Water distribution in insulin-dependent diabetes mellitus in various states of metabolic control.

Alterations in water compartments have been described in insulin-dependent diabetes mellitus (IDDM). Both insulin and lack of natriuretic counteracting response lead to water expansion, while hyperglycemica-induced osmotic diuresis leads to water depletion. Both total body water and water distribution in the extra-intracellular space, as well as their relationships to metabolic control, were investigated in 15 controls (30.1 +/- 1.4 years) and in 26 IDDM patients (31.3 +/- 1.6, diabetes duration 11.3 +/- 1.4 years) who were neither hypertensive nor proteinuric. The amounts of total body water (TBW) and extracellular water (ECW) were predicted by impedance measurements at 100 KHz and at 1 KHz. The amount of intracellular water (ICW) was computed as the difference between the two. Water distribution was estimated by measuring the ratio between low- and high-frequency impedance and by computing the ratio between ECW and ICW. The IDDM patients were divided into four groups on the basis of reference HbA(lc) mean and SD: A < or = mean + 2 SD < B < or = mean + 4 SD < C < or = mean +6SD < D. The groups were comparable with sodium intake, insulin dosage, fasting glycemia and laboratory hydration markers. As compared to controls, impedance values at 1, 5, 10, 50 and 100KHz were significantly lower in diabetic patients and the difference within group D increased as the frequency increased: -3.9% at 1 KHz, -10.1% at 100 KHz. As compared to controls, groups A, B and C showed higher TBW, ECW and ICW while water distribution was normal, and group D showed higher TBW and ICW but normal ECW and a different water distribution. In all IDDM patients, HbA(lc) correlated with ECW (r = -0.49) and distribution ratios (r = 0.42, impedance; r = 0.40, ECW/ICW ratio). These observations suggest that good or moderate long-term control IDDM patients have proportionately normal distributions of ECW and ICW excess. However, water excess in poor control IDDM patients was only found in the ICW space.

Hypertension and non-insulin-dependent diabetes. A comparison between an angiotensin-converting enzyme inhibitor and a calcium antagonist.

The effects of the angiotensin-converting enzyme lisinopril were compared with those of the calcium antagonist nifedipine in 162 non-insulin-dependent diabetic hypertensive patients for a 24-week period. In 83 and 79 patients, respectively, lisinopril and slow-release nifedipine produced similar reductions in blood pressure (systolic/diastolic: -16/-13 mmHg supine and -14/-11 mmHg standing after lisinopril; -15/-12 mmHg supine and -14/-11 mmHg standing nifedipine). Fasting and post-prandial plasma glucose, glycosylated haemoglobin and plasma lipids appeared to be unaffected by either agent. Also, 28% of the patients on lisinopril and 30% of those on nifedipine presented microalbuminuria. Both drugs induced a reduction in the albumin excretion rate (AER). The geometric mean x:tolerance factor of the reduction in AER among the 23 microalbuminuric patients on lisinopril (-10.0 x:1.3 micrograms/min) was greater, though not significantly so, than that observed in the 26 on nifedipine (-0.9 x 1.2 micrograms/min). Moreover, lisinopril appeared to be better tolerated than nifedipine in our study population. Microalbuminuria is an important risk factor for cardiovascular mortality in non-insulin-dependent diabetic patients as well as in the general population. To what extent a reduction in the AER could ameliorate diabetic patients is, at present, unknown. Finally, both lisinopril and nifedipine showed a similar antihypertensive effect in these patients which was not associated with significant differences in plasma glucose, insulin or lipid concentrations. The clinical consequences of the insignificant differences in AER remain unclear.

An instrument for real-time spectral estimation of heart rate variability signals.

A Digital Signal Processor (DSP)-based instrument is proposed for estimating and displaying the Heart Rate Variability (HRV) spectrum in real-time. It consists of an intelligent module which is properly interfaced to an IBM PC and whose operations are independent from the computer's other tasks. In this way, the simultaneous recording of the ECG sequence, needed for the more complete off-line analysis, can be performed by the same host. The employed hybrid spectral estimator (in which a classical FFT analysis follows the autoregressive extrapolation of data) appears to be the most apt for the present fixed point arithmetics implementation. The reliability of the instrument and its accuracy are checked both with suitable test signals and by comparison with the results obtained through off-line analysis of the same ECG tracks. The instrument is presently used for cardiovascular investigations, in particular for quickly picking patients with cardiac autonomic neuropathy (CAN) out of a population of diabetic subjects.

Impairment of central motor conduction in diabetic patients.

In 70 patients with diabetes mellitus (DM) we recorded the motor evoked potentials (MEPs) following magnetic stimulation of the motor cortex and spinal roots. Central motor conduction time (CMCT) was determined as the difference between MEP latencies after cortical and spinal stimulation. The mean CMCTs for the biceps, thenar and tibialis anterior muscles were prolonged in the DM group, as compared to normal controls, and 21 patients exceeded the CMCT upper confidence limit for at least one muscle. CMCT changes and peripheral conduction velocity abnormalities occurred independently and were related to different clinical parameters. We conclude that a subclinical impairment of central motor conduction is present in 30% of DM patients, independently from the occurrence of a diabetic peripheral neuropathy and possibly reflecting different pathophysiological mechanisms.

