Treatment satisfaction with inhaled insulin in patients with type 1 diabetes: a randomized controlled trial.

OBJECTIVE: In patients with type 1 diabetes, glycemic control can be achieved as effectively with an inhaled insulin regimen, comprising preprandial inhaled intrapulmonary insulin plus a bedtime ultralente injection, as with a conventional subcutaneous insulin regimen involving two to three injections per day. Our objective was to compare patient satisfaction between inhaled insulin and subcutaneous insulin. RESEARCH DESIGN AND METHODS: Subjects with type 1 diabetes participated in a 12-week open-label trial and were randomized to either an inhaled insulin regimen or a subcutaneous insulin regimen. Subjects (n = 69) were asked to complete a 15-item self-administered satisfaction questionnaire, the Patient Satisfaction with Insulin Therapy (PSIT) Questionnaire, at baseline and week 12. Outcomes included mean percentage changes in global (overall) satisfaction and two subscales: convenience/ease of use and social comfort. RESULTS: The mean percentage improvement in overall satisfaction with inhaled insulin (35.1%, 95% CI 18.0-52.2) was greater than with subcutaneous insulin (10.6%, 4.7-16.5) (P < 0.01), as was the improvement in convenience/ease of use: inhaled insulin 41.3% (22.9-59.6) versus subcutaneous insulin 11.2% (4.1-18.3; P < 0.01). Improvement in social comfort was greater with inhaled insulin but was not statistically significant. The 12-week change in HbA(1c) was associated with improved overall satisfaction (r = -0.27, P = 0.04). CONCLUSIONS: Inhaled insulin may offer the first practical, noninvasive alternative to insulin injections. For patients with type 1 diabetes, inhaled insulin maintains glycemic control and provides greater overall satisfaction and convenience/ease of use than subcutaneous insulin.

Inhaled human insulin treatment in patients with type 2 diabetes mellitus.

BACKGROUND: Despite demonstrated benefits, intensive insulin therapy has not gained widespread clinical acceptance for several reasons: Multiple daily injections are inconvenient, adherence is a concern, and the time-activity profile may not mimic normal insulin secretion. As such, alternate means of administering insulin are being evaluated. OBJECTIVE: To assess the efficacy and safety of pulmonary delivery of insulin in type 2 diabetic patients who require insulin. DESIGN: Randomized, open-label, 3-month study consisting of a screening visit, a 4-week baseline lead-in phase, and a 12-week treatment phase. SETTING: General clinical research center and outpatient research clinics. PATIENTS: 26 patients (16 men, 10 women) with type 2 diabetes (average age, 51.1 years; average duration of diabetes, 11.2 years). INTERVENTION: Patients received inhaled insulin before each meal plus a bedtime injection of ultralente insulin, performed home glucose monitoring, and had weekly adjustment of insulin dose; target level for preprandial plasma glucose was 5.55 to 8.88 mmol/L (100 to 160 mg/dL). MEASUREMENTS: Glycemic control (hemoglobin A(1c) level) obtained at baseline and monthly for 3 months. Pulmonary function tests were done at baseline and at the end of the study. RESULTS: Inhaled insulin treatment for 3 months significantly improved glycemic control compared with baseline: Mean hemoglobin A(1c) levels decreased by 0.0071 +/- 0.0072 (0.71% +/- 0.72%). Patients experienced an average of 0.83 mild to moderate hypoglycemic event per month; no severe events were recorded. Patients showed no significant weight gain or change in pulmonary function compared with baseline. CONCLUSIONS: Pulmonary delivery of insulin in type 2 diabetic patients who require insulin improved glycemic control, was well tolerated, and demonstrated no adverse pulmonary effects. Larger-scale studies are ongoing to provide long-term efficacy and safety data.

Efficacy of inhaled human insulin in type 1 diabetes mellitus: a randomised proof-of-concept study.

