Differential Effects of Prednisone and Growth Hormone on Fuel Metabolism and Insulin Antagonism in Humans FRITZ F. HORBER, H. MICHAEL MARSH, AND MOREY W. HAYMOND
Human growth hormone (hGH) and prednisone cause insulin resistance and glucose intolerance. However, it is unknown whether hGH and prednisone antagonize insulin action on protein, fat, and carbohydrate metabolism by a common or independent mechanism. Therefore, protein, fat, and carbohydrate metabolism was assessed simultaneously in four groups of eight subjects each after 7 days of placebo, recombinant DNA hGH (rhGH; 0.1 mg k g 1 - day 1 ), prednisone (0.8 mg k g 1 • day 1 ), or rhGH and prednisone administration after an 18-h fast and during gut infusion of glucose and amino acids (fed state). Fasting plasma glucose concentrations were similar during placebo and rhGH but elevated (P < 0.001) during combined treatment, whereas plasma insulin concentrations were higher (237 ± 57 pmol/ml, P < 0.001) during combined than during placebo, rhGH, or prednisone treatment (34, 52, and 91 pM, respectively). In the fed state, plasma glucose concentrations were elevated only during combined treatment (11.3 ± 2.1 mM, P < 0.001). Plasma insulin concentrations were elevated during therapy with prednisone alone and rhGH alone (667 ± 72 and 564 ± 65 pmol/ml, respectively, P < 0.001) compared with placebo (226 ± 44 pmol/ml) but lower than with the combined rhGH and prednisone treatment (1249 ± 54 pmol/ml, P < 0.01). Protein oxidation (["CJleucine) increased (P < 0.001) with prednisone therapy, decreased (P < 0.001) with rhGH treatment, and was normal during the combined treatment. By indirect calorimetry, glucose oxidation was similar in all groups; fasting fat oxidation was decreased by prednisone (P < 0.001) and increased by rhGH, although only significantly in the fed state (P < 0.01). In summary, insulin antagonism of rhGH and prednisone are probably caused by independent mechanisms in From the Departments of Pediatrics and Anesthesiology, Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota; and Medizinische Universitaets Poliklinik, Inselspital Bern, Bern, Switzerland. Address correspondence and reprint requests to M.W. Haymond, MD, Nemours Children's Clinic, PO Box 5720, Jacksonville, FL 32247. Received for publication 5 March 1990 and accepted in revised form 5 September 1990.
DIABETES, VOL. 40, JANUARY 1991
which rhGH and prednisone may reciprocally regulate the oxidation of protein and fat while decreasing the efficiency of glucose disposal. Diabetes 40:141-49, 1991
H
uman growth hormone (hGH; 1-4) and glucocorticosteroids (5-8) induce carbohydrate intolerance and insulin resistance in both hepatic and peripheral tissues. The mechanisms by which these agents affect hepatic glucose release and disposal of both enterally absorbed and endogenously produced glucose remains to be determined. However, the similar effects of hGH and prednisone on carbohydrate metabolism suggest that the insulin antagonism of these two drugs might be operating through a common mechanism. In contrast, long-term treatment with prednisone is known to cause protein wasting (9-11) and accumulation of body fat, whereas hGH treatment is associated with decreased body fat and protein anabolism (12-17). The latter data provide strong evidence that prednisone and hGH may differentially affect the oxidation of fat and protein and are operating through independent mechanisms. Therefore, we examined simultaneously the effects of rhGH and/or prednisone therapy on carbohydrate, fat, and protein metabolism in healthy subjects. RESEARCH DESIGN AND METHODS
This study was carried out in the same subjects and at the same time as our study of the leucine kinetic aspects of these investigations (18). After review and approval of the protocol by the Mayo Institutional Review Board and the Clinical Research Center (CRC) Advisory Committee, informed consent was obtained from 32 healthy adult volunteers between the ages of 18 and 36 yr and within 6% of ideal body weight. None had a family history of diabetes mellitus in first-degree relatives, gastritis, peptic ulcer disease, or history of gastrointestinal bleeding. All had a normal 2-h postprandial plasma glucose concentration after a meal
141
GROWTH HORMONE, PREDNISONE. AND SUBSTRATE METABOLISM
containing at least 100 g of glucose to exclude possible carbohydrate intolerance. Only subjects exhibiting normal hematology, chemistry, urinalysis, and plasma thyroxine values were enrolled in the study. The laboratory values mentioned above did not change as a result of the following drug or placebo therapy (see below). Subjects were randomized to one of four study groups (8 subjects/group) and received oral tablets and injections in a single-blinded fashion under the supervision of the nursing staff of the Mayo CRC (Fig. 1). Group 1 served as controls (placebo lactose tablets and saline injections). Group 2 (prednisone tablets and saline injections) received prednisone orally in three equal doses 15 min before breakfast, lunch, and dinner for 7 days at a dose of 0.8 mg • k g - 1 • d a y 1 . Group 3 (placebo tablets and subcutaneous injections of 0.1 m g - k g - 1 - d a y - 1 recombinant DNA hGH [rhGH]) received rhGH 15 min before dinner (daily alternation between both thighs) for 7 days before the study. Group 4 (prednisone tablets and rhGH injections) received prednisone and rhGH as described above for groups 2 and 3. Subjects consumed only the prescribed diet of 31-35 kcal • kg" 1 • day" 1 containing ~53, 29, and 18% carbohydrate, fat, and protein, respectively, for 7 days in the CRC. On each