1. In an attempt to define the importance of acetate as a metabolic precursor, the activities of acetyl-CoA synthetase (EC 6.2.1.1) and acetyl-CoA hydrolase (Ec 3.1.2.1) were assayed in tissues from rats and sheep. In addition, the concentrations of acetate in blood and liver were measured, as well as the rates of acetate production by tissue slices and mitochondrial fractions of these tissues. 2. Acetyl-CoA synthetase occurs at high activities in heart and kidney cortex of both species as well as in rat liver and the sheep masseter muscle. The enzyme is mostly in the cytosol fraction of liver, whereas it is associated with the mitochondrial fraction in heart tissue. Both mitochondrial and cytosol activities have a K(m) for acetate of 0.3mm. Acetyl-CoA synthetase activity in liver was not altered by changes in diet, age or alloxan-diabetes. 3. Acetyl-CoA hydrolase is widely distributed in rat and sheep tissues, the highest activity being found in liver. Essentially all of the activity in liver and heart is localized in the mitochondrial fraction. Hepatic acetyl-CoA hydrolase activity is increased by starvation in rats and sheep and during the suckling period in young rats. 4. The concentrations of acetate in blood are decreased by starvation and increased by alloxan-diabetes in both species. The uptake of acetate by the sheep hind limb is proportional to the arterial concentration of acetate, except in alloxan-treated animals, where uptake is impaired. 5. Acetate is produced by liver and heart slices and also by heart mitochondrial fractions that are incubated with either pyruvate or palmitoyl-(-)-carnitine. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate. 6. We propose that acetate in the blood of rats or starved sheep is derived from the hydrolysis of acetyl-CoA. Release of acetate from tissues would occur under conditions when the function of the tricarboxylic acid cycle is restricted, so that the circulating acetate serves to redistribute oxidizable substrate throughout the body. This function is analogous to that served by ketone bodies.

1. Rates of degradation of normal and abnormal protein were measured in hepatoma cells after labelling first for 16h with [14C]leucine plus L-arginine and then for 3h with [3H]-leucine plus the arginine analogue, L-canavanine. 2. Over the first 2h of the degradation period, canavanine-containing proteins were degraded at approximately 5 times the average degradation rate of normal proteins. 3. Degradation of normal proteins was inhibited by about 30% by insulin, cycloheximide, puromycin, leupeptin, antipain and foetal calf serum, whereas these agents had a negligible effect on the breakdown of canavanine-containing proteins. 4. Other compounds inhibited degradation of both classes of protein to equal extents. 5. Combination experiments showed no additional inhibitory effects on the degradation of normal proteins over degradation measured in the presence of a single selective inhibitor. 6. In contrast with the results with a 16 h labelling period, the degradation of normal proteins labelled for only 3 h was not inhibited by insulin. 7. These results are explained by a model with two distinct pathways of protein turnover. The first of these pathways involves the formation of autophagic vacuoles and would be completely inhibited by each of the selective inhibitors. Normal and canavanine-containing proteins would be catabolized by this pathway at equal rates. We propose that degradation by a second pathway is not regulated by the agents tested, but by the inherent stability of each protein.

