Steven Nissen

Inverse relationship of leucine flux and oxidation to free fatty acid availability in vivo.

To determine the effect of fatty acid availability on leucine metabolism, 14-h fasted dogs were infused with either glycerol or triglyceride plus heparin, and 46-h fasted dogs were infused with either nicotinic acid or nicotinic acid plus triglyceride and heparin. Leucine metabolism was assessed using a simultaneous infusion of L-[4,5-3H]leucine and alpha-[1-14C]ketoisocaproate. Leucine, alpha-ketoisocaproate (KIC), and totalleucine carbon (leucine plus KIC) flux and oxidation rates were calculated at steady state. In 14-h fasted animals, infusion of triglyceride and heparin increased plasma free fatty acids (FFA) by 0.7 mM (P less than 0.01) and decreased leucine (P less than 0.01), total leucine carbon flux (P less than 0.02), and oxidation (P less than 0.05). The estimated rate of leucine utilization not accounted for by oxidation and KIC flux decreased, but the changes were not significant. During glycerol infusion, leucine and KIC flux and oxidation did not change. In 46-h fasted dogs, nicotinic acid decreased FFA by 1.0 mM (P less than 0.01) and increased (P less than 0.05) the rate of leucine and total leucine carbon flux, but did not affect KIC flux. Leucine oxidation increased (P less than 0.01) by nearly threefold, whereas nonoxidized leucine utilization decreased. Infusion of triglyceride plus heparin together with nicotinic acid blunted some of the responses observed with nicotinic acid alone. In that changes in oxidation under steady state condition reflect changes in net leucine balance, these data suggest that FFA availability may positively affect the sparing of at least one essential amino acid and may influence whole body protein metabolism.

Effects of free fatty acid availability, glucagon excess, and insulin deficiency on ketone body production in postabsorptive man.

The present studies were undertaken to assess the relative effects of free fatty acid (FFA) availability, glucagon excess, and insulin deficiency on ketone body (KB) production in man. To determine whether an increase in FFA availability would augment KB production in the absence of insulin deficiency and glucagon excess, plasma insulin and glucagon were maintained at basal concentrations by infusion of somatostatin and exogenous insulin and glucagon, and plasma FFA were increased from 0.32 +/- 0.06 to 1.4 +/- 0.1 mM by a 2.5-h-infusion of a triglyceride emulsion plus heparin. KB production increased fivefold from 2.2 +/- 0.4 to 11.4 +/- 1.2 mumol . kg-1 . min-1, P less than 0.001. To determine whether insulin deficiency would further augment KB production, analogous experiments were performed but the replacement infusion of insulin was stopped. Despite a greater increase in plasma FFA (from 0.26 +/- 0.04 to 1.95 +/- 0.3 mM), KB production increased (from 1.5 +/- 0.3 to 11.1 +/- 1.8 mumol . kg-1 . min-1) to the same extent as in the absence of insulin deficiency. To determine whether hyperglucagonemia would augment KB production beyond that accompanying an increase in plasma FFA and, if so, whether this required insulin deficiency, similar experiments were performed in which the glucagon infusion rate was increased to produce plasma glucagon concentrations of 450-550 pg/ml with and without maintenance of the basal insulin infusion. When basal plasma insulin concentrations were maintained, hyperglucagonemia did not further increase KB production; however, when the basal insulin infusion was discontinued, hyperglucagonemia increased KB production significantly, whereas no change was observed in saline control experiments. These studies indicate that, in man, FFA availability is a major determinant of rates of KB production; insulin does not appear to influence ketogenesis rates by a direct hepatic effect, and glucagon can further augment KB production when FFA concentrations are increased but only in the setting of insulin deficiency.

Failure of Infused β-Hydroxybutyrate to Decrease Proteolysis in Man

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.

Regulation of Whole-Body Leucine Metabolism With Insulin During Mixed-Meal Absorption in Normal and Diabetic Humans

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.

Measurement of plasma α-ketoisocaproate concentrations and specific radioactivity by high-performance liquid chromatography

Abstract An isocratic high-performance liquid chromatographic technique for the measurement of the specific radioactivity and concentration of α-ketoisocaproate in plasma is described. Plasma proteins are precipitated by additions of acetone, the supernatant is applied to a cation-exchange column, and the resulting eluate is injected into a C18 reverse-phase column. Analysis time is approximately 10 min. Quantitative recovery, specificity, and sensitivity of this method are described and make this system attractive for in vivo α-ketoisocaproate kinetic studies. Using this procedure, the apparent flux of α-ketoisocaproate in postabsorptive dogs was determined during an infusion of α-[U-14C]ketoisocaproate and averaged 2.8 + 0.41 μmol/kg-min.