of the study days, plasma fasting glucose concentrations were determined. Subsequently, all subjects were admitted to the Mayo CRC in the afternoon of the 7th treatment day and were hospitalized for the next 3 consecutive days. At ~1700 on the day of admission, 20 (juCi 3H2O was administered orally to each subject and then urine was collected before and 2, 4, and 6 h after to determine whole-body water specific activity (SA) from which lean body mass (LBM) was calculated as previously described (19). At - 1 7 3 0 on the afternoon of admission, an intravenous catheter was placed in an antecubital vein, and a continuous infusion of prednisolone (0.8 mg - k g " 1 • day" 1 , groups 2 and 4) or 0.9% saline (groups 1 and 3) was started and continued throughout the 3 days of inpatient study. Three different study protocols were carried out over the 3 days of hospitalization study (see below). After completion of each study, subjects received their full daily diet in two meals. Study A. At 0330 after admission, exact body weight was obtained, and another intravenous site was established in a
contralateral hand vein for arterialized venous blood sampling throughout the study as previously described (20,21). At 0400, a primed constant infusion of sodium [14C]bicarbonate (5 |xCi then 0.08 |xCi • kg" 1 • min" 1 ) was initiated and continued for 3.5 h. After 0600, breath samples (but no blood samples) were collected every 20 min. At -0730, a standard nasogastric enteral feeding tube (8 french diam, 109 cm length; Keofeed, IVAC, San Diego, CA) was placed into the duodenum. Primed constant intravenous infusions of [1-14C]leucine (7 (xCi then 15 |xCi/h in groups 1, 2, and 4 and 14 (xCi then 30 jxCi/h in group 3) and [6-3H]glucose (15 (xCi then 14 jxCi/h) were initiated at - 0 8 0 0 and continued for 6 h. In addition, [6,6-2H2]glucose (2300 mg/6 h) was infused at a constant rate via the enteral tube over the 6 h of study. The latter isotope was added to the enteral infusion to trace the systemic entry of glucose and permit the partitioning of the circulating glucose into endogenous and exogenous components. In addition, 0.45% saline was infused at 160 ml/h with a Travenol infusion pump (Baxter, Deerfield, IL), a volume equal to that of the test meal infused in studies B and C (see below). After 4 h of isotope infusion, blood and breath samples were obtained at 20-min intervals for the remaining 2 h (see below). Study B. Studies B and C were carried out in random order. Study B was carried out in identical fashion to that of study A except that 1) a solution containing glucose and mixed amino acids (Travasol, Baxter) was infused to deliver 28.6 and 1.1 n-mol • kg" 1 LBM • min" 1 of glucose and leucine, respectively, over the 6 h of study to simulate the fed state, and 2) the infusion rates of [14C]leucine in study A were doubled in the groups receiving prednisone (2 and 4). Study C. This study was carried out in identical fashion to study B except that 7) no [1-14C]leucine was infused, and 2) sodium [14C]bicarbonate was infused throughout the entire study period (i.e., for 10 h). Study C was necessary to measure 14CO2 recovery from NaH14CO3 in the fed state (0800-1400) to correct for individual meal-induced differences in 14CO2 fixation. Blood and breath sampling. Approximately 17 ml blood was drawn, and breath samples were collected at each sampling time (0, 260, 280,300, 320, 340, and 360 min) of studies A-C. At 0, 240, and 360 min, an additional 6 ml blood was drawn to determine the plasma concentrations of glucose (YSI glucose analyzer, Yellow Springs, OH), insulin, C-pep-
Diet and nitrogen balance studies r-hGH
or
placebo
(0.1 mg/kg SQ daily) Prednisone
or
placebo
(0.8 mg/kg daily t.i.d.) l
H2O
Prednisolone or placebo (0.8 mg/kg daily i.v.) A
•
B l**l
-7
-5
-3
-2
-1
Study days
142
+1
+2
+3
FIG. 1. Overall study design employed in these investigations. See protocol description in METHODS for further details. r-hGH, recombinant human growth hormone; SQ, subcutaneous injection. 'Postabsorptive. "Fed.
DIABETES, VOL. 40, JANUARY 1991
F.F. HORBER, H.M. MARSH. AND M.W. HAYMOND
tide, growth hormone, glucagon, free fatty acids (FFA), ketone bodies, pyruvate, and lactate (22,23). Blood samples were placed on ice and centrifuged at 4°C, and plasma was stored at -70°C until analyzed. Analysis. [6,6-2H2]glucose (Merck Sharp & Dohme, St. Louis, MO) and i_-[1-14C]leucine (>55 mCi/mmol), sodium [14C]bicarbonate, and 3H2O (all from Amersham, Arlington Heights, IL) were determined to be pyrogen free (limulus amoebocyte lysate) and >99% pure and sterile before use. [6,6-2H2]glucose was determined to be >95% enriched by gas chromatography-mass spectrometry (GC-MS; 5985B Hewlett-Packard, Palo Alto, CA). Prednisone (Upjohn, Kalamazoo, Ml) and prednisolone (Hydeltrasol, Merck Sharp & Dohme) were obtained from commercial sources, whereas rhGH was generously supplied by Genentech (Palo Alto, CA). Plasma [14C]-labeled a-ketoisocaproate (a-KIC) SA was determined by high-performance liquid chromatography (24). The enrichment of plasma [2H2]glucose was determined by GC-MS with the dibutylboronic-acetal derivative (25,26). Plasma glucose SAs were determined as previously described (27), except that 0.5 ml of the perchloric acidprecipitated samples were transferred to two 5 x 1-cm columns (QS-Q, Isolab, Akron, OH) arranged in series containing 3 ml of a 50% aqueous solution of an anion-exchange resin (AG1 x 8 , formate form, 100-200 mesh) and a cationexchange resin (AG50W x80, H + form, 100-200 mesh) to remove radiolabeled a-KIC (AG1) and leucine (AG50), respectively. The columns were then