Increased expression of tissue plasminogen activator and its inhibitor and reduced fibrinolytic potential of human endothelial cells cultured in elevated glucose.

In diabetic patients, elevated plasma levels of t-PA and PAI-1 accompany impaired fibrinolysis. To identify mechanisms for these abnormalities, we examined whether vascular endothelial cells exposed to high glucose upregulate t-PA and PAI-1 production and whether ambient PA activity is decreased concomitantly. In 17 cultures of human umbilical vein endothelial cells grown to confluency in 30 mM glucose, the t-PA antigen released to the medium in 24 h was (median) 52 ng/10(6) cells (range 10-384) and the PAI-1 antigen was 872 ng/10(6) cells (range 217-2074)--both greater (P less than 0.02) than the amounts released by paired control cultures grown in 5 mM glucose--29 ng/10(6) cells (range 7.5-216) and 461 ng/10(6) cells (range 230-3215), respectively. In the presence of high glucose, the steady-state levels of t-PA and PAI-1 mRNAs were increased correspondingly (median 142 and 183% of control, respectively, P less than 0.05); high glucose per se and hypertonicity contributed to the upregulation in additive fashion. The PA activity of conditioned medium from cultures exposed to high glucose was 0.4 IU/ml (range 0.2-0.6), which was significantly lower (P less than 0.02) than the PA activity of control medium (0.5 IU/ml, range 0.2-0.9). No difference was observed when comparing the PA activities of acidified conditioned media, expected to be depleted of inhibitors. Thus, high glucose coordinately upregulates endothelial t-PA and PAI-1 expression through effects exerted at the pretranslational level and enhanced by even mild degrees of hypertonicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of carvedilol and nifedipine in patients with essential hypertension and non-insulin-dependent diabetes mellitus.

The safety and tolerability of carvedilol, a new antihypertensive agent with the combined pharmacological activities of beta-blockade and vasodilation, and of nifedipine were investigated in patients with essential hypertension and non-insulin-dependent (type II) diabetes mellitus. Twenty patients were openly randomized to receive 25 mg carvedilol once daily (five men and five women; mean age, 63 years) or 10 mg nifedipine t.i.d. (three men and seven women; mean age, 64 years) for a period of 4 weeks. Baseline mean sitting blood pressures were 168/98 and 169/95 mm Hg in the carvedilol and nifedipine groups, respectively. Baseline mean areas under the curve (AUC) of the intravenous glucose tolerance test (IVGTT) for the carvedilol and nifedipine groups were 6,136 +/- 1,195 and 6,287 +/- 1,228 mg/dl/min, respectively. Demographic and efficacy variables were not statistically different between treatment groups. After 4 weeks of therapy, mean sitting blood pressure was significantly (p less than 0.02) reduced to 144/91 mm Hg in the carvedilol group and to 149/87 mm Hg in the nifedipine group. Week 4 IVGTT AUC values of 5,735 +/- 1,464 mg/dl/min in the carvedilol group and 5,988 +/- 993 mg/dl/min in the nifedipine group, representing mean reductions of 6.14% and 3.17%, respectively, were not statistically different from baseline. Both treatments were well tolerated. No patient experienced adverse events in the carvedilol treatment group, whereas two patients in the nifedipine group reported episodes of headache (one patient) and palpitations (one patient); each episode was mild in severity and considered to be related to study medication.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of pig pancreatic islets by a new method with hydraulic shaking: preliminary report.

The limited availability of human pancreas represents a serious problem in islet transplantation. In the past few years many efforts have been made to isolate pancreatic islets from large mammals in order to achieve valid and reproducible isolation methods. For several reasons swine may be considered an ideal source of islet tissue because of the similarity between human and porcine insulin and because of the easy availability of pig pancreata. Some papers have been published recently on this topic with good results. However, some problems, such as islet dissociation into single cells after collagenase digestion, are not completely solved. In this article, an automated method involving a hydraulic shaking system is described for islet isolation from the pig pancreas, developed in our laboratory and derived from Ricordi's model.

Malondialdehyde (MDA) level in diabetic subjects. Relationship with blood glucose and glycosylated hemoglobin.

