Persistent impairment of insulin secretory response to glucose in adult rats after limited period of protein-calorie malnutrition early in life.

The effect of a limited period of protein-calorie malnutrition in young rats on glucose tolerance, insulin secretory response to glucose, and tissue composition in the adult was studied. Three-week-old rats were weaned onto semisynthetic diets containing either 5% protein (low protein; LP) or 15% protein (control; C) and maintained for 3 wk on their respective diets. At 6 wk of age all rats were returned to a commercial rat chow diet (18% protein). Glucose tolerance, insulin secretory response to glucose, and the protein/DNA ratio in liver, skeletal muscle, heart, kidney, small intestine, and lung were investigated at 3, 6, and 12 wk of age. Rats receiving LP diet failed to gain weight, but growth resumed immediately when they were transferred to commercial rat chow. They did not, however, catch up with C rats. Glucose tolerance and insulin secretory response to glucose remained similar between 3 and 12 wk in C rats. In 6-wk-old LP rats, glucose tolerance was impaired, and the insulin secretory response to glucose was absent. At 12 wk of age the glucose tolerance of the LP rats had normalized, but the insulin secretory response was still blunted. In 6-wk-old LP rats there was an inhibition of the age-dependent increase in cell size, shown by lowered protein/DNA ratios in all tissues studied. This decrease in cell size persisted at 12 wk in liver, skeletal muscle, heart, and lung. We conclude that protein-calorie malnutrition early in life persistently impairs the insulin secretion. The persistently lowered protein/DNA ratios in many tissues may be related to this lowered capacity for insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal rat myoblasts release both rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity in vitro: partial characterization and biological activity of myoblast-derived SM-C/IGF I.

The relative release of rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity (MSA) immunoreactivity and bioactivity from isolated fetal rat myoblasts was assessed by a partial characterization of the SM peptides present in concentrated myoblast-conditioned culture medium (MCM). The SM bioactivity of MCM, measured by [3H]thymidine or [35S]sulfate uptake by fetal rat cartilage explants, eluted with an apparent size of 50-80K on Sephadex G-200 at pH 7.5, and was associated with SM-C/IGF I immunoreactivity. Chromatography of MCM on Bio-Gel P-10 or Sephadex G-75 at acidic pH resulted in a peak of SM bioactivity associated with both SM-C/IGF I and MSA immunoreactivity in the 6-9K region. SM-binding activity, measured by competition with activated charcoal for [125I]SM-C or MSA, eluted in the void volume. When these fractions were incubated with [125I]SM-C and chromatographed on Sephadex G-200 at neutral pH, a heterogeneous pattern of binding proteins was seen, with a major component of 50-80K. After chromatofocusing of proteins in the 6-9K region from Bio-Gel P-10, three peaks of SM bioactivity were recovered, each associated with SM-C immunoreactivity, with pI values of 8.5, 7.1, and 6.5. Although both the basic and neutral peaks enhanced [3H]thymidine uptake by growth-restricted fetal rat myoblasts in vitro, only the bioactivity of the former could be blocked by incubation with a monoclonal antibody to human SM-C. Both human SM-C/IGF I and MSA purified from Buffalo rat liver cell-conditioned medium enhanced thymidine incorporation by growth-restricted fetal rat myoblasts. The results suggest that unlike reports of other fetal rat tissues, fetal rat myoblasts released approximately equal amounts of rat SM-C/IGF I and MSA during culture. The myoblast-derived SM-C/IGF I was biologically active on the cell type of origin and may play a paracrine role in muscle development.

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin.

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.

Intermittent protein-calorie malnutrition in the young rat causes long-term impairment of the insulin secretory response to glucose in vitro.

The effect of a limited period of protein-calorie malnutrition in young rats on insulin secretion in the adult has been studied. Three-week-old rats were weaned onto diets containing 5% protein (low protein; LP) or 15% protein (control; C) and maintained for 3 weeks on their respective diets. A third experimental group was weaned onto standard rat chow (18% protein; normal diet; N). From 6 weeks of age onwards all rats were fed the standard rat chow. Pancreatic islets were isolated from rats aged 3, 6 and 12 weeks and their insulin secretory response to glucose or arginine was tested. At 12 weeks the effects of the secretagogues were also tested using perfusion of isolated pancreatic glands. In islets from 6-week-old LP rats the glucose-stimulated insulin release was only 25% of that of C and N rats of the same age. Islets from C and N rats responded to arginine in the presence of a low glucose concentration with a small increase in insulin secretion, whereas no such response could be demonstrated in islets from 6-week-old LP rats. Islets from 6- and 12-week-old N rats responded to glucose and arginine. Islets from 12-week-old C rats had a similar response to glucose but did not respond to arginine in the presence of a low glucose concentration. In islets from 12-week-old LP rats the secretory response to glucose remained only 40% that of C and N rats and there was no response to arginine in the presence of a low glucose concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogenic actions of insulin on fetal and neonatal rat cells in vitro.

