Recombinant growth hormone enhances muscle myosin heavy-chain ...

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fibrillar protein accrual in vivo. Growth hormone (GH) is presently used in the treatment of pituitary dwarfism (5). Although in vitro data are conflicting with regard ...
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 3371-3374, May 1989 Medical Sciences

Recombinant growth hormone enhances muscle myosin heavy-chain mRNA accumulation and amino acid accrual in humans (protein metabolism/skeletal muscle)

YUMAN FONG*, MICHAEL ROSENBAUMt, KEVIN J. TRACEY*, G. RAMAN*, DAVID G. HESSE*, DWIGHT E. MATTHEWS*§, RUDOLPH L. LEIBELt, JOSEPH M. GERTNERt, DONALD A. FISCHMANt, AND STEPHEN F. LOWRY*¶ *Laboratory of Surgical Metabolism, Departments of tPediatrics, tCell Biology and Anatomy, and WMedicine, The New York Hospital-Cornell Medical

Center, 525 East 68th Street, New York, NY 10021

Communicated by Bernard L. Horecker, January 27, 1989 (receivedfor review August 19, 1988) A potentially lethal complication of trauma, ABSTRACT malignancy, and infection is a progressive erosion of muscle protein mass that is not readily reversed by nutritional support. Growth hormone is capable of improving total body nitrogen balance, but its role in myofibrillar protein synthesis in humans is unknown. The acute, in situ muscle protein response to an infusion of methionyl human growth hormone was investigated in the limbs of nutritionally depleted subjects during a period of intravenous refeeding. A 6-hr methionyl growth hormone infusion achieved steady-state serum levels comparable to normal physiologic peaks and was associated with a significant increase in limb amino acid uptake, without a change in body amino acid oxidation. Myosin heavy-chain mRNA levels, measured by quantitative dot blot hybridization, were also significantly elevated after growth hormone administration. The data indicate that methionyl growth hormone can induce intracellular amino acid accrual and increased levels of myofibrillar protein mRNA during hospitalized nutritional support and suggest growth hormone to be a potential therapy of lean body wasting.

sis, we infused this hormone in hospitalized volunteers during intravenous nutritional repletion.

MATERIALS AND METHODS

Cachexia in hospitalized patients frequently results in severe lean body wasting that may threaten survival. Skeletal muscle myofibrillar protein losses are particularly prominent in patients afflicted with infection, malignancy, and trauma. Under such circumstances, nutritional supplementation may not reverse these losses of skeletal muscle protein, despite massive caloric and protein intake (1). Additional anabolic stimuli have therefore been proposed to enhance the rate of skeletal muscle protein repletion (2-4), but to date none have been shown to positively effect cellular conditions for myofibrillar protein accrual in vivo. Growth hormone (GH) is presently used in the treatment of pituitary dwarfism (5). Although in vitro data are conflicting with regard to the direct anabolic properties of GH (6-8) and suggest that many of the biological activities of this hormone may be mediated by secondary mediators such as insulin-like growth factors (IGFs) (7-9), it is clear that administration of this peptide to animals (10) and humans (11) promotes weight gain and whole body nitrogen retention. However, a mechanistic analysis of these effects in humans has previously been limited partly by the restricted quantities of the hormone and by possible contaminants in pituitary extracts of this hormone (12). Human in vivo experiments are now feasible with the availability of recombinant DNA-produced GH. To examine whether an infusion of recombinant methionyl human GH (Met-hGH) may acutely produce intracellular conditions favorable for myofibrillar protein synthe-

