Paolo Tessari

Differential effects of hyperinsulinemia and hyperaminoacidemia on leucine-carbon metabolism in vivo. Evidence for distinct mechanisms in regulation of net amino acid deposition.

The effects of physiologic hyperinsulinemia and hyperaminoacidemia, alone or in combination, on leucine kinetics in vivo were studied in postabsorptive healthy subjects with primed-constant infusions of L-[4,5-3H]leucine and [1-14C]alpha-ketoisocaproate (KIC) under euglycemic conditions. Hyperinsulinemia (approximately 100 microU/ml) decreased (P less than 0.05 vs. baseline) steady state Leucine + KIC rates of appearance (Ra) from proteolysis, KIC (approximately leucine-carbon) oxidation, and nonoxidized leucine-carbon flux (leucine----protein). Hyperaminoacidemia (plasma leucine, 210 mumol/liter), with either basal hormone replacement or combined to hyperinsulinemia, resulted in comparable increases in leucine + KIC Ra, KIC oxidation, and leucine----protein (P less than 0.05 vs. baseline). However, endogenous leucine + KIC Ra was suppressed only with the combined infusion. Therefore, on the basis of leucine kinetic data, hyperinsulinemia and hyperaminoacidemia stimulated net protein anabolism in vivo by different mechanisms. Hyperinsulinemia decreased proteolysis but did not stimulate leucine----protein. Hyperaminoacidemia per se stimulated leucine----protein but did not suppress endogenous proteolysis. When combined, they had a cumulative effect on net leucine deposition into body protein.

Effect of Metformin on Insulin-Stimulated Glucose Turnover and Insulin Binding to Receptors in Type II Diabetes

Euglycemic insulin glucose-clamp and insulin-binding studies on erythrocytes and monocytes were performed in seven type II (non-insulin-dependent) diabetic subjects before and after 4 wk of metformin treatment (850 mg 3 times/day) and in five obese subjects with normal glucose tolerance. Glucose turnover was also measured at basal insulin concentrations and during hyperinsulinemic euglycemic clamps. During euglycemic insulin-glucose clamps, diabetic subjects showed glucose disposal rates of 3.44 ± 0.42 and 7.34 ± 0.34 mg · kg−1 · min−1 (means ± SD) before metformin at insulin infusion rates of 0.80 and 15.37 mU · kg−1 · min−1, respectively. With the same insulin infusion rates, glucose disposal was 4.94 ± 0.55 (P < .01) and 8.99 ± 0.66 (P < .01), respectively, after metformin treatment. Glucose disposal rates in normal obese subjects were 5.76 ± 0.63 (P < .01) and 10.92 ± 1.11 (P < .01) at 0.80 and 15.37 mU · kg−1 · min−1, respectively. Insulin maximum binding to erythrocytes in diabetics was 9.6 ± 4.2 and 5.8 ± 2.6 × 109 cells (means ± SD) before and after metformin treatment, respectively (NS). Insulin maximum binding to monocytes in diabetics was 6.2 ± 2.3 × 107 cells before and 5.0 ± 1.6% after metformin. Hepatic glucose production was higher in the diabetic patients at basal insulin levels, but not at higher insulin concentrations, and was not significantly changed by drug treatment. Basal glucose and insulin concentrations decreased with metformin. Thus, metformin treatment improved glucose disposal rate without significant effect on insulin-binding capacity on circulating cells. Basal hepatic glucose output was slightly lower after metformin treatment in view of lower (9 vs. 15 μU/ml) insulin levels, potentially indicating increased sensitivity of the liver to insulin.

High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity.

Background To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. Methodology/Principal Findings Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. Conclusions/Significance Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.

Kidney, splanchnic, and leg protein turnover in humans. Insight from leucine and phenylalanine kinetics.

