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Thermal and nutritional influences on tissue levels of insulin-like growth factor-I mRNA and peptide
J. therm. Biol. Vol. 17, No. 2, pp. 89-95, 1992 Printed in Great Britain.All rights reserved
0306-4565/92 $5.00+ 0.00 Copyright © 1992PergamonPress Ltd
THERMAL A N D NUTRITIONAL INFLUENCES ON TISSUE LEVELS OF INSULIN-LIKE GROWTH FACTOR-I mRNA A N D PEPTIDE L. MA, K. A. BURTON,J. C. SAUNDERSand M. J. DAUNCEY* Department of Molecular and Cellular Physiology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, England (Received 1 October 1991; accepted 19 November 1991) Abstract--1. The influence of environmental temperature on tissue levels of IGF-I mRNA and peptide has been investigated in 8 week old pigs living under conditions of controlled energy intake. Animals were acclimated to 35 or 10°C on a high (H) or low (L) energy intake (where H = 2L) and there were thus 4 treatment groups: 35H, 35L, 10H and 10L. 2. The warm temperature was associated with greater levels of IGF-I peptide in plasma and all tissues investigated (liver, kidney, skeletal muscle and heart). Temperature had significant but contrasting influences on IGF-I mRNA in liver and kidney: at 35 compared with 10°C, levels were increased in liver but reduced in kidney. Despite marked differences in growth and protein deposition, there was no effect of temperature on muscle IGF-I mRNA. 3. A high energy intake was associated with increased levels of IGF-I peptide in plasma and tissues. However, although there was a tendency for liver IGF-I mRNA to be elevated in the 35H compared with the 35L group, there was no overall effect of energy intake on IGF-I mRNA in any issue. 4. It is concluded that ambient temperature and energy intake can significantlyaffect the concentration of IGF-I peptide in tissues and that regulation may occur at either transcriptional or translational levels. The probability is that the effects of temperature are mediated via its influence on energy balance. Key Word Index: Insulin-like growth factor-I; mRNA; temperature; energy intake; energy balance; growth; pig
INTRODUCTION Previous investigations have shown that acclimation of young pigs to a warm or cold temperature under conditions of controlled energy intake has a marked influence on growth and morphology (Dauncey and Ingram, 1986; Ingram and Dauncey, 1986). For .example, animals living in the warm have longer extremities than littermates in the cold on the same level of food intake (Dauncey et al., 1983) and there are significant differences in protein and fat deposition (Dauncey and Ingram, 1983). Recent studies have shown that although growth hormone (GH) secretion tends to be increased in animals living in a warm compared with a cold environment on the same food intake, there is wide individual variation and the difference is not statistically significant (Dauncey and Buttle, 1990). By contrast, plasma concentrations of insulin-like growth factor-I (IGF-I) are considerably greater in animals living in the warm than the cold and on a high than a low energy intake (Dauncey et al., 1990). However, the source of this increased IGF-I was not determined. Although liver is considered to be a major source of circulating IGF-I, its mRNA and peptide have been found in a wide variety of tissues (D'Ercole et al., 1984; Lund et al., 1986; Murphy et al., 1987). Together with evidence that IGF-I influences the growth of many cell types, it has been suggested that, in addition to its endocrine role,
IGF-I may function as an autocrine/paracrine regulator of growth (D'Ercole et al., 1980; Daughaday and Rotwein, 1989). Whether long-term changes in environmental temperature and energy intake influence tissue levels of IGF-I mRNA and its peptide is not known. To our knowledge, no investigations have been carried out on the effects of temperature. In addition, many studies on the effect of food intake have been concerned with the acute effects of severe undernutrition, which usually involve a period of fasting for several days (Emler and Schalch, 1987; Lowe et al., 1989; Leaman et al., 1990; Straus and Takemoto, 1990), rather than the response to a prolonged period of reduced food intake. The aim of the present investigation was therefore to determine whether long-term changes in ambient temperature and food intake can influence tissue levels of IGF-I mRNA and peptide in young growing animals. MATERIALS AND METHODS Experimental design Eight litters of pigs from the Large White herd kept at the Institute were used. At the age of 2-3 weeks, 4 male pigs from each litter were weaned and kept in pairs for 2-3 days at 25-30°C. They were then separated into individual cages and, over the next 2-3 weeks, for two pigs the environmental temperature was increased gradually to 35°C, while for the other two animals it was reduced to 10°C. All animals were therefore at the required temperature by 5 weeks of
*Author for correspondence. 89
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age. At each temperature, one pig received a high (H) intake and the other a low (L) intake of standard feed (Ultra-Wean, Dalgety, Bristol, England). Animals were fed once daily, at 09.00 h and although the absolute amount of food was increased as the animals grew, the ratio of H : L was always 2: 1. Final food intakes were 700 and 350 g for the H and L intakes respectively. Water was always available ad libitum. Animals were killed at 8-9 weeks of age, 24 h after the last meal, by intracardiac injection of sodium pentobarbital following intramuscular administration of tranquillizer (Ketamine, Parke-Davis). There were marked differences in growth rate, and mean body weights ( + S E M ) were 17.2+ 1.0, 11.7+0.8, 13.6+0.8 and 7 . 4 + 0 . 7 k g for the 35H, 35L, 10H and 10L treatment groups respectively. The liver, kidney, longissimus dorsi muscle and heart (ventricle) were removed immediately, divided into 5 g portions, frozen in liquid nitrogen and stored at - 4 0 ° C . Whole blood and plasma were stored in 0.5 ml aliquots at - 2 0 ° C . Assay o f specific mRNAs RNA extraction. The guanidium/lithium chloride method was used to isolate total RNA from the various tissues. Approximately 1 g frozen tissue was pulverised in an anvil press which had been cooled in dry ice, homogenized in 8ml lysis buffer ( 5 M guanidine isothiocyanate, 10mM EDTA, 50mM Tris base, pH 7.5) and forced through a 19-gauge needle until the viscosity caused by released chromosomal D N A was reduced. To inhibit denaturation of proteins, fl-mercaptoethanol was added to a final concentration of 0.1 M. The sample was then made up to 50 ml with 4 M LiC1 and left overnight at 4°C. Centrifugation at 12,500g for 20min at 4°C was followed by resuspension of the pellet in 30 ml 3 M LiC1. The suspension was left on ice for 10 rain and centrifuged again for 10min. The pellet was resuspended in 3 ml I% SDS, 20 #! proteinase K (1 #g/ml) and left at room temperature for 20 min. An equal volume of phenol was added and the sample vortexed. The aqueous solution containing R N A was obtained by centrifugation at 12,500g for 10 min at 10°C. This was further purified by mixing with 0.5 vol 7.5 M ammonium acetate and 3 vol 100% ethanol, and precipitation for 1 h on dry ice. After centrifugation at 12,500g for 10min at 4°C, the pellet was resuspended in 0.5 ml water, 50/~1 3 M sodium acetate pH 4.8 and 4 ml 100% ethanol. After overnight precipitation at - 2 0 ° C , the R N A precipitate was sedimented at 12,500g for 10 min at 4°C and redissolved in water. The total RNA concentration of each sample was estimated spectrophotometrically as described by Maniatis et al. (1982) and the samples were then stored at - 7 0 ° C . Dot blot analysis. Total R N A was denatured with a solution containing 1 M deionized glyoxal, 12.5#i dimethyl sulphoxide, 10/~M sodium phosphate, pH 7.0, by incubation at 50°C for 1 h. The mixture was then kept on ice during dot blotting. Serial dilutions of 10 to 1.25/~g RNA with TrisEDTA buffer, pH 8.0, were blotted onto hybond-N membrane fitted into the dot blot apparatus. After washing with buffer, the membrane was baked at 80°C for 1 h between two filter papers. D N A probes
