Massimo Buscema

Comparison of Combined Therapies in Treatment of Secondary Failure to Glyburide

Objective To compare the effectiveness of alternative combined treatments in patients with non-insulin-dependent diabetes mellitus (NIDDM) with secondary failure to sulfonylureas. Research Design and Methods A crossover study was carried out by randomly assigning 16 NIDDM patients to a combined treatment with the addition of either a single low-dose bedtime injection of 0.2 U/kg body wt NPH insulin or an oral three times a day administration of 1.5 g/day metformin to the previously ineffective glyburide treatment. Results Both combined therapies significantly (P <0.01) reduced fasting plasma glucose (FPG), postprandial plasma glucose (PPPG) and percentage of HbA1. The addition of metformin was more effective than the addition of insulin (P <0.01) in improving PPPG in the 8 patients with higher post-glucagon C-peptide levels. In contrast, the efficacy of neither combined therapy was related to patient age, age of diabetes onset, duration of the disease, percentage of ideal body weight, and FPG. The addition of insulin but not metformin caused a significant (P <0.01) increase of mean body weight. Neither combined treatment caused changes in serum cholesterol and triglyceride levels. No symptomatic hypoglycemic episode was reported in any of the 16 patients. CONCLUSIONS The addition of bedtime NPH insulin or metformin was effective in improving the glycemic control in most NIDDM patients with secondary failure to glyburide. The combination of metformin and sulfonylurea was more effective in reducing PPPG and did not induce any increase of body weight.

Genetics of Specific Phenotypes of Congenital Hypothyroidism: A Population-Based Approach

Congenital hypothyroidism (CH) may cause severe and irreversible neurologic and developmental abnormalities when not recognized early. Many millions of newborns have now been screened and many thousands of patients with CH have been identified. Approximately 80%-85% have defects of thyroid gland development, while 15%-20% have congenital errors of thyroid hormone biosynthesis. An entire population screened for CH over a long period of time, was studied in the present report, using a population-based approach. In particular, two CH phenotypes, both presenting with in situ thyroid gland (patients with either goiter or with thyroid gland volume ranging from normal to hypoplasic) were analyzed. Mutations were searched in some of the most likely candidate genes: thyroperoxidase (TPO) in patients with CH goiter, Pax8 and thyrotropin receptor (TSHR) in the other group. In the former group (n = 8), four TPO gene mutations were identified in three patients. One patient was a compound heterozygous. In two cases an already described mutation (1277(insGGCC)) was present; in two other cases mutations not previously described (1996(G-->T) and 2295(G-->A)), which induced aminoacid variations with a Glu --> Stop and Val --> Ile changes, respectively, were identified. In all patients mutations were inherited from one of the parents. In the case of the compound heterozygous patient, one mutation was inherited from the mother (1277(insGGCC)) and the other from the father (1996(G-->T), Glu --> Stop). In the latter group (n = 8), a patient with a 16-base pair C(T)(13)CC deletion in TSHR gene intron 8, 42-bp distal to exon/intron 8 splice junction, was identified. No mutation was identified in Pax8 gene.

Glucose Modulates Glucose Transporter Affinity, Glucokinase Activity, and Secretory Response in Rat Pancreatic β-Cells

Pancreatic islets were cultured for 24 h in medium containing either low (1.4), normal (5.5), or high (16.7 mM) glucose, and then insulin secretion was measured at the end of 1 h incubation at 37°C. Insulin release in the absence of glucose was 64 ± 20,152 ± 11, and 284 ± 30 pg · islet−1 · h−1 (mean ± SE, n = 6, G1.4 and G16.7 vs. G.5.5, P < 0.05) and the response to 22 mM glucose stimulation was 640 ± 136, 2460 ± 276, and 1890 ± 172 pg · islet−1 · h−1, respectively (n = 6, G1.4 vs. G5.5, P < 0.01, G16.7 vs. G5.5, P = 0.065). The 50% maximal response of insulin secretion (increment over baseline) was reached at an average glucose concentration of 9.9 ± 0.7 mM in islets preexposed to G5.5, and at glucose 13.3 ± 0.9 and 4.8 ± 0.4 mM (P < 0.05 in respect to G5.5) in islets preexposed to G1.4 and G16.7, respectively. To investigate the molecular mechanism responsible for this altered glucose sensitivity, we measured, in parallel experiments, the kinetic characteristics of glucose transport, glucokinase, and glucose utilization. Glucose transport was measured by evaluating 3-O-methylglucose uptake. The apparent Km of the low-affinity transporter (GLUT2) was 16.6 ± 2.4 mM in isolated pancreatic cells cultured at 5.5 mM glucose. In cells cultured at both low (1.4 mM) or high (16.7 mM) glucose concentrations, a significant change in the apparent Km of this glucose transporter function was observed (24.4 ± 2.9 and 7.1 ± 0.6 mM, n = 5, P < 0.05 and < 0.01, respectively), with no change in the Vmax of the uptake. Under the same experimental conditions a concomitant change in glucokinase activity was observed: the enzyme Vmax was 4.9 ± 0.32, 8.7 ± 0.79, and 15.8 ± 0.98 μmol · μg DNA−1 · h−1 in islets From the Institute of Internal Medicine, Metabolism and Endocrinologyexposed to either 1.4, 5.5, or 16.7 mM glucose, respectively (mean ± SE, n = 5, G1.4 and G16.7 vs. G.5.5, P < 0.05), with no significant change in the enzyme Km. In control islets glucose utilization, measured by 3H20 production from [5-3H]glucose, had a Km of 8.0 ± 1.7 mM and a Vmax of 8.9 ± 0.41 nmol · μg DNA−1 · 2 h−1. In islets exposed to either G1.4 or G16.7, the glucose utilization Vmax was decreased and increased (5.3 ± 0.56 and 13.8 ± 0.98 nmol · μg DNA−1 · 2 h−1 n = 5, P < 0.05), parallel with the changes observed in glucokinase activity. Moreover, the apparent glucose utilization Km was increased in G1.4 preexposed islets (15.6 ± 1.3 mM) and decreased in G16.7 preexposed islets (3.7 ± 0.9 mM), parallel with the changes observed in the glucose transport Km. These studies indicate that in vitro the sensitivity and responsiveness of pancreatic islets to glucose may be regulated by the ambient glucose concentrations. They support the concept that glucokinase plays a pivotal role in regulating glucose metabolism Vmax, and, therefore, the β-cell responsiveness to glucose. In addition, they also indicate that changes in the affinity (Km) of the glucose transporter are associated to changes in the Km of glucose utilization, thus suggesting a possible role of glucose transport in determining the β-cell sensitivity to glucose.