BACKGROUND: Effective glycaemic control in type 1 diabetes mellitus usually requires two or more insulin injections daily. Inhaled intrapulmonary delivery of insulin offers a potential new way to deliver meal-related insulin, eliminating the need for preprandial injections. METHODS: 73 patients with type 1 diabetes mellitus were studied in an open-label, proof-of-concept, parallel-group randomised trial. Patients in the experimental group received preprandial inhaled insulin plus a bedtime subcutaneous ultralente insulin injection. Patients in the control group received their usual insulin regimen of two to three injections per day. Participants monitored their blood glucose four times daily, and adjusted insulin doses weekly to achieve preprandial glucose targets of 5.6-8.9 mmol/L. The primary outcome measure was change in glycosylated haemoglobin (HbA1c) after 12 weeks. Secondary outcomes were fasting and postprandial glucose response to a mixed meal; hypoglycaemia frequency and severity; pulmonary function; and patients' satisfaction. FINDINGS: Changes in HbA1c were indistinguishable between groups (difference 0.2% [95% CI -0.2 to 0.5]). Changes in fasting and postprandial glucose concentrations, and occurrence and severity of hypoglycaemia were also similar between groups. Inhaled insulin was well tolerated and had no effect on pulmonary function (ie, spirometry, lung volumes, diffusion capacity, and oxygen saturation). INTERPRETATION: This proof-of-concept study shows that preprandial insulin can be given by inhalation in individuals with insulin-deficient type 1 diabetes as a less invasive alternative to conventional preprandial insulin injections.

Development and factor analysis of a questionnaire to measure patient satisfaction with injected and inhaled insulin for type 1 diabetes.

OBJECTIVE: To develop a self-administered questionnaire to address alternative delivery routes of insulin and to investigate aspects of patient satisfaction that may be useful for subsequent assessment and comparison of an inhaled insulin regimen and a subcutaneous insulin regimen. RESEARCH DESIGN AND METHODS: Attributes of patient treatment satisfaction with both inhaled and injected insulin therapy were derived from five qualitative research studies to arrive at a 15-item questionnaire. Each item was analyzed on a five-point Likert scale so that higher item scores indicated a more favorable attitude. There were 69 subjects with type 1 diabetes previously taking injected insulin therapy who were enrolled in a phase II clinical trial. Their baseline responses on the questionnaire were evaluated and subjected to an exploratory factor analysis. Meaningful factors were retained and interpreted based on their psychometric properties. RESULTS: Exploratory factor analysis suggested a two-factor solution accounting for 66 and 20% of the variance, respectively. The first factor contained 10 reliable items (Cronbach's alpha = 0.89) relating to convenience and ease of use, and the second contained 5 reliable items (Cronbach's alpha = 0.82) relating to social comfort. CONCLUSIONS: Among patients with type 1 diabetes, this analysis highlighted and quantified two key factors contributing to patient satisfaction: convenience/ease of use and social comfort. The questionnaire underwent rigorous development, had reliable properties, and an interpretable and rich factor structure. This report is intended to help advance, in subsequent investigations, the understanding and measurement of treatment satisfaction with novel and existing forms of insulin delivery.

Inhibiting cholesterol absorption with CP-88,818 (beta-tigogenin cellobioside; tiqueside): studies in normal and hyperlipidemic subjects.

