0021-972x/92/7505-1192$03.00/0 Journal of Clmical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 75, No. 5 Printed
Low Dose Recombinant Human Factor-I Fails to Affect Protein Islet Cell Secretion in Humans* NELLY
MAURAS,
Nemours
Children’s
FRITZ
F. HORBER,
Clinic, Jacksonuille,
Florida
AND
MOREY
Growth But Inhibits
W. HAYMOND
32207
ABSTRACT
peptide, and glucagon concentrations significantly decreased, whereas plasma free fatty acids were not affected. No changes were observed in the rate of proteolysis (as estimated by the rate of leucine appearance), the rate of leucine oxidation, or the rate of protein synthesis in the absence or presence of glucocorticosteroid treatment. Plasma concentrations of insulin-like growth factor binding protein-3 did not change during the rhIGF-I infusion whereas they increased 50% in subjects who received rhGH, and in whom rhGH caused a potent protein anabolic effect. These results suggest that rhIGF-I may have a somatostatin-like effect. In addition, we found that rhIGF-I infusion is insufficient to promote protein anabolism. This may be due to the failure of rhIGF-I alone to induce a pivotal GH-dependent cofactor(s) necessary for IGFI to elicit an anabolic effect on protein metabolism in humans. (J Clin Endocrinol Metab 75: 1192-1197, 1992)
The in uiuo effects of recombinant human insulin-like growth factorI (rhIGF-I) on whole bodv nrotein metabolism were studied to ascertain whether rhIGF-I has co&arable effects as those reported with rhGH use in humans. The doses of rhIGF-I chosen achieved similar plasma IGF-I concentrations as those achieved after 7 days of rhGH injections. Eight normal volunteers were studied using [I-13C]and [1-“Clleucine tracers, before, 4 h, and 28 h after a continuous infusion of rhIGF-I at 5 pg kg-’ h-’ (n = 6) and 10 fig kg-’ h-’ (n = 2). Two additional subjects were studied in a protein catabolic state after 7 days of high dose (0.8 mg kg-’ day-‘) glucocorticosteroid administration. Plasma concentrations of rhIGF-I were similar using either 5 or 10 pg kg-’ h-’ and increased to values approximately 300% above baseline by 28 h of infusion. No decrease in the plasma glucose concentration was observed during the 28-h infusion; however, plasma insulin, C-
G
Insulin-Like Anabolism
in U.S.A.
H IMPROVES nitrogen
balance in a variety of catabolic conditions (l-5) and stimulates growth in GH-deficient children (6). Since recombinant human GH (rhGH) administration over 7-10 days in normal volunteers, sufficient to increase insulin-like growth factor-I (IGF-I) concentrations 3-fold, decreased the rates of leucine oxidation under both fed and fasted conditions, had no effect on the rate of appearance of leucine from endogenous protein but increased the nonoxidative rate of leucine disappearance (7); these studies provide strong evidence that GH’s protein anabolic effect is primarily mediated as a result of an isolated increase in protein synthesis since estimates of proteolysis were not altered. In addition to its protein anabolic effects rhGH treatment results in glucose intolerance by inducing insulin resistance (8). It has long been thought that GH alone is not directly responsible for its anabolic effect (9). An extensive body of in vitro and in vivo evidence suggests that the protein anabolic effects of GH are mediated by the generation of IGF-I which has a 50% homology with proinsulin. IGF-I circulates bound to one of the several binding proteins (IGFBPs) (lo12) and appears to elicit its biological effects through a membrane receptor similar to that of the insulin receptor (13). When infused alone and at high rates, IGF-I stimulates
glucose disposal and suppresses both hepatic glucose production and lipolysis in rats (14, 15) and humans (16). In addition, it suppresses estimates of whole body proteolysis in rats (17). These effects are similar to those of insulin and not of GH, and suggest that IGF-I at high rates of infusion may interact predominantly with the insulin receptor and not via the IGF-I receptor. If GH’s anabolic effect on protein metabolism is mediated directly by IGF-I, then an infusion of rhIGF-I sufficient to increase plasma IGF-I by 2- to 3-fold should result in protein anabolism. Thus, the present studies were undertaken to determine whether an infusion of rhIGF-I at a rate sufficient to increase the plasma concentrations of IGF-I in a range observed with GH therapy, but insufficient to provoke a lowering of the plasma glucose, would result in a decrease in the rate of leucine oxidation, consistent with the protein anabolic effect observed with GH treatment. Materials
and Methods
Subjects The protocol was reviewed and approved by the Institutional Review Boards at both Baptist Medical Center and University of Florida Health Science Center (Jacksonville, FL). Eight normal healthy volunteers, three females and five males between 19 and 33 yr of age (25 + 2 yr: mean + SE) participated in Study A and two males (ages 26 and 36 yr) participated in Study B after informed written consent. Subjects were within an average 2% of their ideal body weight (65.0 + 3.5 kg) and had normal serum chemistries, blood counts, urinalysis, plasma glucoses (both fasting and 2 h post 75 g oral glucose) and EKG. All females had
October 21, 1991. Address requests for reprints to: Nelly Mauras, M.D., Nemours Children’s Clinic, 807 Nira Street, Jacksonville, Florida 32207. * Supported by Genentech, Inc. (South San Francisco, CA) Nemours Foundation (Jacksonville, FL) and NIH Grant ROl-DK-269-89. -192
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 07:50 For personal use only. No other uses without permission. . All rights reserved.
