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Aim: Aim of this study was to analyse clinical correlates of HbA1c, and of overall, nocturnal, and severe hypoglycaemia, through direct-weighted regressions, as well as the effect of different insulin regimens and insulin analogues, through meta-analysis., Methods: Appropriate methodology (PRISMA statement) was used. Sixty-seven randomized studies, published as full papers were analysed to identify predictors of both HbA1c and hypoglycaemia; studies were included in a meta-analysis to study the effect of different insulin regimens or insulin analogues on HbA1c and hypoglycaemia during the first year of insulin treatment in type 2 diabetes patients., Results: Final HbA1c, change of HbA1c, hypoglycaemia, nocturnal hypoglycaemia and severe hypoglycaemia were associated with intensity of treatment. Final HbA1c was higher with basal than with twice-a-day or prandial, and with twice-a-day than with prandial regimen, with opposite figures for hypoglycaemia. Within basal regimens, detemir and glargine were similar to NPH insulin on HbA1c, with less hypoglycaemia and nocturnal hypoglycaemia; within prandial regimens, new analogues were more effective than regular insulin on HbA1c, and induced less hypoglycaemia. The effect of glargine on HbA1c and on hypoglycaemia vanished with increasing number of insulin injections., Conclusion: Metabolic control and hypoglycaemia are associated with intensity of treatment. Basal regimens have a reduced effect on metabolic control, but are associated with lower frequency of hypoglycaemia. Newer analogues, short- and long-acting, yield better control and less hypoglycaemia than older analogues., (© 2011 Blackwell Publishing Ltd.)

Aim: This systematic review and meta-analysis was to evaluate the body weight increase and its clinical correlates, through direct weighted regressions, as well as the effect of different insulin regimens and insulin analogues, through meta-analysis., Methods: Appropriate methodology according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was adhered to. Forty-six randomized studies, published as full papers, reporting the effect of insulin treatment on change in body weight were identified, and used to identify predictors of weight change; studies were included in a meta-analysis to study the effect of different insulin regimens or insulin analogues on weight change., Results: Intensity of treatment [aim of study (fasting blood glucose, mg/dl), dose of insulin, final HbA1c, change of HbA1c and frequency of hypoglycaemia] was significantly associated with body weight increase, with small differences between basal versus twice-a-day and prandial regimen. At meta-analysis, body weight increase was lower with basal regimen than with twice-a-day regimen and than with a prandial regimen. Within all regimens, body weight increase was lower with detemir than with NPH, with no difference between glargine and NPH; only two studies directly compared detemir and glargine, indicating lower weight gain with the former insulin. Within twice-a-day regimens and within prandial regimens, comparison was between newer analogues and older drugs, with no significant difference in body weight increase., Conclusion: Body weight increase during the first year of insulin treatment is associated with the intensity of treatment; body weight increase also depends on the insulin regimen applied., (© 2011 Blackwell Publishing Ltd.)

We evaluated both insulin release (IR) and insulin sensitivity (IS) through a single oral glucose tolerance test (OGTT) (blood samples at 0, 60, 120 min, as routinely performed in Europe) in subjects with normal and abnormal glucose tolerance. The value 1/HOMA was used as an index of IS and deltaI/deltaG at 60 min was used as an index of IR. In preliminary experiments, 1/HOMA correlated with glucose infusion rate (GIR) at euglycaemic insulin clamp (r=0.495) and with insulin sensitivity index (ISI) at LDIGIT (r=0.714). At OGTT with blood samples at 0, 30, 60 and 120 min, insulin levels at 30 min correlated with insulin levels at 60 min (I30 vs. I60, r=0.584) and deltaI/deltaG at 30 and at 60 min correlated (r=0.365). Values of 1/HOMA from 345 subjects with normal glucose tolerance (NGT), 32 with impaired fasting glucose (IFG), 186 with impaired glucose tolerance (IGT) and 72 with type 2 diabetic mellitus were divided into quartiles. For each quartile, mean (+/- SE) and 95% confidence intervals (CI) of deltaI/deltaG at 60 min were calculated, and subjects were represented by plotting IS vs. IR. Plots of NGT, IGT, and type-2 diabetes mellitus described different curves. Values of subjects with IFG, IGT and type 2 diabetes mellitus fell outside the 95% CI of NGT subjects in all quartiles of IS. To validate this finding, 113 morbidly obese subjects (basal OGTT: 55 NGT, 40 IGT, 18 T2DM) who underwent a major reduction of body weight through bariatric surgery received a second OGTT one year after surgery. Glucose tolerance improved in 40 patients, deteriorated in 8, did not change in 65; the new plots were concordant with the new class of glucose tolerance. OGTT can be used to evaluate both IR and IS in subjects with NGT, IFG, IGT, and type 2 diabetes mellitus in population studies and in follow-up studies. IFG, IGT and type 2 diabetes mellitus are characterized by reduced IR compared to IS.

Recent evidence suggests that insulin signaling through the insulin receptor A type (Ex11-), regulates insulin gene transcription. Because chronic hyperglycemia negatively affects insulin receptor function and regulates alternative splicing of the insulin receptor, we inquired whether chronic exposure of pancreatic beta-cells to high glucose results in alterations in insulin signaling due to changes in insulin receptor expression and relative abundance of its spliced isoforms. Our results demonstrate that the insulin receptor is localized in insulin secretory vescicles in human pancreatic beta-cells. Furthermore, we find that alterations in insulin expression and secretion caused by chronic exposure to high glucose are paralleled by decreased insulin receptor expression and increased relative abundance of the Ex11+ isoform in both human islets and RIN beta-cells. PDX-1 and HMGI(Y) transcription factors are down-regulated by high glucose. These changes are associated with defects in insulin signaling involving insulin receptor-associated PI 3-kinase/Akt/PHAS-I pathway in RIN beta-cells. Re-expression in RIN beta-cells chronically exposed to high glucose of the Ex11-, but not the Ex11+, isoform restored insulin mRNA expression. These data suggest that changes in early steps of insulin receptor signaling may play a role in determining beta-cell dysfunction caused by chronic hyperglycemia.