CP-88,818 (beta-tigogenin cellobioside; tiqueside) is a synthetic saponin developed to treat hypercholesterolemia by inhibiting the absorption of biliary and dietary cholesterol. Two studies are reported here: one in patients to assess safety and efficacy, and one in normal volunteers to explore the mechanism of action. The former included 15 hypercholesterolemic outpatients [low-density lipoprotein cholesterol (LDL-C) > or = 160 mg/dl] treated with 1, 2, and 3 g of tiqueside daily (b.i.d.) in a crossover design for three 2-week treatment periods, each separated by a 3-week placebo period. The mechanistic study was conducted with 24 healthy male subjects who were randomized in a parallel group design to either placebo (n = 6) or tiqueside (2 or 4 g/day; n = 9 each) once daily for 3 weeks. All subjects in this study were fed a low-fat, low-cholesterol diet [National Cholesterol Education Program (NCEP) Step 1]. Fecal steroid excretion rates and plasma lipid levels were determined at baseline and after 3 weeks of treatment. Fractional cholesterol absorption was measured before and after treatment by the continuous feeding, dual-isotope method. Tiqueside produced a dose-dependent reduction in plasma LDL cholesterol levels in the hypercholesterolemic patients. In the mechanistic study, it decreased fractional cholesterol absorption rates and increased fecal neutral sterol excretion rates, changes associated with trends toward lower LDL cholesterol levels. Other lipoprotein levels were unaffected, as were fecal fat and bile acid excretion and fat-soluble vitamin absorption. Thus tiqueside dose-dependently inhibits cholesterol absorption in humans, resulting in a reduction in serum LDL cholesterol levels.

Inhibition of cholesterol absorption with CP-148,623 lowers serum cholesterol in humans.

OBJECTIVE: To determine the effects of the reduction of intestinal cholesterol absorption with CP-148,623 on serum cholesterol levels in men with mild hyperlipidemia. METHODS: In an outpatient study in a university medical center, healthy male volunteers (n = 25) with borderline-high serum cholesterol levels participated in a double-blind, placebo-controlled parallel-group study. A 3-week dietary run-in period was followed by 2 weeks of treatment with either CP-148,623 (300 mg twice a day; n = 12) or placebo (n = 13). RESULTS: Fractional cholesterol absorption (by the dual-isotope, continuous-feeding technique), fecal neutral sterol excretion, and serum lipids were measured after the diet run-in and after the treatment periods. CP-148,623 caused a marked inhibition (by 38%) of fractional cholesterol absorption (50% +/- 2% [baseline] to 31% +/- 1%) and a 71% increase in fecal neutral sterol excretion (481 +/- 39 mg/day [baseline] to 804 +/- 55 mg/day), compared with negligible changes in the placebo group (p < 0.0001 for both). Mean percent reductions from baseline in serum low-density lipoprotein (LDL) cholesterol levels were 11.6% with CP-148,623 (119 +/- 17 mg/dl to 104 +/- 13 mg/dl) versus a nonsignificant 1.8% reduction with placebo (change with CP-148,623 versus placebo, p < 0.0002). CONCLUSIONS: In healthy male volunteers with mild hypercholesterolemia, treatment for 2 weeks with 600 mg/day CP-148,623 inhibited fractional cholesterol absorption by 35% to 40%, increased fecal neutral sterol excretion by 60% to 70%, and reduced serum LDL cholesterol by 10% to 12%.

Metabolic effects of darglitazone, an insulin sensitizer, in NIDDM subjects.

Insulin resistance is a significant pathogenetic factor in the development of non-insulin-dependent diabetes mellitus (NIDDM). A new class of drugs, the thiazolidinediones, have been shown to lower blood glucose levels without stimulating insulin secretion. We report the metabolic effect of the thiazolidinedione, darglitazone, in obese NIDDM subjects. Nineteen subjects were enrolled in a double-blind placebo-controlled study in which 25 mg of darglitazone was given once a day for 14 days. Nine subjects received the active drug and ten subjects received placebo. Darglitazone-treated subjects showed; 1) a decrease in 24-h plasma glucose area under the curve from 292.8 +/- 31.2 to 235.2 +/- 21.6 mmol.h-1.l-1 p = 0.002; 2) a decrease in 24-h serum insulin area under the curve from 1027.2 +/- 254.4 to 765.6 +/- 170.4 microU.h-1.l-1 p = 0.045; 3) a decrease in 24-h non-esterified fatty acid area under the curve from 1900 +/- 236 to 947 +/- 63 g.h-1.l-1 p = 0.002; 4) a decrease in mean 24-h serum triglyceride by 25.9 +/- 6.2% as compared to -3.9 +/- 4.8% for the placebo-treated group, p = 0.012. Placebo-treated subjects showed no change in their metabolic parameters after treatment. Thus, darglitazone is effective in increasing insulin effectiveness in obese NIDDM subjects. The potential for this and similar drugs to treat or prevent NIDDM as well as the insulin-resistance syndrome needs to be explored.