were labelled with [ct-32p]dCTP, by nick translation, to a specific activity of 2-3 x 10 epm/# g (Amersham International pie). Prehybridization was performed at 65°C for 1 h in a shaking water bath, using a solution of 1% BSA, 1 mM EDTA, 7% SDS, 0.5 M sodium phosphate, pH 7.2. Hybridization was performed at 65°C for 24 h in a shaking water bath. The labelled IGF-I eDNA probe was mixed with the prehybridization solution to form the hybridization solution. The membrane was washed twice with solution A (0.5% BSA, 5% SDS, 1 mM EDTA, 50 mM sodium phosphate) for 20 min at 65°C, once with solution B (the same as solution A but without BSA) for 20 min at 65°C, and blotted with filter paper. Autoradiography of the membrane was carried out at - 70°C, for 1, 2 and 4 days for liver, kidney and skeletal muscle respectively. The membrane was rehybridized with a fl-actin eDNA probe, after first removing the I G F probe with 2 x SSC plus 0.1% SDS at 95°C for 15-20 min, and then prehybridizing as described previously. The density of dots on the autoradiograpbed X-ray films was measured with a Chromoscan (JoyceLoebl) and the relative amount of specific m R N A per unit total tissue R N A was estimated. Estimation o f IGF-I peptide The methods were based on those described by Furlanetto et al. (1977) and Corps et al. (1988). IGF-I was first separated from its binding proteins under acid conditions (Daughaday et al., 1980), for both plasma and tissues. For determining the net tissue IGF-I content, the plasma derived IGF-I trapped in the tissue was subtracted from the total tissue IGF-I. This involved determination of the haemoglobin content of the tissues and the measurement of haematocrit. Plasma extraction. Plasma (100#1) was diluted with 400 gl radioimmunoassay (RIA) buffer (100 mg protamine sulphate, 2.07 g NaH2PO4 2H20, 100 mg sodium azide, 10ml EDTA pH 7.5, made up to 500 ml with water, then 250 #1 Tween 20 added) and 4vol of extraction mixture (87.5% ethanol: 12.5% 2 M HCI) were added to 100 #1 of the diluted sample. After mixing and standing for 30 min at room temperature, centrifugation was carried out at 10,000g for 15 min and the supernatant was neutralized with 0.3 vol of 0.8 M Tris base. Tissue extraction. Approximately 3 - 4 g tissue (liver, kidney, skeletal muscle, heart) was pulverized in an ice-cold anvil press, and about 3 g was used for the tissue extraction while 0.5 g was stored at - 4 0 ° C for the haemoglobin assay. The tissue was mixed thoroughly with 10ml I M acetic acid and shaken gently for 2 h at 4°C. The supernatant was obtained by centrifugation at 2000g for 15 min at 4°C. The pellet was re-extracted with 1 M acetic acid, the supernatants combined and freeze-dried, and the dried material reconstituted with RIA buffer. Finally, undissolved material was removed by centrifugation at 20,000g for 20 min at 4°C, and the supernatants stored in 0.5 ml aliquots at - 2 0 ° C . Radioimmunoassay. IGF-I was measured in the plasma and tissue extracts by RIA. The antiserum (NIDDK) was rabbit antibody for hIGF-I; standards were rhIGF-I (Bachem); hIGF-I was labelled by the
Environment and IGF-I
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Table 1. Concentration of IGF-I mRNA (units/10#g total RNA) in liver, kidney and Iongissimua dorsi muscle from animals which had been living at 35 or 10°C on a high (H) or low (L) energy intake Tissue
35H
35L
10H
10L
Liver Kidney Muscle
10.0+0.7 2.5 + 0.4 2.5 ± 0.2
8.6+1.1 3.0 + 0.5 2.6 ± 0.3
6.2+1.7 4.3 + 0.3 2.9 ± 0.4
6.5+0.8 3.6 _+0.6 2.6 + 0.2
P values: Liver Kidney Muscle
Temperature (T) < 0.05 (35 > 10) <0.01 (10 > 35) NS
Energy intake (E) NS NS NS
Interaction (T x E) NS NS NS
Mean values _+ SEM in arbitrary units indicate density of dots on autoradiograph and thus give the relative amount of specific mRNA in a given amount of total tissue RNA; autoradiography was for 1, 2 or 4 days for liver, kidney and muscle respectively, P values are from the analysis of variance.
Iodo-Gen method. Separation of bound from free 125I I G F - I was with a second antibody of sheep antiserum to rabbit IgG (kindly provided by Dr C. G. Prosser), and normal rabbit serum was used as a carrier. In all instances, samples from each animal in a given litter were examined within the same assay. Estimation of tissue plasma content. The whole blood content within each tissue was determined by the method of Klein (1945). This involved mixing 0.5 g powdered tissue with distilled water and toluene and leaving for 1 h at room temperature. Ammonium sulphate-phosphate solution, pH 6.6, was then added and the solution left for 20 min with some mixing. Extracts were filtered and to the filtrate was added 20% potassium ferrocyanide and 5% potassium cyanide. Solutions were mixed and absorbances read at 540 nm. Serial dilutions of whole blood from the same animal were treated in the same way as the tissues, allowing determination of the blood content of the tissue by comparing its absorbance with that of whole blood. Measurement of haematocrit allowed estimation of the plasma content of the tissues. The plasma derived I G F - I content of the tissues was then subtracted from the total I G F - I content to give the net tissue I G F - I peptide concentration. RESULTS
Tissue abundance of IGF-I mRNA and peptide Table 1 shows that the relative levels of I G F - I m R N A in liver were considerably greater than in the two other tissues examined. By contrast, the I G F - I peptide occurred in the descending order: kidney, liver, heart, skeletal muscle (Table 2). This was the case for all treatment groups except 10L, where all
tissue concentrations were very low and slightly lower in liver than in heart.
Environmental influences on IGF-I mRNA and peptide Environmental temperature and food intake tended to have different effects on I G F - I m R N A compared with its peptide. The results for the peptide (Table 2) clearly showed that in all tissues investigated, the concentration was greater in animals living in the warm than in the cold and on the high compared with the low food intake. For all tissues, values were greatest in the 35H, intermediate in the 35L and 10H, and lowest in the 10L animals. These findings were in the same order as those for plasma I G F - I concentrations of 25.2 + 8.4, 8.0 + 2.6, 10.3 + 2.4 and 2.8 + 0.2 nmol/l in the 35H, 35L, 10H and 10L groups respectively. However, no such generalization can be made with respect to the results for mRNA, since the effects of treatment varied from one tissue to the next (Table 1). In the following sections, results are presented in detail for I G F - I m R N A and peptide in liver, kidney and skeletal muscle. Liver. Environmental temperature had a significant influence on both m R N A (P < 0.05) and peptide (P <0.01), with levels being greater at 35 than at 10°C. The concentration of peptide in liver was also found to be significantly greater on the high compared with the low food intake (P < 0.05), and the effect was particularly striking at 10°C. However, there was no statistically significant effect of food intake on mRNA; levels were slightly greater in the 35H than the 35L group, whereas values for the 10H and 10L were similar to each other.
Table 2. Concentration of IGF-I peptide (nmoi/kg tissue) in liver, kidney, Iongissimus dorsi muscle and heart from animals which had been living at 35 or 10°C on a high (H) or low (L) energy intake Tissue
35H
35L
10H
10L
Liver Kidney Muscle Heart
2.45 -t- 0.21 4.65 + 0.62 0.44 + 0.10 1.77 + 0.37
1.90 + 0.47 3.68 _+ 0.94 0.20 ± 0.04 0.85 + 0.19
1.77 + 0.48 3.49 +_0.58 0.27 + 0.05 0.78 ± 0.09
0.54 ± 0.07 1.01 + 0.19 0.22 _+0.04 0.63 ± 0.08
P values: Liver Kidney Muscle Heart
Temperature <0.01 (35 > <0.01 (35 > NS <0.05 (35 >
(T) 10) 10) 10)
Energy intake (E) <0.05 (H > L) <0.01 (H > L) <0.01 (H > L) <0.05 (H > L)
Interaction (T x E) NS NS NS NS
Mean values +_ SEM; P values are from the analysis of variance.
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Table 3. Concentrationof actinmRNA(units/10#g total RNA)in liver,kidney and Iongissimus dorsi muscleof animalswhichhad beenlivingat 35 or 10C on a high (H) or low (L) energyintake Tissue 35H 35L 10H 10L Liver 10.2+ 0.4 8.6_+ 1.2 8.2_+2.5 5.6+ 1.1 Kidney 8.4-+0.8 8.2-+ 1.2 10.8-+0.8 8.8-+0.5 Muscle 8.8+ 1.3 9.2-+ 1.3 9.1 -+ 1.2 8.0-+0.7 P values: Temperature(T) Energyintake (E) Interaction(T x E) Liver NS NS NS Kidney <0.05 (10 > 35) NS NS Muscle NS NS NS Mean values+ SEM in arbitrary units indicatedensityof dots on autoradiograph and thus givethe relativeamountof specificmRNAin a givenamount of total tissueRNA;autoradiographywas for I, 2 or 4 daysfor liver,kidney and musclerespectively;P valuesare from the analysisof variance. Kidney. A significantly higher concentration of peptide was found in the kidney of animals in the warm compared with the cold (P < 0.01) and on the high compared with the low food intake (P < 0.01). By contrast with the results for mRNA in liver, the level of IGF-I mRNA in kidney was found to be significantly lower (P < 0.01) in the warm than in the cold environment. There was, however, no overall effect of energy intake on mRNA, although the interaction between temperature and diet did approach statistical significance (P = 0.075), reflecting the opposing effects of diet at the two temperatures (35H < 35L, 10H > 10L). Skeletal muscle. Concentrations of the peptide were low in all groups, with an average of less than 0.5 nmol/kg. Nevertheless, as with the other tissues, there was a tendency for the value to be greater at 35 than 10°C (P < 0.1) and it was significantly greater on the high than the low food intake (P <0.01). Levels of IGF-I mRNA were similar in the four treatment groups and there were no significant influences of either temperature or nutrition. Environmental influences on actin m R N A
The results for fl-actin mRNA are presented in Table 3. There was a tendency for the treatment effects to be similar to those observed for IGF-I mRNA. For example, actin mRNA in liver was greater both in the warm compared with the cold and on the high compared with the low food intake. However, only the effect of temperature on kidney reached statistical significance (35 < 10°C, P < 0.05). Actin mRNA has been used in a number of studies as a reference, when a specific mRNA such as that for IGF-I is being investigated (Van Neste et al., 1988; Goldstein et al., 1988). In these earlier studies, a constant amount of actin mRNA was always reported. However, the current finding of a tendency for actin m R N A to be affected by temperature and diet suggests that it may not invariably be an appropriate reference. It has been demonstrated previously that the level of actin mRNA is changed when cells are in different states of growth (Tokunaga et al., 1988). The use of actin mRNA as a reference in the present study was considered to be inappropriate, particularly since animals with very different growth rates were being investigated. Results for IGF-I were therefore expressed in relation to a given amount of total tissue R N A .