In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 x 10(6) InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic betaTC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines, and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced betaTC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential beta-cell surrogate because they are more resistant than pancreatic beta cells to different apoptotic insults and might therefore be particularly suitable for encapsulation.

Objectives: To evaluate the frequency of impaired glucose tolerance (IGT) and of Type 2 diabetes mellitus (Type 2 DM) in siblings of patients with Type 2 DM, and to assess insulin release and insulin sensitivity in siblings with normal glucose tolerance (NGT), compared with NGT spouses of probands without family history of Type 2 DM., Design and Methods: We evaluated 87 families including 103 Type 2 DM patients (87 probands), and we carried out an oral glucose tolerance test (OGTT) in 130 siblings and in 60 spouses. Among NGT subjects, 12 siblings and 16 spouses underwent a low-dose insulin-glucose infusion test (LDIGIT) to evaluate C-peptide release and insulin sensitivity., Results: After the OGTT, 24 siblings were classified as having Type 2 DM, 31 as IGT, and only 14 spouses as IGT (P=0.0012 vs siblings). NGT siblings (n=75) showed higher insulin levels at 120 min than NGT spouses (n=46) at OGTT, in spite of identical blood glucose levels; at LDIGIT, NGT siblings secreted more C-peptide and showed a lower insulin sensitivity than NGT spouses., Conclusions: These data indicate that middle-aged siblings of probands with Type 2 DM have a high frequency of IGT and Type 2 DM, and that NGT siblings have increased insulin resistance and increased insulin secretion when compared with adequate controls.

We have recently presented experimental evidence indicating that insulin has a physiologic inhibitory effect on growth hormone (GH) release in healthy humans. The aim of the present study was to determine whether in obesity, which is characterized by hyperinsulinemia and blunted GH release, insulin contributes to the GH defect. To this aim, we used a simplified experimental protocol previously used in healthy humans to isolate the effect of insulin by removing the interference of free fatty acids (FFAs), which are known to block GH release. Six obese subjects (four men and two women; age, 30.8 +/- 5.2 years; body mass index, 36.8 +/- 2.8 kg/m2 [mean +/- SE]) and six normal subjects (four men and two women; age, 25.8 +/- 1.9 years; body mass index, 22.7 +/- 1.1 kg/m2) received intravenous (i.v.) GH-releasing hormone (GHRH) 0.6 microg/kg under three experimental conditions: (1) i.v. 0.9% NaCl infusion and oral placebo, (2) i.v. 0.9% NaCl infusion and oral acipimox, an antilipolytic agent able to reduce FFA levels (250 mg at 6 and 2 hours before GHRH), and (3) euglycemic-hyperinsulinemic clamp (insulin infusion rate, 0.4 mU x kg(-1) x min(-1)). As expected, after placebo, the GH response to GHRH was lower for obese subjects versus normals (488 +/- 139 v 1,755 +/- 412 microg/L x 120 min, P < .05). Acipimox markedly reduced FFA levels and produced a mild reduction of insulin levels; under these conditions, the GH response to GHRH was increased in both groups, remaining lower in obese versus normal subjects (1,842 +/- 360 v 4,871 +/- 1,286 microg/L x 120 min, P < .05). In both groups, insulin infusion yielded insulin levels usually observed under postprandial conditions and reduced circulating FFA to the levels observed after acipimox administration. Again, the GH response to GHRH was lower for obese subjects versus normals (380 +/- 40 v 1,075 +/- 206 microg/L x 120 min, P < .05), and in both groups, it was significantly lower than the corresponding response after acipimox. In obese subjects, as previously reported in normals, the GH response to GHRH was inversely correlated with the mean serum insulin (r = -.70, P < .01). In conclusion, our data indicate that in the obese, as in normal subjects, the GH response to GHRH is a function of insulin levels. The finding that after both the acipimox treatment and the insulin clamp the obese still show higher insulin levels and a lower GH response to GHRH than normal subjects suggests that hyperinsulinemia is a major determinant of the reduced GH release associated with obesity.

Background: There is a great need to learn more about porcine islet physiology because porcine islets represent a promising source of xenogeneic tissue for beta-cell replacement therapy in humans., Methods: We evaluated the effects of two important physiological regulators of insulin secretion, glucagon-like peptide-1 (GLP-1) and pituitary adenylate cyclase-activating peptide (PACAP), on insulin release and intracellular calcium ([Ca++]i) by adult porcine islet cells., Results: Exposure to GLP-1 and PACAP significantly potentiated glucose-induced insulin release and improved the sensitivity to glucose as a secretagogue. About 70% of cells stimulated with 20 mmol/L glucose alone showed an increase in [Ca++]i, whereas the addition of GLP-1 and PACAP induced [Ca++]i increases in 86% and 93% of cells, respectively., Conclusions: The good insulin and [Ca++]i responsiveness of porcine islet cells to both GLP-1 and PACAP provides an additional proof of their suitability for transplantation.