The administration of insulin-like growth factor-I (IGF-I) via subcutaneously implanted osmotic pumps partially reversed a catabolic state produced by the co-administration of 20 micrograms of dexamethasone/day to 150 g male rats. Marked dose-dependent effects on body weight and nitrogen retention were produced, with the highest IGF-I dose, 695 micrograms/day, giving a 6 g increase in body weight over 7 days, compared with a 19 g loss in the dexamethasone-only group and an 18 g gain in pair-fed controls. Two IGF-I analogues that bind poorly to IGF-binding proteins, the truncated form, des(1-3)IGF-I, and a variant with an N-terminal extension as well as arginine at residue 3, LR3IGF-I, were approx. 2.5-fold more potent than IGF-I. The response with LR3IGF-I was particularly striking because this peptide binds 3-fold less well than IGF-I to the type 1 IGF receptor. The increased potencies of the IGF-I variants may relate to the substantially increased plasma levels of IGF-binding proteins, particularly IGFBP-3, produced by the combined treatment of dexamethasone with IGF-I or the variants. These binding proteins would be expected to decrease the transfer of IGF-I, but not that of the variants, from blood to tissue sites of action. Measurements of muscle protein synthesis at the end of the treatment period and muscle protein breakdown by 3-methylhistidine (3MH) excretion throughout the experiment indicated coordinate anabolic effects of the IGF peptides on both processes. Thus 3MH excretion was decreased at the highest IGF-I dose from 83.5 +/- 4.2 (S.E.M.) mumol/kg per 7 days to 65.1 +/- 2.2, compared with 54.9 +/- 1.2 in the pair-fed controls. Part of this response in 3MH excretion may have reflected a decrease in gut protein breakdown, because IGF-I and especially the IGF analogues increased the gut weight by up to 45%. Notwithstanding the effects on protein synthesis and breakdown, the fractional carcass weights remained low in the IGF-treated groups, although the increase in total carcass weight reflected nitrogen rather than fat gain. The dexamethasone-induced changes in liver, spleen and heart weight were restored towards normal by the IGF treatment. The experiment demonstrates the potential of IGF-I treatment of catabolic states and especially the value of modified forms of growth factors that bind weakly to IGF-binding proteins.

No abstract

Incubation of 125I-labelled insulin-like growth factor-I (IGF-I) with rat plasma at 4 degrees C led to the transfer of approximately half the radioactivity to 150 kDa and smaller complexes with IGF-binding proteins. The extent of association was greater with labelled IGF-II and essentially absent with the truncated IGF-I analogue, des(1-3)IGF-I. A greater degree of binding of IGF peptides with binding proteins occurred after i.v. injection of the tracers into rats, but most of the des(1-3)IGF-I radioactivity remained free. Measurement of the total plasma clearances showed the rapid removal of des(1-3)IGF-I compared with IGF-I and IGF-II; the mean clearances were 4.59, 1.20 and 1.34 ml/min per kg respectively. The mean steady-state volume of distribution was larger for des(1-3)IGF-I than for IGF-I and IGF-II (461, 167 and 181 ml/kg respectively), probably because of the differences in plasma protein binding. With all tracers, radioactivity appeared in the kidneys to a greater extent than in other organs. The amount of radioactivity found in the adrenals, brain, skin, stomach, duodenum, ileum plus jejunum and colon was in rank order, des(1-3)IGF-I greater than IGF-I greater than IGF-II. Since this ranking is the opposite of the abilities of the three IGF peptides to form complexes with plasma binding proteins, we propose that the plasma binding proteins inhibit the transfer of the growth factors to their tissue sites of action. Moreover, we suggest that IGF analogues that are cleared rapidly from blood may have greater biological potencies in vivo.

We have examined the influence of nutrition on plasma IGF-I, IGF-II and IGF-binding protein (IGFBP) levels and on hepatic IGF-I gene expression in young meat-type chickens. Plasma IGF concentrations were measured by using RIA with recombinant chicken IGFs as standards. In chickens fed the control diet containing 200 g/kg dietary protein ad libitum for 7 days, plasma IGF-I concentrations increased significantly from those found in the initial control group. Food restriction for either 4 or 7 days decreased plasma IGF-I by 30% from the initial control. When chickens were refed ad libitum for 3 days after 4 days of restricted feeding, plasma IGF-I levels recovered to those of the control birds fed ad libitum. In chickens eating a low protein diet (100 g/kg protein), the plasma IGF-I tended to be lowered but the decrease was not significant. Although the intensity of IGF-I and beta-actin mRNA bands protected in the RNase protection assay was changed by nutrition, no statistical effect of nutrition on the ratio of IGF-I to beta-actin was observed. The nutritional treatments had no effect on plasma IGF-II concentrations. Western ligand blot and chromatographic analyses were used to investigate the influence of nutrition on IGFBP profiles. Both IGF-I and IGF-II ligands in the Western ligand blot revealed the most intense binding at 30 kDa for plasma obtained from chickens with restricted food intake. The 30 kDa band also appeared at a lower intensity in the group fed a low protein diet but not in any other groups. These observations were confirmed by neutral gel chromatography. The chicken IGF-II ligand revealed an intensely labelled band corresponding to 75 kDa and this was not affected by nutrition. IGF-I and IGFBP concentrations in the plasma of young broiler chickens were influenced by nutritional state but IGF-II concentrations were not. The lack of a response in circulating IGF-II levels may have been due to the presence of high concentrations of a 75 kDa specific binding protein which did not respond to nutrition in this experiment.