DISCUSSION The present investigation clearly demonstrates that long-term changes in environmental temperature and energy intake influence not only the plasma level of IGF-I but also its concentration in a variety of tissues. Thus, in plasma, liver, kidney, skeletal muscle and heart, IGF-I levels are greater in young animals living at a warm compared with a cold ambient temperature and on a high compared with a low food intake. Numerous studies have demonstrated a direct correlation between food intake and plasma IGF-I (Clemmons et al., 1981; Merimee et al., 1982; Prewitt et aL, 1982; Lauterio and Scanes, 1987; Phillips et al., 1988). The current investigation, together with that described previously (Dauncey et al., 1990), suggests that it is energy supply in relation to demand rather than the absolute amount of energy intake which is important in determining IGF-I levels. The effect of ambient temperature on plasma and tissue IGF-I is probably mediated indirectly via its influence on energy balance. In a cold environment, extra energy is needed for maintenance of deep body temperature and thus for a given intake, resting metabolic rate increases and growth rate is reduced (Macari et al., 1983). A decrease in energy availability in adult man, caused by vigorous exercise on a fixed intake, is also accompanied by a reduction in plasma IGF-I (Smith et al., 1987). Thus, plasma and tissue levels of IGF-I are probably related to the surplus energy available for growth after other demands for energy have been met. In the present study, there was indeed a close correlation between the IGF-I levels and growth rates of animals in the four treatment groups. The current findings on the long-term influence of energy status on liver IGF-I peptide are similar to those of its more acute effects. Thus, in rats fasted for 3 days, there is a decrease in levels of both serum and extractable hepatic immunoreactive IGF-I (Goldstein et aL, 1991). Similarly, in young pigs of about 40 kg body weight, withdrawal of food for 3 days caused decreases in plasma and liver IGF-I peptide of 80 and 55°,/0 respectively (Leaman et al., 1990). The close correlation between hepatic and circulating levels of IGF-I reported in these studies, together with others on hepatic IGF-I production (Miller et aL, 1981; Schwander et al., 1983; Goldstein et al., 1988), provide strong evidence for liver being the major source of plasma IGF-I.
Environment and IGF-I The highest level of IGF-I peptide occurred in kidney, followed in descending order by liver, heart and skeletal muscle. This concurs with the finding that in the adult rat, kidney also contains the highest concentration of IGF-I peptide (D'Ercole et al., 1984). The possibility is that, whereas the IGF-I peptide in liver, heart and muscle is a reflection of local tissue synthesis, the peptide level in kidney also reflects the role played by this organ in the degradation (D'Ercole et al., 1977) and clearance of IGF-I (Hizuka et al., 1987; Quattrin et al., 1988). By contrast with the results for abundance of peptide in different tissues, IGF-I mRNA was found to occur at a higher level in liver than in kidney or muscle. Liver also contains significantly more IGF-I m R N A than any other tissue in the rat (Murphy et al., 1987). However, the relative expression of IGF-I m R N A in 40 kg pigs was found to be greater in skeletal muscle and heart than in liver (Leaman et al., 1990). The dot blot technique was used in both this and the current investigation, although in this earlier study a specific 580-base pair porcine IGF-I eDNA probe was used, whereas a human IGF-I eDNA probe was used in the present study. However, given the high sequence homology ( > 90%) between the sequences, this is unlikely to have contributed to the differences between the two investigations. Possible differences in relation to methodology, muscle type and age of animal remain to be investigated. The effects of environmental temperature and food intake on transcription and translation were found to be tissue specific. Thus, although the levels of IGF-I peptide in all the tissues were positively correlated with both a warm temperature and a high intake, the results for m R N A varied between tissues. For example, at a high compared with a low ambient temperature, the level of IGF-I mRNA was elevated in liver but reduced in kidney. At 35°C, there was a tendency for liver m R N A to be increased on the high food intake, although the overall effect of intake was not statistically significant for any of the tissues. A number of studies have demonstrated a relation between food intake and IGF-I mRNA in various tissues. However, these studies have usually been concerned with acute food deprivation. Thus, in rats which had been fasted for 30 h, liver m R N A fell to less than 40% of that in controls (Emler and Schalch, 1987). Similarly, the abundance of several IGF-I m R N A species in liver decreased in 6-week old rats fasted for 24, 48 or 72 h (Straus and Takemoto, 1990). A significant decline was also found in the m R N A content of several tissues, including liver, kidney and skeletal muscle from rats which had been fasted for 48 h (Lowe et al., 1989). A recent study in 40 kg pigs fasted for 3 days demonstrated reductions in liver, muscle and heart IGF-I m R N A (Leaman et al., 1990). There was also a direct relation between the abundance of liver mRNA and the availability of energy for growth in the present investigation, although there was no effect on muscle mRNA. The degree of food restriction was much less severe but for a longer period of time than in these earlier studies. The possibility is therefore that the tissue response of IGF-I m R N A to energy status is dependent on whether there has been longTB 17/2--C
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term undernutrition or short-term starvation. Whether this response is affected by age also needs investigating, since changes in IGF-I gene expression induced by protein restriction are age-dependent in growing rats (VandeHaar et al., 1991). Nevertheless, a 3-fold increase in liver mRNA was induced in older pigs of approximately 100 kg body weight when given growth hormone (GH) over a 24 day period, whereas longissimus dorsi muscle mRNA was slightly reduced (Grant et al., 1991). This suggested that the GHinduced increase in muscle growth in these pigs was mediated by an endocrine, as distinct from an autocrine/paracrine, action of IGF-I. The rate of skeletal muscle growth and protein deposition are significantly greater in young pigs living in the warm compared with the cold and on a high compared with a low food intake (Dauncey and Ingram, 1983; Ingram and Dauncey, 1986). The lack of an equally marked increase in muscle IGF-I mRNA and peptide suggests that this muscle hypertrophy is related to the level of circulating IGF-I, which in turn probably reflects the high levels of liver IGF-I mRNA and peptide. The finding of relatively less kidney IGF-I m R N A at the high compared with the low temperature could possibly be related to differences in handling of water loss. In the warm environment, a major route for water loss would be via evaporation from the respiratory tract, whereas in the cold most water loss would be via the urine. Preliminary studies on one litter of animals have shown urine volume/kg body weight/24 h to be considerably greater in those living in the cold than the warm. In addition, the clearance ratio of IGF-I was found to be greater in the cold. It has been demonstrated previously that IGF-I may mediate the GH stimulated increase in renal plasma flow and glomerular filtration rate (Hirshberg and Kopple, 1989) and detailed investigation is now needed on the influences of diet and temperature on kidney function. Studies on rat kidney have demonstrated localization of IGF-I mRNA and peptide in the collecting duct, with the abundance of the mRNA being 10-fold greater here than in whole kidney (Bortz et al., 1988). Future studies therefore need to focus on the effects of environmental factors in relation to isolated components of the nephron. Factors responsible for the regulation of IGF-I gene expression in different tissues, under conditions of altered temperature and food intake, need to be determined. The extent to which endocrine factors including GH could be involved is unclear. Thus, in animals similar to those described in the present study, the overall effects of temperature and diet on circulating concentrations of GH were not statistically significant (Dauncey and Buttle, 1990). However, hepatic GH receptors are influenced by nutritional status (Gluckmann and Breier, 1989), and in fasted rats there is a rapid fall in GH binding to liver (Maes et al., 1983) and a reduction in hepatic GH receptor m R N A (Straus and Takemoto, 1990). Whether there are differences in GH receptor m R N A and GH binding in response to long-term changes in environmental temperature under conditions of controlled food intake now needs to be determined.
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Acknowledgements--We thank Dr C. G. Prosser for advice on the assay of IGF-I peptide in tissues and Dr R. S. Gilmour for advice on estimation of IGF-I mRNA. Anti-IGF-I antiserum was a generous gift from Dr L. Underwood and Dr J. Van Wyk, provided by the National Institute of Diabetes and Digestive and Kidney Diseases through the National Hormone and Pituitary Program. L. M. was a postgraduate student registered with the Council for National Academic Awards.