Direct effects of biguanides on glucose utilization in vitro

The effect of the biguanides metformin and phenformin on glucose utilization in isolated cells was studied with IM-9 human lymphocytes. Both agents stimulated glucose consumption from the incubation media. Detectable effects of metformin were seen at 33 mumol/L and detectable effects of phenformin were seen at 1.7 mumol/L. Both agents, at similar concentrations, also stimulated [3H] 2-deoxy-D-glucose uptake. Studies with phenformin indicated that biguanides increase the Vmax of uptake without changing the Km. In contrast to the biguanides, IM-9 cells insulin did not influence either glucose consumption or [3H] 2-deoxy-D-glucose uptake. These data provide evidence, therefore, that biguanides may directly influence the cellular utilization of glucose.

Copper addition prevents the inhibitory effects of interleukin 1-β on rat pancreatic islets

SummarySince copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-Iβ (IL-1β), we studied whether CU(II) addition could prevent the inhibitory effect of IL-1β on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1β, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-l-histidyl-l-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO4 (1–1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0±11.4 pg · islet−1 h−1 (mean ± SEM,n=7) and glucose-induced release was 2824.0±249.0 pg · islet−1 h−1. In islets pre-exposed to 50 U/ml IL-1β, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2±76.9,n=7,p<0.005). In islets incubated with IL-1β and Cu-GHL (0.4 μmol/l, maximal effect) basal secretion was 119.0±13.1 pg · islet−1 h−1 and glucose-induced release was 2797.2±242.2, (n=7,p<0.01 in respect to islets exposed to IL-1β alone). In contrast to data obtained with Cu(II)-GHL, increasing concentrations of CuSO4 (up to 10 μmol/l) did not influence the inhibitory effect of IL-1β on glucose-stimulated insulin release. Glucose oxidation (in the presence of 16.7 mmol/l glucose) was 31.5±2.4 pmol · islet−1·90min−1 in control islets and 7.0±0.9 (p<0.01) in IL-1β-exposed islets. In islets exposed to IL-1β and Cu-GHL glucose oxidation was similar to control islets (31.9±1.9). In contrast, Cu-GHL did not prevent the IL-1β-induced increase in nitric oxide production. Nitrite levels were 5±1.7, 26±5 and to 29±4 pmol · islet−1·48 h−1 (mean ± SEM,n=5) in the culture medium from control IL-1β and IL-1β+Cu-GHL exposed islets, respectively. These data indicate that the Cu(II) complexed to GHL is able to prevent the inhibitory effects of IL-1β on insulin secretion and glucose oxidation, but not on NO production. The mechanism of action of Cu-GHL is still unclear, but it might restore the activity of the enzymatic systems inhibited by IL-1β. [Diabetologia (1995) 38∶39–45]

Metformin enhances certain insulin actions in cultured rat hepatoma cells

SummaryThe effect of the oral antidiabetic agent metformin on insulin regulation of glycogen metabolism, tyrosine-aminotransferase activity, and [1-14C]aminoisobutyric acid uptake was studied in H4IIE cultured rat hepatoma cells. Metformin enhanced both basal (from 0.213±0.016 to 0.262±0.024 nmol/mg protein,p<0.01) and insulin stimulated [3H] glucose incorporation into glycogen in a time-dependent and dose-dependent manner. A small effect of metformin was seen at 1 μmol/l, and its greatest effects were obtained at 10 μmol/l. At the same concentrations, metformin did not influence basal tyrosine-aminotransferase activity but it potentiated insulin stimulated tyrosine-aminotransferase activity (+ 29.2±1.4%,p<0.01) and prevented the loss of tyrosine-aminotransferase responsiveness to insulin in H4IIE cells desensitised by a previous exposure to insulin. In contrast, metformin had no effect on basal or insulin-stimulated [1-14C]aminoisobutyric acid uptake. Over the concentrations of metformin that enhanced insulin action in H4IIE cells, the drug had no significant effect on insulin binding to its receptor. These studies suggest, therefore, that metformin may influence cellular metabolism by potentiating certain insulin actions through mechanisms that may be beyond insulin receptor binding.