METABOLIC negative pregnancy
serum pregnancy test the morning
tests 1 week before of the first study.
and
negative
EFFECTS urine
Study design Study A. All subjects were instructed to follow a weight-maintaining diet consisting of 35 kcal/kg and distributed as approximately 50% carbohydrates, 30% fat, and 20% protein for 5 days before the study. On the afternoon before the first study day (day l), subjects were admitted for 3 days to our Research Unit at the Jacksonville Wolfson Children’s Hospital and fed their evening meal at 1800 h and subsequently fasted except for ad libitum water until completion of the study on day 1 at approximately 1300 h. On the morning of day 1, two iv catheters were placed, one in an antecubital vein for the infusion of isotopes and rhIGF-I and another in a retrograde fashion in a contralateral hand vein inserted for arterializedblood sampling (16, 17). At approximately 0930 h (time 0), primed doseconstant infusions of L-[l-‘3C]leucine (-3.6 pmol kg-‘; 0.06 pmol kg-’ min-‘), L-[l-‘4C]leucine (15 FCi, 0.25 FCi min-‘) were initiated and continued for the 240 min of the study. Arterialized blood samples and breath samples were collected at frequent intervals as well (see below). VC02 (pm01 kg-’ min-‘) was determined using a CPX-MAX calorimeter (Medical Graphics Corp. St. Paul, MN), three times during the study and also by dividing the rate of 14C02 in expired air by the i4C0, specific activities (SA) (7). After completion of the study subjects were fed half of their total daily intake at 1300 h and half at 1800 h and subsequently kept NPO except for water until completion of the study on day 2. The study on day 2 was identical to that on day 1 except that six subjects received rhIGF-I at 5 pg kg-’ h-’ and two subjects received 10 pg kg-’ h-’ beginning at 0 min. Upon completion of the study on day 2 (min 240), the rhIGF-I infusion was continued for a total of 28 h. During the infusion of rhIGF-I, plasma glucose was monitored at frequent intervals. The rhIGF-I infusion was continued through day 3. On the morning of day 3, a study identical to that of day 2 was carried out. Upon completion of the study on day 3, the rhIGF-I and isotope infusions were discontinued, the subjects fed, and their plasma glucose was documented to be normal before discharge. Study B. To determine whether rhIGF-I could offset the catabolic effects of pharmacologic doses of glucocorticosteroids as has been demonstrated with GH (7), study B was conducted in an identical fashion as study A except that the subjects received 0.8 mg kg-’ day-’ of oral prednisone divided into three equal doses for 6 days before the study and were admitted to the hospital the afternoon of the 6th day (day 0). At 1800 h on day 0 a constant infusion of methylprednisolone (0.8 mg kg-’ day-‘) was started and continued throughout the duration of all 3 days of study.
Blood and breath samples Blood was obtained at min -20, -5, 30, 60, 90, 120, 150, 180, 200, 210, and 235 during each study for plasma enrichments of [1-“Clleucine and [1-‘3C]KIC, and plasma SA of [1-“C]leucine and [1-‘4C]KIC, and plasma concentrations of glucose, acetoacetate, B-hydroxybutyrate, and free fatty acids (FFA). Plasma glucose was also measured at 360, 480, 720, 1080 on day 2 and at 330 min on day 3. Plasma insulin, C-peptide, glucagon, IGF-I, and IGFBP-3 concentrations were obtained at variable intervals during each of the studies. Expired air samples were collected to calculate the rate of expiratory loss of i4C02, 13C02, and the SA of “CO2 at -50, -30, -10,150,180,200,210, and 235 min on each study day.
