Pietro Castellino

Effect of insulin and plasma amino acid concentrations on leucine metabolism in man. Role of substrate availability on estimates of whole body protein synthesis.

We examined the effect of insulin and plasma amino acid concentrations on leucine kinetics in 15 healthy volunteers (age 22 +/- 2 yr) using the euglycemic insulin clamp technique and an infusion of [1-14C]leucine. Four different experimental conditions were examined: (a) study one, high insulin with reduced plasma amino acid concentrations; (b) study two, high insulin with maintenance of basal plasma amino acid concentrations; (c) study three, high insulin with elevated plasma amino acid concentrations; and (d) study four, basal insulin with elevated plasma amino acid concentrations. Data were analyzed using both the plasma leucine and alpha-ketoisocaproate (the alpha-ketoacid of leucine) specific activities. In study one total leucine flux, leucine oxidation, and nonoxidative leucine disposal (an index of whole body protein synthesis) all decreased (P less than 0.01) regardless of the isotope model utilized. In study two leucine flux did not change, while leucine oxidation increased (P less than 0.01) and nonoxidative leucine disposal was maintained at the basal rate; endogenous leucine flux (an index of whole body protein degradation) decreased (P less than 0.01). In study three total leucine flux, leucine oxidation, and nonoxidative leucine disposal all increased significantly (P less than 0.01). In study four total leucine flux, leucine oxidation, and nonoxidative leucine disposal all increased (P less than 0.001), while endogenous leucine flux decreased (P less than 0.001). We conclude that: (a) hyperinsulinemia alone decreases plasma leucine concentration and inhibits endogenous leucine flux (protein breakdown), leucine oxidation, and nonoxidative leucine disposal (protein synthesis); (b) hyperaminoacidemia, whether in combination with hyperinsulinemia or with maintained basal insulin levels decreases endogenous leucine flux and stimulates both leucine oxidation and nonoxidative leucine disposal.

Hyperglucagonemia and insulin-mediated glucose metabolism.

The effect of chronic physiologic hyperglucagonemia on basal and insulin-mediated glucose metabolism was evaluated in normal subjects, using the euglycemic insulin clamp technique (+50, +100, and +500 microU/ml). After glucagon infusion fasting glucose increased from 76 +/- 4 to 93 +/- 2 mg/dl and hepatic glucose production (HGP) rose from 1.96 +/- 0.08 to 2.25 +/- 0.08 mg/kg X min (P less than 0.001). Basal glucose oxidation after glucagon increased (P less than 0.05) and correlated inversely with decreased free fatty acid concentrations (r = -0.94; P less than 0.01) and decreased lipid oxidation (r = -0.75; P less than 0.01). Suppression of HGP and stimulation of total glucose disposal were impaired at each insulin step after glucagon (P less than 0.05-0.01). The reduction in insulin-mediated glucose uptake was entirely due to diminished non-oxidative glucose utilization. Glucagon infusion also caused a decrease in basal lipid oxidation and an enhanced ability of insulin to inhibit lipid oxidation and augment lipid synthesis. These results suggest that hyperglucagonemia may contribute to the disturbances in glucose and lipid metabolism in some diabetic patients.

Leucine metabolism in IDDM: Role of insulin and substrate availability

The effect of insulin on plasma amino acid concentrations and leucine metabolism was examined in 18 healthy nondiabetic young volunteers and in 7 subjects with insulin-dependent diabetes mellitus (IDDM) with the euglycemic insulin-clamp technique (40 mU · m−2 · min−1) in combination with [1-14C]leucine. All diabetic subjects were studied while in poor metabolic control (fasting glucose 13.3 ± 1.1 mM; HbA1c 10.8 ± 0.2%) and again after 2 mo of intensified insulin therapy (fasting glucose 7.2 ± 0.5 mM; HbA1c 8.0 ± 0.2%). Insulin-mediated total-body glucose uptake in poorly controlled diabetic subjects (3.6 ± 0.5 mg · kg−1 · min−1) was significantly reduced compared with control subjects (7.5 ± 0.2 mg · kg−1 · min−1; P < .001) and improved slightly after insulin therapy (4.8 ± 0.3 mg · kg−1 · min−1; P < .05), although it still remained significantly lower than in control subjects (P < .01). During the insulin-clamp study performed in subjects with poorly controlled IDDM, endogenous leucine flux (ELF), leucine oxidation (LO), and nonoxidative leucine disposal (NOLD) all decreased (50.1 ± 2.0 to 26.4 ± 0.4; 9.2 ± 0.4 to 6.0 ± 0.3; 40.9 ± 2.0 to 20.4 ± 2.0 μmol · m−2 · min−1, respectively) to the same extent as in control subjects. After 2 mo of intensified insulin therapy, the effect of acute hyperinsulinemia on ELF, LO, and NOLD was comparable to that of control subjects, whereas insulin-stimulated glucose metabolism was still impaired. To examine the effect of substrate availability on leucine turnover, well-regulated IDDM and control subjects underwent a repeat insulin-clamp study combined with a balanced amino acid infusion designed to increase circulating plasma amino acid levels approximately twofold. Under these conditions, NOLD was equally enhanced above baseline in both control and IDDM subjects (P < .01), whereas ELF was inhibited to a greater extent (P < .01) than during the insulin clamp performed without amino acid infusion (control vs. diabetic subjects, NS). In conclusion, insulin-mediated glucose metabolism is severely impaired in subjects with both poorly controlled and well-controlled IDDM, whereas the effect of acute insulin infusion on leucine turnover is normal, and combined hyperaminoacidemia/hyperinsulinemia stimulated NOLD to a similar extent in both IDDM and control subjects.

