Haymond, Morey W.

It is well established that patients with non-insulindependent diabetes mellitus (NIDDM) are resistant to insulin. However, the contribution of hepatic and extrahepatic tissues to insulin resistance remains controversial. The uncertainty may be at least in part due to errors introduced by the unknowing use in previous studies of impure isotopes to measure glucose turnover. To determine hepatic and extrahepatic responses to insulin in the absence of these errors, steady-state glucose turnover was measured simultaneously with [6-3H]- and [6-14C]glucose during sequential 5- and 4-h infusions of insulin at rates of 0.4 and 10 mil · kg−1 · min−1 in diabetic and nondiabetic subjects. At low insulin concentrations, [6-3H]- and [6-14C]glucose gave similar estimates of glucose turnover. Hepatic glucose release was equal to but not below zero in the nondiabetic subjects, but persistent glucose release (P < 0.001) and decreased glucose uptake (P < 0.001) was observed in the diabetic patients. At high insulin concentrations, both isotopes underestimated glucose turnover during the 1st h after initiation of the highdose insulin infusion. More time (P < 0.05) was required to reachieve steady state in NIDDM than nondiabetic subjects. At steady state, [6-3H] but not [6-14C]glucose systematically underestimated (P < 0.05) glucose turnover in both groups due to the presence of a tritiated nonglucose contaminant. The percentage of radioactivity in plasma due to tritiated contaminants was linearly related to turnover. When plasma [6-3H]glucose specific activity was corrected for the presence of the contaminant in each subject, both [6-3H]- and [6-14C]glucose indicated that diabetic patients had hepatic and extrahepatic insulin resistance. We conclude that in these experiments, although the presence of a low percentage of tritiated nonglucose contaminant in the tracer may alter estimates of the severity of hepatic and extrahepatic insulin resistance in patients with NIDDM, it does not obscure their presence

To determine the effects of acute metabolic acidosis and alkalosis on leucine metabolism in vivo, mongrel dogs were infused with [1-14C] leucine for 8 h, along with NaCI, HCI, or NaHCO3 over the last 4 h. Arterial pH did not change from the basal value during NaCI infusion but decreased (P <.01) and increased (P <.01) during HCI and NaHCO3 infusions, respectively. Total leucine carbon entry did not change from the basal value during saline infusion but increased (P <.01) with acidosis and decreased (P <.05) with alkalosis. Compared with saline controls, acidosis increased (P <.01) leucine oxidation, whereas alkalosis decreased (P <.01) leucine oxidation. During acidosis, total plasma essential and nonessential amino acid concentrations increased (P <.05), whereas during alkalosis, total plasma essential and nonessential amino acid concentrations decreased (P <.05). These studies suggest that acute alterations in arterial pH may affect the regulation of protein metabolism in vivo and must be considered in the interpretation of results from experiments in which alterations of acid-base homeostasis may have occurred.

To determine the role insulin resistance may play in the catabolic effect of high-dose prednisone therapy, healthy volunteers were studied on four occasions with the hormone-clamp technique at two insulin infusion rates. Subjects were studied after 5 days of prednisone (60 mg/day) or no steroid treatment and were infused with somatostatin, glucagon, growth hormone, [3H]glucose, [14C]leucine, and insulin (0.1 or 0.2 mil · kg1 · min1). At each rate of insulin infusion, the rate of leucine oxidation was increased (P < .001) after steroid treatment. Leucine flux, an indicator of whole-body proteolysis, was similar in the presence or absence of steroid treatment (2.26 ± 0.08 vs. 2.13 ± 0.04 μmol · kg1 · min1 respectively) at the lower rate of insulin infusion but was higher during steroid treatment (2.18 ± 0.06 vs. 1.84 ± 0.13 μmol · kg1 · min1) at the 0.2-mU · kg1 · min1 insulin infusion. Steroid pretreatment had no significant effect on the nonoxidative rates of leucine disappearance. These data provide strong evidence that the protein wasting associated with glucocorticosteroid therapy is in part the result of steroid-induced resistance to the antiproteolytic effect of insulin and an increase in the oxidation (and thus wasting) of one essential amino acid, leucine.