Isotopes L-[l-i3C]Leucine (99% enriched, Merck, Sharpe & Dohme, St. Louis, MO) and L-[l-‘4C]leucine (Amershan Corp) were determined to be sterile and pyrogen free and prepared using sterile 0.9% nonbacterostatic saline. rhIGF-I was provided by Genentech (South San Francisco, CA), and mixed using sterile saline.
OF rhIGF-I
1193
Assays Plasma concentrations and SA of KIC and leucine were determined bv HPLC as oreviouslv described (18). The plasma enrichments of L-ll“Clleucine and [1-i3C]KIC were determined by gas chromatographymass spectrometry (GC-MS) (5970 Hewlett-Packard GC-MS, HewlettPackard, Co., Palo Alto, CA) (19). Acetoacetate, B-hydroxybutyrate, and FFA were determined by microfluorometric methods (20, 21). Plasma glucose was determined using a glucose oxidase method (Beckman glucose analyzer, Beckman Instruments, Palo Alto, CA). The plasma total IGF-I concentrations were kindly determined by Dr. A. Celnikker at Genentech by RIA after acid-ethanol extraction, using the modified method of Daughaday (22). Insulin (23), glucagon (24), and C-peptide (25) were measured by standard RIA (kindly performed by Dr. J. Gerich’s Laboratory, Pittsburgh, PA). IGFBP-3 was measured by RIA at Endocrine Science Laboratories (Calabasas Hills, CA). The 2-min rate of 14C02 expired and the SA of 14C02 in expired air were determined as previously described (26). Radioactivity was determined using a scintillation counter (LS 9800 series; Beckman Instruments, Palo Alto, CA). ‘CO2 enrichments were determined using an automated isotope ratio MS (27-29).
Calculations The plasma enrichment or SA of KIC was used as an index of the intracellular enrichment or SA of leucine (the reciprocal pool model) (30, 31). Estimates of whole body leucine metabolism were made at’near substrate and isotopic steady state, between min 180 to 240 of each study. Calculations of the rate of leucine oxidation, rate of leucine appearance (Ra), and nonoxidative leucine disappearance (NOLD) have been previously described (7, 25, 27).
Statistics Results are expressed as mean + SE. Repeated measures analysis of variance with past-hoc comparisons using Duncan’s multiple range test was used to calculate differences between means, Nonpaired Student’s t testing was used to analyze differences in plasma IGF-I and IGFBP-3 concentrations between different groups. Significance was established at P < 0.05.
Results Study A Plasma IGF-1, glucose, insulin, C-peptide, and @wagon. During rhIGF-I infusion, total IGF-I plasma concentrations increased from of 145 f 14 to 484 % 19 pg/L during the last 4 h of infusion and were remarkably similar whether 5 or 10 pg kg-’ h-’ of rhIGF-I were administered (Fig. 1). The IGF-I concentration in the infusates was documented to be doubled between the two rates used. Since similar plasma rhIGF-I concentrations were obtained, data on all eight patients were grouped for analysis. Plasma glucose concentrations were stable during the 3-day study and did not decrease with the rhIGF-I infusion (Table 1). Plasma insulin, C-peptide, and glucagon concentrations decreased during rhIGF-I administration as compared to the control study day (day 1) (Table 1). Significant differences were observed in the plasma Cpeptide concentrations among all three study days (P < 0.002). In addition, plasma glucagon concentrations also declined on day 3 during the rhIGF-I infusion as compared to day 1 [F(2, 14 = 17.51, P < O.OOl)]. Mean IGFBP-3 concentrations did not change significantly using either 5 or 10 PcLgkg-’ h-’ of rhIGF-I infusion (3.3 f 0.2 VS. 3.5 f 0.3 pg/L, day 1 US. day 3, respectively). For comparison, we measured the IGFBP-3 plasma concentrations in control and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 07:50 For personal use only. No other uses without permission. . All rights reserved.
ET AL.
MAURAS
1194 600,
JCE & M. 1992 Vol75.No5
rates of leucine during the rhIGF-I infusion (Table 3). The R,, as an estimate of the rate of proteolysis decreased during the rhIGF-I infusion but did not achieve statistical significance. The NOLD did not increase during the rhIGF-I infusion; it actually decreased slightly but significantly.