The ability of insulin-like growth factor-I (IGF-I) to protect against losses of body protein during periods of dietary nitrogen restriction has been evaluated in young rats. Recombinant human IGF-I was administered by osmotic pumps at dose rates of 0, 1.2 or 2.9 mg/kg per day over a 7-day period beginning with the transfer of animals from an 18% to a 4% protein diet. A fourth group received the potent truncated IGF-I analogue, des(1-3)IGF-I, at a dose of 1.2 mg/kg per day over a comparable 7-day period. Plasma IGF-I levels were reduced by 60% following nitrogen restriction, a reduction that was partly prevented by IGF-I administration, especially at the higher dose, but not measurably by des(1-3)IGF-I. The major IGF-binding protein circulating in blood, IGFBP-3, demonstrated a similar pattern of change. A significant (P less than 0.05) protection of body weight was achieved in the low dose IGF-I and des(1-3)IGF-I groups, but only after differences in food intake had been eliminated by analysis of covariance. Nitrogen balances were not significantly different unless analysis of covariance was used to adjust for the nitrogen intakes, whereupon all treatment groups showed improved balance, especially the animals treated with the low IGF-I dose and des(1-3)IGF-I (both P less than 0.01). The rate of muscle protein breakdown calculated from the urinary excretion of 3-methyl-histidine was not significantly altered by the treatments, but fell progressively throughout the 7 days. The fractional rate of muscle protein synthesis measured on the final day was increased by 31,26 and 21% respectively by the low and high doses of IGF-I and by des(1-3)IGF-I. Organ weights (g/kg body weight) showed no effects of IGF-I treatment except for 16% increases in the weight of kidneys in the high dose IGF-I and the des(1-3)IGF-I groups. Carcass analyses demonstrated higher water and lower fat contents (all P less than 0.01) in the same groups. These results suggest that exogenous IGF-I and especially des(1-3)IGF-I can partly protect body protein reserves during nitrogen restriction.

Administration of IGF-I over a 14-day period to growing female rats via s.c. implanted osmotic pumps led to an increased body weight gain, an improved N retention and a greater food conversion efficiency. The effects were dose-dependent, with the highest daily dose tested, 278 micrograms/day, producing 18-26% increases in these measurements. LR3IGF-I, a variant of human IGF-I that contains an amino terminal extension peptide as well as glutamate-3 replaced by arginine and exhibits very weak binding to IGF-binding proteins, was substantially more potent than the natural growth factor, in the 44 micrograms/day of this peptide produced similar effects to the high IGF-I dose. Organ weight and carcass composition measurements showed that the two IGF peptides generally maintained body proportions at those existing when the experiment began. Muscle protein synthesis and myofibrillar protein breakdown were both slightly increased by IGF treatment, so that the observed improvement in N retention could not be explained through protein accretion rates calculated from these measures. Infusion of human GH at a dose of 213 micrograms/day did not stimulate body growth. This investigation establishes that IGF peptides stimulate the growth of normal growing animals, with IGF-I variants that bind less well to IGF-binding proteins being more active than IGF-I.