Overnight branched-chain amino acid infusion causes sustained suppression of muscle proteolysis.

Short-term (3 to 4 hours) infusion of branched-chain amino acids (BCAA) has been shown to suppress muscle protein breakdown. Whether these effects are sustained with chronic elevations of BCAA is not known. In the present study, we examined the effect of an overnight (16-hour) systemic BCAA infusion on whole-body and skeletal muscle amino acid metabolism, as assessed by simultaneously measured 3H-phenylalanine and 14C-leucine kinetics in eight normal volunteers; 10 overnight-fasted subjects studied during a 4-hour saline infusion served as controls. Overnight BCAA infusion increased plasma BCAA concentrations by fivefold to eightfold, and this was associated with a 20% to 60% decline in arterial concentrations of other amino acids. For Phe, this decline was mediated by a reduction in the systemic rate of appearance ([Ra] 0.38 +/- 0.03 v 0.60 +/- 0.01 mumol/kg/min for BCAA and saline, respectively, P < .001). Endogenous Leu Ra, calculated more indirectly as the difference between the total Leu Ra and the unlabeled Leu infusion rate, did not differ between groups. In the forearm, overnight BCAA infusion resulted in a diminished net release of Phe (-3 +/- 2 v -18 +/- 4 [saline] nmol/min/100 mL, P < .02), and BCAA balance became markedly positive (751 +/- 93 v -75 +/- 30, P < .001). The diminished net forearm Phe release was accounted for by a decrease in local Phe Ra (P < .02). As with the systemic endogenous Leu Ra, forearm Leu Ra was not reproducibly affected by infused BCAA.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 induces up-regulation of murine macrophage beta-endorphin receptors.

Cultured murine bone marrow macrophages specifically bound 125I-labeled beta-endorphin. Binding was displaceable by 100 times molar excess of full-length beta-endorphin but was insensitive to the opioid receptor antagonist, naloxone. Binding was inhibited by beta-endorphin's C-terminal tetrapeptide, lys-lys-gly-glu, but not by the truncated N-terminal 27 amino acid fragment, indicating that binding of beta-endorphin to this receptor is dependent on its C-terminus. Macrophages incubated for 24 h with 10(-8)-10(-5) M prostaglandin E2 showed a dose-dependent increase in beta-endorphin binding, implying receptor up-regulation. This was also observed in response to the phosphodiesterase inhibitor, isobutylmethylxanthine, indicating that regulation of these receptors may be mediated through a cAMP-dependent process. This is the first demonstration that beta-endorphin receptor expression can be positively regulated.

Effects of epinephrine on human muscle glucose and protein metabolism.

Systemic epinephrine infusion causes hypoaminoacidemia and inhibits whole body leucine flux (proteolysis) in humans. Its specific action on muscle protein is not known and is difficult to assess during systemic epinephrine infusions, which affect plasma insulin, amino acid, and free fatty acid concentrations. During a steady-state infusion of L-[ring-2,6-3H]phenylalanine, we examined the effect of locally infused epinephrine on the metabolism of protein and glucose in forearm muscle of 10 healthy human volunteers. During local epinephrine infusion, systemic concentrations of glucose, phenylalanine, insulin, and epinephrine were unchanged and lactate declined (P < 0.02). Compared with baseline, epinephrine induced significant increases in forearm blood flow (P < 0.01) and net lactate release (P < 0.001) and a decrease in glucose uptake (P < 0.01) at both 2 and 4 h. At 2 and 4 h phenylalanine release from muscle proteolysis was suppressed (P < 0.01), and at 4 h the net phenylalanine balance was less negative than baseline (P < 0.02), indicating an anticatabolic effect on muscle protein. We conclude that in human forearm muscle epinephrine, at physiological concentrations, has a catabolic effect on muscle glycogen but an anticatabolic action on muscle protein. The mechanism of this latter effect is not known.