Study B
01,
,
0
1
,
400
,
800
,
1200
TIME
(
2000
1600
(mid
1. Mean plasma IGF-I concentrations volunteers before (time 0) and during infusion. Six subjects received 5 pg kg” kg-’ h-’ rhIGF-I.
FIG.
,
Table 4 shows the summary for the leucine R,s, oxidation rates, and NOLD in the two subjects treated with glucocorticosteroids. As was previously observed (7), glucocorticosteroids increased the rate of leucine R, and oxidation and did not alter the rate of protein synthesis. In contrast to rhGH (7), rhIGF-I administration had no effect on any of these parameters.
in a group eight of healthy a continuous 28-h rhIGF-I h-‘, and two subjects 10 pg
Discussion Using infusions of carbon-labeled tracers of the essential amino acid leucine, estimates of whole body rates of proteolysis and protein synthesis can be determined. Since the only source of leucine in the postabsorptive state is endogenous protein, the R, of leucine provides an estimate of whole body rate of leucine released from body protein. Under steady state conditions and using I-14C-labeled tracers, the rate of disappearance of leucine can be partitioned into the oxidative and nonoxidative losses; hence the NOLD (calculated as the difference between the R, and the oxidation rate of leucine) provides an indicator of the rate of leucine entering into protein (30, 31). Using this technique, the present studies have failed to demonstrate a protein anabolic effect of rhIGF-I in either healthy humans, or healthy humans in a protein catabolic state induced by glucocorticosteroid administration. This observation contrasts with the protein anabolic effect observed after rhGH administration in healthy subjects reported previously (7) in whom rhGH had no effect on the R, of leucine but caused a 50% decrease in the oxidation rate of leucine and a significant increase in protein synthesis (as calculated by NOLD). The lack of anabolic effect observed in the present study cannot be attributed to differences in the plasma IGF-I concentration since comparable increases in the plasma IGF-I concentrations were observed in both the
rhGH-treated subjects (at a dose of 0.1 mg/kg day) previously reported (7). In contrast to our present study, rhGH treatment increased serum IGFBP-3 concentrations (2.7 f 0.2 pg/L in controls at baseline DS. 4.2 + 0.3 after rhGH treatment, P < 0.001) despite a similar increase in plasma rhIGF-I concentrations as compared to the rhIGF-I infused subjects (194 + 18 controls, 677 f 82 &L, rhGH-treated patients). &Hydroxybutyrate, acetoacetate, FFA. Plasma P-hydroxybutyrate, FFA, and acetoacetate concentrations declined during the rhIGF-I infusions but only the /I-hydroxybutyrate changed significantly. Leucine kinetics. Six of the eight patients studied received both stable and radioactive tracers of leucine. One of the subjects who only got r3C-leucine had an iv pump failure on day 1; hence her data were excluded from this analysis. A summary of the isotopic enrichments and SA of plasma KIC and breath CO, are shown on Table 2. Table 3 provides a summary of the R,, the rate of leucine oxidation, and NOLD (as an estimate of protein synthesis) using the stable or radioactive tracers of leucine. Despite the differences in methodologies, the mean numbers are quite similar in both groups. There was no significant change in the oxidation TABLE 1. Plasma concentration of glucose, 2) and 28 h (day 3) of rhIGF infusion Time
Glucose (rnM) C-Peptide hM) Insulin (PM)
Glucagon (rig/L) Analysis
of variance
(min)
0 120 240 0 120 240 0 120 240 0 120 240 was carried
out using
C-peptide,
Day
4.2 4.2 4.1 3.9 4.2 3.3 45.2 44.2 43.8 162 176 193
insulin,
Day
1
f + f -c f + f + + + + +
0.1 0.2 0.2 0.6 0.6 0.3 3.6 3.6 3.6 14 25 20
the last samples
and glucagon
5.2 5.0 4.1 6.1 3.9 2.4 60.3 38.1 33.7 227 175 142 from
at baseline,
2
f + * + + + f + + f f +
Day
0.2 0.1 0.2 0.3 0.6 0.3 4.3 2.9 3.6 12 12 14
each study
2 h, and 4 h of each study
4.9 4.8 4.9 2.4 2.4 1.5 34.5 32.3 29.4 131 131 122
3
+ 0.1 AZ 0.2 + 0.2 k 0.6 + 0.3 + 0.3 + 3.6 rt 4.3 + 4.3 f 17 + 15 + 17
before
(day
l), at 4 h (day
F
P
(2,14)
= 10.8
Vol. 75, No. 5 Printed
Low Dose Recombinant Human Factor-I Fails to Affect Protein Islet Cell Secretion in Humans* NELLY
MAURAS,
Nemours
Children’s
FRITZ
F. HORBER,
Clinic, Jacksonuille,
Florida
AND
MOREY
Growth But Inhibits
W. HAYMOND
32207
ABSTRACT
peptide, and glucagon concentrations significantly decreased, whereas plasma free fatty acids were not affected. No changes were observed in the rate of proteolysis (as estimated by the rate of leucine appearance), the rate of leucine oxidation, or the rate of protein synthesis in the absence or presence of glucocorticosteroid treatment. Plasma concentrations of insulin-like growth factor binding protein-3 did not change during the rhIGF-I infusion whereas they increased 50% in subjects who received rhGH, and in whom rhGH caused a potent protein anabolic effect. These results suggest that rhIGF-I may have a somatostatin-like effect. In addition, we found that rhIGF-I infusion is insufficient to promote protein anabolism. This may be due to the failure of rhIGF-I alone to induce a pivotal GH-dependent cofactor(s) necessary for IGFI to elicit an anabolic effect on protein metabolism in humans. (J Clin Endocrinol Metab 75: 1192-1197, 1992)