REFERENCES
Bortz J. D., Rotwein P., DeVol D., Bechtel P. J., Hansen V. A. and Hammerman M. R. (1988) Focal expression of insulin-like growth factor I in rat kidney collecting duct. J. Cell. Biol. 107, 811-819. Clemmons D. R., Klibanski A., Underwood L. E., McArthur J. W., Ridgway E. C., Beitins I. Z. and Van Wyk J. J. (1981) Reduction of plasma immunoreactive somatomedin C during fasting in humans. J. din. Endocr. Metab. 53, 1247-1250. Corps A. N., Brown K. D., Rees L. H., Carr J. and Prosser C. G. (1988) The insulin-like growth factor I content in human milk increases between early and full lactation. J. clin. Endocrinol. Metab. 67, 25-29. Daughaday W. H. and Rotwein P. (1989) Insulin-like growth factors I and If. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocrine Rev. 10, 68-91. Daughaday W. H., Mariz I. K. and Blethen S. L. (1980) Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: a comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J. clin. Endocrinol. Metab. 51, 781-788. Dauncey M. J. and Buttle H. L. (1990) Differences in growth hormone and prolactin secretion associated with environmental temperature and energy intake. Horm. Metab. Res. 22, 524-527. Dauncey M. J. and Ingrain D. L. (1983) Evaluation of the effects of environmental temperature and nutrition on body composition. J. agric. Sci. 101, 351-358. Dauncey M, J. and Ingram D. L. (1986) Acclimatization to warm or cold temperatures and the role of food intake. J. therm. Biol. 11, 89-93. Dauncey M. J., Ingram D. L., Waiters D. E. and Legge K. F. (1983) Evaluation of the effects of environmental temperature and nutrition on growth and development. J. agric. Sci. 101, 291-299. Dauncey M, J., Shakespear R. A., Rudd B. T. and Ingram D. L. (1990) Variations in somatomedin-C/insulin-like growth factor-I associated with environmental temperature and nutrition. Horm. Metab. Res. 22, 261-264. D'Ercole A. J., Applewhite G. T. and Underwood L. E. (1980) Evidence that somatomedin is synthesized by multiple tissues in the fetus. Devl. Biol. 75, 315-328. D'Ercole A. J., Decedue C. J., Furnaletto R. W., Underwood L. E. and Van Wyk J. J. (1977) Evidence that somatomedin-C is degraded by the kidney and inhibits insulin degradation. Endocrinology 101, 577-586. D'Ercole A. J., Stiles A. D. and Underwood L. E. (1984) Tissue concentrations of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc. natn. Acad. Sci. USA 81, 935-939. Emler C. A. and Schalch D. S. (1987) Nutritionally-induced changes in hepatic insulin-like growth factor I (IGF-I) gene expression in rats. Endocrinology 120, 832-834. Furlanetto R. W., Underwood L. E., Van Wyk J. J. and D'Ercole A. J. (1977) Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J. clin. Invest. 60, 648-657.
Gluckman P. D. and Breier B. H. (1989) The regulation of the growth hormone receptor. In Biotechnology in Growth Regulation (Edited by Heap R. B., Prosser C. G. and Lamming G. E.), pp. 27-33. Butterworths, London. Goldstein S., Harp J. B. and Phillips L. S. (1991) Nutrition and somatomedin XXII: Molecular regulation of insulinlike growth factor-I during fasting and refeeding in rats. J. molec. Endocr. 6, 33-43. Goldstein S., Sertich G. J., Levan K. R. and Phillips L. S. (1988) Nutrition and somatomedin. XIX. Molecular regulation of insulin-like growth factor-I in streptozotocin diabetic rats. Molec. Endocr. 2, 1093-1100. Grant A. L., Helferich W. G., Kramer S. A., Merkel R. A. and Bergen W. G. (1991) Administration of growth hormone to pigs alters the relative amount of insulin-like growth factor-I mRNA in liver and skeletal muscle. J. Endocr. 130, 331-338. Hirschberg R. and Kopple J. D. (1989) Effects of growth hormone and IGF-I on renal function. Kidney Int. 36, S-20-S-26. Hizuka N., Takano K., Tanaka I., Asakawa K., Miyakawa M., Horikawa R. and Shizume K. (1987) Demonstration of insulin-like growth factor-I in human urine. J. din. Endocr. Metab. 64, 1309-1312. Ingram D. L. and Dauncey M. J. (1986) Environmental effects on growth and development. In Control and Manipulation o f Animal Growth (Edited by Buttery P. J., Haynes N. B. and Lindsay D. B.), pp. 5-20. Butterworths, London. Klein J. R. (1945) Estimation of blood in tissue. Arch. Biochem. 8, 421-424. Lauterio T. J. and Scanes C. G. (1987) Hormonal responses to protein restriction in two strains of chickens with different growth characteristics. J. Nutr. 117, 758-763. Leaman D. W., Simmen F. A., Ramsay T. G. and White M. E. (1990) Insulin-like growth factor-I and -II messenger RNA expression in muscle, heart and liver of streptozotocin-diabetic swine. Endocrinology 126, 2850-2857. Lowe W. L., Adamo M., Werner H., Roberts C. T. and LeRoith D. (1989) Regulation by fasting of rat insulin-like growth factor-I and its receptor. Effects on gene expression and binding. J. clin. Invest. 84, 619-626. Lund P. K., Moats-Staats B. M., Hynes M. A., Simmons J. G., Jansen M., D'Ercole A. J. and Van Wyk J. J. (1986) Somatomedin/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J. biol. Chem. 261, 14539-14544. Macari M., Ingram D. L. and Dauncey M. J. (1983) Influence of thermal and nutritional acclimatization on body temperatures and metabolic rate. Comp. Biochem. Physiol. 74A, 549-553. Maes M., Underwood L. E. and Ketelslegers J. M. (1983) Plasma somatomedin-C in fasted and refed rats: close relationship with changes in liver somatogenic but not lactogenic binding sites. J. Endocrinol. 97, 243-252. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning. A Laboratory Manual. Laboratory Press, Cold Spring Harbor. Merimee T. J., Zapf J. and Froesch E. R. (1982) Insulin-like growth factors in the fed and fasted states. J. clin. Endocr. Metab. 55, 999-1002. Miller L. L., Schalch D. S. and Draznin B. (1981) Role of the liver in regulating somatomedin activity: effects of streptozotocin diabetes and starvation on the synthesis and release of insulin-like growth factor and its carrier protein by the isolated perfused rat liver. Endocrinology 108, 1265-1271. Murphy L. J., Bell G. I. and Friesen H. G. (1987) Tissue distribution of insulin-like growth factor-I and -II messenger ribonucleic acid in the adult rat. Endocrinology 120, 1279-1282.
Environment and IGF-I Phillips L. S., Goldstein S. and Gavin J. R. (1988) Nutrition and somatomedin XVI: Somatomedins and somatomedin inhibitors in fasted and refed rats. Metabolism 37, 209-216. Prewitt T. E., D'Erc,ole A. J., Switzer B. R. and Van Wyk J. J. (1982) Relationship of serum immunoreactive somatomedin-C to dietary protein and energy in growing rats. J. Nutr. 112, 144-150. Quattrin T., Albini C. H., Cara J. F., Vandlen R. L., Mills B. J. and MacGillivray M. H. (1988) Quantitation of urinary somatomedin-C and growth hormone in preterm and fullterm infants and normal children. J. clin. Endocr. Metab. 66, 792-797. Schwander J. C., Hauri C., Zapf J. and Froesch E. R. (1983) Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status. Endocrinology 113, 297-305. Smith A. T., Clemmons D. R., Underwood L. E., Ben-Ezra V. and McMurray R. (1987) The effect of exercise on plasma somatomedin-C/insulinlike growth factor I concentrations. Metabolism 36, 533-537.
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Straus D. S. and Takemoto C. D. (1990) Effect of fasting on insulin-like growth factor-I (IGF-I) and growth hormone receptor mRNA levels and IGF-I gene transcription in rat liver. Molec. Endocr. 4, 91-100. Tokunaga K., Takeda K., Kamiyama K., Kageyama H., Takenage K. and Sakiyama S. (1988) Isolation of cDNA clones for mouse cytoskeletal 3,-actin and differential expression of eytoskeletal actin mRNAs in mouse cells. Molec. Cell. Biol. 8, 3929-3933. VandeHaar M. J., Moats-Staats B. M., Davenport M. L., Walker J. L., Ketelslegers J.-M., Sharma B. K. and Underwood L. E. (1991) Reduced serum concentrations of insulin-like growth factor-I (IGF-I) in proteinrestricted growing rats are accompanied by reduced IGF-I mRNA levels in liver and skeletal muscle. J. Endocrinol. 130, 305-312. Van Neste L., Husman B., Moiler C., Andersson G. and Norstedt G. (1988) Cellular distribution of somatogenic receptors and insulin-like growth factor-I mRNA in rat liver. J. EndocrinoL 119, 69-74.