Differential Responsiveness of Protein Synthesis and Degradation to Amino Acid Availability in Humans

We investigated the effects of graded hyperaminoacidemia on protein metabolism in eight healthy, young (25 ± 2 years), normal weight (BMI = 25 ± 1 kg/m2), overnight-fasted human subjects. A balanced amino acid solution was infused for 180 min at five different rates: 0.5 (study I), 1.0 (study II), 2.0 (study III), 4.0 (study IV), and 6.0 (study V) mg · kg−1 · min−1 on separate days in random order. Studies were performed with [1-14C]leucine infusion and indirect calorimetry to calculate leucine oxidation (LOX), nonoxidative leucine disposal (NOLD) (an index of protein synthesis), and endogenous leucine flux (ELF) (an index of proteolysis). Basal total plasma amino acid concentrations averaged 1.85 ± 0.1 nunol/1 and increased to 2.27 ± 0.1, 2.70 ± 0.2, 3.84 ± 0.2, 5.87 ± 0.4, and 7.52 ± 0.3 mmol/1 in studies I–V, respectively. ELF decreased from a basal value of 2.27 ± 0.2 to 2.12 ± 0.2, 1.97 ± 0.1, 1.73 ± 0.2, 1.67 ± 0.3, and 1.65 ± 0.1 μmol · kg−1 · min−1 in studies I–V, respectively (P < 0.05 for study I vs. basal, P < 0.01 for studies II–V vs. basal, and NS for studies IV and V vs. study III). LOX increased from a basal value of 0.31 ± 0.04 to 0.38 ± 0.05, 0.41 ± 0.02, 0.64 ± 0.04, 1.11 ± 0.07, and 1.56 ± 0.05 μmol kg−1 · min−1 in studies I–V (all P < 0.01 vs. basal; P < 0.05–0.01 for each study vs. preceding study). Basal NOLD averaged 1.96 ± 0.2 and did not change significantly in studies I and II (2.03 ± 0.2 and 2.10 ± 0.1 μmol · kg−1 · min−1). In contrast, a significant increase in NOLD was observed in studies III, IV, and V (to 2.3 ± 0.15, 2.74 ± 0.2, and 3.25 ± 0.7 (μmol · kg−1 · min−1 , respectively; all P < 0.01 vs. basal; P < 0.05–0.01 for each study vs. preceding study). The net leucine balance (difference between ELF and NOLD) (−0.31 ± 0.06 (μmol · kg−1 · min−1) became less negative in study I (P < 0.01 vs. basal) and positive during studies II–V when the rise in plasma total amino acid levels was ≥50% above basal level (P < 0.01 vs. each preceding study). In conclusion, NOLD, ELF, and LOX exhibit a differential responsiveness to acute changes in substrate availability: 1) small increments (25–50%) in plasma amino acid levels inhibit ELF and stimulate LOX but have no effect on NOLD; 2) stimulation of NOLD is observed only with increments in plasma amino acid levels ≥100% above basal values; and 3) increments in plasma amino acid concentrations >100% above basal values cause a progressive dose-related increase in LOX and NOLD but do not induce any further inhibition of ELF.