Subjects. Six normal male adult volunteers (26.1 ± 1.2 years, 180 ± 2 cm, 73 ± 3 kg) were admitted to the Clinical Research Center of The New York Hospital-Cornell Medical Center (NYH-CMC) after giving informed written consent. The protocol was reviewed and approved by the Institutional Review Board of the NYH-CMC. Study Protocol. Subjects underwent 10 days of total protein and calorie starvation followed by 10 days of intravenous hypercaloric feeding regimen [41 kcal/kg per day (1 cal = 4.18 J), 0.33 gm of nitrogen per kg per day, no lipid emulsions]. Body weight and urinary nitrogen excretion were monitored daily. During the 10th day of intravenous feeding, amino acid flux across a lower extremity, whole body leucine oxidation rate, and skeletal muscle myosin heavy-chain (MHC) mRNA content were measured prior to and at the completion of a 6-hr intravenous infusion of recombinant Met-hGH (Genentech) at 2 ,ug/kg per hr. Hormone Levels. Radial arterial plasma specimens were obtained prior to and during the GH infusion. These specimens were collected in tubes containing EDTA. The plasma fractions were immediately separated by centrifugation and stored at -700C for later determination of circulating GH (13) and insulin (14) levels by radioimmunoassay. IGF-I levels were also measured (15). Extremity Amino Acid Flux. For determinations of extremity amino acid flux, percutaneous teflon radial artery and retrograde femoral venous catheters were placed. Simultaneous arterial and venous samples were drawn just prior to and at the end of the Met-hGH infusion. Plasma fractions were isolated and immediately stored at -700C for later analysis of amino acid concentrations by column chromatography (Beckman) using a three-buffer lithium system with N-ethylcysteine as an internal standard (16). Blood flow to the extremity was determined before and after blood sampling by using electrocapacitance plethysmography (17). Plasma amino acid flux was calculated as (arterial concentration - venous concentration) x blood flow x (1 hematocrit). A positive value represents net uptake, whereas a negative value represents net efflux. Leucine Oxidation. Whole body leucine oxidation was determined with L-[1-13C]leucine according to described methods (18). After a priming bolus of NaH13C03 (0.8 mg/kg) and L-[1-13C]leucine (1 mg/kg), a constant infusion (1 mg/kg per hr) of L-[1-13C]leucine was begun 6 hr before and

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Abbreviations: GH, growth hormone; Met-hGH, methionyl human GH; MHC, myosin heavy chain; IGF, insulin-like growth factor. ITo whom reprint requests should be addressed. 3371

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continued through the Met-hGH infusion (12-hr total tracer infusion). Prior to the [13C]leucine infusion and at 30-min intervals between hours 4 and 6 and again between hours 10 and 12 of the tracer infusion, expired CO2 was trapped in evacuated glass tubes for later determination of '3C enrichment. Rates of CO2 production were determined simultaneously by 7-10 1-min recordings of expired air on a metabolic measurement cart (Beckman). Simultaneous arterial blood specimens were collected for determination of the 13C enrichment of plasma leucine. The rate of leucine oxidation (C) was calculated as C=

F13c02'(1/Ep - 1/E)100,

where F13co2 is the rate of 1'CO2 production, Ep is the [13C]leucine enrichment in plasma, and Ei is the enrichment of the infused [13C]leucine. MHC mRNA Levels. Percutaneous muscle biopsy specimens were obtained from the vastus lateralis of the subjects just prior to and at the end of the 6-hr infusion of GH by using the method of Bergstrom (19). The specimens were immediately immersed in liquid nitrogen and subsequently stored at -70'C. Total RNA was then isolated from these specimens by using the protocol of Auffray and Rougeon (20). Three samples of total RNA (0.25 gg, 0.50 Ag, and 1.0 pug) from muscle biopsies obtained before and after met-hGH infusion were immobilized on GeneScreen (NEN). Relative amounts of mRNA for MHC were then determined by dot hybridization with a 32P-labeled cDNA probe (PSMHC-A) specific for human MHC (21). The PSMHC-A clone is a 2.7-kilobase insert that codes for the 635 carboxyl-terminal amino acid residues of MHC. A 2-kilobase portion of the PSMHC-A was isolated from the pBR322 vector by digestion with the restriction endonucleases Xba I and BamHI and electrophoresis on a 1% agarose gel. The isolated insert was then labeled with [32P]CTP by using an oligonucleotide labeling kit (Prime Time C, International Biotechnologies). Prehybridizations (12 hr) and hybridizations (18 hr) were performed in a solution of 50% (vol/vol) formamide/0.2% polyvinylpyrrolidone/0.2% bovine serum albumin/0.2% Ficoll/0.05 M TrisHCl, pH 7.5/0.1% sodium pyrophosphate/1.0% SDS/salmon sperm DNA at 100 ,ug/ml (45°C). The filter was washed sequentially in 2x SSC (lx SSC = 150 mM NaCl/15 mM sodium citrate, pH 7.0) (260C), 2x SSC/1% SDS (650C), and 0.1x SSC (26°C). The filters were then exposed to Kodak X-OMAT AR film. The extent of hybridization was quantified by densitometry and scintillation counting. Statistics. All data are presented as the mean ± SEM. Comparisons between groups were performed by the twotailed Students t test. Statistical analysis on hybridization