Insulin has been implicated in the regulation of fetal growth. The aim of these studies was to determine if insulin has a direct mitogenic effect on fetal and neonatal rat cells in vitro. Myoblasts and fibroblasts were isolated from skeletal muscle and grown until myotube formation began or until fibroblasts were confluent. The cultures were then incubated in the presence of insulin (10(-5)-10(-1) units/ml) and its effects were measured by the cellular incorporation of [3H]-thymidine. Myoblasts from fetuses of 21 days of gestation showed a marked, linear dose-response to insulin, significant increases over control values being observed at 2 X 10(-5) units/ml or 2 X 10(-4) units/ml in five out of seven experiments. Neither myoblasts from 19-day fetuses or neonates nor fibroblasts from animals of any of the three ages showed a significant thymidine uptake response to insulin. Myoblasts released immunoreactive somatomedin-C-like activity into the culture medium, but this was not related to fetal age nor to the presence of insulin in the culture medium. The results suggest that insulin may have a direct role in fetal muscle growth.

Long-term follow-up after early protein-calorie malnutrition in young rats: sex difference in glucose tolerance and serum insulin levels.

Acute protein-calorie malnutrition impairs both glucose tolerance and insulin secretion, and long-term pancreatic damage leading to malnutrition diabetes has been postulated. The present study has investigated this association in rats weaned onto 5% protein (LP) or 18% protein (normal, N) diet from age 3 weeks to 6 weeks. From 6 weeks both LP and N rats were fed N diet for the remainder of the experiment. LP rats did not grow while on the LP diet and remained significantly lighter for several weeks. Nose to tail tip length was identical for the two groups in both sexes at both 24 and 48 weeks, and mean body weight was not significantly less in LP than N after 18 weeks in either sex. Protein/DNA ratios in LP (an index of cell size) remained lower than N in heart, skeletal muscle, and lung at 24 and 48 weeks, but not in gut, liver, or kidney tissues. Thus, skeletal growth was apparently not impaired by the early malnutrition, but muscle tissue did not catch up. The similarity in final body weight implies greater adipose stores in older LP rats. At 12 weeks there was no difference in glucose tolerance tests (GTT) either between males and females within a dietary group or between N and LP, despite impaired insulin secretion in LP. Both fasting glucose levels and GTT deteriorated markedly between 12 and 48 weeks in all rats, but especially in LP males. Serum insulin levels following glucose injection were lower at 48 weeks than 12 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects on glucose tolerance and insulin secretory response to glucose following a limited period of severe protein or energy malnutrition in young rats.

The long-term effects on growth, glucose tolerance and insulin secretory response to glucose of temporary malnutrition early in life have been investigated. Rats were weaned onto either normal diet (18% protein), a protein-restricted diet (5% protein) or a diet adequate in protein but restricted in amount to equal the energy intake of protein-restricted rats ("energy restriction"). From 6 weeks of age and onwards all rats were fed normal diet. Body weight gain was inhibited by both protein and energy restriction but growth was resumed when rats were transferred to normal diet. Protein restriction impaired glucose tolerance and blunted insulin secretory response to glucose. Following refeeding glucose tolerance was normalized but insulin secretory response remained impaired at 12 weeks of age. Energy restriction did not initially affect glucose tolerance and insulin secretion. However, after refeeding male energy restricted rats developed a delayed and exaggerated insulin secretory response to glucose without concomitant deterioration of glucose tolerance. It is suggested that temporary protein restriction at a young age impairs pancreatic B-cell function and decreases peripheral sensitivity to insulin. By contrast, temporary energy restriction does not directly affect B-cell function but confers insulin resistance and compensatory increases of the insulin secretory response to glucose later in life. These models of malnutrition offer possibilities to further study long-term effects of early nutritional insults.