0306-4565/92 $5.00+ 0.00 Copyright © 1992PergamonPress Ltd
THERMAL A N D NUTRITIONAL INFLUENCES ON TISSUE LEVELS OF INSULIN-LIKE GROWTH FACTOR-I mRNA A N D PEPTIDE L. MA, K. A. BURTON,J. C. SAUNDERSand M. J. DAUNCEY* Department of Molecular and Cellular Physiology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, England (Received 1 October 1991; accepted 19 November 1991) Abstract--1. The influence of environmental temperature on tissue levels of IGF-I mRNA and peptide has been investigated in 8 week old pigs living under conditions of controlled energy intake. Animals were acclimated to 35 or 10°C on a high (H) or low (L) energy intake (where H = 2L) and there were thus 4 treatment groups: 35H, 35L, 10H and 10L. 2. The warm temperature was associated with greater levels of IGF-I peptide in plasma and all tissues investigated (liver, kidney, skeletal muscle and heart). Temperature had significant but contrasting influences on IGF-I mRNA in liver and kidney: at 35 compared with 10°C, levels were increased in liver but reduced in kidney. Despite marked differences in growth and protein deposition, there was no effect of temperature on muscle IGF-I mRNA. 3. A high energy intake was associated with increased levels of IGF-I peptide in plasma and tissues. However, although there was a tendency for liver IGF-I mRNA to be elevated in the 35H compared with the 35L group, there was no overall effect of energy intake on IGF-I mRNA in any issue. 4. It is concluded that ambient temperature and energy intake can significantlyaffect the concentration of IGF-I peptide in tissues and that regulation may occur at either transcriptional or translational levels. The probability is that the effects of temperature are mediated via its influence on energy balance. Key Word Index: Insulin-like growth factor-I; mRNA; temperature; energy intake; energy balance; growth; pig
INTRODUCTION Previous investigations have shown that acclimation of young pigs to a warm or cold temperature under conditions of controlled energy intake has a marked influence on growth and morphology (Dauncey and Ingram, 1986; Ingram and Dauncey, 1986). For .example, animals living in the warm have longer extremities than littermates in the cold on the same level of food intake (Dauncey et al., 1983) and there are significant differences in protein and fat deposition (Dauncey and Ingram, 1983). Recent studies have shown that although growth hormone (GH) secretion tends to be increased in animals living in a warm compared with a cold environment on the same food intake, there is wide individual variation and the difference is not statistically significant (Dauncey and Buttle, 1990). By contrast, plasma concentrations of insulin-like growth factor-I (IGF-I) are considerably greater in animals living in the warm than the cold and on a high than a low energy intake (Dauncey et al., 1990). However, the source of this increased IGF-I was not determined. Although liver is considered to be a major source of circulating IGF-I, its mRNA and peptide have been found in a wide variety of tissues (D'Ercole et al., 1984; Lund et al., 1986; Murphy et al., 1987). Together with evidence that IGF-I influences the growth of many cell types, it has been suggested that, in addition to its endocrine role,
IGF-I may function as an autocrine/paracrine regulator of growth (D'Ercole et al., 1980; Daughaday and Rotwein, 1989). Whether long-term changes in environmental temperature and energy intake influence tissue levels of IGF-I mRNA and its peptide is not known. To our knowledge, no investigations have been carried out on the effects of temperature. In addition, many studies on the effect of food intake have been concerned with the acute effects of severe undernutrition, which usually involve a period of fasting for several days (Emler and Schalch, 1987; Lowe et al., 1989; Leaman et al., 1990; Straus and Takemoto, 1990), rather than the response to a prolonged period of reduced food intake. The aim of the present investigation was therefore to determine whether long-term changes in ambient temperature and food intake can influence tissue levels of IGF-I mRNA and peptide in young growing animals. MATERIALS AND METHODS Experimental design Eight litters of pigs from the Large White herd kept at the Institute were used. At the age of 2-3 weeks, 4 male pigs from each litter were weaned and kept in pairs for 2-3 days at 25-30°C. They were then separated into individual cages and, over the next 2-3 weeks, for two pigs the environmental temperature was increased gradually to 35°C, while for the other two animals it was reduced to 10°C. All animals were therefore at the required temperature by 5 weeks of
*Author for correspondence. 89
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age. At each temperature, one pig received a high (H) intake and the other a low (L) intake of standard feed (Ultra-Wean, Dalgety, Bristol, England). Animals were fed once daily, at 09.00 h and although the absolute amount of food was increased as the animals grew, the ratio of H : L was always 2: 1. Final food intakes were 700 and 350 g for the H and L intakes respectively. Water was always available ad libitum. Animals were killed at 8-9 weeks of age, 24 h after the last meal, by intracardiac injection of sodium pentobarbital following intramuscular administration of tranquillizer (Ketamine, Parke-Davis). There were marked differences in growth rate, and mean body weights ( + S E M ) were 17.2+ 1.0, 11.7+0.8, 13.6+0.8 and 7 . 4 + 0 . 7 k g for the 35H, 35L, 10H and 10L treatment groups respectively. The liver, kidney, longissimus dorsi muscle and heart (ventricle) were removed immediately, divided into 5 g portions, frozen in liquid nitrogen and stored at - 4 0 ° C . Whole blood and plasma were stored in 0.5 ml aliquots at - 2 0 ° C . Assay o f specific mRNAs RNA extraction. The guanidium/lithium chloride method was used to isolate total RNA from the various tissues. Approximately 1 g frozen tissue was pulverised in an anvil press which had been cooled in dry ice, homogenized in 8ml lysis buffer ( 5 M guanidine isothiocyanate, 10mM EDTA, 50mM Tris base, pH 7.5) and forced through a 19-gauge needle until the viscosity caused by released chromosomal D N A was reduced. To inhibit denaturation of proteins, fl-mercaptoethanol was added to a final concentration of 0.1 M. The sample was then made up to 50 ml with 4 M LiC1 and left overnight at 4°C. Centrifugation at 12,500g for 20min at 4°C was followed by resuspension of the pellet in 30 ml 3 M LiC1. The suspension was left on ice for 10 rain and centrifuged again for 10min. The pellet was resuspended in 3 ml I% SDS, 20 #! proteinase K (1 #g/ml) and left at room temperature for 20 min. An equal volume of phenol was added and the sample vortexed. The aqueous solution containing R N A was obtained by centrifugation at 12,500g for 10 min at 10°C. This was further purified by mixing with 0.5 vol 7.5 M ammonium acetate and 3 vol 100% ethanol, and precipitation for 1 h on dry ice. After centrifugation at 12,500g for 10min at 4°C, the pellet was resuspended in 0.5 ml water, 50/~1 3 M sodium acetate pH 4.8 and 4 ml 100% ethanol. After overnight precipitation at - 2 0 ° C , the R N A precipitate was sedimented at 12,500g for 10 min at 4°C and redissolved in water. The total RNA concentration of each sample was estimated spectrophotometrically as described by Maniatis et al. (1982) and the samples were then stored at - 7 0 ° C . Dot blot analysis. Total R N A was denatured with a solution containing 1 M deionized glyoxal, 12.5#i dimethyl sulphoxide, 10/~M sodium phosphate, pH 7.0, by incubation at 50°C for 1 h. The mixture was then kept on ice during dot blotting. Serial dilutions of 10 to 1.25/~g RNA with TrisEDTA buffer, pH 8.0, were blotted onto hybond-N membrane fitted into the dot blot apparatus. After washing with buffer, the membrane was baked at 80°C for 1 h between two filter papers. D N A probes
were labelled with [ct-32p]dCTP, by nick translation, to a specific activity of 2-3 x 10 epm/# g (Amersham International pie). Prehybridization was performed at 65°C for 1 h in a shaking water bath, using a solution of 1% BSA, 1 mM EDTA, 7% SDS, 0.5 M sodium phosphate, pH 7.2. Hybridization was performed at 65°C for 24 h in a shaking water bath. The labelled IGF-I eDNA probe was mixed with the prehybridization solution to form the hybridization solution. The membrane was washed twice with solution A (0.5% BSA, 5% SDS, 1 mM EDTA, 50 mM sodium phosphate) for 20 min at 65°C, once with solution B (the same as solution A but without BSA) for 20 min at 65°C, and blotted with filter paper. Autoradiography of the membrane was carried out at - 70°C, for 1, 2 and 4 days for liver, kidney and skeletal muscle respectively. The membrane was rehybridized with a fl-actin eDNA probe, after first removing the I G F probe with 2 x SSC plus 0.1% SDS at 95°C for 15-20 min, and then prehybridizing as described previously. The density of dots on the autoradiograpbed X-ray films was measured with a Chromoscan (JoyceLoebl) and the relative amount of specific m R N A per unit total tissue R N A was estimated. Estimation o f IGF-I peptide The methods were based on those described by Furlanetto et al. (1977) and Corps et al. (1988). IGF-I was first separated from its binding proteins under acid conditions (Daughaday et al., 1980), for both plasma and tissues. For determining the net tissue IGF-I content, the plasma derived IGF-I trapped in the tissue was subtracted from the total tissue IGF-I. This involved determination of the haemoglobin content of the tissues and the measurement of haematocrit. Plasma extraction. Plasma (100#1) was diluted with 400 gl radioimmunoassay (RIA) buffer (100 mg protamine sulphate, 2.07 g NaH2PO4 2H20, 100 mg sodium azide, 10ml EDTA pH 7.5, made up to 500 ml with water, then 250 #1 Tween 20 added) and 4vol of extraction mixture (87.5% ethanol: 12.5% 2 M HCI) were added to 100 #1 of the diluted sample. After mixing and standing for 30 min at room temperature, centrifugation was carried out at 10,000g for 15 min and the supernatant was neutralized with 0.3 vol of 0.8 M Tris base. Tissue extraction. Approximately 3 - 4 g tissue (liver, kidney, skeletal muscle, heart) was pulverized in an ice-cold anvil press, and about 3 g was used for the tissue extraction while 0.5 g was stored at - 4 0 ° C for the haemoglobin assay. The tissue was mixed thoroughly with 10ml I M acetic acid and shaken gently for 2 h at 4°C. The supernatant was obtained by centrifugation at 2000g for 15 min at 4°C. The pellet was re-extracted with 1 M acetic acid, the supernatants combined and freeze-dried, and the dried material reconstituted with RIA buffer. Finally, undissolved material was removed by centrifugation at 20,000g for 20 min at 4°C, and the supernatants stored in 0.5 ml aliquots at - 2 0 ° C . Radioimmunoassay. IGF-I was measured in the plasma and tissue extracts by RIA. The antiserum (NIDDK) was rabbit antibody for hIGF-I; standards were rhIGF-I (Bachem); hIGF-I was labelled by the
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Table 1. Concentration of IGF-I mRNA (units/10#g total RNA) in liver, kidney and Iongissimua dorsi muscle from animals which had been living at 35 or 10°C on a high (H) or low (L) energy intake Tissue
35H
35L
10H
10L
Liver Kidney Muscle
10.0+0.7 2.5 + 0.4 2.5 ± 0.2
8.6+1.1 3.0 + 0.5 2.6 ± 0.3
6.2+1.7 4.3 + 0.3 2.9 ± 0.4
6.5+0.8 3.6 _+0.6 2.6 + 0.2
P values: Liver Kidney Muscle
Temperature (T) < 0.05 (35 > 10) <0.01 (10 > 35) NS
Energy intake (E) NS NS NS
Interaction (T x E) NS NS NS
Mean values _+ SEM in arbitrary units indicate density of dots on autoradiograph and thus give the relative amount of specific mRNA in a given amount of total tissue RNA; autoradiography was for 1, 2 or 4 days for liver, kidney and muscle respectively, P values are from the analysis of variance.