(1989)

data was performed by the Wilcoxin matched pairs signedrank test. A value of P < 0.05 was considered statistically significant. RESULTS Weight loss comparable to that frequently observed in patients was induced in healthy volunteers subjected to 10 days of total protein and calorie starvation (-5 ± 1 kg). During the refeeding period, the subjects gained weight steadily (0.4 ± 0.1 kg/day) and achieved positive urinary nitrogen balance (7.0 ± 0.4 g of nitrogen per day). Hormone Levels. GH levels rose from a baseline level of 1.1 ± 0.2 ng/ml to steady-state levels within 2 hr after the beginning of the GH infusion (Fig. 1). The mean level at the time of the amino acid flux determinations was 15.1 ± 1.2 ng/ml. The subjects were hyperinsulinemic at baseline because of the high dextrose content in the intravenous feedings. The GH infusion further increased the arterial plasma insulin level to 154 ± 18 microunits per milliliter (P < 0.05). No significant change was noted in circulating IGF-I levels in response to this 6-hr infusion of GH. Extremity Amino Acid Flux. Despite the positive urinary nitrogen balance, total extremity amino acid balance was still negative (-95 ± 120 nmol/min per 100 ml of tissue) (Fig. 2). With the 6-hr Met-hGH infusion, the lower extremity became a site of significant total amino acid uptake (387 ± 103 nmol/min per 100 ml of tissue). This increase in nitrogen accrual involved branched-chain as well as the other essential amino acids. There were no significant changes in glutamine or alanine flux across the extremity. Leucine Oxidation. Branched-chain amino acids are mainly oxidized in the skeletal muscle (22). Measurement of leucine oxidation was used as an index of the peripheral utilization of amino acids in catabolic pathways. No change was noted in whole body leucine oxidation (Fig. 3). Myosin mRNA Levels. There were no complications associated with the percutaneous muscle biopsies. GH infusion was associated with a significant increase in the level of MHC mRNA (Fig. 4) in the muscle biopsy specimens obtained at the end of the 6-hr hormone infusion as compared to paired, baseline specimens from the same individual (mean change = 65%, median change = 64%, SD = 26, interquantile range = 25, P = 0.03).

DISCUSSION The current starvation and refeeding protocol simulates the net catabolic losses of peripheral protein commonly noted in hospitalized and/or malnourished patients (23). On day 10 of refeeding, prior to GH infusion, the isolated lower extremity 15 r-

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Proc. NatL. Acad. Sci. USA 86 (1989)

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was in net negative amino acid balance, despite concurrent positive body nitrogen balance. This is consistent with previous observations demonstrating that hypercaloric feeding can replete total body nitrogen stores without specifically restoring skeletal muscle protein mass in hospitalized patients (23, 24). Following Met-hGH infusion, there was a marked increase in lower extremity amino acid uptake (Fig. 2). In vitro data differ concerning the ability of GH to mediate enhanced cellular amino acid uptake (8, 25). The current study shows, however, that GH administration in vivo is capable of promoting cellular amino acid uptake. It may well be that secondary mediators released in vivo in response to GH, such as IGFs (8, 26), may be the direct mediator of this cellular nitrogen accrual. There are four main fates for the amino acids taken up into the skeletal muscle cells: (i) deamination and transamination to form predominantly glutamine or alanine, which are then released; (ii) consumption through oxidation; (iii) retention as free intracellular amino acids; or (iv) incorporation into new proteins. Under normal conditions, nitrogen from intra-