Because the pituitary contains hormones with beta-cell trophic activity, we evaluated whether cotransplantation of pituitary tissue with pancreatic islets might be beneficial for islet graft function and survival. Streptozotocin diabetic nude mice were transplanted under the kidney capsule with 150 handpicked islets alone or mixed with two diced pituitaries and were then followed for 4 weeks. Mice transplanted with mixed islet/pituitary grafts had higher levels of circulating prolactin (PRL) than mice transplanted with islets only, while serum cortisol, growth hormone, and follicle-stimulating hormone were similar in the two groups. After transplantation, recipients of mixed islet/pituitary grafts showed a more pronounced decrease in glycemic levels and higher systemic insulin levels than mice transplanted only with islets. Mixed islet/pituitary grafts were macroscopically characterized by an excellent vascularization and were biochemically characterized by higher insulin and PRL content than pure islet grafts. Histologically, posttransplantation remodeling originated a hybrid organ in which healthy, well-vascularized islets were adjacent to pituitary cell clusters. Transplantations performed to address the specific effect of the anterior versus the intermediate pituitary lobes indicated the former as responsible for the improved function of cotransplanted islets. Mixed islet/pituitary grafts composed of anterior lobes were also the best vascularized and were histologically characterized by the presence of many folliculo-stellate cells. In conclusion, we obtained evidence that pituitary cotransplantation significantly improves the function, insulin content, and vascularization of suboptimal islet grafts. Evidence suggesting that ectopically produced PRL and/or locally released angiogenic peptides might play a causal role is provided.

Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm X 50 microm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85+/-14% (n = 5, mean+/-standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.

Eighteen patients with non-insulin-dependent (type 2) diabetes mellitus of normal body weight [body mass index (BMI) <25 kg/m2] without signs of autoimmunity [negative for islet cell antibodies (ICA)], with secondary failure of sulphonylureas, defined as persistent hyperglycaemia in spite of maximal doses of sulphonylureas, were evaluated for C-peptide release under basal conditions and 6 min after i.v. glucagon, for glycosylated haemoglobin (HbA1C), and for fasting and mean daily blood glucose levels. They entered a 6-month, single-blind study in which they were randomly assigned to one of three treatments: (1) insulin plus nicotinamide (group 1, 0.5 g, three tablets/day); (2) insulin plus placebo (group 2, 3 tablets/day); (3) current sulphonylureas plus nicotinamide (group 3, 0.5 g, three tablets/day). They were re-evaluated for C-peptide, HbA1C, and fasting and mean daily blood glucose levels after 6 months. Compared with group 2, C-peptide release increased in both groups 1 and 3, while HbA1C, fasting and mean daily blood glucose levels improved in the three groups to the same extent. With multiple regression analysis, nicotinamide administration was the only significant factor for the improvement of C-peptide release. These data indicate that nicotinamide improves C-peptide release in type 2 diabetic patients with secondary failure of sulphonylureas, leading to a metabolic control similar to patients treated with insulin.

It has been previously reported that in healthy subjects, the acute reduction of free fatty acids (FFA) levels by acipimox enhances the GH response to GHRH. In the present study, the GH response to GHRH was evaluated during acute blockade of lipolysis obtained either by acipimox or by insulin at different infusion rates. Six healthy subjects (four men and two women, 25.8 +/- 1.9 yrs old, mean +/- SE) underwent three GHRH tests (50 micrograms iv, at 1300 h) during: 1) iv 0.9% NaCl infusion (1200-1500 h) after oral acipimox administration (250 mg) at 0700 h and at 1100 h; 2) 0.1 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral acipimox administration (250 mg at 0700 h and at 1100 h); 3) 0.4 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral placebo administration (at 0700 and 1100 h). Serum insulin (immunoreactive insulin) levels were significantly different in the three tests (12 +/- 2, 100 +/- 10, 194 +/- 19 pmol/L, P < 0.06), plasma FFA were low and similar (0.04 +/- 0.003, 0.02 +/- 0.005, 0.02 +/- 0.003, not significant), and the GH response to GHRH was progressively lower (4871 +/- 1286, 2414 +/- 626, 1076 +/- 207 micrograms/L 120 min), although only test 3 was significantly different from test 1 (P < 0.05). Pooling the three tests together, a significant negative regression was observed between mean serum immunoreactive insulin levels and the GH response to GHRH (r = -0.629, P < 0.01). Our results indicate that in healthy subjects, acipimox and hyperinsulinemia produce a similar decrease in FFA levels and that at similar low FFA, the GH response to GHRH is lower during insulin infusion than after acipimox. These data suggest that insulin exerts a negative effect on GH release. Because the insulin levels able to reduce the GH response to GHRH are commonly observed during the day, for instance during the postprandial period, we conclude that the insulin negative effect on GH release may have physiological relevance.

Hyperglucagonemia is commonly found in insulin-dependent as well as in non-insulin-dependent diabetes mellitus, and is likely to be caused by absolute or relative insulin deficiency. The aim of the present study was to evaluate whether a chronic glucagon exposure (1.0 microM for 4 h) modifies the insulin response to acute stimuli with glucagon (1.0 microM), arginine (10.0 mM) and glucose (16.7 mM), or the glucagon response to arginine and glucose, in human islets. Chronic exposure to glucagon did not affect the insulin response to glucose and arginine, but inhibited its response to glucagon (44.6 +/- 9.3 vs 168.6 +/- 52.3 pg/islet per 20 min, P < 0.05); the latter effect was not observed when exposure to glucagon was discontinuous (2.0 microM glucagon alternated with control medium for 30 min periods). The chronic exposure to glucagon also reduced the glucagon response to arginine (- 4.9 +/- 5.7 vs 19.9 +/- 7.9 pg/islet per 20 min, P < 0.05) without affecting the inhibition of glucagon release exerted by glucose. These data indicate that chronic exposure to glucagon desensitizes pancreatic alpha and beta cells in response to selected stimuli.

Calcium ion entry through voltage-operated calcium channels is a crucial step in the coupling of beta cell depolarization with insulin secretion. Various calcium channel subtypes have been shown to be coexpressed in single neurons and endocrine cells. Using the patch-clamp technique, we investigated the biophysical and pharmacological properties of calcium channels in freshly dispersed human pancreatic beta cells. Both low and high voltage activated currents were expressed, the two current types being easily distinguishable on the basis of biophysical criteria. The high voltage activated currents were not homogeneous: one component was affected by the dihydropyridine antagonist nitrendipine and the agonist Bay-K-8644; the other was insensitive to both dihydropyridines and omega-conotoxin GVIA. In line with this pharmacology, nitrendipine reduced and Bay-K-8644 increased glucose-induced insulin secretion from perifused human islets, whereas omega-conotoxin GVIA had no effect. However, about 20% of the glucose-induced insulin release was found to be resistant to high nitrendipine concentrations. These data show that human pancreatic beta cells express heterogeneous voltage-operated calcium channels, only one of which is dihydropyridine-sensitive (L type). The L type channels are clearly involved in the control of insulin secretion, but our data suggest that dihydropyridine- and omega-conotoxin GVIA-insensitive channels may also play a role in the stimulus-secretion coupling of human beta cells.