Recent studies indicate that hydrogen-labeled glucose tracers underestimate glucose turnover in humans under conditions of high flux. The cause of this underestimation is unknown. To determine whether the error is time-, pool-, model-, or insulin-dependent, glucose turnover was measured simultaneously with [6-3H]-, [6,6-2H2]-, and [6-14C]glucose during a 7-h infusion of either insulin (1 mU · kg−1 · min−1) or saline. During the insulin infusion, steady-state glucose turnover measured with both [6-3H]glucose (8.0 ± 0.5 mg · kg−1 · min−1) and [6,6-2H2]glucose (7.6 ± 0.5 mg kg−1 min−1) was lower (P < .01) than either the glucose infusion rate required to maintain euglycemia (9.8 ± 0.6 mg · kg−1 · min−1) or glucose turnover determined with [6-14 C]glucose and corrected for Cori cycle activity (9.8 ± 0.7 mg · kg−1 · min1). Consequently “negative” glucose production rates (P <.01) were obtained with either [6-3 H]- or [6,6-2 H2]-but not [6-14 C]glucose. The difference between turnover estimated with [6-3 H]glucose and actual glucose disposal (or 14C glucose flux) did not decrease with time and was not dependent on duration of isotope infusion. During saline infusion, estimates of glucose turnover were similar regardless of the glucose tracer used. High-performance liquid chromatography of the radioactive glucose tracer and plasma revealed the presence of a tritiated nonglucose contaminant. Although the contaminant represented only 1.5% of the radioactivity in the [6-3H]glucose infusate, its clearance was 10-fold less (P <.001) than that of [6-3H]glucose. This resulted in accumulation in plasma, with the contaminant accounting for 16.6 ± 2.09 and 10.8 ± 0.9% of what customarily is assumed to be plasma glucose radioactivity during the insulin or saline infusion, respectively (P < .01). When corrected for the presence of the contaminant, glucose turnover determined with [6-3H]glucose during insulin infusion (9.5 ± 0.6 mg · kg−1 · min−1) no longer differed from either the glucose infusion rate or that determined with [6-14C]glucose. Therefore, the underestimation of glucose turnover during insulin infusion and negative glucose production rates observed with traditional methods to analyze plasma radioactivity and commercially available tracers is the result of an artifactual increase in [6-3H]glucose specific activity. The etiology of the underestimation of glucose turnover with [6,6-2H2]glucose remains to be determined.