The in uiuo effects of recombinant human insulin-like growth factorI (rhIGF-I) on whole bodv nrotein metabolism were studied to ascertain whether rhIGF-I has co&arable effects as those reported with rhGH use in humans. The doses of rhIGF-I chosen achieved similar plasma IGF-I concentrations as those achieved after 7 days of rhGH injections. Eight normal volunteers were studied using [I-13C]and [1-“Clleucine tracers, before, 4 h, and 28 h after a continuous infusion of rhIGF-I at 5 pg kg-’ h-’ (n = 6) and 10 fig kg-’ h-’ (n = 2). Two additional subjects were studied in a protein catabolic state after 7 days of high dose (0.8 mg kg-’ day-‘) glucocorticosteroid administration. Plasma concentrations of rhIGF-I were similar using either 5 or 10 pg kg-’ h-’ and increased to values approximately 300% above baseline by 28 h of infusion. No decrease in the plasma glucose concentration was observed during the 28-h infusion; however, plasma insulin, C-
G
Insulin-Like Anabolism
in U.S.A.
H IMPROVES nitrogen
balance in a variety of catabolic conditions (l-5) and stimulates growth in GH-deficient children (6). Since recombinant human GH (rhGH) administration over 7-10 days in normal volunteers, sufficient to increase insulin-like growth factor-I (IGF-I) concentrations 3-fold, decreased the rates of leucine oxidation under both fed and fasted conditions, had no effect on the rate of appearance of leucine from endogenous protein but increased the nonoxidative rate of leucine disappearance (7); these studies provide strong evidence that GH’s protein anabolic effect is primarily mediated as a result of an isolated increase in protein synthesis since estimates of proteolysis were not altered. In addition to its protein anabolic effects rhGH treatment results in glucose intolerance by inducing insulin resistance (8). It has long been thought that GH alone is not directly responsible for its anabolic effect (9). An extensive body of in vitro and in vivo evidence suggests that the protein anabolic effects of GH are mediated by the generation of IGF-I which has a 50% homology with proinsulin. IGF-I circulates bound to one of the several binding proteins (IGFBPs) (lo12) and appears to elicit its biological effects through a membrane receptor similar to that of the insulin receptor (13). When infused alone and at high rates, IGF-I stimulates
glucose disposal and suppresses both hepatic glucose production and lipolysis in rats (14, 15) and humans (16). In addition, it suppresses estimates of whole body proteolysis in rats (17). These effects are similar to those of insulin and not of GH, and suggest that IGF-I at high rates of infusion may interact predominantly with the insulin receptor and not via the IGF-I receptor. If GH’s anabolic effect on protein metabolism is mediated directly by IGF-I, then an infusion of rhIGF-I sufficient to increase plasma IGF-I by 2- to 3-fold should result in protein anabolism. Thus, the present studies were undertaken to determine whether an infusion of rhIGF-I at a rate sufficient to increase the plasma concentrations of IGF-I in a range observed with GH therapy, but insufficient to provoke a lowering of the plasma glucose, would result in a decrease in the rate of leucine oxidation, consistent with the protein anabolic effect observed with GH treatment. Materials
and Methods
Subjects The protocol was reviewed and approved by the Institutional Review Boards at both Baptist Medical Center and University of Florida Health Science Center (Jacksonville, FL). Eight normal healthy volunteers, three females and five males between 19 and 33 yr of age (25 + 2 yr: mean + SE) participated in Study A and two males (ages 26 and 36 yr) participated in Study B after informed written consent. Subjects were within an average 2% of their ideal body weight (65.0 + 3.5 kg) and had normal serum chemistries, blood counts, urinalysis, plasma glucoses (both fasting and 2 h post 75 g oral glucose) and EKG. All females had
October 21, 1991. Address requests for reprints to: Nelly Mauras, M.D., Nemours Children’s Clinic, 807 Nira Street, Jacksonville, Florida 32207. * Supported by Genentech, Inc. (South San Francisco, CA) Nemours Foundation (Jacksonville, FL) and NIH Grant ROl-DK-269-89. -192
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 07:50 For personal use only. No other uses without permission. . All rights reserved.
