Paolo Cecchetti

Kinetic Analysis of Plasma Insulin Disappearance in Nonketotic Diabetic Patients and in Normal Subjects: A TRACER STUDY WITH 125I-INSULIN

The studies so far reported on the metabolic clearance rate of insulin in human diabetes mellitus have given conflicting results, probably because they have been conducted on few patients and have used a variety of experimental techniques and data treatments. We investigated the kinetics of insulin distribution and degradation in 35 normal subjects and in 42 nonketotic, nonobese, overtly diabetic patients, of whom 26 were above 40 yr old and 16 were 40 yr old or less at diagnosis. The design of the study combined (a) the use of a tracer to perturb minimally the steady state and to avoid glucose infusion; (b) the preparation of purified [(125)I]-monoiodoinsulin, which has a metabolic behavior similar to that of native insulin; and (c) noncompartmental analysis of the plasma immunoprecipitable (125)I-insulin disappearance curves, which were recorded for 2 h after pulse i.v. injection of the tracer.Metabolic clearance rate was found to be similar in diabetics (404+/-18 ml/min.m(2), mean+/-SEM) and in normals (420+/-14), although the latter-onset patients had slightly, if not significantly, lower metabolic clearance rate values than the earlier-onset diabetics (385+/-19 and 443+/-36, respectively). The initial distribution volume of the hormone also did not significantly differ in diabetics and normals and was similar to plasma volume. The reentry rate into the initial distribution volume of the hormone and the total, plasma-equivalent distribution volume of insulin were both significantly raised in diabetics (251+/-12 ml/min.m(2) and 10.3+/-0.5 liters/m(2)) in comparison with normals (195+/-8 and 7.5+/-0.3). The posthepatic delivery rate of insulin was found to be slightly raised in later-onset diabetics (194+/-20 mU/h.m(2)), but somewhat reduced in earlier-onset diabetics (133+/-15) in comparison with normals (172+/-14); these differences reflected the different basal plasma insulin concentrations in these three groups. Chronic treatment with oral hypoglycemic drugs, age, duration of the disease, and degree of metabolic control appeared to have only little effect on the kinetics of insulin.On the basis of these results, we conclude that insulin-independent adult diabetics show, already in the fasting state, a combination of insulin resistance and insulin deficiency and a derangement in insulin distribution, the precise significance of which is uncertain.

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFα

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor α (TNFα) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) Ay, ob/ob and goldthioglucose‐treated mice (10‐, 8‐, and 7‐fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFα in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFα in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFα function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFα is an important signal for this regulation. J. Cell. Physiol. 190: 251–258, 2002.

Agonist‐Induced Internalization and Recycling of the Human A3 Adenosine Receptors

Abstract: A3 adenosine receptors have been proposed to play an important role in the pathophysiology of cerebral ischemia with a regimen‐dependent nature of the therapeutic effects probably related to receptor desensitization and down‐regulation. Here we studied the agonist‐induced internalization of human A3 adenosine receptors in transfected Chinese hamster ovary cells, and then we evaluated the relationship between internalization and signal desensitization and resensitization. Binding of N6‐(4‐amino‐3‐[125I]iodobenzyl)adenosine‐5′‐N‐methyluronamide to membranes from Chinese hamster ovary cells stably transfected with the human A3 adenosine receptor showed a profile typical of these receptors in other cell lines (KD = 1.3 ± 0.08 nM; Bmax = 400 ± 28 fmol/mg of proteins). The iodinated agonist, bound at 4°C to whole transfected cells, was internalized by increasing the temperature to 37°C with a rate constant of 0.04 ± 0.034 min‐1. Agonist‐induced internalization of A3 adenosine receptors was directly demonstrated by immunogold electron microscopy, which revealed the localization of these receptors in plasma membranes and intracellular vesicles. Moreover, short‐term exposure of these cells to the agonist caused rapid desensitization as tested in adenylyl cyclase assays. Subsequent removal of the agonist led to restoration of the receptor function and recycling of the receptors to the cell surface. The rate constant of receptor recycling was 0.02 ± 0.0017 min‐1. Blockade of internalization and recycling demonstrated that internalization did not affect signal desensitization, whereas recycling of internalized receptors was implicated in the signal resensitization.

Improvement with metformin in insulin internalization and processing in monocytes from NIDDM patients.