Measurement of L-[1-14C]Leucine Kinetics in Splanchnic and Leg Tissues In Humans: Effect of Amino Acid Infusion

Although whole-body leucine flux is widely measured to study body protein turnover in humans, the contribution of specific tissues to the total-body measurement remains unknown. By combining the organ-balance technique with the systemic infusion of L-[1-14C]leucine, we quantitated leucine production and disposal by splanchnic and leg tissues and by the whole body, simultaneously, in six normal men before and during amino acid infusion. At steady state, disposal of arterial leucine by splanchnic and leg tissues was calculated from the percent extraction (E) of L-[1-14C]leucine counts: uptake = E × [Leu]a × flow. Tissue release of cold leucine (from protein turnover) into vein was calculated as the difference between leucine uptake and the net tissue leucine balance. In the postabsorptive state, despite substantial (P < .01) extraction of L-[1-14C]leucine by splanchnic (23 ± 1%) and leg (18 ± 2%) tissues, net leucine balance across both tissue beds was small, indicating active simultaneous disposal and production of leucine at nearly equivalent rates. Splanchnic tissues accounted for −50% of the measured total-body leucine flux. During amino acid infusion, the net leucine balance across splanchnic and leg tissues became positive, reflecting not only an increase in leucine uptake but also a marked suppression (by ∼50%, P < .02) of cold leucine release. This reduction in splanchnic and leg leucine release was indicated by a sharp decline in whole-body endogenous leucine flux. Conclusions: 1) combining the organ-balance method with systemic L-[1-14C]leucine infusion enables leucine kinetics to be measured simultaneously in the whole body and in specific tissues; 2) splanchnic tissues account for −50% of whole-body leucine flux in postabsorptive humans; and 3) amino acid infusion markedly suppresses leg and splanchnic tissue leucine release, which may indicate inhibition of proteolysis.

Effect of Glyburide on Glycemic Control, Insulin Requirement, and Glucose Metabolism in Insulin-Treated Diabetic Patients

Glycemic control and glucose metabolism were examined in 5 patients with insulin-dependent diabetes mellitus (IDDM) and 8 insulin-treated non-insulin-dependent diabetes mellitus (NIDDM) patients before and after 2 mo of therapy with glyburide (20 mg/day). Glycemic control was assessed by daily insulin requirement, 24-h plasma glucose profile, glucosuria, and glycosylated hemoglobin. Insulin secretion was evaluated by glucagon stimulation of C-peptide secretion, and insulin sensitivity was determined by a two-step euglycemic insulin clamp (1 and 10 mU ·kg−1 · min−1) performed with indirect calorimetry and [3−3H]glucose. In the IDDM patients, the addition of glyburide produced no change in daily insulin dose (54 ± 8 vs. 53 ± 7 U/day), mean 24-h glucose level (177 ± 20 vs. 174 ± 29 mg/dl), glucosuria (20 ± 6 vs. 35 ± 12 g/day) or glycosylated hemoglobin (10.1 ± 1.0 vs. 9.5 ± 0.7%). Furthermore, there was no improvement in basal hepatic glucose production (2.1 ± 0.2 vs. 2.4 ± 0.1 mg · kg−1 · min−1), suppression of hepatic glucose production by low- and high-dose insulin infusion, or in any measure of total, oxidative, or nonoxidative glucose metabolism in the basal state or during insulin infusion. C-peptide levels were undetectable (<0.01 pmol/ml) in the basal state and after glucagon infusion and remained undetectable after glyburide therapy. In contrast to the IDDM patients, the insulin-treated NIDDM subjects exhibited significant reductions in daily insulin requirement (72 ± 6 vs. 58 ± 9 U/day), mean 24-h plasma glucose concentration (153 ± 10 vs. 131 ± 5 mg/dl), glucosuria (14 ± 5 vs. 4 ± 1 g/day), and glycosylated hemoglobin (10.3 ± 0.7 vs. 8.0 ± 0.4 %) after glyburide treatment (all P ≤ .05). However, there was no change in basal hepatic glucose produc-tion (1.7 ± 0.1 vs. 1.7 ± 0.1 mg kg−1 min−1), suppression of hepatic glucose production by insulin, or insulin sensitivity during the two-step insulin-clamp study. Both basal (0.14 ± 0.05 vs. 0.32 ± 0.05 pmol/ ml, P < .05) and glucagon-stimulated (0.24 ± 0.07 vs. 0.44 ± 0.09 pmol/ml) C-peptide levels rose after 2 mo of glyburide therapy and both were correlated with the decrease in insulin requirement (basal: r = .65, P = .08; glucagon stimulated: r = .93, P < .001). These data indicate that in IDDM subjects, the addition of glyburide to insulin does not affect insulin requirement, glycemic control, or insulin sensitivity. In contrast, in insulin-treatedNIDDM patients, glyburide produces a modest decrease in insulin dose and improves glycemic control without altering insulin sensitivity. This improvement in glucose metabolism primarily reflectsan increase in endogenous insulin secretion.