ß-endorphin binding in cultured adrenal cortical cells.

The polypeptide ß-endorphin binds to cultured bovine adrenal cortical cells in a naloxone insensitive manner, ß-endorphin and N-Acetyl-ß-endorphin are equipotent in inhibiting binding. The amino terminal 27 amino acid fragment referred to as ß-endorphin[1-27] shows no ability to inhibit binding, whereas the carboxy-terminal tetrapeptide Lys-Lys-Gly-Glu partially inhibits binding. ACTH, angiotensin II and met-enkephalin show little or no ability to inhibit ß-endorphin binding. Competition bin-ding reveals an apparently single affinity class with Kd of 33 nM. Molecular cross linking experiments reveal putative receptor subunits of 85 kD, 64 kD, 54 kD and 44 kD. The lower molecular weight bands are preferentially cross-linked by a hydrophobic cross linking reagent, in contrast to the two higher molecular weight bands, which are cross linked equally by hydrophobic and water soluble cross linking reagents. The ß-endorphin binding characteristics of adrenal cortical cells revealed here are quite similar to those of a class of non-opioid ß-endorphin receptors previously shown to exist in cells of the immune system.

Glucocorticoids antagonize insulin's antiproteolytic action on skeletal muscle in humans.

Although chronic glucocorticoid elevations cause net skeletal muscle protein loss in man, the kinetic mechanisms responsible for this catabolic effect and the capacity of insulin to overcome it remain unclear. To examine this issue, we measured basal and insulin-stimulated rates of protein synthesis and breakdown in muscle using the phenylalanine forearm kinetic method in eight normal volunteers studied postabsorptively and after 4 days of dexamethasone treatment (8 mg/day). To avoid the confounding effects of systemic insulinization, local forearm insulin levels were raised by approximately 430 pmol/L using a 150-min brachial arterial infusion of insulin (0.251 pmol/kg.min). Postabsorptively, dexamethasone produced mild hyperglycemia (P < 0.003) and a 3-fold rise in plasma insulin (P < 0.001), but no change in forearm phenylalanine balance or kinetics. Before dexamethasone treatment, local hyperinsulinemia increased forearm glucose uptake 2.5-fold and caused a positive net balance of phenylalanine due to a marked 40% inhibition of proteolysis. After dexamethasone treatment, forearm glucose uptake was modestly reduced. However, forearm net phenylalanine balance remained negative due to a striking reduction in insulin's inhibitory effect on proteolysis. We conclude that 1) the effects of glucocorticoid on basal muscle protein turnover are minimized by compensatory hyperinsulinemia, and 2) glucocorticoids cause muscle resistance to insulin's antiproteolytic action.

Insulin sensitivity of protein and glucose metabolism in human forearm skeletal muscle.