This study investigated the relative effect of obesity alone and in combination with non-insulin-dependent diabetes mellitus (NIDDM) on the intracellular processing of insulin and evaluated the effect of metformin therapy on this process. Monocytes from 11 obese hyperinsulinemic subjects, 13 obese hyperinsulinemic NIDDM patients, and 7 nondiabetic control subjects were incubated with A14-125I-labeled insulin for 60 min at 37 degrees C, and intracellular insulin degradation was characterized by high-performance liquid chromatography. Total cell-associated insulin (insulin binding) and internalized and degraded insulin were decreased in obese subjects and significantly decreased in obese NIDDM patients compared with nondiabetic control subjects. In NIDDM patients, intracellular insulin degradation was inversely correlated with fasting plasma glucose (P less than 0.01). Eight obese subjects and 9 obese NIDDM patients were restudied after 4 wk of therapy with metformin (850 mg twice a day). Plasma levels of the drug were superimposable in the two groups. Metformin therapy did not change glucose and insulin levels in obese subjects but caused a decrease in blood glucose in obese NIDDM patients. Total cell-associated radioactivity (insulin binding) significantly increased in both groups (P less than 0.01). On the contrary, internalized radioactivity increased (0.83 +/- 0.3 vs. 1.31 +/- 0.35%, P less than 0.01), and similarly, insulin degradation was enhanced (54.6 +/- 8.9 vs. 74.22 +/- 9.15%, P less than 0.01) only in monocytes from obese NIDDM patients. However, the levels of these parameters were still lower than in control subjects (internalization, 2.94 +/- 0.68%; degradation, 93.03 +/- 3.7%).(ABSTRACT TRUNCATED AT 250 WORDS)

Salivary insulin concentrations in type 2 (non-insulin-dependent) diabetic patients and obese non-diabetic subjects: relationship to changes in plasma insulin levels after an oral glucose load

SummaryThe presence of immunoreactive insulin in saliva and its relationship to plasma immunoreactive insulin was investigated in healthy subjects, newly diagnosed non-obese Type 2 (non-insulin-dependent) diabetic patients and obese non-diabetic subjects, basally and after an oral glucose tolerance test. The mean ± SEM fasting values of plasma and salivary immunoreactive insulin were significantly higher in diabetic patients and obese non-diabetic subjects than in normal volunteers (p<0.05). During the glucose challenge, the increase of salivary insulin was related with that of plasma in the three groups of subjects, with a time lag in normal and obese subjects. In normal volunteers, plasma and salivary peak values were respectively 49.5 ± 13.4 μU/ml (p<0.05 vs obese subjects) at 60 min and 12.0±3.3μU/min (p<0.05 vs obese subjects) at 120 min; in diabetic patients, the values were 51.7 ± 5.6 μU/ml (p<0.05 vs obese subjects) and 14.6±4.1 μU/min at 120 min; in obese subjects, the peak value for plasma insulin was 111.5±40.1 μU/ml at 90 min and for salivary insulin 15.6 ± 5.1 μU/min at 120 min. A positive linear relationship was shown between plasma and salivary insulin during the oral glucose tolerance test. The identity of salivary insulin was assessed by reversed-phase HPLC. We conclude that salivary immunoreactive insulin can be found in Type 2 diabetic patients and in obese non-diabetic subjects, as well as normal volunteers, that plasma and salivary insulin are related after a glucose load, and that differences exist in salivary insulin secretion patterns among the three groups of subjects.

Plasma biguanide levels are correlated with metabolic effects in diabetic patients.

Metabolic abnormalities occur in biguanide‐treated diabetic patients. We investigated the relationship between plasma metformin and phenformin concentrations and metabolic effects. Drug levels were measured in 37 type II diabetic patients by HPLC. The method was sensitive, specific, and linear over a wide range of drug concentrations. Metformin and phenformin values ranged from 236 to 718 ng/ml and from 28 to 114 ng/ml, respectively. The plasma metformin level was correlated with triglycerides (r = −0.55; P < 0.05) but not with drug dosage, plasma glucose, HbA1, creatinine, creatinine clearance, lactate, pyruvate, lipid, and clinical parameters. Plasma phenformin concentrations correlated with lactate (r = 0.49; P < 0.05) and HbA1 (r = 0.50; P < 0.05) but not with drug dosage, parameters of diabetes control, creatinine, creatinine clearance, pyruvate, and clinical parameters. The clinical usefulness of this HPLC method, the evidence that the increase of lactate is related to the circulating phenformin levels, and the demonstration that the metformin effect on triglyceride metabolism is correlated to plasma drug levels are the positive findings of this work.

Insulin permeability across an in vitro dynamic model of endothelium

AbstractPurpose. Endothelium insulin permeability was investigated using in vitro, dynamic culture of endothelial cells. Methods. Endothelial cells were cultured in a hollow fiber apparatus and continuously exposed to a flow. Transendothelial electrical resistance and permeability to [14C]sucrose and [14C]inulin were used to monitor the integrity of the endothelial monolayer. Results. Under these experimental conditions, measurements of insulin permeability, investigated at increasing hormone concentrations, suggested that the predominant transendothelial insulin fluxes were attributable to bidirectional convective transport rather than to a saturable transport mechanism, in agreement with in vivo experiment results published earlier. Analytical determinations of insulin catabolism demonstrated a low percent of insulin degradation by the endothelium, leading to production of insulin metabolites qualitatively identical to those produced by human monocytes. Conclusions. The findings of this paper indicated that (a) insulin crosses the endothelial monolayer by paracellular “leak” and endothelial insulin receptors have a minor (if any) role in insulin transport; (b) degradation of the hormone by BAEC is minimal; (c) the in vitro, dynamic culture of endothelial cells presented here should represent a valuable transport model system to study permeability mechanisms of insulin and many other drugs.