washed four times with 2-ml aliquots of deionized distilled H2O. The effluent plus washing were collected in scintillation vials (Research Products, Mount Prospect, IL) and thereafter processed as previously described (27). Tracer from each study was added to a sample of each subject's plasma obtained before infusion of isotope and processed in parallel with the experimental sample to correct for any systematic losses of labeled glucose during the isolation procedure. Expired rates of 14CO2 were determined by collecting expired air over 2-min periods in 50-L Douglas bags and slowly aspirating through 250 ml 1 M ethanolamine solution to determine quantitatively 14CO2 expired during isotopic steady state. Triplicate 1-ml aliquots of the resultant solution were diluted in 7 ml of Safety Solve (Research Products) (28). In addition, the SA of breath 14CO2 was determined at each breath sampling time by slowly aspirating expired air through a scintillation vial containing 2 ml 0.5 M Hyamine Hydroxide in ethanol with thymolphthalein as a pH indicator. Fourteen milliliters of Safety Solve was added and the 14C radioactivity determined by scintillation spectrometry (28). Indirect calorimetry was carried out with a Beckman metabolic cart (Fullerton, CA) at 240, 300, and 360 min of studies A-C (29). The 14C radioactivity in a-KIC and CO2 and 3H radioactivity in glucose and H2O were determined with a Beckman LS9800 series liquid-scintillation counter with dual-counting mode for glucose, which corrects the radioactivity for both quench and the spillover of 14C radioactivity into the 3H energy spectrum. Calculations. The actual rate of stable isotope infusion was determined as the product of the infusate concentration, isotope enrichment, and the pump infusion rate. Rates of radiolabeled isotope administration were determined by mul-
DIABETES, VOL. 40, JANUARY 1991
tiplying the dpm per milliliter of infusate by the infusion rate (ml • min"1). Estimates of whole-body leucine oxidation ((xmol • kg~1 • min-1) were made at near-substrate and isotopic steady state between 260 and 360 min after starting the leucine tracer infusion with the reciprocal pool model as previously described (30,31). The rate of leucine oxidation was corrected for the individual CO2 recovery obtained during NaH14CO3 infusions in the postabsorptive (mean of studies A-C used) and the fed states during the course of study protocol C (see above). CO2 recovery was calculated by dividing the NaH14CO3 infusion rate (dpm • kg- 1 • min"1) by the 14CO2 extraction rate (dpm • kg- 1 • min~1) at steady state (i.e., after 120 min of isotope infusion). Protein oxidation (mg • kg~1 LBM • min) was calculated with the equation leu oxidation (ixmol • k g - ' • mirr 1 ) x (131.2/0.08) x 10'
(1)
where 131.2 is the molecular weight of leucine, 0.08 is the fraction of leucine in body proteins (32), and 10" 6 converts micromoles to moles. These values for protein oxidation were used in the calculations of the oxidation rates of fat and carbohydrate with Vco2 and Vo2 values obtained by indirect calorimetry (29). If the rate of appearance (Ra) of endogenous glucose during meal infusion (ftafedendo) was zero, then the enrichment of [2H2]glucose in plasma and the enterally infused glucose would be identical. Because this was not the case (Table 1), the total (endogenous and total enterally infused) glucose fta (Atoned) was calculated as atotfed =
[ 2 H 2 ]glu i n ,, a t e /[ 2 H 2 ]glu e r sys
(2)
where [2H2]gluinlrate is the rate of infusion of the [2H2]glucose into the duodenum, and [2H2]gluersys is the systemic plasma glucose enrichment. The meal-derived Ra into the systemic circulation (meal Ra) was calculated (derived from the standard dual-isotope formula) as meal Ra = Raledsys x [ 2 H 2 ]glu ersys /[ 2 H 2 ]glu erme ai
(3)
wherefta(edsysis the Ra calculated from the systemically infused [6-3H]glucose tracer, and [2H2]gluermeai is the enrichment of glucose in the meal. Thefta,edendo°f glucose was calculated as = [ 2 H 2 ]glu in([ale x [(1/[ 2 H 2 ]glu ersys ) -
(i/[ 2 H 2 ]glu e r m e a l )]
(4) Systemic availability of glucose was calculated in the absorptive state with the equation (5) where ftameaisys is the Ra calculated from the systemically infused [3H]glucose tracer, and meal Ra is derived from Eq. 3. Splanchnic glucose uptake was calculated as Plotted -
meal glUCOSein|raie - ftafedendo
(6)
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GROWTH HORMONE, PREDNISONE, AND SUBSTRATE METABOLISM
TABLE 1 Infusion rates, specific activities (SA), and enrichment in 4 treatment groups
[3H]glucose infusion (dpm • kg~1LBM • min~1) Fasting Fed 2 [ H]glucose infusion (nmol • kg^LBM • min~1) Fasting Fed 3 [ H]glucose SA (102 dpm • M-mol"1) Fasting Fed 2 [ H]glucose enrichment (mol%) Fasting Fed Test meal a-[t4C]KIC SA (dpm • nmol"1) Fasting Fed 14 CO2 recovery* (%) Fasting Fed
Control
Prednisone
Human growth hormone
Prednisone + human growth hormone
5558 ± 407 5423 ± 392
5191 ± 397 4833 ± 367
4990 ± 4 1 3 5378 ± 476
4950 ± 204 4714 ± 266
0.44 ± 0.03 0.44 ± 0.03
0.42 ± 0.03 0.42 ± 0.03
0.42 ± 0.03 0.42 ± 0.03
0.40 ± 0.02 0.40 ± 0.02
5.3 ± 0.5 2.0 ± 0.1
4.9 ± 0.3 1.8 ± 0.1
5.2 ± 0.3 2.2 ± 0.1
4.3 ± 0.2 1.9 ±0.1
3.39 ± 0.23 1.19 ± 0.08 1.45 ± 0.10
3.08 ± 0.17 1.12 ± 0.06 1.43 ± 0.06
3.48 ± 0.26 1.23 ±0.09 1.50 ± 0.11
2.75 ±0.15 1.07 ±0.09 1.43 ± 0.08
4.83 ± 0.28 3.49 ± 0.26
4.69 ± 0.67 5.40 ± 0.73
7.21 ± 0.68 5.70 ± 0.65
5.03 ± 0.57 5.41 ± 0.40
73 ± 2 f 85 ± 4
68 ± 2f 84 ± 4
70 ± 2f 85 ± 3
71 ± 3 f 93 ± 3
Values are means ± SE. LBM, lean body mass, a-KIC, a-ketoisocaproate. *During NaHMCO3 infusion. tP < 0.01 vs. fed.
