Effects of in-vivo administration of insulin-like growth factor-I on the rate of glucose utilization in the soleus muscle of the rat G. Dimitriadis, M. Parry-Billings, D. Dunger, S. Bevan, A. Colquhoun, A. Taylor, P. Calder, U. Krause, G. Wegener and E. A. Newsholme Departments of Biochemistry and *Pediatrics, University of Oxford, South Parks Road, Oxford oxi 3QU, U.K. fDepartment of Growth and Development, Institute of Child Health, London, U.K. {Institut für Zoologie der Johannes Gutenberg-Universität, Saarstrasse 21, D-6500 Mainz, Germany (Requests for offprints should be addressed to G Dimitriadis now at 'Evangelismos' General Hospital, 103-105 Aegeou Pelagous Street, GR-15342, Agia Paraskevi, Greece or to E. A. Newsholme) revised manuscript received

5 November 1991

ABSTRACT

study investigated the effects of insulin-like growth factor-I (IGF-I) administered to rats in vivo on

utilization by IGF-I at physiological concentrations of insulin (10 or 100 mU/l) was similar to that observed at maximal concentrations of insulin (1000 mU/l). Similar results were obtained after prolonged treatment of the rats with IGF-I; however, the increase in the rate of glucose utilization was less pronounced than when IGF-I was given acutely and the muscles were still capable of responding to insulin. These results suggest that: (a) acute or chronic increases in the serum level of IGF-I in the rat in vivo increase the basal rate of glucose utilization in skeletal muscle; this increase is independent of insulin; (b) the effects of insulin on the rate of glucose utilization are not additive to those of IGF-I; however, this may depend on the level of IGF-I in serum. Journal of Endocrinology (1992) 133, 37\p=n-\43

INTRODUCTION

or rat

This

the soleus muscle isolated from these rats. In order to study the interactions between IGF-I and insulin, the soleus muscles were incubated in the presence of various concentrations of insulin. IGF-I (190\p=n-\200\g=m\g) was given twice daily; the rats were killed 1 h after one injection of IGF-I (acute administration) or after treatment with IGF-I for 10 days (prolonged administration). The level of IGF-I in plasma was increased by \m=~\100% after acute administration and by around 30% after 10 days of treatment with IGF-I. Acute administration of IGF-I to the rats increased the flux of glucose to hexose monophosphate and the rates of lactate formation and glycogen synthesis in the soleus muscles; however, the responsiveness of these muscles to insulin was lost: the increase in the rate of glucose

Insulin-like growth factor-I (IGF-I), which has a close structural homology with insulin, is considered to play a role in mediating the growth-promoting effects of growth hormone (GH) (Rinderknecht & Humbel, 1978; Schoenle, Zapf, Humbel & Froesch, 1982). Invitro studies with isolated incubated rat and human adipocytes demonstrate that IGF-I increases the rates of glucose transport and oxidation in this tissue (Schoenle, Zapf & Froesch, 1977; Zapf, Hauri, Waldvogel & Froesch, 1986; Bolinder, Lindblad, Engfeldt & Amer, 1987; Kern, Svoboda, Eckel & Van Wyk, 1989), and studies with isolated incubated mouse

soleus muscle and human muscle demonstrate that IGF-I increases the rates of glucose transport (Dimitriadis, Parry-Billings, Piva et al. 1990), glucose

phosphorylation, glycolysis and glycogen synthesis (Poggi, Le Marchand-Brustel, Zapf et al. 1979; Dimitriadis et al. 1990; Dohm, Elton, Radju et al. 1990). Intravenous administration of IGF-I as a bolus injection to rats (Zapf et al. 1986; Jacob, Barrett, Plewe et al. 1989), dogs (Giacca, Gupta, Efendic et al. 1990) or humans (Guler, Zapf & Froesch, 1987) lowers the plasma glucose level acutely; in the rat such

has been shown to increase the rates of utilization and glycogen synthesis in the glucose diaphragm and rectus muscle (Zapfe? al. 1986; Jacob treatment