Physiologic increases of insulin promote net amino acid uptake and protein anabolism in forearm skeletal muscle by restraining protein degradation. The sensitivity of this process to insulin is not known. Using the forearm perfusion method, we infused insulin locally in the brachial artery at rates of 0.00 (saline control), 0.01, 0.02, 0.035, or 0.05 mU/min per kg for 150 min to increase local forearm plasma insulin concentration by 0, approximately 20, approximately 35, approximately 60, and approximately 120 microU/ml (n = 35). L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine were infused systemically, and the net forearm balance, rate of appearance (Ra) and rate of disposal (R(d)) of phenylalanine and leucine, and forearm glucose balance were measured basally and in response to insulin infusion. Compared to saline, increasing rates of insulin infusion progressively increased net forearm glucose uptake from 0.9 mumol/min per 100 ml (saline) to 1.0, 1.8, 2.4, and 4.7 mumol/min per 100 ml forearm, respectively. Net forearm balance for phenylalanine and leucine was significantly less negative than basal (P < 0.01 for each) in response to the lowest dose insulin infusion, 0.01 mU/min per kg, and all higher rates of insulin infusion. Phenylalanine and leucine R(a) declined by approximately 38 and 40% with the lowest dose insulin infusion. Higher doses of insulin produced no greater effect (decline in R(a) varied between 26 and 42% for phenylalanine and 30-50% for leucine). In contrast, R(d) for phenylalanine and leucine did not change with insulin. We conclude that even modest increases of plasma insulin can markedly suppress proteolysis, measured by phenylalanine R(a), in human forearm skeletal muscle. Further increments of insulin within the physiologic range augment glucose uptake but have little additional effect on phenylalanine R(a) or balance. These results suggest that proteolysis in human skeletal muscle is more sensitive than glucose uptake to physiologic increments in insulin.

Growth hormone stimulates skeletal muscle protein synthesis and antagonizes insulin's antiproteolytic action in humans.

We examined the effects of a combined, local intra-arterial infusion of growth hormone (GH) and insulin on forearm glucose and protein metabolism in seven normal adults. GH was infused into the brachial artery for 6 h with a dose that, in a previous study, stimulated muscle protein synthesis (phenylalanine Rd) without affecting systemic GH, insulin, or insulinlike growth factor I concentrations. For the last 3 h of the GH infusion, insulin was coinfused with a dose that, in the absence of infused GH, suppressed forearm muscle proteolysis by 30-40% without affecting systemic insulin levels. Measurements of forearm glucose, amino acid balance, and [3H]phenylalanine and [14C]leucine kinetics were made at 3 and 6 h of the infusion. Glucose uptake by forearm tissues in response to GH and insulin did not change significantly between 3 and 6 h. By 6 h, the combined infusion of GH and insulin promoted a significantly more positive net balance of phenylalanine, leucine, isoleucine, and valine (all P less than 0.05). The change in net phenylalanine balance was due to a significant increase in phenylalanine Rd (51%, P less than 0.05) with no observable change in phenylalanine Ra. For leucine, a stimulation of leucine Rd (50%, P less than 0.05) also accounted for the change in leucine net balance, with no suppression of leucine Ra. The stimulation of Rd, in the absence of an observed effect on Ra, suggests that GH blunts the action of insulin to suppress proteolysis in addition to blunting insulin's action on Rd.

Recurrent Cushing's disease with low adrenal androgen production.

A 33 year old woman presented with recurrent Cushing's disease 4 years after complete remission induced by pituitary surgery. On relapse she exhibited the unusual pattern of elevated indices of cortisol secretion with markedly suppressed serum DHEA-S; urinary 17-ketosteroid excretion was also below the normal range. Biochemical testing was otherwise consistent with ACTH-mediated hypercortisolism, and adrenal histopathology showed bilateral hyperplasia with no evidence of tumor. This case illustrates that serum DHEA-S is not an infallible guide to the differential diagnosis of Cushing's syndrome, and it supports the existence of a pituitary-secreted adrenal androgen stimulating factor that is distinct from ACTH.

Brain and atrial natriuretic peptides bind to common receptors in brain capillary endothelial cells.

The recent discovery of brain natriuretic peptides (BNP) that stimulates natriuresis, diuresis, and vascular smooth muscle relaxation in a manner similar to that of atrial natriuretic peptide (ANP) suggests the possibility that these endocrine hormones function via some common mechanism. Indirect evidence from several laboratories suggests that BNP and ANP may bind to the same receptors. We examined whether ANP and BNP bind to a common set of receptors in cultured bovine brain capillary endothelial cells and in bovine aortic endothelial cells. Scatchard plot analysis of binding data shows a similar dissociation constant (KD) of approximately 0.3 nM and a maximal binding capacity (Bmax) of 50 fmol/mg protein for both natriuretic peptides in brain capillary cells and 0.6 nM and 80 fmol/mg protein, respectively, in the aortic endothelial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity cross-linked receptor-ligand complex shows a strongly labeled 65-kDa receptor and a 125-kDa band that is likely to be a receptor of the guanylate cyclase type. ANP and BNP cross compete equally for binding to the two receptors identified on the gels. ANP and BNP also stimulate guanosine 3', 5'-cyclic monophosphate production in these cells, consistent with the presence of a functional guanylate cyclase-linked B receptor. We conclude that ANP and BNP share common receptors in brain capillary and aortic endothelial cells.