where Ra[0[fecj is derived from Eq. 2, meal glucoseinfrate is the absolute rate of duodenally infused unlabeled glucose, and flafedendo is derived from Eq. 4. Results are given as means ± SE. Groups were compared with analysis of variance (ANOVA), and statistical differences among the groups were established with a post hoc Newman-Keuls test. Comparisons within groups were done with the Student's t test for paired observations. RESULTS
Body weight, height, and LBM were not significantly different among the four groups of subjects studied (mean body weight, body height, and LBM for the entire group of subjects were 69 ± 3 kg, 174 ± 3 cm, and 61 ± 3 kg, respectively) and did not change as a result of any of the treatments. Calorie intake was not different among the groups investigated. Isotope studies. After 4 h of isotope infusion, plasma
a-[14C]KIC SA, [3H]glucose SA, [2H2]glucose enrichment, and 14CO2 recovery were at steady state. The mean values for each study group obtained during min 260-360 in the postabsorptive (study A) and fed (mean of studies B and C) states are depicted in Table 1. In the postabsorptive state, 14 CO2 recovery in expired air from the infused NaH14CO3 was similar in the four groups of subjects investigated and increased by - 1 0 - 1 5 % as a consequence of feeding (P < 0.01, Table 1). Plasma concentrations of hormones and nonglucose substrates. In the postabsorptive state, plasma concentrations of pyruvate were increased (P < 0.05), whereas FFA and ketone bodies were decreased (P < 0.01) in subjects treated with prednisone alone (Table 2). As a consequence of meal infusion, plasma concentrations of lactate and pyruvate increased as expected in all groups of subjects investigated (P < 0.01), whereas FFA and ketone body concentrations decreased (P < 0.01). In subjects treated
TABLE 2 Plasma concentrations of lactate, pyruvate, total ketone bodies, and free fatty acids in 4 treatment groups
Lactate (mM) Fasting Fed Pyruvate (|xM) Fasting Fed Total ketone bodies (mM) Fasting Fed Free fatty acids (mM) Fasting Fed
Control
Prednisone
Human growth hormone
Prednisone + human growth hormone
1.24 ± 0.10 1.90 ±0.20
1.47 ± 0.07 2.40 ±0.10*
1.31 ± 0.12 1.90 ± 0.08
1.24 ± 0.12 2.72 ± 0.16*
57 ± 7 80 ± 15
150 ± 19*
2.04 ± 0.69 0.07 ± 0.01
0.46 ± 0.05* 0.11 ± 0.02
2.24 ± 0.21 0.06 ± 0.01
1.14 ± 0.12 0.23 ± 0.11
1.27 ± 0.17 0.16 ± 0.01
0.86 ± 0.08* 0.19 ± 0.03
1.39 ± 0.10 0.21 ± 0.01
1.03 ± 0.05 0.50 ± 0.14*
85 ± 7t
61 ± 4 109 ± 6
62 ± 6 177 ± 22*
Values are means ± SE. *P < 0.001, fP < 0.05, *P < 0.01, vs. control by analysis of variance.
144
DIABETES, VOL. 40, JANUARY 1991
F.F. HORBER, H.M. MARSH, AND M.W. HAYMOND
with prednisone (with or without hGH), plasma lactate and pyruvate concentrations were greater (P < 0.001) than in subjects treated with placebo or rhGH alone during meal infusion. In the fed state, FFA concentrations were increased (P < 0.01) in subjects on combined hormone treatment. In the postabsorptive state, plasma insulin concentrations were similar in the control subjects and subjects treated with prednisone or rhGH alone but elevated (237 ± 57 pM, P < 0.001) in the combined-treatment group (Table 3). Plasma C-peptide concentrations were elevated (P < 0.001) in the subjects treated with prednisone alone and the combinedtreatment subjects (Table 3). During meal infusion (mean values from studies B and C), plasma insulin and C-peptide concentrations were elevated (P < 0.001 by ANOVA) in subjects treated with prednisone or rhGH alone compared with the control subjects. In addition, plasma concentrations of insulin and C-peptide were higher (P < 0.01 by ANOVA) in the combined-treatment group compared with all other groups. In the postabsorptive state, plasma hGH concentrations measured 19 h after the last injection of saline or rhGH were similar in the control subjects, subjects treated with prednisone alone, and combined-treatment group, whereas after rhGH alone, this value was elevated (P < 0.01). During meal infusion, values and statistical relationships similar to those obtained in the postabsorptive state were observed (Table 3). In subjects treated with prednisone alone or the combined treatment, plasma concentrations of glucagon were elevated during meal infusion (P < 0.01) but not in the postabsorptive state compared with placebo-treated subjects. Indirect calorimetry. Vco2, Vo2, RQ, and resting energy expenditure (REE) were not different in the postabsorptive state among the four groups studied (Table 4). Vco2 and RQ increased (P < 0.01) in all subjects as a consequence of meal infusion, whereas Vo2 and REE increased (P < 0.01) only in prednisone-treated subjects (groups 2 and 4). During infusion of the test meal, RQ was lower (P < 0.05) in both groups treated with rhGH compared with subjects treated with prednisone alone or placebo. Glucose kinetics. Plasma glucose concentrations (control, 5.3 ± 0.3 mM; prednisone, 5.5 ± 0.3 mM; hGH, 5.1 ± 0.34
mM; and rhGH plus prednisone, 5.4 ± 0.2 mM) were similar in all four groups investigated before initiation of drug and/ or placebo treatment (Table 5). During the 2 h of blood sampling in study A (19-21 h of fasting), the plasma glucose concentrations were increased in the groups treated with prednisone and prednisone plus rhGH (P < 0.001 by ANOVA) compared with placebo-treated subjects (Table 5). Plasma glucose concentrations were increased (P < 0.001) in all four groups investigated during administration of the test meal (28.6 ixmol glucose • kg" 1 LBM • min"1) on both study days (only averaged data from studies B and C). No significant differences (by ANOVA) in plasma glucose concentrations during meal infusion were observed among the control subjects and subjects treated with prednisone or rhGH alone; however, during combined treatment with rhGH and prednisone, plasma glucose concentrations were elevated (11.3 ± 2.1 mM,P