To determine the effects of insulin on dietary and endogenous leucine metabolism, five normal subjects, seven insulin-insufficient insulin-dependent (IDDM) diabetic patients, and five diabetic patients controlled with continuous subcutaneous insulin infusion (CSII) were studied before and for 8 h after ingestion of a chemically defined elemental test meal (10 cal/kg) containing crystalline amino acids. L-[1-14C]leucine was included in the meal to trace the entry and oxidation of the dietary leucine. Total (meal + endogenous) entry of leucine into the circulation was estimated with a constant infusion of [2H3]leucine. Postabsorptive and meal-related increases in the plasma leucine concentration were greater (P < .05) in the insulin-insufficient IDDM than in the normal subjects but returned to near-normal values with CSII. Baseline leucine flux was ∼40% greater in the insulin-insufficient IDDM than in normal subjects (2.17 ± 0.17 vs. 1.55 ± 0.15 μmol · kg−1 · min−1, respectively; .05 < P < .01) but were near normal during CSII treatment (1.85 ± 0.25 μmol · kg−1 · min−1). Furthermore, total leucine entry during meal absorption was greater in the insulin-insufficient IDDM (1.41 ± 0.10 mmol · kg−1.8 h−1) than in either normal (0.96 ± 0.08 mmol · kg−1 · 8 h−1, P < .01) or IDDM subjects during CSII treatment (1.09 ± 0.11 mmol · kg−1 · 8 h−1, P < .05). Fractional oxidation (∼40–50%) and entry of dietary leucine were similar in all three groups. The calculated rate of leucine entry from endogenous proteins over the 8 h of study in insulin-insufficient IDDM (1.04 ± 0.12 mmol · kg−1 · 8 h−1) was significantly greater than in normal subjects (0.64 ± 0.07 mmol · kg−1 · 8 h−, P < .01) or in IDDM subjects during CSII treatment (0.76 ± 0.10 mmol · kg−1 · 8 h−1, P < .05). However, during meal absorption, the calculated rate of leucine entry from endogenous protein was significantly suppressed below baseline only in the normal subjects. In summary, during absorption of a mixed meal, 1) one-third of the leucine entering the peripheral plasma space is derived from the diet in normal humans; 2) the rate of absorption and oxidation of dietary leucine in IDDM is normal regardless of adequacy of insulin therapy; and 3) postprandial hyperleucinemia in poorly controlled diabetic patients is at least partly the result of inadequate suppression of the entry rate of leucine from endogenous protein, suggesting persistence of excessive rates of proteolysis, which is at least partly corrected by intensified insulin treatment. Thus, insulin-mediated suppression of endogenous proteolysis is an integral part of protein anabolism during meal absorption.

Competition between glucose and free fatty acids as metabolic fuels is supported by both in vitro and in vivo data, but whether amino acids can also compete with glucose as a source of energy in vivo remains to be established. To determine the effect of increased availability of an amino acid on whole-body glucose flux and glucose carbon uptake by the human forearm, five groups of overnight-fasted normal subjects were infused with either saline, leucine (at 0.5 or 1.0μmol · kg−1 · min−1), isoleucine (0.5 μmol · kg−1 · min−1), or threonine (0.5 μmol · kg−1 · min−1). Plasma glucose concentrations and glucose flux decreased similarly in all groups. No significant changes in forearm output of leucine carbon, isoleucine carbon, or threonine were seen during saline infusion. In contrast, during leucine infusion there was a dose-dependent increase (r = .86, P < .001) in leucine carbon uptake with increased arterial leucine and a-ketoisocaproate concentrations. During infusions of isoleucine and threonine, increases (P < .05) in isoleucine carbon uptake and threonine uptake, respectively, were observed. Glucose uptake by forearm tissues did not change during the saline infusion, but it decreased (P < .05) in all four groups receiving an amino acid infusion. Changes in leucine carbon uptake were strongly correlated (r = −.76, P < .001) with changes in glucose uptake. Therefore, amino acids affect glucose uptake in human forearm tissue and presumably compete as oxidative fuels.

Type A insulin resistance, associated with acanthosis nigricans and menstrual irregularity, has been ascribed to a decreased concentration of insulin receptors. We now report four affected females from one family, a mother and three daughters (including identical twins) who appear to have the type A syndrome. Two of the kindred had no apparent ovarian dysfunction, while the other two had hyperprolactinemia without other findings of polycystic ovary disease, suggesting a genetic disease with variable penetrance. All had normal erythrocyte and monocyte insulin binding. Insulin dose-response studies to assess glucose metabolism and insulinsensitivity were performed in the affected twins. The dose response to insulin was shifted to the right with a decrease in maximal response. These results are consistent with a postbinding defect in insulin action in these patients.