The purpose of the study was to evaluate fasting endothelin-1 levels in subjects with syndrome X, in subjects with insulinoma, and in normal subjects. The single and synergistic contributions of insulin and triglyceride levels to endothelin-1 release were studied in normal subjects. This was achieved by the evaluation of endothelin-1 levels in response to an insulin bolus combined with a euglycemic clamp (protocol A) and during intralipid (test 1) or saline (test 2) infusions lasting 360 min (protocol B). In protocol B, a euglycemic two-step hyperinsulinemic (25 and 125 mU x kg-1 x h-1) clamp was started at 120 min. Subjects with syndrome X showed significantly higher endothelin-1 levels than normal subjects and subjects with insulinoma (7.22 +/- 0.89 vs. 2.61 +/- 0.38 and 2.49 +/- 0.24 pg/ml, P < 0.01). After an insulin bolus, endothelin-1 levels peaked at 10 min (3.71 +/- 0.96 pg/ml). The incremental area of endothelin-1 was significantly higher after insulin than after a saline bolus. In test 1, an acute increase in triglyceride levels significantly enhanced endothelin-1 levels, with were further increased by the synergistic contribution of high insulin and triglyceride levels. In test 2, endothelin-1 release was achieved at high insulin levels but remained significantly lower than in test 1. In conclusion, subjects with syndrome X showed higher endothelin-1 levels than normal subjects and subjects with insulinoma. These levels were reproduced in normal subjects by a simultaneous increase in insulin and triglyceride levels.

Insulin release was investigated in parallel with changes in cytosolic calcium concentration, [Ca2+]i, in pig islets stimulated by glucose. After two days in culture, glucose stimulation failed to induce insulin release, and caused limited [Ca2+]i changes in few cells. After ten days, insulin response was partially restored and [Ca2+]i recordings revealed a slow oscillatory activity of the whole islet. Slow oscillations appeared to be due to the average [Ca2+]i variations resulting from the spreading of waves throughout the islet. These waves demonstrate the reestablishment of functional cell coupling, which appears to play a critical role in insulin release.

It has been shown that peripheral glucagon secreting cells (A-cells) are lost during most of the isolation procedures employed for pig islets. Loss of A-cells decreases intra-islet glucagon levels and cAMP levels in B-cells and might reduce glucose-induced insulin release. This study was designed to test this hypothesis, by evaluating the effects of culture of porcine islets with exogenous glucagon on insulin secretion and on insulin and cAMP content in islets. Islets were isolated from adult 2-year old Large White pigs using an automated method. The number of A-cells was calculated by immunostaining for glucagon in islets before and after isolation and a significant decrease in A-cells was observed. After an overnight culture, islets were cultured for 48 h in a standard medium (CMRL 1066, 10% foetal calf serum, 1% antibiotics, 1% glutamine) alone or in the presence of glucagon at two different concentrations (1.0 and 10.0 microM); exposure to glucagon was either continuous or alternated with periods of incubation in CMRL 1066 alone. After the 48-h culture in standard medium, the islet glucagon response to arginine was almost negligible and significantly lower than that observed in human islets.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study we investigated a simple nonlabor-intensive method to evaluate insulin sensitivity and beta-cell function which is suitable for application in population studies. The method is a refinement of the modified Harano test and consists of a continuous low dose insulin (25 mU/kg.h) and glucose (4 mg/kg.min) infusion test (LDIGIT) lasting 150 min. Insulin sensitivity was evaluated as the MCR of glucose divided by the steady state serum insulin level achieved at the end of the test. Insulin secretion was expressed as the incremental area for C-peptide concentration during the first 15 min of the test. We compared the indices of insulin sensitivity and insulin secretion yielded by LDIGIT with those derived from the euglycemic clamp and the hyperglycemic clamp, respectively. Fifty-four subjects underwent a LDIGIT (33 with normal glucose tolerance and 21 with impaired glucose tolerance); of the 54, 19 were submitted to a euglycemic clamp, 18 to a hyperglycemic clamp, and 10 to a modified Harano test (insulin infusion, 50 mU/kg.h; glucose infusion, 6 mg/kg.min). LDIGIT overcame the drawbacks associated with the modified Harano test because it resulted in more stable final glucose levels and prevented the occurrence of hypoglycemic episodes. No significant differences were found between the insulin sensitivity index (ISI) of the LDIGIT and that of the euglycemic clamp for each group of subjects. Moreover, there was a strong correlation between the ISI determined by LDIGIT and the ISI determined by clamp (r = 0.90; P < 0.0001), and the best regression line was not different from the identity line, suggesting that the two indices are equivalent. The index of insulin secretion provided by LDIGIT correlated well with that of the hyperglycemic clamp (r = 0.82; P < 0.001) and was significantly higher in overweight subjects than in normal weight subjects. In conclusion, LDIGIT is a simple and accurate method to assess insulin sensitivity and secretion. It can be useful in population studies and in situations when more complex techniques are not feasible.