Human growth hormone (hGH) and prednisone cause insulin resistance and glucose intolerance. However, it is unknown whether hGH and prednisone antagonize insulin action on protein, fat, and carbohydrate metabolism by a common or independent mechanism. Therefore, protein, fat, and carbohydrate metabolism was assessed simultaneously in four groups of eight subjects each after 7 days of placebo, recombinant DNA hGH (rhGH; 0.1 mg k g 1 - day 1 ), prednisone (0.8 mg k g 1 • day 1 ), or rhGH and prednisone administration after an 18-h fast and during gut infusion of glucose and amino acids (fed state). Fasting plasma glucose concentrations were similar during placebo and rhGH but elevated (P < 0.001) during combined treatment, whereas plasma insulin concentrations were higher (237 ± 57 pmol/ml, P < 0.001) during combined than during placebo, rhGH, or prednisone treatment (34, 52, and 91 pM, respectively). In the fed state, plasma glucose concentrations were elevated only during combined treatment (11.3 ± 2.1 mM, P < 0.001). Plasma insulin concentrations were elevated during therapy with prednisone alone and rhGH alone (667 ± 72 and 564 ± 65 pmol/ml, respectively, P < 0.001) compared with placebo (226 ± 44 pmol/ml) but lower than with the combined rhGH and prednisone treatment (1249 ± 54 pmol/ml, P < 0.01). Protein oxidation (["CJleucine) increased (P < 0.001) with prednisone therapy, decreased (P < 0.001) with rhGH treatment, and was normal during the combined treatment. By indirect calorimetry, glucose oxidation was similar in all groups; fasting fat oxidation was decreased by prednisone (P < 0.001) and increased by rhGH, although only significantly in the fed state (P < 0.01). In summary, insulin antagonism of rhGH and prednisone are probably caused by independent mechanisms in From the Departments of Pediatrics and Anesthesiology, Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota; and Medizinische Universitaets Poliklinik, Inselspital Bern, Bern, Switzerland. Address correspondence and reprint requests to M.W. Haymond, MD, Nemours Children's Clinic, PO Box 5720, Jacksonville, FL 32247. Received for publication 5 March 1990 and accepted in revised form 5 September 1990.
DIABETES, VOL. 40, JANUARY 1991
which rhGH and prednisone may reciprocally regulate the oxidation of protein and fat while decreasing the efficiency of glucose disposal. Diabetes 40:141-49, 1991
H
uman growth hormone (hGH; 1-4) and glucocorticosteroids (5-8) induce carbohydrate intolerance and insulin resistance in both hepatic and peripheral tissues. The mechanisms by which these agents affect hepatic glucose release and disposal of both enterally absorbed and endogenously produced glucose remains to be determined. However, the similar effects of hGH and prednisone on carbohydrate metabolism suggest that the insulin antagonism of these two drugs might be operating through a common mechanism. In contrast, long-term treatment with prednisone is known to cause protein wasting (9-11) and accumulation of body fat, whereas hGH treatment is associated with decreased body fat and protein anabolism (12-17). The latter data provide strong evidence that prednisone and hGH may differentially affect the oxidation of fat and protein and are operating through independent mechanisms. Therefore, we examined simultaneously the effects of rhGH and/or prednisone therapy on carbohydrate, fat, and protein metabolism in healthy subjects. RESEARCH DESIGN AND METHODS
This study was carried out in the same subjects and at the same time as our study of the leucine kinetic aspects of these investigations (18). After review and approval of the protocol by the Mayo Institutional Review Board and the Clinical Research Center (CRC) Advisory Committee, informed consent was obtained from 32 healthy adult volunteers between the ages of 18 and 36 yr and within 6% of ideal body weight. None had a family history of diabetes mellitus in first-degree relatives, gastritis, peptic ulcer disease, or history of gastrointestinal bleeding. All had a normal 2-h postprandial plasma glucose concentration after a meal
141
GROWTH HORMONE, PREDNISONE. AND SUBSTRATE METABOLISM
containing at least 100 g of glucose to exclude possible carbohydrate intolerance. Only subjects exhibiting normal hematology, chemistry, urinalysis, and plasma thyroxine values were enrolled in the study. The laboratory values mentioned above did not change as a result of the following drug or placebo therapy (see below). Subjects were randomized to one of four study groups (8 subjects/group) and received oral tablets and injections in a single-blinded fashion under the supervision of the nursing staff of the Mayo CRC (Fig. 1). Group 1 served as controls (placebo lactose tablets and saline injections). Group 2 (prednisone tablets and saline injections) received prednisone orally in three equal doses 15 min before breakfast, lunch, and dinner for 7 days at a dose of 0.8 mg • k g - 1 • d a y 1 . Group 3 (placebo tablets and subcutaneous injections of 0.1 m g - k g - 1 - d a y - 1 recombinant DNA hGH [rhGH]) received rhGH 15 min before dinner (daily alternation between both thighs) for 7 days before the study. Group 4 (prednisone tablets and rhGH injections) received prednisone and rhGH as described above for groups 2 and 3. Subjects consumed only the prescribed diet of 31-35 kcal • kg" 1 • day" 1 containing ~53, 29, and 18% carbohydrate, fat, and