Growth hormone acutely stimulates forearm muscle protein synthesis in normal humans.

The short-term effects of growth hormone (GH) on skeletal muscle protein synthesis and degradation in normal humans are unknown. We studied seven postabsorptive healthy men (age 18-23 yr) who received GH (0.014 micrograms.kg-1.min-1) via intrabrachial artery infusion for 6 h. The effects of GH on forearm amino acid and glucose balances and on forearm amino acid kinetics [( 3H]Phe and [14C]Leu) were determined after 3 and 6 h of the GH infusion. Forearm deep vein GH rose to 35 +/- 6 ng/ml in response to GH, whereas systemic levels of GH, insulin, and insulin-like growth factor I (IGF-I) were unchanged. Forearm glucose uptake did not change during the study. After 6 h, GH suppressed forearm net release (3 vs. 6 h) of Phe (P less than 0.05), Leu (P less than 0.01), total branched-chain amino acids (P less than 0.025), and essential neutral amino acids (0.05 less than P less than 0.1). The effect on the net balance of Phe and Leu was due to an increase in the tissue uptake for Phe (71%, P less than 0.05) and Leu (37%, P less than 0.005) in the absence of any significant change in release of Phe or Leu from tissue. In the absence of any change in systemic GH, IGF-I, or insulin, these findings suggest that locally infused GH stimulates skeletal muscle protein synthesis. These findings have important physiological implications for both the role of daily GH pulses and the mechanisms through which GH can promote protein anabolism.

Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man.

1. Using the forearm balance method, together with systemic infusions of L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine, we examined the effects of infused branched-chain amino acids on whole-body and skeletal muscle amino acid kinetics in 10 postabsorptive normal subjects; 10 control subjects received only saline. 2. Infusion of branched-chain amino acids caused a four-fold rise in arterial branched-chain amino acid levels and a two-fold rise in branched-chain keto acids; significant declines were observed in circulating levels of most other amino acids, including phenylalanine, which fell by 34%. Plasma insulin levels were unchanged from basal levels (8 +/- 1 mu-units/ml). 3. Whole-body phenylalanine flux, an index of proteolysis, was significantly suppressed by branched-chain amino acid infusion (P less than 0.002), and forearm phenylalanine production was also inhibited (P less than 0.03). With branched-chain amino acid infusion total leucine flux rose, with marked increments in both oxidative and non-oxidative leucine disposal (P less than 0.001). Proteolysis, as measured by endogenous leucine production, showed a modest 12% decrease, although this was not significant when compared with saline controls. The net forearm balance of leucine and other branched-chain amino acids changed from a basal net output to a marked net uptake (P less than 0.001) during branched-chain amino acid infusion, with significant stimulation of local leucine disposal. Despite the rise in whole-body non-oxidative leucine disposal, and in forearm leucine uptake and disposal, forearm phenylalanine disposal, an index of muscle protein synthesis, was not stimulated by infusion of branched-chain amino acids. 4. The results suggest that in normal man branched-chain amino acid infusion suppresses skeletal muscle proteolysis independently of any rise of plasma insulin. Muscle branched-chain amino acid uptake rose dramatically in the absence of any apparent increase in muscle protein synthesis, as measured by phenylalanine disposal, or in branched-chain keto acid release. Thus, an increase in muscle branched-chain amino acid concentrations and/or local branched-chain amino acid oxidation must account for the increased disposal of branched-chain amino acids.