The aim of this study was to investigate the effect of hyperinsulinemia on the first and second phase of arginine-induced insulin release in humans. Seven healthy subjects underwent three studies (lasting 360 min): a control study using saline infusion and two euglycemic clamps using a low-dose (0.33 mU.kg-1.min-1) and a high-dose (1.20 mU.kg-1.min-1) insulin infusion. After a 3-h equilibration period, arginine (25 g) was infused for 30 min, and insulin and C-peptide responses to arginine were followed for 180 min. At the end of the equilibration period, before arginine administration, steady-state insulin levels were (means +/- SE) 60.0 +/- 2.4, 165.6 +/- 1.8, and 455.4 +/- 7.8 pmol/l during saline, low-dose, and high-dose insulin infusions, respectively. The time course of insulin release during the arginine test was calculated from C-peptide concentrations by using C-peptide kinetic modeling and deconvolution. In particular, first-phase and second-phase insulin response was obtained by integrating the time course of the insulin release during either the first 5 min or the following 40 min of the arginine test, respectively. Whereas first-phase insulin release was independent of any effect induced by either insulin infusion, second-phase insulin release was reduced in a similar degree by both insulin infusion doses. First phase was 75.5 +/- 10.1, 73.7 +/- 12.8, and 73.4 +/- 10.3 pmol/kg, whereas second phase was 266.1 +/- 46.0, 143.1 +/- 33.5, and 133.0 +/- 30.2 pmol/kg for saline, low-dose, and high-dose insulin infusions, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin inhibits its own release (autofeedback), and growth hormone (GH) inhibits the GH response to a variety of stimuli. The aim of this study was to evaluate whether glucagon (G) can modify pancreatic G (IRG) release in humans. Seven healthy men received intravenous (i.v.) arginine (30 g in 30 minutes) 240 minutes after the beginning of a 0.9% NaCI saline infusion and a 2.5-, 4.0-, and 8.0-ng/kg.min-1 porcine G infusion, with each infusion lasting 360 minutes. All G infusions yielded stable and dose-related plasma IRG levels, and the 4.0- and 8.0-ng/kg.min-1 G infusions decreased plasma free fatty acids (FFA) and blood glycerol and beta-OH-butyrate levels and elicited insulin (IRI) release, and the 8.0-ng/kg.min-1 G infusion elicited GH release and increased blood glucose (BG) levels; somatostatin (SRIF) levels were not affected by G infusions. At 240 minutes, plasma IRG levels were higher during G infusion than during saline infusion, whereas serum IRI and BG levels had returned to preinfusion levels. At this point, G infusions decreased the integrated (240 to 300 minutes) IRG, IRI, BG, and SRIF responses, but not the GH response to arginine. These data indicate that prolonged G infusions decrease the IRG response to arginine; in addition, G decreases plasma FFA levels, and higher G doses stimulate IRI release and exert a self-limited hyperglycemic effect. The fact that the IRI response to arginine was decreased by G could be due to a refractoriness of beta cells to subsequent stimuli; the decreased SRIF response to arginine is likely due to G itself or to a decrease of plasma FFA levels.

The ideal hypocaloric diet should reduce body weight, decrease fat more than muscle tissue, and ameliorate insulin sensitivity and lipid levels. The aim of this study was to investigate the effect of three hypocaloric diets with different carbohydrate (CHO) and fat contents on body weight reduction, insulin release and sensitivity, and lipid levels in patients with simple obesity. Twenty-five obese subjects with normal glucose tolerance were randomly allocated to three hypocaloric (800 kcal) diets containing: 60% high complex/high starch and fibre (HC/HSF-CHO) and 20% fat (group 1;11 subjects); 60% high simple/high natural fibre (HS/HNF-CHO) and 20% fat (group 2; 7 subjects); or 20% CHO (L-CHO) and 60% fat (group 3; 7 subjects). The remaining 20% of the diet was protein. In all cases the duration of the diet was 21 days. Before and after the diet, seven subjects from each group underwent a hyperglycemic clamp, and the other four subjects of group 1 underwent a euglycemic-hyperinsulinemic clamp, combined with a glucose turnover study. A similar decrease in body weight, fat-free mass, fat mass, total cholesterol, LDL cholesterol and apo B levels was observed in the three groups. The M/I ratio during hyperglycemic and euglycemic-hyperinsulinemic clamp and the glucose turnover rate during euglycemic-hyperinsulinemic clamp significantly decreased, and FFA levels significantly increased only after the HC/HSF-CHO diet. HDL cholesterol and apo A1 significantly increased only during the HS/HNF-CHO diet.(ABSTRACT TRUNCATED AT 250 WORDS)

The capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 microM for hGH and 1.3 microM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained.

In this study, in vitro responsiveness to glucose of fresh and cultured islets from adult pigs was tested under both static (incubation) and dynamic (perifusion) conditions. Islets were isolated by an automated method from pancreases of 24-month-old animals and cultured overnight in CMRL 1066 and 10% FCS plus antibiotics; islets, perifused immediately after the overnight culture, showed a paradoxical decrease in insulin release when exposed to an acute glucose stimulus (16.7 mmol/L), and a normal response to acute glucose when isobutylmethylxanthine (IBMX) was added to the perifusing buffer. In addition, an acute reduction of glucose concentration in the perifusate elicited a paradoxical insulin release. At the microscope, islets appeared loose and irregularly shaped after the overnight culture; immunohistochemistry showed loss of peripheral A and other mantle cells. After the overnight culture, islets were divided into 5 groups and were cultured for a further 48 hr in different tissue culture media: CMRL 1066; RPMI 1640 (without glucose); RPMI 1640 (plus 11.1 mmol/L glucose); Ham's F12; and medium 199 (all media were supplemented with 10% FCS and antibiotics). During this period, insulin release was 11.4 +/- 1.1 pg/islet/min in islets cultured in CMRL 1066, 16.2 +/- 2.4 in islets cultured in RPMI 1640 (11.1 mmol/L glucose), 1.8 +/- 0.2 (P < 0.001 vs. all the other groups), and 9.0 +/- 0.6 and 8.4 +/- 0.9 pg/islet/min in islets cultured in RPMI 1640 (without glucose), Ham's F12, and medium 199, respectively. After the 48-hr culture in different media, the islets' responsiveness to an acute glucose stimulus (16.7 mmol/L; static incubation) was evaluated: islets cultured in CMRL 1066 and in RPMI 1640 (with and without glucose) showed no insulin response to the acute glucose stimulus; in contrast, insulin release rose from 0.42 +/- 0.06 to 0.60 +/- 0.12 pg/islet/min (NS) in islets cultured in Ham's F12, and from 0.24 +/- 0.06 to 0.48 +/- 0.06 pg/islet/min (P < 0.001) in islets cultured in medium 199. During perifusions, the paradoxical insulin release in response to an acute fall in glucose concentration disappeared, but a significant increase in response to high (16.7 mmol/L) glucose was observed only in islets previously cultured in medium 199. To assess the possible role of glucagon and of cAMP, additional perifusions were done in islets cultured for 48 hr in CMRL 1066 in the presence of glucagon (10 mumol/L) and IBMX (10 mumol/L); glucagon and IBMX were unable to modify the insulin response to 16.7 mmol/L glucose.(ABSTRACT TRUNCATED AT 400 WORDS)