Iodo-Gen method. Separation of bound from free 125I I G F - I was with a second antibody of sheep antiserum to rabbit IgG (kindly provided by Dr C. G. Prosser), and normal rabbit serum was used as a carrier. In all instances, samples from each animal in a given litter were examined within the same assay. Estimation of tissue plasma content. The whole blood content within each tissue was determined by the method of Klein (1945). This involved mixing 0.5 g powdered tissue with distilled water and toluene and leaving for 1 h at room temperature. Ammonium sulphate-phosphate solution, pH 6.6, was then added and the solution left for 20 min with some mixing. Extracts were filtered and to the filtrate was added 20% potassium ferrocyanide and 5% potassium cyanide. Solutions were mixed and absorbances read at 540 nm. Serial dilutions of whole blood from the same animal were treated in the same way as the tissues, allowing determination of the blood content of the tissue by comparing its absorbance with that of whole blood. Measurement of haematocrit allowed estimation of the plasma content of the tissues. The plasma derived I G F - I content of the tissues was then subtracted from the total I G F - I content to give the net tissue I G F - I peptide concentration. RESULTS
Tissue abundance of IGF-I mRNA and peptide Table 1 shows that the relative levels of I G F - I m R N A in liver were considerably greater than in the two other tissues examined. By contrast, the I G F - I peptide occurred in the descending order: kidney, liver, heart, skeletal muscle (Table 2). This was the case for all treatment groups except 10L, where all
tissue concentrations were very low and slightly lower in liver than in heart.
Environmental influences on IGF-I mRNA and peptide Environmental temperature and food intake tended to have different effects on I G F - I m R N A compared with its peptide. The results for the peptide (Table 2) clearly showed that in all tissues investigated, the concentration was greater in animals living in the warm than in the cold and on the high compared with the low food intake. For all tissues, values were greatest in the 35H, intermediate in the 35L and 10H, and lowest in the 10L animals. These findings were in the same order as those for plasma I G F - I concentrations of 25.2 + 8.4, 8.0 + 2.6, 10.3 + 2.4 and 2.8 + 0.2 nmol/l in the 35H, 35L, 10H and 10L groups respectively. However, no such generalization can be made with respect to the results for mRNA, since the effects of treatment varied from one tissue to the next (Table 1). In the following sections, results are presented in detail for I G F - I m R N A and peptide in liver, kidney and skeletal muscle. Liver. Environmental temperature had a significant influence on both m R N A (P < 0.05) and peptide (P <0.01), with levels being greater at 35 than at 10°C. The concentration of peptide in liver was also found to be significantly greater on the high compared with the low food intake (P < 0.05), and the effect was particularly striking at 10°C. However, there was no statistically significant effect of food intake on mRNA; levels were slightly greater in the 35H than the 35L group, whereas values for the 10H and 10L were similar to each other.
Table 2. Concentration of IGF-I peptide (nmoi/kg tissue) in liver, kidney, Iongissimus dorsi muscle and heart from animals which had been living at 35 or 10°C on a high (H) or low (L) energy intake Tissue
35H
35L
10H
10L
Liver Kidney Muscle Heart
2.45 -t- 0.21 4.65 + 0.62 0.44 + 0.10 1.77 + 0.37
1.90 + 0.47 3.68 _+ 0.94 0.20 ± 0.04 0.85 + 0.19
1.77 + 0.48 3.49 +_0.58 0.27 + 0.05 0.78 ± 0.09
0.54 ± 0.07 1.01 + 0.19 0.22 _+0.04 0.63 ± 0.08
P values: Liver Kidney Muscle Heart
Temperature <0.01 (35 > <0.01 (35 > NS <0.05 (35 >
(T) 10) 10) 10)
Energy intake (E) <0.05 (H > L) <0.01 (H > L) <0.01 (H > L) <0.05 (H > L)
Interaction (T x E) NS NS NS NS
Mean values +_ SEM; P values are from the analysis of variance.
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Table 3. Concentrationof actinmRNA(units/10#g total RNA)in liver,kidney and Iongissimus dorsi muscleof animalswhichhad beenlivingat 35 or 10C on a high (H) or low (L) energyintake Tissue 35H 35L 10H 10L Liver 10.2+ 0.4 8.6_+ 1.2 8.2_+2.5 5.6+ 1.1 Kidney 8.4-+0.8 8.2-+ 1.2 10.8-+0.8 8.8-+0.5 Muscle 8.8+ 1.3 9.2-+ 1.3 9.1 -+ 1.2 8.0-+0.7 P values: Temperature(T) Energyintake (E) Interaction(T x E) Liver NS NS NS Kidney <0.05 (10 > 35) NS NS Muscle NS NS NS Mean values+ SEM in arbitrary units indicatedensityof dots on autoradiograph and thus givethe relativeamountof specificmRNAin a givenamount of total tissueRNA;autoradiographywas for I, 2 or 4 daysfor liver,kidney and musclerespectively;P valuesare from the analysisof variance. Kidney. A significantly higher concentration of peptide was found in the kidney of animals in the warm compared with the cold (P < 0.01) and on the high compared with the low food intake (P < 0.01). By contrast with the results for mRNA in liver, the level of IGF-I mRNA in kidney was found to be significantly lower (P < 0.01) in the warm than in the cold environment. There was, however, no overall effect of energy intake on mRNA, although the interaction between temperature and diet did approach statistical significance (P = 0.075), reflecting the opposing effects of diet at the two temperatures (35H < 35L, 10H > 10L). Skeletal muscle. Concentrations of the peptide were low in all groups, with an average of less than 0.5 nmol/kg. Nevertheless, as with the other tissues, there was a tendency for the value to be greater at 35 than 10°C (P < 0.1) and it was significantly greater on the high than the low food intake (P <0.01). Levels of IGF-I mRNA were similar in the four treatment groups and there were no significant influences of either temperature or nutrition. Environmental influences on actin m R N A
The results for fl-actin mRNA are presented in Table 3. There was a tendency for the treatment effects to be similar to those observed for IGF-I mRNA. For example, actin mRNA in liver was greater both in the warm compared with the cold and on the high compared with the low food intake. However, only the effect of temperature on kidney reached statistical significance (35 < 10°C, P < 0.05). Actin mRNA has been used in a number of studies as a reference, when a specific mRNA such as that for IGF-I is being investigated (Van Neste et al., 1988; Goldstein et al., 1988). In these earlier studies, a constant amount of actin mRNA was always reported. However, the current finding of a tendency for actin m R N A to be affected by temperature and diet suggests that it may not invariably be an appropriate reference. It has been demonstrated previously that the level of actin mRNA is changed when cells are in different states of growth (Tokunaga et al., 1988). The use of actin mRNA as a reference in the present study was considered to be inappropriate, particularly since animals with very different growth rates were being investigated. Results for IGF-I were therefore expressed in relation to a given amount of total tissue R N A .