METABOLIC negative pregnancy
serum pregnancy test the morning
tests 1 week before of the first study.
and
negative
EFFECTS urine
Study design Study A. All subjects were instructed to follow a weight-maintaining diet consisting of 35 kcal/kg and distributed as approximately 50% carbohydrates, 30% fat, and 20% protein for 5 days before the study. On the afternoon before the first study day (day l), subjects were admitted for 3 days to our Research Unit at the Jacksonville Wolfson Children’s Hospital and fed their evening meal at 1800 h and subsequently fasted except for ad libitum water until completion of the study on day 1 at approximately 1300 h. On the morning of day 1, two iv catheters were placed, one in an antecubital vein for the infusion of isotopes and rhIGF-I and another in a retrograde fashion in a contralateral hand vein inserted for arterializedblood sampling (16, 17). At approximately 0930 h (time 0), primed doseconstant infusions of L-[l-‘3C]leucine (-3.6 pmol kg-‘; 0.06 pmol kg-’ min-‘), L-[l-‘4C]leucine (15 FCi, 0.25 FCi min-‘) were initiated and continued for the 240 min of the study. Arterialized blood samples and breath samples were collected at frequent intervals as well (see below). VC02 (pm01 kg-’ min-‘) was determined using a CPX-MAX calorimeter (Medical Graphics Corp. St. Paul, MN), three times during the study and also by dividing the rate of 14C02 in expired air by the i4C0, specific activities (SA) (7). After completion of the study subjects were fed half of their total daily intake at 1300 h and half at 1800 h and subsequently kept NPO except for water until completion of the study on day 2. The study on day 2 was identical to that on day 1 except that six subjects received rhIGF-I at 5 pg kg-’ h-’ and two subjects received 10 pg kg-’ h-’ beginning at 0 min. Upon completion of the study on day 2 (min 240), the rhIGF-I infusion was continued for a total of 28 h. During the infusion of rhIGF-I, plasma glucose was monitored at frequent intervals. The rhIGF-I infusion was continued through day 3. On the morning of day 3, a study identical to that of day 2 was carried out. Upon completion of the study on day 3, the rhIGF-I and isotope infusions were discontinued, the subjects fed, and their plasma glucose was documented to be normal before discharge. Study B. To determine whether rhIGF-I could offset the catabolic effects of pharmacologic doses of glucocorticosteroids as has been demonstrated with GH (7), study B was conducted in an identical fashion as study A except that the subjects received 0.8 mg kg-’ day-’ of oral prednisone divided into three equal doses for 6 days before the study and were admitted to the hospital the afternoon of the 6th day (day 0). At 1800 h on day 0 a constant infusion of methylprednisolone (0.8 mg kg-’ day-‘) was started and continued throughout the duration of all 3 days of study.
Blood and breath samples Blood was obtained at min -20, -5, 30, 60, 90, 120, 150, 180, 200, 210, and 235 during each study for plasma enrichments of [1-“Clleucine and [1-‘3C]KIC, and plasma SA of [1-“C]leucine and [1-‘4C]KIC, and plasma concentrations of glucose, acetoacetate, B-hydroxybutyrate, and free fatty acids (FFA). Plasma glucose was also measured at 360, 480, 720, 1080 on day 2 and at 330 min on day 3. Plasma insulin, C-peptide, glucagon, IGF-I, and IGFBP-3 concentrations were obtained at variable intervals during each of the studies. Expired air samples were collected to calculate the rate of expiratory loss of i4C02, 13C02, and the SA of “CO2 at -50, -30, -10,150,180,200,210, and 235 min on each study day.