The rate of kidney protein turnover in humans is not known. To this aim, we have measured kidney protein synthesis and degradation in postabsorptive humans using the arterio-venous catheterization technique combined with 14C-leucine, 15N-leucine, and 3H-phenylalanine tracer infusions. These measurements were compared with those obtained across the splanchnic bed, the legs (approximately muscle) and in the whole body. In the kidneys, protein balance was negative, as the rate of leucine release from protein degradation (16.8 +/- 5.1 mumol/min.1.73 m2) was greater (P < 0.02) than its uptake into protein synthesis (11.6 +/- 5.1 mumol/min. 1.73 m2). Splanchnic net protein balance was approximately 0 since leucine from protein degradation (32.1 +/- 9.9 mumol/min. 1.73 m2) and leucine into protein synthesis (30.8 +/- 11.5 mumol/min. 1.73 m2) were not different. In the legs, degradation exceeded synthesis (27.4 +/- 6.6 vs. 20.3 +/- 6.5 mumol/min. 1.73 m2, P < 0.02). The kidneys extracted alpha-ketoisocaproic acid, accounting for approximately 70% of net splanchnic alpha-ketoisocaproic acid release. The contributions by the kidneys to whole-body leucine rate of appearance, utilization for protein synthesis, and oxidation were approximately 11%, approximately 10%, and approximately 26%, respectively; those by the splanchnic area approximately 22%, approximately 27%, and approximately 18%; those from estimated total skeletal muscle approximately 37%, approximately 34%, and approximately 48%. Estimated fractional protein synthetic rates were approximately 42%/d in the kidneys, approximately 12% in the splanchnic area, and approximately 1.5% in muscle. This study reports the first estimates of kidney protein synthesis and degradation in humans, also in comparison with those measured in the splanchnic area, the legs, and the whole-body.

Defective suppression by insulin of leucine-carbon appearance and oxidation in type 1, insulin-dependent diabetes mellitus. Evidence for insulin resistance involving glucose and amino acid metabolism.

To determine whether a resistance to insulin in type 1, insulin-dependent diabetes mellitus (IDDM) is extended to both glucose and amino acid metabolism, six normal subjects and five patients with IDDM, maintained in euglycemia with intravenous insulin administration, were infused with L-[4,5-3H]leucine (Leu) and [1-14C]alpha ketoisocaproate (KIC). Steady-state rates of leucine-carbon appearance derived from protein breakdown (Leu + KIC Ra) and KIC (approximately leucine) oxidation were determined at basal and during sequential euglycemic, hyperinsulinemic (approximately 40, approximately 90 and approximately 1,300 microU/ml) clamps. In the euglycemic postabsorptive diabetic patients, despite basal hyperinsulinemia (24 +/- 6 microU/ml vs. 9 +/- 1 microU/ml in normals, P less than 0.05), Leu + KIC Ra (2.90 +/- 0.18 mumol/kg X min), and KIC oxidation (0.22 +/- 0.03 mumol/kg X min) were similar to normal values (Leu + KIC Ra = 2.74 +/- 0.25 mumol/kg X min) (oxidation = 0.20 +/- 0.02 mumol/kg X min). During stepwise hyperinsulinemia, Leu + KIC Ra in normals decreased to 2.08 +/- 0.19, to 2.00 +/- 0.17, and to 1.81 +/- 0.16 mumol/kg X min, but only to 2.77 +/- 0.16, to 2.63 +/- 0.16, and to 2.39 +/- 0.08 mumol/kg X min in the diabetic patients (P less than 0.05 or less vs. normals at each clamp step). KIC oxidation decreased in normal subjects to a larger extent than in the diabetic subjects. Glucose disposal was reduced at all insulin levels in the patients. In summary, in IDDM: (a) Peripheral hyperinsulinemia is required to normalize both fasting leucine metabolism and blood glucose concentrations. (b) At euglycemic hyperinsulinemic clamps, lower glucose disposal rates and a defective suppression of leucine-carbon appearance and oxidation were observed. We conclude that in type 1 diabetes a resistance to the metabolic effects of insulin on both glucose and amino acid metabolism is present.

Effects of acute systemic hyperinsulinemia on forearm muscle proteolysis in healthy man.

To investigate the mechanism(s) of insulin-induced suppression of plasma amino acid concentration and release, we studied forearm as well as whole-body leucine and phenylalanine uptake and release during a peripheral insulin infusion in postabsorptive normal subjects using isotope-dilution methods. Before insulin, leucine and phenylalanine release exceeded uptake (P less than 0.01 and P less than 0.07, respectively). A net output of alpha-ketoisocaproate (KIC) was also observed. During insulin, arterial plasma leucine, KIC and phenylalanine concentrations decreased (P less than 0.05 or less vs. basal), despite ongoing net output of these substrates by the forearm, that persisted after correction for the mean transit time spent through the extracellular muscular space. By the end of insulin, whole-body leucine and phenylalanine concentrations and rate of appearance were decreased (P less than 0.01 vs. basal). However, release and uptake of both amino acids by the forearm were not significantly decreased vs. the preinsulin values. These data indicate that systemic hyperinsulinemia acutely decreases plasma amino acid concentrations by acting primarily at sites other than skeletal muscle.