The aim of this study was to evaluate the effects of long term in vitro exposure of human pancreatic islets to different secretagogues on their subsequent secretory activity. Therefore, groups of 100 islets were cultured for 48 h in standard tissue culture medium (CMRL 1066) in the presence of 1 of the following: 5.5 mmol/L glucose, 16.7 mmol/L glucose, 5.5 mmol/L glucose plus 10 mmol/L L-arginine, or 5.5 mmol/L glucose plus 100 mumol/L tolbutamide. Insulin levels in the culture medium declined with time under all culture conditions. Islets were then perifused and acutely stimulated with glucose (16.7 mmol/L), L-arginine (10 mmol/L), and tolbutamide (100 mumol/L). Islets cultured in 16.7 mmol/L glucose showed no response to 16.7 mmol/L glucose [net area under the curve (delta AUC), 11% of control], and a reduced response to acute tolbutamide (delta AUC, 35% of control), but responded to L-arginine (delta AUC, 75% of control). Islets cultured in the presence of 10 mmol/L L-arginine had reduced responses to glucose (delta AUC, 11% of control) and tolbutamide (delta AUC, 27% of control), but responded to L-arginine (delta AUC, 75% of control). Islets cultured in tolbutamide did not respond to tolbutamide (delta AUC, 14% of control) and showed a reduced responses to acute glucose (delta AUC, 36% of control) and L-arginine (delta AUC, 24% of control). In a second set of experiments, islets cultured in 5.5 or 16.7 mmol/L glucose showed an insulin response to a supramaximal glucose stimulation (30 mmol/L glucose plus 0.5 mmol/L isobutylmethylxanthine) that was not statistically different. Similarly, islets that were cultured in the presence of 100 mumol/L tolbutamide still responded to 1 mmol/L tolbutamide. In conclusion, all stimuli evaluated in this study, chronically applied, reduced the insulin response to further acute stimulations. The different patterns of unresponsiveness observed together with the finding of a preserved insulin content in the islets after perifusions and a maintained capability to release insulin in response to supramaximal stimulations suggest that after chronic exposure to different stimuli, human islets become selectively desensitized to the same stimuli given acutely and do not become exhausted.

In normal men, plasma free fatty acids (FFA) are influenced by feeding and by fasting; in addition, iv infusions of methionyl growth hormone (met-GH), so far performed during morning hours, induce an FFA rise that can be blocked by acipimox (ACX), a nicotinic acid analogue. The aims of the present study were to evaluate, in the absence of food interferences, the following aspects: i) Fasting FFA, glycerol, and insulin (IRI) in the morning and in the evening, and their response to met-GH ii) The effect of ACX and of a sustained release ACX formulation (ACX-SR) on fasting and met-GH induced lipolysis and on IRI levels. In a double blind study, 6 normal men received 50% cal at 09:30 h and 50% at 21:30 h; during placebo administrations FFA, glycerol, and IRI levels were lower at 06:30 h than at 18:30 h, and an iv met-GH infusion (160 ng/kg/min lasting 180 min) had a similar effect on FFA and on glycerol at 06:30 h and at 18:30 h. ACX-SR, administered at 21:30 h and 09:30 h, lowered FFA and glycerol in the morning as well as in the evening, and prevented met-GH induced lipolysis in the morning, but not in the evening.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to evaluate the insulin (IRI) response to different stimuli and insulin sensitivity in Type 2 diabetic patients responsive to oral hypoglycaemic agents (OHA) and in Type 2 diabetic patients with secondary failure of OHA (SF), all patients being of normal body weight (relative body weight less than 120%), and the possible role of cyclic AMP in the reduced IRI release. SF patients, without islet cell antibodies (ICA), with hyperglycaemia lasting more than 3 months, underwent tests with i.v. tolbutamide (n = 21), i.v. glucose (n = 14), i.v. glucagon (n = 19), i.v. arginine infusion (n = 18); the arginine infusion was repeated in 12 patients during administration of aminophylline, an inhibitor of phosphodiesterase. The same tests were performed in groups of 8 to 15 OHA patients and in groups of 6 to 17 healthy subjects. During all the tests, blood glucose levels were higher in SF patients, than in OHA patients and in healthy subjects. Both SF patients and OHA patients had no IRI response to glucose; SF patients, in contrast to OHA patients, had a reduced IRI response to tolbutamide and to glucagon. The IRI response to arginine was not different in OHA, in SF patients and in healthy controls, but was significantly enhanced by aminophylline only in healthy controls. Insulin infusions (1.66 mU/Kg/min for 90 min) were performed in OHA patients and in SF patients at blood glucose levels of 150 and of 250 mg/dl: during the last 60 min, the amount of glucose metabolized (M), and the insulin sensitivity (M/I) index were greater in OHA than in SF patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle can utilize glucose by two different mechanisms, one non-insulin-mediated and the other insulin-mediated. The aim of this study was to investigate and to quantify the influence of high and low free fatty acids (FFA) levels on muscle non-insulin-mediated glucose uptake (MNIMGU) and muscle insulin-mediated glucose uptake (MIMGU) and on muscle metabolism during euglycemia and hyperglycemia. Six healthy volunteers were submitted, in a random order, to a 2-hour euglycemic clamp (EC) followed by a 2-hour hyperglycemic (11 mmol/L) clamp (HC) under five different conditions: (1) somatostatin infusion (SRIF, 500 micrograms/h); (2) SRIF infusion preceded by a nicotinic acid analogue (acipimox, 250 mg orally, (3) SRIF plus insulin infusion; (4) SRIF plus insulin plus intralipid infusion; and (5) SRIF plus insulin infusion plus acipimox. In the postabsorptive state MNIMGU represented 71% of the total muscle glucose uptake (MGU) and during the EC a sharp reduction of FFA levels increased the MNIMGU by 10% (P less than .05), and an acute increase in FFA levels decreased the MNIMGU by 26% (P less than .05). MIMGU was significantly increased by 103% after acipimox administration (P less than .05) and was decreased by 65% during intralipid infusion (P less than .05). During HC, MNIMGU was not significantly influenced by low or high FFA levels, and MIMGU was not affected by a sharp lowering of FFA levels, but was significantly decreased (85%) during intralipid infusion. There was no significant difference in the lactate, pyruvate, and alanine balance across the forearm during EC and HC.(ABSTRACT TRUNCATED AT 250 WORDS)