al. 1989). The hypoglycaemic effect of IGF-I is con¬ sidered to be due to an increase in the rate of glucose uptake by peripheral tissues, since hepatic glucose production is not affected (Jacob et al. 1989; Giacca étal. 1990). Although skeletal muscle is quantitatively the most important tissue for glucose disposal in response to insulin (De Fronzo, Jequier, Maeder & Felber, 1981), the effects of IGF-I on this tissue after in-vivo administration have not been adequately studied. The present studies were therefore undertaken to examine the effects of both acute and prolonged administration of IGF-I to rats on the flux of glucose to hexose monophosphate, and on the subsequent disposition of glucose (i.e. glycolysis and glycogen synthesis) at various insulin concentrations in the incubated soleus muscle preparation isolated from these animals. et

MATERIALS AND METHODS

5-5 mmol

D-glucose/1. Defatted bovine serum albumin a final concentration of 1-5% (w/v) and the pH was adjusted to 7-30. The medium was gassed with 02/C02 (95/5, v/v) during preparation and during the incubation which was carried out at 37 °C. was

added to

After 30 min, the muscles were transferred into other flasks containing 3-5 ml identical Krebs-Ringer bicarbonate buffer except that pyruvate, succinate and L-glutamate were omitted and insulin was added (see Results). In each experiment, additional flasks which contained an identical amount of incubation buffer but no muscles were included as controls. After 60 min of incubation, the muscles were quickly removed, blotted, freeze-clamped and processed for determination of metabolites. Flux of glucose to hexose monophosphate, rates of lactate formation and glycogen synthesis and content of fructose 2,6-bisphosphate in muscle

The flux of

(representing

Animals and treatments

(140-160 g)

obtained from Olac, Bicester, Oxon, U.K. All chemicals, biochemicals, enzymes and radiolabelled substances were obtained from sources given previously (Dimitriadis, Leighton, Parry-Billings et al. 1988). Biosynthetic IGF-I (Kabi Pharmacia, Stockholm, Sweden) was given as a s.c. injection except in the experiments examining acute effects, in which it was given i.p. The experiments were started with an injection of IGF-I at 08.00 h on day 1. Injections of IGF-I (190-200 pg) were given daily at 08.00 and 20.00 h. Rats were killed by cervical dislocation for removal of the soleus muscle and collection of blood, 1 h (acute exper¬ iments) or 10 days (prolonged experiments) after the first injection of IGF-I. In experiments in which rats were treated for 10 days they were killed 12 h after the last IGF-I injection. Control rats were given injections of saline. For each individual experiment, six to ten rats were used. All rats were fasted for 12 h before the experiments. From each soleus muscle, two strips were prepared; each one of these strips was incubated at a different concentration of insulin. All results are expressed as means ± s.e.m. The number of rats or muscle strips used on each occasion is given in parentheses next to results. Male Wistar rats

were

Muscle incubations

Strips of soleus muscles were prepared as previously described (Espinal, Dohm & Newsholme, 1983). The isolated muscles were transferred into siliconized 25-ml Erlenmeyer flasks with 3-5 ml Krebs-Ringer bicarbonate buffer containing 1 mmol pyruvate/1, 1 mmol succinate/1, 1 mmol L-glutamate/1 and

was

&

glucose

to

the rate of measured as described

Newsholme, 1984).

hexose monophosphate glucose phosphorylation) previously (Challiss, Arch

For experiments in which the rates of [l4C]lactate formation and glycogen synthesis were measured, [Ul4C]glucose was added to the incubation medium to a final specific radioactivity of 0-4 pCi/ml. To deter¬ mine the rate of incorporation of l4C into glycogen, after incubation of the muscles and freezing, the muscles were digested in 0-5 ml 1 mol KOH/1 for 1 h at 70 °C. After digestion, unlabelled glycogen (100 pi of a solution containing 25 mg/ml), saturated Na2S04 (50 pi) and absolute ethanol (1-5 ml) were added to samples, and the tubes mixed and stored overnight at 20 °C to precipitate glycogen. Samples were centri¬ fuged at 1500 g (4 °C for 15 min), the supernatant was removed by aspiration and the pellet dissolved in 0-5 ml distilled water at 70 °C. Absolute ethanol was added once more, samples were stored at 20 °C for 2 h and the above procedure was repeated. After dis¬ solving in 0-5 ml water, the contents of all tubes were transferred to scintillation vials; each tube was rinsed once with 0-5 ml distilled water and this was also transferred to the vial. Radioactivity was measured in a liquid scintillation counter after addition of 10 ml scintillation fluid (Guendet, Loten, Jeanrenaud & Renold, 1976). For measurement of the rate of —