Nitric Oxide Synthesis Is Reduced in Subjects With Type 2 Diabetes and Nephropathy

OBJECTIVE Nitric oxide (NO) is a key metabolic and vascular regulator. Its production is stimulated by insulin. A reduced urinary excretion of NO products (NOx) is frequently found in type 2 diabetes, particularly in association with nephropathy. However, whether the decreased NOx excretion in type 2 diabetes is caused by a defective NOx production from arginine in response to hyperinsulinemia has never been studied. RESEARCH DESIGN AND METHODS We measured NOx fractional (FSR) and absolute (ASR) synthesis rates in type 2 diabetic patients with diabetic nephropathy and in control subjects, after l-[15N2-guanidino]-arginine infusion, and use of precursor–product relationships. The study was conducted both before and after an euglycemic hyperinsulinemic (∼1,000–1,200 pmol/l) clamp. RESULTS In type 2 diabetes, NOx FSR was reduced both under basal (19.3 ± 3.9% per day, vs. 22.9 ± 4.5% per day in control subjects) and hyperinsulinemic states (24.0 ± 5.6% per day, vs. 37.9 ± 6.4% per day in control subjects; P < 0.03 by ANOVA). Similarly, in type 2 diabetes, NOx ASR was lower than in control subjects under both conditions (basal, 0.32 ± 0.06 vs. 0.89 ± 0.34 mol per day; hyperinsulinemia, 0.35 ± 0.07 vs. 1.15 ± 0.38 mol per day; P = 0.01 by ANOVA). In type 2 diabetes, the ability of insulin to stimulate both the FSR (4.7 ± 3.2% per day) and the ASR (0.03 ± 0.04 mol per day) of NOx was several-fold lower than that in control subjects (15.0 ± 2.9% per day and 0.25 ± 0.07 mol per day, P < 0.03 and P < 0.02, respectively). Also the fraction of arginine flux converted to NOx (basal, 0.22 ± 0.05% vs. 0.65 ± 0.25%; hyperinsulinemia, 0.32 ± 0.06% vs. 1.03 ± 0.33%) was sharply reduced in the patients (P < 0.01 by ANOVA). CONCLUSIONS In type 2 diabetic patients with nephropathy, intravascular NOx synthesis from arginine is decreased under both basal and hyperinsulinemic states. This defect extends the concept of insulin resistance to NO metabolism.

Hyperaminoacidaemia reduces insulin-mediated glucose disposal in healthy man

SummaryTo determine whether hyperaminoacidaemia may modify insulin-mediated glucose disposal, normal subjects were studied with the euglycaemic glucose-clamp technique, with or without an amino acid infusion, at a rate sufficient to duplicate the plasma concentration of most amino acids. Steady-state glucose infusion rates to maintain euglycaemia were 36% lower during hyperaminoacidaemia (7.3±1.0 versus 11.4±0.8mg· kg−1· min−1, p<0.01) at comparable insulin concentrations (92±6 versus 93±7 mU/l respectively). Thus, under conditions of hyperinsulinaemia, amino acids could compete with glucose as metabolic fuels.

Slow versus fast proteins in the stimulation of beta-cell response and the activation of the entero-insular axis in type 2 diabetes.

We tested whether ingestion of whey protein can induce greater post‐prandial amino acid (AA) levels in the plasma and a higher beta‐cell response than casein ingestion in type 2 diabetes mellitus patients.

Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach

Antimicrobial photodynamic therapy (PDT) is a promising tool to combat antibiotic-resistant bacterial infections. During PDT, bacteria are killed by reactive oxygen species generated by a visible light absorbing photosensitizer (PS). We used a classical proteomic approach that included two-dimensional gel electrophoresis and mass spectrometry analysis, to identify some proteins of Staphylococcus aureus that are damaged during PDT with the cationic PS meso-tetra-4-N-methyl pyridyl porphine (T4). Suspensions of S. aureus cells were incubated with selected T4 concentrations and irradiated with doses of blue light that reduced the survival to about 60% or 1%. Proteomics analyses of a membrane proteins enriched fraction revealed that these sub-lethal PDT treatments affected the expression of several functional classes of proteins, and that this damage is selective. Most of these proteins were found to be involved in metabolic activities, in oxidative stress response, in cell division and in the uptake of sugar. Subsequent analyses revealed that PDT treatments delayed the growth and considerably reduced the glucose consumption capacity of S. aureus cells. This investigation provides new insights towards the characterization of PDT induced damage and mechanism of bacterial killing using, for the first time, a proteomic approach.