Chronic exposure to glibenclamide impairs insulin secretion in isolated rat pancreatic islets

We investigated the effect of 24 h exposure to 100 nmol/l glibenclamide on insulin secretion in isolated rat pancreatic islets. The insulin content was similar in control islets and in islets preincubated with 100 nmol/l glibenclamide for 24 h. In islets preexposed to glibenclamide: 1) the subsequent response to a maximal glibenclamide stimulatory concentration (10 µmol/l, 1 h at 37 C) was greatly reduced in comparison to control islets (0.69 ± 0.20 % vs 2.16 ± 0.41%; mean ± SE; n=14; p < 0.001); 2) the response to 100 µmol/l tolbutamide stimulation was also reduced (0.55 ± 0.15% vs 2.38 ± 0.44 %; n=8; p < 0.001); 3) the response to 16.7 mmo/l glucose, both in the presence or in the absence of 1 mmol/l IBMX, a phosphodiesterase inhibitor, was also diminished by about 50% (1.79 ± 0.39% vs. 3.22 ± 0.42%; n= 14, p < 0.001). In glibenclamide pretreated islets, blunted responses to stimuli were confirmed also by dynamic studies using a perifusion system. The effect of glibenclamide preincubation was fully reversible: when islets cultured in the presence of glibenclamide were transferred to a glibenclamide-free medium for further 24 h, insulin release in response to glibenclamide stimulation returned to control values. We conclude that prolonged exposure of rat pancreatic islets to glibenclamide induces a reversible desensitization to a variety of metabolic stimuli. The inhibition by prolonged glibenclamide exposure of a common pathway in the mechanism of insulin release is one possible explanation for these results.

Nicotinamide partially reverses the interleukin-1β inhibition of glucose-induced insulin release in pancreatic islets

Interleukin-1 beta (IL-1 beta) is known to inhibit glucose-induced insulin release by pancreatic islets. We studied the effect of nicotinamide, an inhibitor of poly[adenosine diphosphate (ADP)-ribose] synthetase and a free-radical scavenger, on this IL-1 beta-induced inhibition using rat pancreatic islets. In static experiments, groups of five islets were incubated for 24 hours in culture medium CMRL-1066, with or without 50 U/mL IL-1 beta, in the presence or absence of nicotinamide (dose range, 0 to 50 mmol/L), and then exposed for 1 hour to either 1.4 or 19.4 mmol/L glucose, 10 mmol/L arginine, or 10 mumols/L glyburide. Basal insulin secretion was 183 +/- 32 pg/islet/h (mean +/- SE, n = 7) and 176 +/- 39 (n = 7) in control islets and in islets exposed to 50 U/mL IL-1 beta, respectively. Glucose-stimulated insulin secretion was significantly reduced (185 +/- 41) in IL-1 beta-exposed islets in comparison to control islets (2,037 +/- 363). In parallel, arginine-stimulated insulin release was inhibited by IL-1 beta exposure (166 +/- 31 pg/islet/h, mean +/- SE, n = 3) in comparison to control islets (1,679 +/- 307). In contrast, IL-1 beta exposure did not significantly reduce glyburide-induced insulin secretion (1,516 +/- 231 and 1,236 +/- 214 in control and IL-1 beta-exposed islets, respectively; mean +/- SE, n = 3). When islets were simultaneously exposed to IL-1 beta and increasing concentrations of nicotinamide, a dose-dependent recovery of glucose-induced insulin secretion was observed, with the maximum effect at 25 mmol/L nicotinamide (1,007 +/- 123, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic Exposure to High Glucose and Impairment of K+-Channel Function in Perifused Rat Pancreatic Islets

Prolonged exposure to high glucose levels impairs the ability of pancreatic islets to secrete insulin as a response to that stimulus. Because glucose, like other insulin secretagogues, elicits insulin secretion by inhibiting the ATP-sensitive K+ channels, in this study, we investigated the effect of prolonged (24-h) exposure of rat pancreatic islets to high (16.7 mM) glucose concentration on 86Rb efflux (used as a tracer for K+). The data obtained indicate that islets exposed to high glucose concentration have impaired function of the glucose-sensitive K+ channel, this phenomenon is temporarily related to a defective response of glucose-induced insulin release, and these alterations are reversible.

Efficacy of real-time continuous glucose monitoring on glycaemic control and glucose variability in type 1 diabetic patients treated with either insulin pumps or multiple insulin injection therapy: a randomized controlled crossover trial.

The aim of this study was to determine the efficacy of real‐time continuous glucose monitoring in T1D patients treated with insulin pump therapy or multiple daily insulin therapy.