In the experimental animal chronic hyperglycemia alters the islet's sensitivity to glucose. In the present study the glucose sensitivity of human pancreatic islets, isolated and purified, obtained from seven human pancreases using an automated method was evaluated. After a 12-h stabilization period, islets were cultured for 48 h in normal (5.5 mmol/L) or high glucose (16.7 mmol/L) medium. Islets were then perifused to study their insulin response to glucose. Islets cultured in the high glucose medium lost glucose-induced insulin release and, when challenged with an acute fall of glucose concentration in the perifusate, showed a paradoxical insulin release. Insulin release in response to 10 mmol/L L-arginine was preserved in these islets, suggesting a selective reduction of the insulin response to glucose. An additional 48-h culture in 5.5 mmol/L glucose medium partially restored the sensitivity to glucose of the previously unresponsive islets. These findings indicate that short term exposure to high glucose concentrations induces a selective glucose insensitivity of human islets, which can be partially reversed by an additional culture in normal glucose medium.

Clofibrate 3 g/die for 10 consecutive days, significantly reduced arginine-induced IRI release in chemical diabetics and in normal controls, whether of normal body weight or obese. Blood glucose levels were not affected by clofibrate. In agreement with previous findings, it would appear that clofibrate, administered for short periods does not lead to a decrease in glucose tolerance. However, studies relating to the effect of chronic clofibrate administration in chemical diabetes are needed in order to be sure that prolonged inhibition of IRI secretion does not lead to overt diabetes.

The present study was aimed at evaluating the role of the Autonomic Nervous System (ANS) in the insulin (IRI) response to arginine in humans. Nine patients who were recipients of simultaneous segmental pancreatic and renal grafts (6 receiving steroids and azathioprine as immuno suppression therapy, 3 treated with Cyclosporine A), 3 non-diabetic patients with kidney grafts (receiving steroid and azathioprine) and 10 normal subjects were studied. Arginine induced a clear IRI release in all subjects with no significant difference among the groups. Somatostatin (SRIF) inhibited IRI release to a similar degree in all subjects. Since the transplanted pancreas is completely denervated, these data suggest that the integrity of the ANS is not essential to the IRI response to arginine, nor for the inhibitory effect of SRIF on IRI release.

Regular or crystalline insulin with sodium glycocholate as surfactant administered intranasally to normal volunteers induced hypoglycaemia and an increase in serum immunoreactive insulin concentrations. Serum C-peptide concentrations decreased or remained unchanged. Insulin administered intravenously to three of these subjects yielded a potency ratio of 1:8 for intranasal and intravenous insulin. In four insulin-dependent diabetics a cross-over study was performed on different days, insulin being administered once intranasally and once subcutaneously in a ratio of 1:9. In these patients the intranasal insulin was more effective than the subcutaneous insulin in preventing hyperglycaemia after breakfast. In four other insulin-dependent diabetics 11-hours monitoring was performed twice on two different days, insulin being administered in divided dosage sufficient to achieve a reasonable glycaemic profile. The administration during the morning, whereas subcutaneous insulin was more effective than intranasal during the afternoon.

Indirect evidence suggests that sulfonylureas, in addition to stimulating insulin release, exert additional effects at extrapancreatic levels which are of value in the management of type 2 diabetes. In order to characterize in vivo some of these effects, insulin sensitivity was studied in 9 type 1 diabetics with no residual insulin secretory activity, during treatment with chlorpropamide (250 mg b.i.d. for 8 days) and with glipizide (5 mg t.i.d. for 8 days). Employing the glucose clamp technique with the aid of an artificial pancreas (Biostator), glucose disposal during insulin infusion (0.1 U/kg in 60 min) was calculated by the amount of glucose required to keep the blood glucose at preinfusion levels. Chlorpropamide and glipizide administration was accompanied by a significant increase of the amount of glucose required to clamp blood glucose levels, while serum (free) insulin levels were superimposable during the different clamping studies. In the absence of endogenous insulin release, these data strongly suggest that the two sulfonylureas employed enhance in vivo the peripheral sensitivity to insulin. Further studies are required to indicate a preferential site of action (liver, muscle, adipose tissue) of sulfonylureas.