Insulin Degradation In Vitro and In Vivo: A Comparative Study in Men: Evidence That Immunoprecipitable, Partially Rebindable Degradation Products Are Released From Cells and Circulate in Blood

The products of insulin metabolism generated in vitro and in vivo were compared in this study. Monocytes from 10 control subjects were incubated with 125IA14-labeled insulin, acid washed, and solubilized or reincubated in insulin-free binding buffer to study both intracellular radioactivity or radioactivity released from cells to medium. To evaluate in vivo insulin metabolism, labeled insulin (100–120 μCi) was injected as a single intravenous bolus in 5 of the 10 subjects. Cellular and plasma radioactivity was characterized by high-performance liquid chromatography (HPLC). The results of the study show the following: 1) Products with superimposable HPLC elution profiles are found within cells and in medium. Two new labeled products are observed in the latter, suggesting that a membrane degradation process exists in monocytes. 2) Intermediates found within monocytes, in medium from monocytes, and in plasma have identical elution profiles, supporting the possibility that insulin is metabolized in various cells by a common pathway. 3) Insulin metabolism produces intermediates that bind well to anti-insulin antibody. The presence in plasma of these products induces a significant difference in the value of the metabolic clearance rate of insulin when HPLC or immunoprecipitation is used to detect intact insulin. 4) Immunoprecipitable products maintain, in part, the capacity to bind to insulin receptors and to be internalized into monocytes.

A14-[125I] monoiodoinsulin purified by different high-performance liquid chromatographic procedures and by polyacrylamide gel electrophoresis: Preparation, immunochemical properties and receptor binding affinity

Reversed-phase high-performance liquid chromatography (RP-HPLC) allows the rapid separation of A14-[125I]monoiodoinsulin directly from the iodination mixtures. It remains to be clarified, however, whether the RP-HPLC chromatographic conditions affect the properties of the purified tracer. In this study we prepared A14-[125I]insulin purified by polyacrylamide gel electrophoresis (PAGE) and by three different RP-HPLC mobile phases containing, respectively, ammonium acetate, sodium perchlorate and trifluoroacetic acid. The binding characteristics of all these tracers were examined using an insulin antiserum and insulin cell receptors. The specific radioactivity corresponded to the theoretical maximum for the RP-HPLC-purified tracers and was significantly lower for the PAGE-purified tracers. Significant differences were found in the binding of different tracers to the insulin antiserum: maximum binding ranged from 94 to 99% and was significantly lower for tracers purified by RP-HPLC eluents B and C; antiserum dilution giving 50% tracer binding was lower for tracers purified by RP-HPLC eluent B. The four insulin derivatives showed no difference in non-specific precipitation and in the affinity constant values calculated from the Scatchard analysis. No significant difference was found in the binding of the four insulin derivatives to the human-cultured IM-9 lymphocytes and to the human circulating monocytes. In conclusion, the present work demonstrates that the immunological properties of the A14-[125I]monoiodoinsulin purified by RP-HPLC may be partially affected by the composition of the mobile phase. In order to obtain a fully potent A14-[125I]insulin derivative and to have the possibility of comparing data from different laboratories, the chromatographic conditions must be taken into account.

A prevalence study of known diabetes mellitus in Tuscany assessed from pharmaceutical prescriptions and other independent sources.

This study evaluates the prevalence of diabetes mellitus (DM) in Pisa (Tuscany, Italy) using four independent data sources. The main source, represented by computerized prescriptions for anti-diabetic agents collected over a 4-month period, was validated using three secondary sources: (a) the list of diabetic patients who receive material of self-care from the National Health Service; (b) the clinical records of diabetic patients obtained from a random sample of family doctors; (c) the clinical records of diabetic patients attending our outpatient clinic. The main source provided 3806 patients, and 697 patients were added from the secondary sources, thus identifying a total number of 4503. The prevalence of known DM in the “Pisa area” exclusively reckoned by the main source, was 2.01%, and the prevalence corrected by the addition of the various sources resulted in 2.4%. The capture-recapture method showed a completeness of ascertainment of the survey of 90.1%, and thus an estimated prevalence of known diabetes of 2.64%. Of these, 141 patients had insulin-dependent diabetes mellitus (IDDM) corresponding to 3.2% of identified diabetic subjects (prevalence 0.07% inhabitants); 4362 patients had non-insulin-dependent diabetes mellitus (NIDDM), 96.8% of identified diabetic subjects (prevalence 2.36%). Of patients with NIDDM 10.5% was treated by diet, 65% with oral hypoglycaemic agents (OHA), 23% with insulin and 1.5% with insulin plus OHA. This study shows that the method used in this survey is suitable for epidemiological studies because it does not demand the cooperation of the diabetic patients, is addressed to the entire diabetic population without age discrimination and singles out the diabetic population in a very reliable way.