protein, respectively, for 7 days in the CRC. On each of the study days, plasma fasting glucose concentrations were determined. Subsequently, all subjects were admitted to the Mayo CRC in the afternoon of the 7th treatment day and were hospitalized for the next 3 consecutive days. At ~1700 on the day of admission, 20 (juCi 3H2O was administered orally to each subject and then urine was collected before and 2, 4, and 6 h after to determine whole-body water specific activity (SA) from which lean body mass (LBM) was calculated as previously described (19). At - 1 7 3 0 on the afternoon of admission, an intravenous catheter was placed in an antecubital vein, and a continuous infusion of prednisolone (0.8 mg - k g " 1 • day" 1 , groups 2 and 4) or 0.9% saline (groups 1 and 3) was started and continued throughout the 3 days of inpatient study. Three different study protocols were carried out over the 3 days of hospitalization study (see below). After completion of each study, subjects received their full daily diet in two meals. Study A. At 0330 after admission, exact body weight was obtained, and another intravenous site was established in a
contralateral hand vein for arterialized venous blood sampling throughout the study as previously described (20,21). At 0400, a primed constant infusion of sodium [14C]bicarbonate (5 |xCi then 0.08 |xCi • kg" 1 • min" 1 ) was initiated and continued for 3.5 h. After 0600, breath samples (but no blood samples) were collected every 20 min. At -0730, a standard nasogastric enteral feeding tube (8 french diam, 109 cm length; Keofeed, IVAC, San Diego, CA) was placed into the duodenum. Primed constant intravenous infusions of [1-14C]leucine (7 (xCi then 15 |xCi/h in groups 1, 2, and 4 and 14 (xCi then 30 jxCi/h in group 3) and [6-3H]glucose (15 (xCi then 14 jxCi/h) were initiated at - 0 8 0 0 and continued for 6 h. In addition, [6,6-2H2]glucose (2300 mg/6 h) was infused at a constant rate via the enteral tube over the 6 h of study. The latter isotope was added to the enteral infusion to trace the systemic entry of glucose and permit the partitioning of the circulating glucose into endogenous and exogenous components. In addition, 0.45% saline was infused at 160 ml/h with a Travenol infusion pump (Baxter, Deerfield, IL), a volume equal to that of the test meal infused in studies B and C (see below). After 4 h of isotope infusion, blood and breath samples were obtained at 20-min intervals for the remaining 2 h (see below). Study B. Studies B and C were carried out in random order. Study B was carried out in identical fashion to that of study A except that 1) a solution containing glucose and mixed amino acids (Travasol, Baxter) was infused to deliver 28.6 and 1.1 n-mol • kg" 1 LBM • min" 1 of glucose and leucine, respectively, over the 6 h of study to simulate the fed state, and 2) the infusion rates of [14C]leucine in study A were doubled in the groups receiving prednisone (2 and 4). Study C. This study was carried out in identical fashion to study B except that 7) no [1-14C]leucine was infused, and 2) sodium [14C]bicarbonate was infused throughout the entire study period (i.e., for 10 h). Study C was necessary to measure 14CO2 recovery from NaH14CO3 in the fed state (0800-1400) to correct for individual meal-induced differences in 14CO2 fixation. Blood and breath sampling. Approximately 17 ml blood was drawn, and breath samples were collected at each sampling time (0, 260, 280,300, 320, 340, and 360 min) of studies A-C. At 0, 240, and 360 min, an additional 6 ml blood was drawn to determine the plasma concentrations of glucose (YSI glucose analyzer, Yellow Springs, OH), insulin, C-pep-
Diet and nitrogen balance studies r-hGH
or
placebo
(0.1 mg/kg SQ daily) Prednisone
or
placebo
(0.8 mg/kg daily t.i.d.) l
H2O
Prednisolone or placebo (0.8 mg/kg daily i.v.) A
•
B l**l
-7
-5
-3
-2
-1
Study days
142
+1
+2
+3
FIG. 1. Overall study design employed in these investigations. See protocol description in METHODS for further details. r-hGH, recombinant human growth hormone; SQ, subcutaneous injection. 'Postabsorptive. "Fed.
DIABETES, VOL. 40, JANUARY 1991
F.F. HORBER, H.M. MARSH. AND M.W. HAYMOND
tide, growth hormone, glucagon, free fatty acids (FFA), ketone bodies, pyruvate, and lactate (22,23). Blood samples were placed on ice and centrifuged at 4°C, and plasma was stored at -70°C until analyzed. Analysis. [6,6-2H2]glucose (Merck Sharp & Dohme, St. Louis, MO) and i_-[1-14C]leucine (>55 mCi/mmol), sodium [14C]bicarbonate, and 3H2O (all from Amersham, Arlington Heights, IL) were determined to be pyrogen free (limulus amoebocyte lysate) and >99% pure and sterile before use. [6,6-2H2]glucose was determined to be >95% enriched by gas chromatography-mass spectrometry (GC-MS; 5985B Hewlett-Packard, Palo Alto, CA). Prednisone (Upjohn, Kalamazoo, Ml) and prednisolone (Hydeltrasol, Merck Sharp & Dohme) were obtained from commercial sources, whereas rhGH was generously supplied by Genentech (Palo Alto, CA). Plasma [14C]-labeled a-ketoisocaproate (a-KIC) SA was determined by high-performance liquid chromatography (24). The enrichment of plasma [2H2]glucose was determined by GC-MS with the dibutylboronic-acetal derivative (25,26). Plasma glucose SAs were determined as previously described (27), except that 0.5 ml of the perchloric acidprecipitated samples were transferred to two 5 x 1-cm columns (QS-Q, Isolab, Akron, OH) arranged in series containing 3 ml of a 50% aqueous solution of an anion-exchange resin (AG1 x 8 , formate form, 100-200 mesh) and a cationexchange resin (AG50W x80, H + form, 100-200 mesh) to remove radiolabeled a-KIC (AG1) and leucine (AG50), respectively. The