Attempts to achieve a fair metabolic equilibrium in a young woman with brittle diabetes by continuous subcutaneous, intramuscular, and continuous intravenous administration of insulin were unsuccessful. Continuous intraperitoneal administration of insulin through a permanently inserted polyethylene catheter connected to an open-loop peristaltic pump led to an appreciable improvement in mean blood glucose concentration, mean amplitude of glycaemic excursions, and M value and to normalisation of intermediate metabolic products. The peritoneal catheter was well tolerated for over 120 days without appreciable adverse effects. This case suggests that long-term intraperitoneal administration of insulin is a feasible therapeutic approach in the management of brittle diabetes.

In order to evaluate the spontaneous variability of prolactin (PRL) release in response to various stimuli applied repeatedly on different occasions, groups of 5 to 12 subjects each underwent consecutive identical tests with one of the following stimuli applied at 3-6 days' intervals: sulpiride (100 mg im), benserazide (50 mg po), insulin hypoglycaemia (0.1 U/kg b. w. iv) and arginine infusion (25 g iv in 30 min). When repeated in the same subjects, arginine and benserazide yielded superimposable results. In contrast to this, insulin hypoglycaemia yielded significantly lower PRL release, while the PRL response to the second sulpiride test was significantly higher than to the first one. When an interval of 10 days was left between two consecutive sulpiride tests, an identical PRL release was observed. These results indicate that arginine and benserazide are reproducible tests for PRL secretion and it is possible that the decreasing effect of insulin hypoglycaemia on PRL release is due to the stressful effect of the stimulus. Finally, sulpiride probably enhances both PRL release and synthesis thus making greater amounts of PRL available to a subsequent stimulus. Since some of the above stimuli are usual tools for the study of the neuroendocrine control of PRL secretion, our findings suggest that caution appears necessary in attributing to any (neuroactive) drug an effect which might be merely due to a lack of reproducibility of the stimulus employed.

Intranasal administration of glucagon, 1 mg, plus sodium glycocholate 15 mg as a surfactant, raised blood glucose levels and plasma levels of immunoreactive glucagon (IRG) and immunoreactive insulin (IRI). Spray solutions were more effective than drops, and neither the surfactant alone nor glucagon alone had any effect. Blood glucose levels were similarly affected by intravenous glucagon, while intramuscular glucagon was slightly more effective. The highest IRG concentrations were reached after intravenous administration, while intramuscular injection of glucagon was accompanied by the highest IRI release. These data indicate that intranasal administration of glucagon exerts metabolic effects similar to intramuscular and intravenous administrations. Further studies are needed to improve bioavailability and efficacy of intranasally administered glucagon.

The present study was aimed to investigate the metabolic effects of acute and chronic oral administration of maltitol (4-O-i-D-glucopyranosyl-D-glucitol), a hypocaloric sweetener obtained from maltose by catalytic hydrogenation. Fifty grams of maltitol induced a increase lower glycemic and insulinemic than the same dose of glucose or sucrose. No variations of plasma glucose and serum insulin levels were observed after 180 min. A slight increase of plasma maltitol was observed 45 min after maltitol. The chronic administration of maltitol (10 g 3 X daily for 5 days) induced no variations of glycemia or insulinemia when compared with the same dose of sucrose. Plasma sorbitol levels were slightly higher after maltitol than after sucrose. Low amounts of maltitol were detected in the urine and feces.

The aim of the present study was to evaluate the insulin and glucagon responses to various stimuli in patients following pancreatic transplantation. Four Type 1 (insulin-dependent) diabetic patients with end-stage renal failure who had received a cadaveric segmental, neoprene-injected, pancreas transplant, in association with kidney transplantation, were investigated. Free-insulin, pancreatic glucagon, and growth hormone concentrations were measured after both oral and intravenous glucose tolerance tests, and following tolbutamide, arginine and arginine plus somatostatin infusions. Tests were performed 1 month (three cases) and 30 months (one case) after surgery, when no insulin administration was required. Four non-diabetic kidney grafted patients, matched for duration of graft survival and immunosuppressive treatment (steroids, azathioprine and anti-lymphocyte-globulins), served as control subjects. Impaired glucose tolerance was present in all diabetic and control patients. This was possibly related to immunosuppressive treatment. In comparison with control subjects, insulin release was normal in response to arginine and tolbutamide but was reduced in response to oral and intravenous glucose, while glucagon and growth hormone release were similar in both groups. Somatostatin was less effective in diabetic patients than in control subjects in suppressing insulin and glucagon release.

The frequency of secondary failure to oral hypoglycaemic agents (OHA) in patients with non-insulin dependent diabetes (NIDDM) is still unknown, despite more than 30 years of use of OHA. The term secondary failure should be limited to patients who, despite maximal dosages of OHA and despite full compliance with diet and therapy, are no longer controlled and require insulin to obtain an acceptable glucose metabolism. We evaluated 248 out-patients, either on OHA, or on insulin because of poor metabolic control with OHA, in order to assess duration of treatment with OHA since diagnosis, by means of actuarial curves (Mantel-Cox test). Patients with low relative body weight (RBW less than or equal to 100) experienced secondary failure earlier and more often than obese patients (RBW greater than 120) or overweight (RBW 101-120) patients. In 66 of the above out-patients, 33 OHA-treated and 33 insulin-treated, matched for age at onset and duration of disease, islet-cell-antibodies (ICA) and C-peptide release at fasting, 6 min after i.v. glucagon and post prandially were evaluated. Only among lean and overweight patients, was C-peptide release significantly lower in insulin-treated than in OHA-treated patients; differences disappeared in obese patients. ICA were found in only 7 patients (10.6%). HLA phenotype was different from that of healthy blood donors for the loci HLA B5, B13, CW4, with no differences between OHA-treated and insulin-treated patients. These data indicate that secondary failure is more frequent in lean patients with NIDDM, and is related to reduced insulin release.