Isotopes L-[l-i3C]Leucine (99% enriched, Merck, Sharpe & Dohme, St. Louis, MO) and L-[l-‘4C]leucine (Amershan Corp) were determined to be sterile and pyrogen free and prepared using sterile 0.9% nonbacterostatic saline. rhIGF-I was provided by Genentech (South San Francisco, CA), and mixed using sterile saline.
OF rhIGF-I
1193
Assays Plasma concentrations and SA of KIC and leucine were determined bv HPLC as oreviouslv described (18). The plasma enrichments of L-ll“Clleucine and [1-i3C]KIC were determined by gas chromatographymass spectrometry (GC-MS) (5970 Hewlett-Packard GC-MS, HewlettPackard, Co., Palo Alto, CA) (19). Acetoacetate, B-hydroxybutyrate, and FFA were determined by microfluorometric methods (20, 21). Plasma glucose was determined using a glucose oxidase method (Beckman glucose analyzer, Beckman Instruments, Palo Alto, CA). The plasma total IGF-I concentrations were kindly determined by Dr. A. Celnikker at Genentech by RIA after acid-ethanol extraction, using the modified method of Daughaday (22). Insulin (23), glucagon (24), and C-peptide (25) were measured by standard RIA (kindly performed by Dr. J. Gerich’s Laboratory, Pittsburgh, PA). IGFBP-3 was measured by RIA at Endocrine Science Laboratories (Calabasas Hills, CA). The 2-min rate of 14C02 expired and the SA of 14C02 in expired air were determined as previously described (26). Radioactivity was determined using a scintillation counter (LS 9800 series; Beckman Instruments, Palo Alto, CA). ‘CO2 enrichments were determined using an automated isotope ratio MS (27-29).
Calculations The plasma enrichment or SA of KIC was used as an index of the intracellular enrichment or SA of leucine (the reciprocal pool model) (30, 31). Estimates of whole body leucine metabolism were made at’near substrate and isotopic steady state, between min 180 to 240 of each study. Calculations of the rate of leucine oxidation, rate of leucine appearance (Ra), and nonoxidative leucine disappearance (NOLD) have been previously described (7, 25, 27).
Statistics Results are expressed as mean + SE. Repeated measures analysis of variance with past-hoc comparisons using Duncan’s multiple range test was used to calculate differences between means, Nonpaired Student’s t testing was used to analyze differences in plasma IGF-I and IGFBP-3 concentrations between different groups. Significance was established at P < 0.05.
Results Study A Plasma IGF-1, glucose, insulin, C-peptide, and @wagon. During rhIGF-I infusion, total IGF-I plasma concentrations increased from of 145 f 14 to 484 % 19 pg/L during the last 4 h of infusion and were remarkably similar whether 5 or 10 pg kg-’ h-’ of rhIGF-I were administered (Fig. 1). The IGF-I concentration in the infusates was documented to be doubled between the two rates used. Since similar plasma rhIGF-I concentrations were obtained, data on all eight patients were grouped for analysis. Plasma glucose concentrations were stable during the 3-day study and did not decrease with the rhIGF-I infusion (Table 1). Plasma insulin, C-peptide, and glucagon concentrations decreased during rhIGF-I administration as compared to the control study day (day 1) (Table 1). Significant differences were observed in the plasma Cpeptide concentrations among all three study days (P < 0.002). In addition, plasma glucagon concentrations also declined on day 3 during the rhIGF-I infusion as compared to day 1 [F(2, 14 = 17.51, P < O.OOl)]. Mean IGFBP-3 concentrations did not change significantly using either 5 or 10 PcLgkg-’ h-’ of rhIGF-I infusion (3.3 f 0.2 VS. 3.5 f 0.3 pg/L, day 1 US. day 3, respectively). For comparison, we measured the IGFBP-3 plasma concentrations in control and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 07:50 For personal use only. No other uses without permission. . All rights reserved.
ET AL.
MAURAS
1194 600,
JCE & M. 1992 Vol75.No5
rates of leucine during the rhIGF-I infusion (Table 3). The R,, as an estimate of the rate of proteolysis decreased during the rhIGF-I infusion but did not achieve statistical significance. The NOLD did not increase during the rhIGF-I infusion; it actually decreased slightly but significantly.