DISCUSSION The present investigation clearly demonstrates that long-term changes in environmental temperature and energy intake influence not only the plasma level of IGF-I but also its concentration in a variety of tissues. Thus, in plasma, liver, kidney, skeletal muscle and heart, IGF-I levels are greater in young animals living at a warm compared with a cold ambient temperature and on a high compared with a low food intake. Numerous studies have demonstrated a direct correlation between food intake and plasma IGF-I (Clemmons et al., 1981; Merimee et al., 1982; Prewitt et aL, 1982; Lauterio and Scanes, 1987; Phillips et al., 1988). The current investigation, together with that described previously (Dauncey et al., 1990), suggests that it is energy supply in relation to demand rather than the absolute amount of energy intake which is important in determining IGF-I levels. The effect of ambient temperature on plasma and tissue IGF-I is probably mediated indirectly via its influence on energy balance. In a cold environment, extra energy is needed for maintenance of deep body temperature and thus for a given intake, resting metabolic rate increases and growth rate is reduced (Macari et al., 1983). A decrease in energy availability in adult man, caused by vigorous exercise on a fixed intake, is also accompanied by a reduction in plasma IGF-I (Smith et al., 1987). Thus, plasma and tissue levels of IGF-I are probably related to the surplus energy available for growth after other demands for energy have been met. In the present study, there was indeed a close correlation between the IGF-I levels and growth rates of animals in the four treatment groups. The current findings on the long-term influence of energy status on liver IGF-I peptide are similar to those of its more acute effects. Thus, in rats fasted for 3 days, there is a decrease in levels of both serum and extractable hepatic immunoreactive IGF-I (Goldstein et aL, 1991). Similarly, in young pigs of about 40 kg body weight, withdrawal of food for 3 days caused decreases in plasma and liver IGF-I peptide of 80 and 55°,/0 respectively (Leaman et al., 1990). The close correlation between hepatic and circulating levels of IGF-I reported in these studies, together with others on hepatic IGF-I production (Miller et aL, 1981; Schwander et al., 1983; Goldstein et al., 1988), provide strong evidence for liver being the major source of plasma IGF-I.
Environment and IGF-I The highest level of IGF-I peptide occurred in kidney, followed in descending order by liver, heart and skeletal muscle. This concurs with the finding that in the adult rat, kidney also contains the highest concentration of IGF-I peptide (D'Ercole et al., 1984). The possibility is that, whereas the IGF-I peptide in liver, heart and muscle is a reflection of local tissue synthesis, the peptide level in kidney also reflects the role played by this organ in the degradation (D'Ercole et al., 1977) and clearance of IGF-I (Hizuka et al., 1987; Quattrin et al., 1988). By contrast with the results for abundance of peptide in different tissues, IGF-I mRNA was found to occur at a higher level in liver than in kidney or muscle. Liver also contains significantly more IGF-I m R N A than any other tissue in the rat (Murphy et al., 1987). However, the relative expression of IGF-I m R N A in 40 kg pigs was found to be greater in skeletal muscle and heart than in liver (Leaman et al., 1990). The dot blot technique was used in both this and the current investigation, although in this earlier study a specific 580-base pair porcine IGF-I eDNA probe was used, whereas a human IGF-I eDNA probe was used in the present study. However, given the high sequence homology ( > 90%) between the sequences, this is unlikely to have contributed to the differences between the two investigations. Possible differences in relation to methodology, muscle type and age of animal remain to be investigated. The effects of environmental temperature and food intake on transcription and translation were found to be tissue specific. Thus, although the levels of IGF-I peptide in all the tissues were positively correlated with both a warm temperature and a high intake, the results for m R N A varied between tissues. For example, at a high compared with a low ambient temperature, the level of IGF-I mRNA was elevated in liver but reduced in kidney. At 35°C, there was a tendency for liver m R N A to be increased on the high food intake, although the overall effect of intake was not statistically significant for any of the tissues. A number of studies have demonstrated a relation between food intake and IGF-I mRNA in various tissues. However, these studies have usually been concerned with acute food deprivation. Thus, in rats which had been fasted for 30 h, liver m R N A fell to less than 40% of that in controls (Emler and Schalch, 1987). Similarly, the abundance of several IGF-I m R N A species in liver decreased in 6-week old rats fasted for 24, 48 or 72 h (Straus and Takemoto, 1990). A significant decline was also found in the m R N A content of several tissues, including liver, kidney and skeletal muscle from rats which had been fasted for 48 h (Lowe et al., 1989). A recent study in 40 kg pigs fasted for 3 days demonstrated reductions in liver, muscle and heart IGF-I m R N A (Leaman et al., 1990). There was also a direct relation between the abundance of liver mRNA and the availability of energy for growth in the present investigation, although there was no effect on muscle mRNA. The degree of food restriction was much less severe but for a longer period of time than in these earlier studies. The possibility is therefore that the tissue response of IGF-I m R N A to energy status is dependent on whether there has been longTB 17/2--C
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term undernutrition or short-term starvation. Whether this response is affected by age also needs investigating, since changes in IGF-I gene expression induced by protein restriction are age-dependent in growing rats (VandeHaar et al., 1991). Nevertheless, a 3-fold increase in liver mRNA was induced in older pigs of approximately 100 kg body weight when given growth hormone (GH) over a 24 day period, whereas longissimus dorsi muscle mRNA was slightly reduced (Grant et al., 1991). This suggested that the GHinduced increase in muscle growth in these pigs was mediated by an endocrine, as distinct from an autocrine/paracrine, action of IGF-I. The rate of skeletal muscle growth and protein deposition are significantly greater in young pigs living in the warm compared with the cold and on a high compared with a low food intake (Dauncey and Ingram, 1983; Ingram and Dauncey, 1986). The lack of an equally marked increase in muscle IGF-I mRNA and peptide suggests that this muscle hypertrophy is related to the level of circulating IGF-I, which in turn probably reflects the high levels of liver IGF-I mRNA and peptide. The finding of relatively less kidney IGF-I m R N A at the high compared with the low temperature could possibly be related to differences in handling of water loss. In the warm environment, a major route for water loss would be via evaporation from the respiratory tract, whereas in the cold most water loss would be via the urine. Preliminary studies on one litter of animals have shown urine volume/kg body weight/24 h to be considerably greater in those living in the cold than the warm. In addition, the clearance ratio of IGF-I was found to be greater in the cold. It has been demonstrated previously that IGF-I may mediate the GH stimulated increase in renal plasma flow and glomerular filtration rate (Hirshberg and Kopple, 1989) and detailed investigation is now needed on the influences of diet and temperature on kidney function. Studies on rat kidney have demonstrated localization of IGF-I mRNA and peptide in the collecting duct, with the abundance of the mRNA being 10-fold greater here than in whole kidney (Bortz et al., 1988). Future studies therefore need to focus on the effects of environmental factors in relation to isolated components of the nephron. Factors responsible for the regulation of IGF-I gene expression in different tissues, under conditions of altered temperature and food intake, need to be determined. The extent to which endocrine factors including GH could be involved is unclear. Thus, in animals similar to those described in the present study, the overall effects of temperature and diet on circulating concentrations of GH were not statistically significant (Dauncey and Buttle, 1990). However, hepatic GH receptors are influenced by nutritional status (Gluckmann and Breier, 1989), and in fasted rats there is a rapid fall in GH binding to liver (Maes et al., 1983) and a reduction in hepatic GH receptor m R N A (Straus and Takemoto, 1990). Whether there are differences in GH receptor m R N A and GH binding in response to long-term changes in environmental temperature under conditions of controlled food intake now needs to be determined.
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L. MA et aL
Acknowledgements--We thank Dr C. G. Prosser for advice on the assay of IGF-I peptide in tissues and Dr R. S. Gilmour for advice on estimation of IGF-I mRNA. Anti-IGF-I antiserum was a generous gift from Dr L. Underwood and Dr J. Van Wyk, provided by the National Institute of Diabetes and Digestive and Kidney Diseases through the National Hormone and Pituitary Program. L. M. was a postgraduate student registered with the Council for National Academic Awards.