—

[l4C]lactate formation, [l4C]lactate

was

separated

from other labelled compounds in the incubation medium using ion-exchange chromatography, and the radioactivity was measured in a liquid scintillation counter (Hammerstedt, 1980; Challiss et al. 1984; Dimitriadis et al. 1988). For measurements of the content of fructose 2,6bisphosphate, the frozen muscle strips were powdered

in liquid nitrogen, 10 volumes (about 300 pi) of 50 mmol NaOH/1 were added and the mixture was immediately sonicated (3 20 s). Samples were incu¬ bated for 6 min at 80 °C. After incubation, 0-75 ml distilled water was added (at this stage the pH of the homogenate was between 10 and 12), samples were centrifuged at 10 000 # for 30 min and the supernatant 20 °C until assay (Van Schaftigen, was stored at & Thuy, 1990). Krause 1984; Wegener,

table

1. Effects of in-vivo administration of insulin-like

growth factor-I (IGF-I) on serum concentrations of IGF-I, glucose and free fatty acids (FFA) measured in at least five rats, 1 h after a single injection of IGF-I and after 10 days of daily injections of IGF-I. Values are presented as means

±

s.e.m.

glucose and free fatty acids in serum IGF-I in serum was measured by radioimmunoassay using a polyclonal antibody (A/B R557A) raised in rabbits against purified human IGF-I (Morell, Ray, Holder et al. 1986). Acid-ethanol extraction of serum samples was undertaken prior to assay (Daughaday, Mariz & Blethen, 1980; Taylor, Dünger, Preece et al. 1990). 125I-Labelled IGF-I was prepared by the method of Morrell, Dadi, Mori & Taylor (1989), to a specific radioactivity of 300 pCi/pg. The maximum sensitivity of the assay was 10 U/l and 50% inhibition of binding of ,25I-labelled IGF-I was observed at a concentration of 60 U/l. In this assay system, acid-extracted rat serum cross-reacts in parallel with acid-extracted human serum. The interassay coefficients of variation were 9%, 4-5% and 6-2% at analyte levels of 69 ng/ ml, 206 ng/ml and 672 ng/ml respectively and the intra-assay variation was 4% at 206 ng/ml. All serum samples were assayed against a human serum standard which contained 182 ng/ml when assayed against natural sequence recombinant IGF-I (Amgen, obtained from Amersham International pic, Amersham, Bucks, U.K.). Therefore, the units of measurement of the results can be converted from U/ml to ng/ml by multiplying with the conversion

factor 182. Levels of glucose

(glucose oxidase method; Yellow Springs Instruments, Yellow Springs, OH, USA) and free fatty acids (Okabe, Uji, Nagashima & Noma, 1980) were measured as described. Statistical

analysis The statistical significance of the difference between results was tested using a non-paired Student's /-test. RESULTS

The serum concentration of IGF-I was increased by 100% 1 h after administration of IGF-I and by around 30% after 10 days of treatment with IGF-I ~

(Table 1). The body weight of the treated rats, in comparison with that of the controls, was unchanged after 10 days of treatment with IGF-I (188 + 9 (10) vs 182 + 7 (8)). The concentration of free fatty acids in serum was

IGF-I/ml 182 ng IGF-I/ml =

Serum

Serum LGF-I

—

Levels of IGF-I,

1 U

(U/ml)

glucose (mmol/1)

1-76 + 0-12 3-68 + 0-27** 2-29 + 0-22*

4-50 + 0-11 2-53 + 0-12** 508 + 007**

Serum FFA

(mmol/1)

Time after start of

experiment Control lh 10 Days

0147 + 001 0-155 + 0-01 0-262 + 001*

*/>