Study B
01,
,
0
1
,
400
,
800
,
1200
TIME
(
2000
1600
(mid
1. Mean plasma IGF-I concentrations volunteers before (time 0) and during infusion. Six subjects received 5 pg kg” kg-’ h-’ rhIGF-I.
FIG.
,
Table 4 shows the summary for the leucine R,s, oxidation rates, and NOLD in the two subjects treated with glucocorticosteroids. As was previously observed (7), glucocorticosteroids increased the rate of leucine R, and oxidation and did not alter the rate of protein synthesis. In contrast to rhGH (7), rhIGF-I administration had no effect on any of these parameters.
in a group eight of healthy a continuous 28-h rhIGF-I h-‘, and two subjects 10 pg
Discussion Using infusions of carbon-labeled tracers of the essential amino acid leucine, estimates of whole body rates of proteolysis and protein synthesis can be determined. Since the only source of leucine in the postabsorptive state is endogenous protein, the R, of leucine provides an estimate of whole body rate of leucine released from body protein. Under steady state conditions and using I-14C-labeled tracers, the rate of disappearance of leucine can be partitioned into the oxidative and nonoxidative losses; hence the NOLD (calculated as the difference between the R, and the oxidation rate of leucine) provides an indicator of the rate of leucine entering into protein (30, 31). Using this technique, the present studies have failed to demonstrate a protein anabolic effect of rhIGF-I in either healthy humans, or healthy humans in a protein catabolic state induced by glucocorticosteroid administration. This observation contrasts with the protein anabolic effect observed after rhGH administration in healthy subjects reported previously (7) in whom rhGH had no effect on the R, of leucine but caused a 50% decrease in the oxidation rate of leucine and a significant increase in protein synthesis (as calculated by NOLD). The lack of anabolic effect observed in the present study cannot be attributed to differences in the plasma IGF-I concentration since comparable increases in the plasma IGF-I concentrations were observed in both the
rhGH-treated subjects (at a dose of 0.1 mg/kg day) previously reported (7). In contrast to our present study, rhGH treatment increased serum IGFBP-3 concentrations (2.7 f 0.2 pg/L in controls at baseline DS. 4.2 + 0.3 after rhGH treatment, P < 0.001) despite a similar increase in plasma rhIGF-I concentrations as compared to the rhIGF-I infused subjects (194 + 18 controls, 677 f 82 &L, rhGH-treated patients). &Hydroxybutyrate, acetoacetate, FFA. Plasma P-hydroxybutyrate, FFA, and acetoacetate concentrations declined during the rhIGF-I infusions but only the /I-hydroxybutyrate changed significantly. Leucine kinetics. Six of the eight patients studied received both stable and radioactive tracers of leucine. One of the subjects who only got r3C-leucine had an iv pump failure on day 1; hence her data were excluded from this analysis. A summary of the isotopic enrichments and SA of plasma KIC and breath CO, are shown on Table 2. Table 3 provides a summary of the R,, the rate of leucine oxidation, and NOLD (as an estimate of protein synthesis) using the stable or radioactive tracers of leucine. Despite the differences in methodologies, the mean numbers are quite similar in both groups. There was no significant change in the oxidation TABLE 1. Plasma concentration of glucose, 2) and 28 h (day 3) of rhIGF infusion Time
Glucose (rnM) C-Peptide hM) Insulin (PM)
Glucagon (rig/L) Analysis
of variance
(min)
0 120 240 0 120 240 0 120 240 0 120 240 was carried
out using
C-peptide,
Day
4.2 4.2 4.1 3.9 4.2 3.3 45.2 44.2 43.8 162 176 193
insulin,
Day
1
f + f -c f + f + + + + +
0.1 0.2 0.2 0.6 0.6 0.3 3.6 3.6 3.6 14 25 20
the last samples
and glucagon
5.2 5.0 4.1 6.1 3.9 2.4 60.3 38.1 33.7 227 175 142 from
at baseline,
2
f + * + + + f + + f f +
Day
0.2 0.1 0.2 0.3 0.6 0.3 4.3 2.9 3.6 12 12 14
each study
2 h, and 4 h of each study
4.9 4.8 4.9 2.4 2.4 1.5 34.5 32.3 29.4 131 131 122
3
+ 0.1 AZ 0.2 + 0.2 k 0.6 + 0.3 + 0.3 + 3.6 rt 4.3 + 4.3 f 17 + 15 + 17
before
(day
l), at 4 h (day
F
P
(2,14)
= 10.8