Tissue and serum insulin-like growth factor I (IGF I) concentrations in rats subjected to temporary protein-energy malnutrition early in life.

Rats subjected to temporary protein-energy malnutrition and subsequent nutritional rehabilitation remain smaller than adequately fed animals, have a subnormal insulin secretion and persisting cellular hypoplasia in several tissues. This investigation studies whether impaired production of insulin-like growth factor I (IGF I) is another persisting consequence of malnutrition. Rats were subjected to severe protein-energy malnutrition between 3 and 6 weeks of age and subsequently fed adequate diet up to 12 weeks of age. Serum and tissue samples for analysis of IGF I were obtained at 12 weeks of age. IGF I concentrations were similar in serum, heart, liver and lung of previously malnourished and control rats. In the kidneys of previously-malnourished rats the IGF I concentration was twice that of control rats. Results suggest that during protein-energy malnutrition and subsequent nutritional rehabilitation IGF I tissue concentrations are primarily regulated by the prevailing plane of nutrition. It is speculated that the temporary protein-energy malnutrition blunts the cellular capacity for IGF I production and, except in the kidney, prevents increased IGF I tissue concentrations and associated compensatory growth.

Incorporation of [3H]thymidine by isolated fetal myoblasts and fibroblasts in response to human placental lactogen (HPL): possible mediation of HPL action by release of immunoreactive SM-C.

We investigated the actions of human placental lactogen (HPL) and human growth hormone (HGH) on [3H]thymidine incorporation and the release of immunoassayable somatomedin-C (SM-C) by isolated myoblasts, dermal fibroblasts, and costal cartilage explants taken from human fetuses at 11-21 weeks of gestation. The incorporation of [3H]thymidine by myoblasts and fibroblasts was significantly increased after incubation for 20 hr or 44 hr, and cell number after incubation for 7 days, in the presence of 50-250 ng/ml HPL. Incubation with HPL did not increase [3H]thymidine incorporation into cartilage explants, whereas incubation with HGH failed to enhance the uptake of this isotope by any of the tissues. Following extraction with acid-ethanol, culture medium conditioned by exposure to myoblasts or fibroblasts for 44 hr, and to cartilage explants for 7 days, contained radioimmunoassayable SM-C. Myoblast-conditioned medium contained significantly more SM-C [1,609 +/- 953 mU/mg cell protein (mean +/- SD); n = 10] than did that conditioned by fibroblasts (637 +/- 323; n = 5; P less than 0.02). In 1 week of culture, cartilage explants released 4.1 +/- 1.1 mU/mg wet weight (n = 7). The release of immunoassayable SM-C from cultured cells was significantly increased in the presence of 250 ng/ml HPL in five of eight experiments with myoblasts and two of four experiments with fibroblasts. Neither fibroblasts or myoblasts showed increased SM-C release following exposure to HGH. The results suggest that HPL, but not HGH, is growth-promoting for some human fetal tissues in vitro and that this action is mediated, at least in part, by an increased release of somatomedins.

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin.

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.

Persistent reduction of pancreatic beta-cell mass after a limited period of protein-energy malnutrition in the young rat.

Kwashiorkor, the human disease of protein-energy malnutrition, has been implicated in the aetiology of malnutrition-related diabetes mellitus, a form of diabetes not uncommon in developing countries. We have previously demonstrated that temporary protein-energy malnutrition in young rats causes a persisting impairment of insulin secretion. The present study investigates whether this secretory deficiency is accompanied by structural alterations of the endocrine pancreas. Three-week-old rats were weaned onto semi-synthetic diets containing either 15% or 5% protein and these diets were maintained for 3 weeks. From 6 weeks of age all rats were fed a commercial chow containing 18% protein. The endocrine pancreas was investigated by light and electron microscopic morphometry at 3, 6 and 12 weeks of age. In rats not subjected to protein-energy malnutrition there was a progressive increase, with age, of total pancreatic Beta-cell weight and individual Beta-cell size. In 6-week-old rats fed the low protein diet total pancreatic Beta-cell weight and individual Beta-cell size were diminished. In 12-week-old rats previously fed the low protein diet total Beta-cell weight remained lower compared to control rats. It is concluded that protein-energy malnutrition early in life may result in a diminished reserve for insulin production. This may predispose to glucose intolerance or even diabetes in situations with an increased insulin demand.