REFERENCES
Bortz J. D., Rotwein P., DeVol D., Bechtel P. J., Hansen V. A. and Hammerman M. R. (1988) Focal expression of insulin-like growth factor I in rat kidney collecting duct. J. Cell. Biol. 107, 811-819. Clemmons D. R., Klibanski A., Underwood L. E., McArthur J. W., Ridgway E. C., Beitins I. Z. and Van Wyk J. J. (1981) Reduction of plasma immunoreactive somatomedin C during fasting in humans. J. din. Endocr. Metab. 53, 1247-1250. Corps A. N., Brown K. D., Rees L. H., Carr J. and Prosser C. G. (1988) The insulin-like growth factor I content in human milk increases between early and full lactation. J. clin. Endocrinol. Metab. 67, 25-29. Daughaday W. H. and Rotwein P. (1989) Insulin-like growth factors I and If. Peptide, messenger ribonucleic acid and gene structures, serum and tissue concentrations. Endocrine Rev. 10, 68-91. Daughaday W. H., Mariz I. K. and Blethen S. L. (1980) Inhibition of access of bound somatomedin to membrane receptor and immunobinding sites: a comparison of radioreceptor and radioimmunoassay of somatomedin in native and acid-ethanol-extracted serum. J. clin. Endocrinol. Metab. 51, 781-788. Dauncey M. J. and Buttle H. L. (1990) Differences in growth hormone and prolactin secretion associated with environmental temperature and energy intake. Horm. Metab. Res. 22, 524-527. Dauncey M. J. and Ingrain D. L. (1983) Evaluation of the effects of environmental temperature and nutrition on body composition. J. agric. Sci. 101, 351-358. Dauncey M, J. and Ingram D. L. (1986) Acclimatization to warm or cold temperatures and the role of food intake. J. therm. Biol. 11, 89-93. Dauncey M. J., Ingram D. L., Waiters D. E. and Legge K. F. (1983) Evaluation of the effects of environmental temperature and nutrition on growth and development. J. agric. Sci. 101, 291-299. Dauncey M, J., Shakespear R. A., Rudd B. T. and Ingram D. L. (1990) Variations in somatomedin-C/insulin-like growth factor-I associated with environmental temperature and nutrition. Horm. Metab. Res. 22, 261-264. D'Ercole A. J., Applewhite G. T. and Underwood L. E. (1980) Evidence that somatomedin is synthesized by multiple tissues in the fetus. Devl. Biol. 75, 315-328. D'Ercole A. J., Decedue C. J., Furnaletto R. W., Underwood L. E. and Van Wyk J. J. (1977) Evidence that somatomedin-C is degraded by the kidney and inhibits insulin degradation. Endocrinology 101, 577-586. D'Ercole A. J., Stiles A. D. and Underwood L. E. (1984) Tissue concentrations of somatomedin C: Further evidence for multiple sites of synthesis and paracrine or autocrine mechanisms of action. Proc. natn. Acad. Sci. USA 81, 935-939. Emler C. A. and Schalch D. S. (1987) Nutritionally-induced changes in hepatic insulin-like growth factor I (IGF-I) gene expression in rats. Endocrinology 120, 832-834. Furlanetto R. W., Underwood L. E., Van Wyk J. J. and D'Ercole A. J. (1977) Estimation of somatomedin-C levels in normals and patients with pituitary disease by radioimmunoassay. J. clin. Invest. 60, 648-657.
Gluckman P. D. and Breier B. H. (1989) The regulation of the growth hormone receptor. In Biotechnology in Growth Regulation (Edited by Heap R. B., Prosser C. G. and Lamming G. E.), pp. 27-33. Butterworths, London. Goldstein S., Harp J. B. and Phillips L. S. (1991) Nutrition and somatomedin XXII: Molecular regulation of insulinlike growth factor-I during fasting and refeeding in rats. J. molec. Endocr. 6, 33-43. Goldstein S., Sertich G. J., Levan K. R. and Phillips L. S. (1988) Nutrition and somatomedin. XIX. Molecular regulation of insulin-like growth factor-I in streptozotocin diabetic rats. Molec. Endocr. 2, 1093-1100. Grant A. L., Helferich W. G., Kramer S. A., Merkel R. A. and Bergen W. G. (1991) Administration of growth hormone to pigs alters the relative amount of insulin-like growth factor-I mRNA in liver and skeletal muscle. J. Endocr. 130, 331-338. Hirschberg R. and Kopple J. D. (1989) Effects of growth hormone and IGF-I on renal function. Kidney Int. 36, S-20-S-26. Hizuka N., Takano K., Tanaka I., Asakawa K., Miyakawa M., Horikawa R. and Shizume K. (1987) Demonstration of insulin-like growth factor-I in human urine. J. din. Endocr. Metab. 64, 1309-1312. Ingram D. L. and Dauncey M. J. (1986) Environmental effects on growth and development. In Control and Manipulation o f Animal Growth (Edited by Buttery P. J., Haynes N. B. and Lindsay D. B.), pp. 5-20. Butterworths, London. Klein J. R. (1945) Estimation of blood in tissue. Arch. Biochem. 8, 421-424. Lauterio T. J. and Scanes C. G. (1987) Hormonal responses to protein restriction in two strains of chickens with different growth characteristics. J. Nutr. 117, 758-763. Leaman D. W., Simmen F. A., Ramsay T. G. and White M. E. (1990) Insulin-like growth factor-I and -II messenger RNA expression in muscle, heart and liver of streptozotocin-diabetic swine. Endocrinology 126, 2850-2857. Lowe W. L., Adamo M., Werner H., Roberts C. T. and LeRoith D. (1989) Regulation by fasting of rat insulin-like growth factor-I and its receptor. Effects on gene expression and binding. J. clin. Invest. 84, 619-626. Lund P. K., Moats-Staats B. M., Hynes M. A., Simmons J. G., Jansen M., D'Ercole A. J. and Van Wyk J. J. (1986) Somatomedin/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues. J. biol. Chem. 261, 14539-14544. Macari M., Ingram D. L. and Dauncey M. J. (1983) Influence of thermal and nutritional acclimatization on body temperatures and metabolic rate. Comp. Biochem. Physiol. 74A, 549-553. Maes M., Underwood L. E. and Ketelslegers J. M. (1983) Plasma somatomedin-C in fasted and refed rats: close relationship with changes in liver somatogenic but not lactogenic binding sites. J. Endocrinol. 97, 243-252. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning. A Laboratory Manual. Laboratory Press, Cold Spring Harbor. Merimee T. J., Zapf J. and Froesch E. R. (1982) Insulin-like growth factors in the fed and fasted states. J. clin. Endocr. Metab. 55, 999-1002. Miller L. L., Schalch D. S. and Draznin B. (1981) Role of the liver in regulating somatomedin activity: effects of streptozotocin diabetes and starvation on the synthesis and release of insulin-like growth factor and its carrier protein by the isolated perfused rat liver. Endocrinology 108, 1265-1271. Murphy L. J., Bell G. I. and Friesen H. G. (1987) Tissue distribution of insulin-like growth factor-I and -II messenger ribonucleic acid in the adult rat. Endocrinology 120, 1279-1282.
Environment and IGF-I Phillips L. S., Goldstein S. and Gavin J. R. (1988) Nutrition and somatomedin XVI: Somatomedins and somatomedin inhibitors in fasted and refed rats. Metabolism 37, 209-216. Prewitt T. E., D'Erc,ole A. J., Switzer B. R. and Van Wyk J. J. (1982) Relationship of serum immunoreactive somatomedin-C to dietary protein and energy in growing rats. J. Nutr. 112, 144-150. Quattrin T., Albini C. H., Cara J. F., Vandlen R. L., Mills B. J. and MacGillivray M. H. (1988) Quantitation of urinary somatomedin-C and growth hormone in preterm and fullterm infants and normal children. J. clin. Endocr. Metab. 66, 792-797. Schwander J. C., Hauri C., Zapf J. and Froesch E. R. (1983) Synthesis and secretion of insulin-like growth factor and its binding protein by the perfused rat liver: dependence on growth hormone status. Endocrinology 113, 297-305. Smith A. T., Clemmons D. R., Underwood L. E., Ben-Ezra V. and McMurray R. (1987) The effect of exercise on plasma somatomedin-C/insulinlike growth factor I concentrations. Metabolism 36, 533-537.
95
Straus D. S. and Takemoto C. D. (1990) Effect of fasting on insulin-like growth factor-I (IGF-I) and growth hormone receptor mRNA levels and IGF-I gene transcription in rat liver. Molec. Endocr. 4, 91-100. Tokunaga K., Takeda K., Kamiyama K., Kageyama H., Takenage K. and Sakiyama S. (1988) Isolation of cDNA clones for mouse cytoskeletal 3,-actin and differential expression of eytoskeletal actin mRNAs in mouse cells. Molec. Cell. Biol. 8, 3929-3933. VandeHaar M. J., Moats-Staats B. M., Davenport M. L., Walker J. L., Ketelslegers J.-M., Sharma B. K. and Underwood L. E. (1991) Reduced serum concentrations of insulin-like growth factor-I (IGF-I) in proteinrestricted growing rats are accompanied by reduced IGF-I mRNA levels in liver and skeletal muscle. J. Endocrinol. 130, 305-312. Van Neste L., Husman B., Moiler C., Andersson G. and Norstedt G. (1988) Cellular distribution of somatogenic receptors and insulin-like growth factor-I mRNA in rat liver. J. EndocrinoL 119, 69-74.