19). The total duration of effect on blood glucose was 25.1 +/- 0.7 h (range, 20--29). These values are similar to those in standard textbooks. Although the total insulin levels increased after the injection of NPH insulin, there was very little if any elevation in free insulin. Recognition of the prolonged effect of regular insulin is important in establishing an insulin treatment regime for diabetic patients.     

Overnight normalization of glucose concentrations improves hepatic but not extrahepatic insulin action in subjects with type 2 diabetes mellitus

Subjects with poorly controlled type 2 diabetes are both hyperglycemic and insulin resistant. To determine whether short term restoration of normoglycemia improves insulin action, hyperinsulinemic (approximately 300 pmol/L) euglycemic clamps were performed in diabetic subjects after either overnight infusion of saline or overnight infusion of insulin in amounts sufficient to maintain euglycemia throughout the night. Fasting glucose concentrations (5.2 +/- 0.2 vs. 11.9 +/- 1.4 mmol/L; P < 0.01) and rates of endogenous glucose production (13.0 +/- 1.1 vs. 18.6 +/- 1.6 mumol/kg.min; P < 0.05) were both lower after overnight insulin than overnight saline. Insulin-induced stimulation of glucose uptake (to 34.9 +/- 6.8 vs. 28.8 +/- 3.4 mumol/kg.min; P = 0.2) and inhibition of free fatty acids (to 0.13 +/- 0.03 vs. 0.12 +/- 0.04 mmol/L; P = 0.6) did not differ after overnight saline and overnight insulin. In contrast, endogenous glucose production during the final hour of the hyperinsulinemic clamps (i.e. when glucose concentrations were the same) remained higher (P = 0.05) after overnight saline than after overnight insulin (5.5 +/- 1.5 vs. 0.02 +/- 1.4 mumol/kg.min). Thus, acute restoration of euglycemia by means of an overnight insulin infusion improves hepatic (and perhaps renal) but not extrahepatic insulin action.     

Amylin-mediated reduction in insulin sensitivity corresponds to reduced insulin receptor kinase activity in the rat in vivo

Studies were undertaken to elucidate further the mechanism whereby the pancreatic peptide amylin induces insulin resistance. Sixteen male Sprague-Dawley rats underwent hyperinsulinemic (14 pmol/kg/min, 0 to 120 minutes) euglycemic clamps in the presence or absence of amylin (500 pmol/kg/min, 60 to 120 minutes). Amylin induced insulin resistance at both the hepatic level (mean +/- SE: hepatic glucose output [HGO] with amylin 1.4 +/- 0.2 v without amylin -1.9 +/- 0.3 mmol/kg/h, P < .001) and peripheral level (glucose disposal [Rd] with amylin 5.0 +/- 0.2 v without amylin 8.5 +/- 0.6 mmol/kg/h, P < .001). Serum insulin levels were similar in the presence or absence of amylin alone (661 +/- 89 v 636 +/- 50 pmol/L, respectively, P = NS), but were significantly less when somatostatin (SRIF) was simultaneously infused (408 +/- 15 pmol/L, P < .02 v the other two groups). This suggests that endogenous insulin production was not suppressed by amylin under these study conditions. Similar findings were obtained in 18 animals in the absence of exogenous insulin infusion. In vitro kinase activity toward histone of skeletal muscle insulin receptors (IRs) activated by insulin in vivo was reduced in the presence of amylin to 6.0 +/- 0.8 versus 9.1 +/- 1.2 fmol phosphate into histone (insulin-infused) and 3.9 +/- 0.7 versus 6.9 +/- 1.4 (non-insulin-infused; P < .03 by ANOVA). Serum calcium was significantly decreased in amylin-treated animals (1.93 +/- 0.04 v 2.30 +/- 0.05 mmol/L, P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Transgenic overproduction of islet amyloid polypeptide (amylin) is not sufficient for islet amyloid formation

Islet amyloid polypeptide forms islet amyloid deposits in non-insulin-dependent diabetes mellitus. We have generated transgenic mice which express human islet amyloid polypeptide in their pancreatic beta cells yet do not develop islet amyloid deposits despite producing levels of the amyloidogenic human peptide 2 - 3 fold higher than the native (mouse) peptide. To determine whether marked overproduction of islet amyloid polypeptide is a potential cause of islet amyloid formation, we increased expression of this transgene by producing homozygous transgenic animals and by making heterozygous mice experimentally insulin resistant with nicotinic acid. Pancreatic content of islet amyloid polypeptide-like immunoreactivity in homozygous and nicotinic acid-treated mice was 2-fold (25 +/- 7 fmol/microg; n = 6) and 3.5-fold (47 +/- 20 fmol/microg; n = 3) higher, respectively, than that of untreated heterozygous animals (13+/-2 fmol/microg; n = 11; both p < 0.05). Despite this marked increase in production of islet amyloid polypeptide, neither group of mice developed gross islet amyloid deposits even after 16 months of age. We conclude that overproduction of islet amyloid polypeptide, even as produced by extreme insulin resistance, is not in itself sufficient for islet amyloid formation.     

Determination of optimal conditions for synthesis of peroxynitrite by mixing acidified hydrogen peroxide with nitrite

Decreased retinal blood flow has been measured in streptozotocin (STZ)-induced diabetes of 1 week's duration, and primary insulin intervention was effective in maintaining normal retinal blood flow in diabetic rats. Retinal blood-flow abnormalities precede clinical diabetic retinopathy in both diabetic animals and patients. An important characteristic of diabetic retinopathy is the difficulty of reversibility once it has been established. Because altered retinal hemodynamics is a possible marker of early diabetic retinopathy, we investigated in this study whether retinal blood-flow changes in rats can be normalized by secondary insulin intervention following short and chronic periods of untreated STZ-induced diabetes. Subcutaneous insulin pumps were placed into diabetic rats for 1 week after 1 week of diabetes (2-week group) and after 3 weeks of diabetes (4-week group). Retinal circulatory parameters were determined using image analysis of video fluorescein angiogram recordings. For the 2-week group, retinal blood flow was significantly (P < 0.05) reduced in the untreated diabetic rats compared with nondiabetic and insulin-treated diabetic rats (80.6+/-29.2, 131.9+/-50.1, and 151.3+/-54.0 pixels2/s respectively). Retinal blood flow was also significantly (P < 0.05) reduced in the 4-week untreated diabetic rats compared with nondiabetic rats (95.7+/-22.2 vs. 125.7+/-29.5 pixels2/s). In contrast to the shorter-duration group, insulin treatment for 1 week after 3 weeks of diabetes did not totally normalize retinal blood flow (117.5+/-32.4 pixels2/s). These results suggest that vascular abnormalities could become more resistant to normalization following short-term (1 week) insulin treatment after longer periods of untreated diabetes.