Catabolic effects of thyroid hormone excess: The contribution of adrenergic activity to hypermetabolism and protein breakdown

Although patients with thyrotoxicosis improve clinically after treatment with beta-adrenergic blocking drugs, it has never been established whether the hypermetabolism and body protein wasting caused by thyroid hormone excess are actually mediated by adrenergic mechanisms. To evaluate this issue, we measured basal energy expenditure, epinephrine-stimulated calorigenesis, and leucine kinetics (an index of body protein catabolism) in six normal volunteers before and after triiodothyronine (T3) administration (150 micrograms/d for 1 week). Serum T3 rose nearly threefold (P less than 0.001) during T3 administration, producing significant increases in basal metabolic rate (21%, P less than 0.001), nitrogen excretion (45%, P less than 0.001), and leucine flux (45%, P less than 0.01). In response to epinephrine infusion, the absolute rise in metabolic rate above basal was 57% greater in the thyrotoxic condition (P less than 0.02). Although beta-adrenergic blockade with intravenous propranolol totally abolished the calorigenic response to epinephrine, it had no detectable effect on either the accelerated basal metabolic rate or the augmented body protein catabolism caused by thyroid horomone excess. Our data suggest that in the basal, resting state, the increased metabolic rate and accelerated protein breakdown caused by thyroid hormone are not adrenergically mediated. However, under nonbasal conditions (when sympathetic activity is stimulated), enhanced responsiveness to catecholamine calorigenesis may exaggerate the hypermetabolic state and thereby contribute to weight loss and other clinical manifestations of thyrotoxicosis. This mechanism may explain the clinical efficacy of beta-adrenergic blocking agents in the treatment of thyrotoxicosis.

Effect of alpha-adrenergic blockers, ACE inhibitors, and calcium channel antagonists on renal function in hypertensive non-insulin-dependent diabetic patients

In the present study we investigated the effect of a selective alpha 1-adrenergic blocker (doxazosin), an angiotensin-converting enzyme (ACE) inhibitor (captopril), and a calcium channel antagonist (nifedipine) on renal function in hypertensive non-insulin-dependent diabetic patients. 30 NIDD hypertensive patients (age = 50 +/- 3 years; BMI = 30 +/- 1 kg/m2) (mean +/- SEM) were studied before and after a 12-week period of antihypertensive treatment. Ten patients were treated with doxazosin (Cardura) (2-8 mg once daily or 8 mg b.i.d.), 9 with captopril (Capoten) (25-50 mg b.i.d.), and 11 with nifedipine (Procardia-XL) (30-60 mg once daily). Blood pressure, creatinine clearance, 24-hour urinary protein excretion, fasting plasma glucose concentration and glycosylated hemoglobin were measured before and after drug treatment. Fasting plasma glucose and glycosylated hemoglobin (HbA1c) were similar in all three groups prior to the start of antihypertensive therapy and did not change significantly from baseline in any treatment groups. In the doxazosin group creatinine clearance rose from 99 +/- 8 to 122 +/- 8 ml/1.73 m2.min (p < 0.01), while 24-hour urinary protein excretion declined from 2.66 +/- 0.05 to 1.76 +/- 0.02 mg/day/ml/1.73 m2.min (p < 0.01). In diabetics treated with captopril creatinine clearance rose from 93 +/- 6 to 109 +/- 9 ml/1.73 m2.min (p < 0.05), while the 24-hour urinary protein excretion fell from 2.70 +/- 0.05 to 2.03 +/- 0.04 mg/day/ml/1.73 m2.min (p < 0.05). In patients treated with nifedipine creatinine clearance did not change (97 +/- 6 vs. 94 +/- 7 ml/1.73 m2.min), while 24-hour urinary protein excretion decreased from 2.84 +/- 0.04 to 1.95 +/- 0.03 mg/day/ml/1.73 m2.min. Systolic and diastolic blood pressure were similar in doxazosin (150 +/- 3/95 +/- 2 mm Hg), captopril (153 +/- 3/93 +/- 1), and nifedipine (155 +/- 4/93 +/- 1) groups prior to the start of antihypertensive therapy and declined to 143 +/- 3/84 +/- 3 (doxazosin), 139 +/- 3/82 +/- 3 (captopril), and 141 +/- 3/84 +/- 1 (nifedipine) mm Hg (all p < 0.01 vs. pretreatment). In summary, both doxazosin and captopril treatment were associated with significant rises in GFR, while all three antihypertensive agents caused a significant decline in proteinuria. These results indicate that alpha-adrenergic blockers, ACE inhibitors, and calcium channel antagonists can safely and effectively be used in the clinical management of non-insulin-dependent diabetic patients with hypertension.