cellular amino acids, particularly the branched-chain amino acids, is used to transaminate pyruvate and glutamate to form alanine and glutamine, which then leave the muscle cell as the major transporters of nitrogen from the extremities to the splanchnic organs (27). In the present study, GH infusion did not induce an increased efflux of alanine and glutamine from the extremity (Fig. 2), suggesting that the additional amino acids taken into the cell during GH infusion were not utilized primarily in the transamination pathways. During the oxidation of leucine, irreversible decarboxylation occurs early; the carboxyl carbon is lost as bicarbonate and is ultimately excreted as carbon dioxide in expired air. Simultaneous measurements of serum [1-13C]leucine and expired 13CO2 enrichments during an infusion of tracer quantities of leucine labeled at the carboxyl carbon 13C permit an estimation of the whole body amino acid oxidation rate (18). In the present study, whole body leucine oxidation was not affected by the Met-hGH infusion (Fig. 3). Since Met-hGH induces an increased extremity uptake of amino acids without evidence of accelerated transamination or oxidation, the data suggest intracellular peptide accrual. As an indicator of myofibrillar protein synthesis, we assessed MHC mRNA levels with a cDNA probe (PSMHCA) specific for the sarcomeric isoforms of the protein subunit (Fig. 4) (21). These data imply that met-hGH not only mediates increased amino acid uptake into myofibers but may also be stimulating the utilization of those amino acids in protein synthesis. The biosynthesis of myofibrillar proteins (MHC is the most abundant myofibrillar protein) in skeletal muscle is rapidly enhanced during incubation of this tissue S

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FIG. 4. MHC mRNA levels before (BL) and at the end of the 6-hr infusion of Met-hGH. Three amounts of total cellular RNA (0.25, 0.5, and 1.0 ,ug) were immobilized on nylon media and were hybridized. A representative dot blot is shown along with the positive controls, human and chicken muscle RNA, and the negative controls, chicken brain and muscle poly(A)-RNA. On the right is the summary of the results from the six subjects presented as percent change in hybridization. Zero represents no change in MHC mRNA levels.

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with GH in vitro (28). Using a specific cDNA probe, we observed a significant increase in levels of mRNA for MHC within 6 hr of GH infusion. Thus, it appears that increased transcription or decreased degradation of mRNA for myofibrillar proteins may participate in the acute anabolic response to GH in vivo. Increased amino acid uptake and elevated MHC mRNA after GH infusion may reflect either a direct response to the hormone or result from secondary GH-induced hormonal changes. Insulin levels rise significantly following GH infusion (Fig. 1), but the metabolic changes we observed cannot be attributed solely to hyperinsulinemia. Insulin can stimulate intracellular amino acid transport (29), but prior studies from our laboratory, with subjects made chronically hyperinsulinemic, have shown that further increases in serum insulin levels following additional carbohydrate loads does not appear to increase extremity amino acid uptake (23, 30). Moreover, insulin infusion predominantly exerts an anticatabolic effect on muscle protein turnover without significantly stimulating protein synthesis (31). IGFs may also participate in the response to GH. It has been established that IGF-I is the secondary mediator for many of the chronic effects ofGH (9, 15). Whether IGF-I may also mediate the acute effects of GH is unclear, however. The circulating levels of IGF-I do not change within the 6-hr study period examined in this report. This is in agreement with previous observations that circulating levels of IGF-I do not rise until 6 hr after GH administration (32). However, circulating levels of IGF-I may not reflect tissue levels of this hormone (33, 34); tissue levels of IGF-I may rise before circulating levels in response to GH administration (33). It has been demonstrated in rats that tissue levels of IGF-I in cardiac muscle are significantly elevated by 4 hr after an intraperitoneal injection of GH (33) and that transcription of IGF-I is significantly increased in skeletal muscle within 6 hr of GH treatment (35). Both of these changes precede elevations of circulating IGF-I levels. It is still unknown whether skeletal muscle IGF-I levels in humans rise within 6 hr in response to the dose of GH used in the current experiments. The potential roles of IGF-I, insulin, and other factors as direct cellular mediators of the anabolic response to GH require further investigation. No model can exactly reproduce the cachexia associated with illness, but the present study shows that myofibrillar protein mRNA levels in vivo can be influenced by GH infusion. To our knowledge, GH is the only agent, thus far identified, that promotes positive nitrogen balance, increased extremity amino acid uptake, and elevated myosin mRNA levels during nutritional support in hospitalized humans. These encouraging observations support further study of GH as an anabolic adjunct to hospitalized nutritional support in injured humans. We thank Dr. L. Leinwand for the PSMHC-A probe and Dr. D. R. Clemmons for the IGF-I analysis. This work was supported by National Institutes of Health Grants GM34695, KO4GM-00505, AR32147, and RR00047; a Clinical Fellowship from the American Cancer Society (Y.F.); the Muscular Dystrophy Association (D.A.F.); the American Heart Association (R.L.L.); and by Norman and Rosita Winston Fellowships in biomedical research (M.R. and

G.R.).

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