Ketone bodies have been suggested to have a proteinsparing effect, since infusion of Na-β-hydroxybutyrate in man decreases plasma alanine concentrations and urinary nitrogen (N) excretion. To test this hypothesis, six normal postabsorptive volunteers were infused with Na-β-hydroxybutyrate for 3 h. Rates of glucose, leucine carbon, and alanine appearance and disappearance from the plasma space were traced with [3-3H]glucose, L-[6,6,6-2H3]leucine, and [2,3,3,3-2H4]alanine. Rates of leucine N appearance and disappearance and the rate of transfer of leucine N to alanine were assessed with [15N]leucine. During ketone body infusion, plasma alanine decreased (P < 0.05), whereas plasma leucine increased (P < 0.05). Rates of alanine appearance increased (5.3 ± 0.3 to 7.8 ± 0.6 μmol/kg · min), but the increase in its rate of disappearance was sightly greater, accounting for the decrease in plasma alanine concentration. Leucine N flux and the rate and percent of leucine N transferred to alanine increased, whereas leucine carbon flux was unchanged. To determine the effect of the alkalemia induced by Na-β-hydroxybutyrate, four additional subjects were infused with NaHCO3. Alkalemia had no effect on leucine N or carbon flux or on the rate of appearance of alanine, but increased the rate of alanine disappearance, resulting in a decrease in the plasma alanine concentration. Since the rate of appearance of leucine carbon was unaltered during the infusion of Na-β-hydroxybutyrate, it is unlikely that hyperketonemia per se decreases proteolysis in postabsorptive man.

The optimal route for insulin administration by insulin infusion devices has not been established. To assess the differences between the peripheral venous and portal venous routes of insulin administration on postprandial metabolic responses, six alloxan-diabetic dogs were studied on four occasions. On the first, the insulin infusion rates given by the Biostator for disposal of a mixed meal were recorded electronically. On two subsequent occasions, these insulin infusion profiles were administered by a peripheral vein. On an additional occasion, the identical insulin infusion profile was given via the portal vein. No differences were observed in basal or peak plasma glucose, insulin, glucagon, branched chain amino acids, and lactate concentrations between portal and peripheral venous insulin administration. Furthermore, no differences in isotopically ([2-3H]glucose) determined rates of systemic glucose appearance and disappearance were observed between the two routes. Although preprandial plasma alanine concentrations were greater when insulin was infused via the portal vein, the postprandial increments did not differ from those observed during infusion of insulin by a peripheral vein. These studies suggest that under the current experimental conditions, there appears to be no difference in the disposition of a mixed meal, measured as net whole body glucose appearance and disappearance rates, when insulin is administered in an open-loop fashion via either a peripheral or the portal vein.

In vitro perfusion and incubation studies and recent investigations in dogs suggest that branched chain amino acids (BCAA) may be a major source of alanine nitrogen. To determine the contribution of BCAA nitrogen to the formation of alanine in man, seven postabsorptive adults received prime-dose constant infusions of 15N-leucine, L-[6,6,6-2H3] leucine, and L-[2,3,3,3-2H4] alanine; isotopic enrichment was determined in arterialized venous plasma samples by gas chromatography-mass spectroscopy. At substrate and isotope steady state, alanine flux and the rate of 15N alanine appearance were 5.4 ± 0.3 μmol/kg-min and 32 ± 2 nmol/kg·min, respectively. Leucine nitrogen flux was significantly > that of leucine carbon flux (2.54 ± 0.25 vs. 1.90 ± 0.10 μmol/kg·min, respectively; P < 0.001). The 30% greater flux of leucine nitrogen when compared with leucine carbon suggests significant recycling of the leucine carbon in vivo. The percent of circulating alanine nitrogen derived from leucine was 12.5 ± 1.5%; however, the rate of leucine nitrogen transferred to alanine was 0.66 ± 0.05 mumol/kg.min, and represents a minimum of 28% of leucine nitrogen going to alanine. On the basis of these data, together with the percent of alanine and leucine in body protein, only 40% of circulating plasma alanine could come from endogenous protein, whereas 60% is derived from de novo synthesis. In addition, at least 20% of the nitrogen required for alanine synthesis is derived solely from leucine following an overnight fast. Therefore, if the contribution of isoleucine and valine nitrogen is similar to that of leucine, the BCAA may contribute to a minimum of 60% of the nitrogen required for alanine synthesis in postabsorptive man.