Effect of starvation on human muscle protein metabolism and its response to insulin.

Although starvation is known to impair insulin-stimulated glucose disposal, whether it also induces resistance to insulin's antiproteolytic action on muscle is unknown. To assess the effect of fasting on muscle protein turnover in the basal state and in response to insulin, we measured forearm amino acid kinetics, using [3H]phenylalanine (Phe) and [14C]leucine (Leu) infused systemically, in eight healthy subjects after 12 (postabsorptive) and 60 h of fasting. After a 150-min basal period, forearm local insulin concentration was selectively raised by approximately 25 muU/ml for 150 min by intra-arterial insulin infusion (0.02 mU.kg-1. min-1). The 60-h fast increased urine nitrogen loss and whole body Leu flux and oxidation (by 50-75%, all P less than 0.02). Post-absorptively, forearm muscle exhibited a net release of Phe and Leu, which increased two- to threefold after the 60-h fast (P less than 0.05); this effect was mediated exclusively by accelerated local rates of amino acid appearance (Ra), with no reduction in rates of disposal (Rd). Local hyperinsulinemia in the postabsorptive condition caused a twofold increase in forearm glucose uptake (P less than 0.01) and completely suppressed the net forearm output of Phe and Leu (P less than 0.02). After the 60-h fast, forearm glucose disposal was depressed basally and showed no response to insulin; in contrast, insulin totally abolished the accelerated net forearm release of Phe and Leu. The action of insulin to reverse the augmented net release of Phe and Leu was mediated exclusively by approximately 40% suppression of Ra (P less than 0.02) rather than a stimulation of Rd. We conclude that in short-term fasted humans 1) muscle amino acid output accelerates due to increased proteolysis rather than reduced protein synthesis, and 2) despite its catabolic state and a marked impairment in insulin-mediated glucose disposal, muscle remains sensitive to insulin's antiproteolytic action.

In vivo measurement of myocardial protein turnover using an indicator dilution technique.

We applied a nondestructive tracer technique, previously developed for measuring skeletal muscle protein turnover, to the measurement of myocardial protein turnover in vivo. During a continuous infusion of L-[ring-2,6-3H]phenylalanine to anesthetized, overnight-fasted dogs, we measured the uptake of radiolabeled phenylalanine from plasma and the release of unlabeled phenylalanine from myocardial proteolysis using arterial and coronary sinus catheterization and analytic methods previously applied to skeletal muscle. Using these measurements, together with a model of myocardial protein synthesis that assumes rapid equilibration of tracer specific activity between myocardial phenylalanyl-tRNA and circulating phenylalanine, we estimated the rates of heart protein synthesis and degradation. The rate of heart protein synthesis was also estimated directly from the incorporation of labeled phenylalanine into tissue protein. The use of [3H]phenylalanine was compared with L-[1-14C]leucine in the measurement of heart protein turnover in dogs given simultaneous infusion of both tracers. Leucine uptake and release by the myocardium exceeded that of phenylalanine by 3.1 +/- 0.4- and 1.7 +/- 0.3-fold, respectively, consistent with leucine's 2.4-fold greater abundance in heart protein and its metabolism via other pathways. Phenylalanine is the preferred tracer for use with this method because of its limited metabolic fate in muscle. One theoretical limitation to the method, slow equilibration of circulating labeled phenylalanine with myocardial phenylalanyl-tRNA, was resolved by comparison of these specific activities after a 30-minute infusion of labeled phenylalanine in the rat. A second, empirical limitation involves precision in the measurement of the small decrements in phenylalanine specific activity that occur with each pass of blood through the coronary circulation. This was addressed by improving the precision of both the measurements of phenylalanine concentration and phenylalanine specific activity using high-performance liquid chromatography. We conclude that the in vivo measurement of phenylalanine tracer exchange across the myocardium permits the nondestructive estimation of heart protein turnover in the intact animal.