columns were then washed four times with 2-ml aliquots of deionized distilled H2O. The effluent plus washing were collected in scintillation vials (Research Products, Mount Prospect, IL) and thereafter processed as previously described (27). Tracer from each study was added to a sample of each subject's plasma obtained before infusion of isotope and processed in parallel with the experimental sample to correct for any systematic losses of labeled glucose during the isolation procedure. Expired rates of 14CO2 were determined by collecting expired air over 2-min periods in 50-L Douglas bags and slowly aspirating through 250 ml 1 M ethanolamine solution to determine quantitatively 14CO2 expired during isotopic steady state. Triplicate 1-ml aliquots of the resultant solution were diluted in 7 ml of Safety Solve (Research Products) (28). In addition, the SA of breath 14CO2 was determined at each breath sampling time by slowly aspirating expired air through a scintillation vial containing 2 ml 0.5 M Hyamine Hydroxide in ethanol with thymolphthalein as a pH indicator. Fourteen milliliters of Safety Solve was added and the 14C radioactivity determined by scintillation spectrometry (28). Indirect calorimetry was carried out with a Beckman metabolic cart (Fullerton, CA) at 240, 300, and 360 min of studies A-C (29). The 14C radioactivity in a-KIC and CO2 and 3H radioactivity in glucose and H2O were determined with a Beckman LS9800 series liquid-scintillation counter with dual-counting mode for glucose, which corrects the radioactivity for both quench and the spillover of 14C radioactivity into the 3H energy spectrum. Calculations. The actual rate of stable isotope infusion was determined as the product of the infusate concentration, isotope enrichment, and the pump infusion rate. Rates of radiolabeled isotope administration were determined by mul-
DIABETES, VOL. 40, JANUARY 1991
tiplying the dpm per milliliter of infusate by the infusion rate (ml • min"1). Estimates of whole-body leucine oxidation ((xmol • kg~1 • min-1) were made at near-substrate and isotopic steady state between 260 and 360 min after starting the leucine tracer infusion with the reciprocal pool model as previously described (30,31). The rate of leucine oxidation was corrected for the individual CO2 recovery obtained during NaH14CO3 infusions in the postabsorptive (mean of studies A-C used) and the fed states during the course of study protocol C (see above). CO2 recovery was calculated by dividing the NaH14CO3 infusion rate (dpm • kg- 1 • min"1) by the 14CO2 extraction rate (dpm • kg- 1 • min~1) at steady state (i.e., after 120 min of isotope infusion). Protein oxidation (mg • kg~1 LBM • min) was calculated with the equation leu oxidation (ixmol • k g - ' • mirr 1 ) x (131.2/0.08) x 10'
(1)
where 131.2 is the molecular weight of leucine, 0.08 is the fraction of leucine in body proteins (32), and 10" 6 converts micromoles to moles. These values for protein oxidation were used in the calculations of the oxidation rates of fat and carbohydrate with Vco2 and Vo2 values obtained by indirect calorimetry (29). If the rate of appearance (Ra) of endogenous glucose during meal infusion (ftafedendo) was zero, then the enrichment of [2H2]glucose in plasma and the enterally infused glucose would be identical. Because this was not the case (Table 1), the total (endogenous and total enterally infused) glucose fta (Atoned) was calculated as atotfed =
[ 2 H 2 ]glu i n ,, a t e /[ 2 H 2 ]glu e r sys
(2)
where [2H2]gluinlrate is the rate of infusion of the [2H2]glucose into the duodenum, and [2H2]gluersys is the systemic plasma glucose enrichment. The meal-derived Ra into the systemic circulation (meal Ra) was calculated (derived from the standard dual-isotope formula) as meal Ra = Raledsys x [ 2 H 2 ]glu ersys /[ 2 H 2 ]glu erme ai
(3)
wherefta(edsysis the Ra calculated from the systemically infused [6-3H]glucose tracer, and [2H2]gluermeai is the enrichment of glucose in the meal. Thefta,edendo°f glucose was calculated as = [ 2 H 2 ]glu in([ale x [(1/[ 2 H 2 ]glu ersys ) -
(i/[ 2 H 2 ]glu e r m e a l )]
(4) Systemic availability of glucose was calculated in the absorptive state with the equation (5) where ftameaisys is the Ra calculated from the systemically infused [3H]glucose tracer, and meal Ra is derived from Eq. 3. Splanchnic glucose uptake was calculated as Plotted -
meal glUCOSein|raie - ftafedendo
(6)
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GROWTH HORMONE, PREDNISONE, AND SUBSTRATE METABOLISM
TABLE 1 Infusion rates, specific activities (SA), and enrichment in 4 treatment groups
[3H]glucose infusion (dpm • kg~1LBM • min~1) Fasting Fed 2 [ H]glucose infusion (nmol • kg^LBM • min~1) Fasting Fed 3 [ H]glucose SA (102 dpm • M-mol"1) Fasting Fed 2 [ H]glucose enrichment (mol%) Fasting Fed Test meal a-[t4C]KIC SA (dpm • nmol"1) Fasting Fed 14 CO2 recovery* (%) Fasting Fed
Control
Prednisone
Human growth hormone
Prednisone + human growth hormone
5558 ± 407 5423 ± 392
5191 ± 397 4833 ± 367
4990 ± 4 1 3 5378 ± 476
4950 ± 204 4714 ± 266
0.44 ± 0.03 0.44 ± 0.03
0.42 ± 0.03 0.42 ± 0.03
0.42 ± 0.03 0.42 ± 0.03
0.40 ± 0.02 0.40 ± 0.02
5.3 ± 0.5 2.0 ± 0.1
4.9 ± 0.3 1.8 ± 0.1
5.2 ± 0.3 2.2 ± 0.1
4.3 ± 0.2 1.9 ±0.1
3.39 ± 0.23 1.19 ± 0.08 1.45 ± 0.10
3.08 ± 0.17 1.12 ± 0.06 1.43 ± 0.06
3.48 ± 0.26 1.23 ±0.09 1.50 ± 0.11
2.75 ±0.15 1.07 ±0.09 1.43 ± 0.08
4.83 ± 0.28 3.49 ± 0.26
4.69 ± 0.67 5.40 ± 0.73
7.21 ± 0.68 5.70 ± 0.65
5.03 ± 0.57 5.41 ± 0.40
73 ± 2 f 85 ± 4
68 ± 2f 84 ± 4
70 ± 2f 85 ± 3
71 ± 3 f 93 ± 3
Values are means ± SE. LBM, lean body mass, a-KIC, a-ketoisocaproate. *During NaHMCO3 infusion. tP < 0.01 vs. fed.