Na+/Li+ and Na+/H+ countertransport activity in hypertensive non- insulin-dependent diabetic patients: Role of insulin resistance and antihypertensive treatment

We measured erythrocyte Na+/Li+ and Na+/H+ countertransport (CT) activity (millimoles per liter per cell per hour) in 10 healthy control subjects (age, 38 +/- 4 years; body mass index, 25 +/- 1 kg/m2) and in 25 hypertensive patients with non-insulin-dependent diabetes mellitus ([NIDDM] age, 49 +/- 3 years; body mass index, 29 +/- 1 kg/m2; fasting plasma glucose, 157 +/- 12 mg/dL) 4 weeks after discontinuation of previous antihypertensive treatment. Na+/Li+ CT was significantly increased in hypertensive NIDDM patients compared with controls (0.56 +/- 0.04 v 0.30 +/- 0.03, P < .01), whereas Na+/H+ CT was similar to control levels (21 +/- 1 v 20 +/- 2). A positive correlation was found between Na+/Li+ CT and the severity of insulin resistance (r = .69, P < .01), mean arterial pressure ([MAP] r = .64, P < .01), plasma triglyceride concentration (r = .46, P < .05), and plasma total cholesterol (r = .41, P < .05). An inverse correlation was found between Na+/Li+ CT activity and plasma insulin concentration (r = -.47, P < .05). No relationship was observed between Na+/Li+ CT activity and either creatinine clearance or proteinuria. Stepwise multiple regression analysis for all metabolic variables and blood pressure showed that only the severity of insulin resistance was positively correlated with increased Na+/Li+ CT activity. Na+/H+ and Na+/Li+ CT activity were not altered by 3 hours of euglycemic physiologic hyperinsulinemia (84 +/- 3 microU/mL). Hypertensive NIDDM subjects were treated for 3 months with captopril, nifedipine, or doxazosin. After captopril, a reduction of Na+/H+ CT was observed (22 +/- 4 v 13 +/- 2, P < .05); Na+/Li+ CT decreased after doxazosin (0.56 +/- 0.06 v 0.45 +/- 0.05, P < .05) and nifedipine (0.52 +/- 0.06 v 0.42 +/- 0.05, P < .05). In conclusion, in hypertensive NIDDM subjects, (1) Na+/Li+ CT is increased and is correlated with the level of insulin resistance and the MAP; (2) acute physiologic hyperinsulinemia does not affect Na+/Li+ or Na+/H+ CT activity; and (3) Na+/H+ CT activity is reduced by captopril, and Na+/Li+ CT is decreased by doxazosin and nifedipine.

Pulse Pressure Is an Independent Predictor of Aortic Stiffness in Patients with Mild to Moderate Chronic Kidney Disease

Background: In patients with end-stage renal disease pulse wave velocity (PWV) has been widely assessed, but its behavior in mild to moderate chronic kidney disease (CKD) has been less investigated. We evaluated PWV in mild to moderate CKD. Methods: We studied 31 patients with grade II–IV CKD. Aortic PWV (aPWV), aortic and upper limb augmentation index, creatinine clearance, C-reactive protein, serum fibrinogen, interleukin-1, interleukin-6, tumor necrosis factor, albumin, total and high-density lipoprotein cholesterol and blood pressure were evaluated. Results: aPWV (7.95 ± 0.64 m/s), but not augmentation index was significantly higher (p = 0.03) in CKD patients than age-matched healthy subjects (aPWV: 6.24 ± 0.43 m/s; upper limb: 32.8 ± 1.9; aortic: 27.7 ± 1.9). At univariate regression analysis, aPWV was significantly correlated with age (r = 0.44; p = 0.013), interleukin-6 (r = 0.43; p = 0.027), pulse (r = 0.39; p = 0.029), systolic blood pressure (r = 0.37; p = 0.038) and tumor necrosis factor (r = 0.39; p = 0.029). At multivariate analysis, pulse pressure was the only significant independent determinant (β = 0.37; p = 0.05) of aPWV. Conclusion: The results of this study confirm an aPWV increase in mild to moderate CKD and emphasize association between pulse pressure and PWV, independently of renal failure.