The relationships between plasma malondialdehyde (MDA) and metabolic parameters in type I and II diabetic subjects have been studied at different levels of glycemic control. In 67 diabetics (20 type I, 47 type II, aged 53 +/- 1.2) and 40 healthy subjects (aged 47 +/- 1.75), triglycerides (TG), total cholesterol (CT) and C-HDL, fasting blood glucose (FBG), glycosylated hemoglobin (GHb) and MDA were measured. Diabetic population as a whole showed higher MDA plasma levels compared to controls, together with higher FBG, TG, GHb. MDA showed a significant correlation with both FBG and GHb, but was not correlated to plasma lipids. The patients with a poor metabolic control showed the highest plasma MDA concentrations, significantly different from the group with a better control: GHb less than 10% = MDA 2.77 +/- 0.28 nmol/ml - GHb greater than 10% = MDA 4.22 +/- 0.39 nmol/ml (z = 2.10, a less than 0.02); FBG less than 150 mg/dl = MDA 2.74 +/- 0.32 nmol/ml - FBG greater than 150 mg/dl = MDA 4.15 +/- 0.37 nmol/ml (z = 2.22, a less than 0.02). Glycemic equilibrium seemed to influence plasma MDA, increasing free radical production. This phenomenon probably occurred either because of enhanced glycosylation and platelet aggregation, or impairment of cellular antioxidant protective systems. The increased free radical production may play a role in the pathogenesis of metabolic vasculopathy.

Prevention of diabetes-increased aging effect on rat collagen-linked fluorescence by aminoguanidine and rutin.

Products from the advanced Maillard reaction, which increase during aging and diabetes, may contribute to the development of the typical pathology of aging and diabetes. These compounds are detectable only by their characteristic fluorescence, and few data based on long-term studies are available. For this reason, we studied subcutaneous skin collagen fluorescence in 57 nondiabetic (10- to 110-wk-old) and 74 streptozocin-induced diabetic (10- to 22-wk-old) rats. An exponential increase (r = 0.969, P less than 0.001) of collagen-linked fluorescence (excitation at 370 nm, emission at 440 nm) was observed with aging; after a lag, diabetes induced an earlier dramatic elevation of the fluorescence, suggesting a more complicated phenomenon than simple accumulation. To prevent such increases, the effects of 1 g.kg-1.day-1 aminoguanidine, suggested to be an inhibitor of the advanced glycosylation reaction, and 1 g.kg-1.day-1 rutin, an aldose reductase inhibitor, in drinking water were tested. Both treatments had a significant lowering effect on collagen fluorescence in diabetic rats. The mechanisms by which aminoguanidine and rutin prevent the accumulation of fluorescence are unknown, but these observations raise the question of whether they could be identical. If fluorescence is a marker for age-related pathologies and diabetic sequelae, aminoguanidine and rutin could have therapeutic effects in their prevention.

Antipeptide antibodies toward the extracellular domain of insulin receptor beta-subunit.

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.

[Insulin secretion and hepatic extraction in juvenile non-insulin-dependent diabetes]. / Secrezione insulinica ed estrazione epatica nel diabete non insulino dipendente giovanile.

Beta-cell secretion is still a point of controversy. As the liver is the major site of insulin metabolism, evaluation of hepatic insulin extraction is crucial for correct measurement of beta-cell secretion. Methods for calculating the secretion and hepatic extraction of insulin indirectly from peripheral C-peptide concentration have been proposed by some investigators. To characterize the low insulin response of a group of young non-insulin-dependent diabetics we evaluated secretion and hepatic insulin extraction during an oral glucose tolerance test by peripheral IRCP determination and IRCP/IRI molar ratio. Our data show that in this population of young non-insulin-dependent diabetics, the low peripheral insulin response to an oral glucose challenge is a possible consequence of diminished beta-cell secretion, as hepatic insulin extraction is at near normal value.

Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells.

Glucose is known to affect mRNA levels of several genes. In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose. Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations. These data suggest that glucose affects insulin receptor gene expression.

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase.

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.

Platelet and clotting activities after cold stress in diabetic patients.

Platelet and clotting abnormalities have been described in diabetes, but little is known about their relationship to daily stresses. In order to evaluate whether states of abnormal carbohydrate metabolism modify the hemostatic response to stress, 12 subjects with type I diabetes mellitus, 9 with type II, 7 with impaired glucose tolerance and 10 healthy controls were exposed to a cold pressor test. Plasma concentrations of beta-thromboglobulin (index of platelet activation) and of fibrinopeptide A (index of thrombin formation) were measured before and 15 minutes after forearm immersion in melting ice. Basal levels of both proteins were significantly elevated (p less than 0.02) in the combined group of patients with diabetes and impaired glucose tolerance. While in healthy controls cold exposure failed to modify plasma concentration of either protein, obvious changes occurred in the diabetic and impaired glucose tolerance groups. In the combined patients group, beta-thromboglobulin levels decreased from 1.37 +/- 0.44 nmol/l to 1.03 +/- 0.39 (mean +/- SD, p less than 0.01), after the cold test, possibly in consequence of enhanced vascular permeability; while fibrinopeptide A levels increased from 1.52 +/- 1.03 nmol/l to 3.45 +/- 4.19 (p less than 0.02). The degree and pattern of abnormalities observed in basal as well as stimulated levels of fibrinopeptide A differed somewhat among the three groups of patients. These studies indicate that, in the basal state, patients with diabetes or simple carbohydrate intolerance are more susceptible than controls to platelet activation and that after stress thrombin formation can occur although some variability exists among and within groups of patients. The consequences of such increased thrombotic activity may have a bearing on the pathogenesis of large vessel disease, a complication common to diabetes and impaired glucose tolerance.