where Ra[0[fecj is derived from Eq. 2, meal glucoseinfrate is the absolute rate of duodenally infused unlabeled glucose, and flafedendo is derived from Eq. 4. Results are given as means ± SE. Groups were compared with analysis of variance (ANOVA), and statistical differences among the groups were established with a post hoc Newman-Keuls test. Comparisons within groups were done with the Student's t test for paired observations. RESULTS
Body weight, height, and LBM were not significantly different among the four groups of subjects studied (mean body weight, body height, and LBM for the entire group of subjects were 69 ± 3 kg, 174 ± 3 cm, and 61 ± 3 kg, respectively) and did not change as a result of any of the treatments. Calorie intake was not different among the groups investigated. Isotope studies. After 4 h of isotope infusion, plasma
a-[14C]KIC SA, [3H]glucose SA, [2H2]glucose enrichment, and 14CO2 recovery were at steady state. The mean values for each study group obtained during min 260-360 in the postabsorptive (study A) and fed (mean of studies B and C) states are depicted in Table 1. In the postabsorptive state, 14 CO2 recovery in expired air from the infused NaH14CO3 was similar in the four groups of subjects investigated and increased by - 1 0 - 1 5 % as a consequence of feeding (P < 0.01, Table 1). Plasma concentrations of hormones and nonglucose substrates. In the postabsorptive state, plasma concentrations of pyruvate were increased (P < 0.05), whereas FFA and ketone bodies were decreased (P < 0.01) in subjects treated with prednisone alone (Table 2). As a consequence of meal infusion, plasma concentrations of lactate and pyruvate increased as expected in all groups of subjects investigated (P < 0.01), whereas FFA and ketone body concentrations decreased (P < 0.01). In subjects treated
TABLE 2 Plasma concentrations of lactate, pyruvate, total ketone bodies, and free fatty acids in 4 treatment groups
Lactate (mM) Fasting Fed Pyruvate (|xM) Fasting Fed Total ketone bodies (mM) Fasting Fed Free fatty acids (mM) Fasting Fed
Control
Prednisone
Human growth hormone
Prednisone + human growth hormone
1.24 ± 0.10 1.90 ±0.20
1.47 ± 0.07 2.40 ±0.10*
1.31 ± 0.12 1.90 ± 0.08
1.24 ± 0.12 2.72 ± 0.16*
57 ± 7 80 ± 15
150 ± 19*
2.04 ± 0.69 0.07 ± 0.01
0.46 ± 0.05* 0.11 ± 0.02
2.24 ± 0.21 0.06 ± 0.01
1.14 ± 0.12 0.23 ± 0.11
1.27 ± 0.17 0.16 ± 0.01
0.86 ± 0.08* 0.19 ± 0.03
1.39 ± 0.10 0.21 ± 0.01
1.03 ± 0.05 0.50 ± 0.14*
85 ± 7t
61 ± 4 109 ± 6
62 ± 6 177 ± 22*
Values are means ± SE. *P < 0.001, fP < 0.05, *P < 0.01, vs. control by analysis of variance.
144
DIABETES, VOL. 40, JANUARY 1991
F.F. HORBER, H.M. MARSH, AND M.W. HAYMOND
with prednisone (with or without hGH), plasma lactate and pyruvate concentrations were greater (P < 0.001) than in subjects treated with placebo or rhGH alone during meal infusion. In the fed state, FFA concentrations were increased (P < 0.01) in subjects on combined hormone treatment. In the postabsorptive state, plasma insulin concentrations were similar in the control subjects and subjects treated with prednisone or rhGH alone but elevated (237 ± 57 pM, P < 0.001) in the combined-treatment group (Table 3). Plasma C-peptide concentrations were elevated (P < 0.001) in the subjects treated with prednisone alone and the combinedtreatment subjects (Table 3). During meal infusion (mean values from studies B and C), plasma insulin and C-peptide concentrations were elevated (P < 0.001 by ANOVA) in subjects treated with prednisone or rhGH alone compared with the control subjects. In addition, plasma concentrations of insulin and C-peptide were higher (P < 0.01 by ANOVA) in the combined-treatment group compared with all other groups. In the postabsorptive state, plasma hGH concentrations measured 19 h after the last injection of saline or rhGH were similar in the control subjects, subjects treated with prednisone alone, and combined-treatment group, whereas after rhGH alone, this value was elevated (P < 0.01). During meal infusion, values and statistical relationships similar to those obtained in the postabsorptive state were observed (Table 3). In subjects treated with prednisone alone or the combined treatment, plasma concentrations of glucagon were elevated during meal infusion (P < 0.01) but not in the postabsorptive state compared with placebo-treated subjects. Indirect calorimetry. Vco2, Vo2, RQ, and resting energy expenditure (REE) were not different in the postabsorptive state among the four groups studied (Table 4). Vco2 and RQ increased (P < 0.01) in all subjects as a consequence of meal infusion, whereas Vo2 and REE increased (P < 0.01) only in prednisone-treated subjects (groups 2 and 4). During infusion of the test meal, RQ was lower (P < 0.05) in both groups treated with rhGH compared with subjects treated with prednisone alone or placebo. Glucose kinetics. Plasma glucose concentrations (control, 5.3 ± 0.3 mM; prednisone, 5.5 ± 0.3 mM; hGH, 5.1 ± 0.34
mM; and rhGH plus prednisone, 5.4 ± 0.2 mM) were similar in all four groups investigated before initiation of drug and/ or placebo treatment (Table 5). During the 2 h of blood sampling in study A (19-21 h of fasting), the plasma glucose concentrations were increased in the groups treated with prednisone and prednisone plus rhGH (P < 0.001 by ANOVA) compared with placebo-treated subjects (Table 5). Plasma glucose concentrations were increased (P < 0.001) in all four groups investigated during administration of the test meal (28.6 ixmol glucose • kg" 1 LBM • min"1) on both study days (only averaged data from studies B and C). No significant differences (by ANOVA) in plasma glucose concentrations during meal infusion were observed among the control subjects and subjects treated with prednisone or rhGH alone; however, during combined treatment with rhGH and prednisone, plasma glucose concentrations were elevated (11.3 ± 2.1 mM,P
