Metabolic and Hormonal Response to a Feed-challenge Test in Lean and Overweight Dogs.

BACKGROUND: Obese dogs risk poor life quality, creating a need for increased knowledge of metabolism in overweight dogs. OBJECTIVES: Investigate postprandial metabolic and hormonal responses to a high-fat mixed-meal in dogs and responses of lean versus overweight dogs. ANIMALS: Twenty-eight healthy intact male Labrador Retrievers were included. METHODS: Prospective observational study. Twelve dogs were grouped as lean (body condition score (BCS 4-5), 10 as slightly overweight (BCS 6), and 6 as overweight (BCS 6.5-8) on a 9-point scale. After an overnight fast, urine and blood samples were collected. Dogs were then fed a high-fat mixed-meal, and blood was collected hourly for 4 hours and urine after 3 hours. RESULTS: Postprandial concentrations of insulin and glucagon were increased at 1 hour (both P < 0.0001), triglycerides at 2 hours (P < 0.0001), and glucose at 3 hours (P = 0.004); and all remained increased throughout the feed-challenge in all dogs. Postprandial urine cortisol/creatinine ratio was higher than fasting values (P = 0.001). Comparing between groups, there was an overall higher triglyceride response in overweight compared to lean (P = 0.001) and slightly overweight (P = 0.015) dogs. Overweight dogs also had higher fasting cortisol/creatinine ratio compared to lean dogs (P = 0.024). CONCLUSIONS AND CLINICAL IMPORTANCE: Postprandial responses of dogs to a high-fat mixed-meal were similar to those previously reported in people. The higher postprandial triglyceride response and fasting cortisol/creatinine ratio in the overweight dogs could be early signs of metabolic imbalance. Thus, although overweight dogs often appear healthy, metabolic alterations might be present.

Influence of a selective histamine H3 receptor antagonist on hypothalamic neural activity, food intake and body weight.

OBJECTIVE: This study was conducted to elucidate whether antagonistic targeting of the histamine H3 receptor increases hypothalamic histamine levels, in parallel with decreases in food intake and body weight. METHODS: The competitive antagonist potency of a recently synthesized histamine H3 receptor antagonist, NNC 38-1049, was studied in intact HEK293 cells expressing human or rat histamine H3 receptor, in which NNC 38-1049 was allowed to antagonize the effect of the H3 receptor agonist R-alpha-methylhistamine on isoprenaline-induced accumulation of cAMP. The affinity of NNC 38-1049 for a number of variants of the histamine receptor was also determined. Following single dosing of normal rats with NNC 38-1049, hypothalamic histamine levels were assessed by means of microdialysis. Plasma and brain levels of NNC 38-1049 and acute effects on food intake and energy expenditure were followed after oral doses of 3-60 mg/kg. Potential side effects were examined with rat models of behaviour satiety sequence (BSS), pica behaviour and conditioned taste aversion (CTA). Intakes of food and water together with body weight were recorded for 15 days during daily dosing of dietary obese rats. RESULTS: NNC 38-1049 was found to be a highly specific and competitive antagonist towards both human and rat histamine H3 receptors, and measurable amounts of NNC 38-1049 were found in the plasma of rats following single oral doses of 3-60 mg/kg and in the brain after 15-60 mg/kg. Following single intraperitoneal injections of NNC 38-1049 (20 mg/kg), significant increases in extracellular histamine concentrations were observed. The same dose did not change BSS or pica behaviour acutely, nor did it induce CTA following repeated administration for 7 days. Reductions in food intake were seen very soon after administration, and occurred in a dose-dependent fashion. Energy expenditure was unchanged, but the respiratory quotient (RQ) tended to decrease at higher doses, indicating an increase in lipid oxidation. Twice daily administration of 20 mg/kg of NNC 38-1049 in old and dietary obese rats resulted in sustained reduction of food intake throughout a 2-week study, and was associated with a highly significant (P<0.01) decrease in body weight compared with controls (-18.4+/-3.4 vs +0.4+/-2.7 g). The same dose of NNC 38-1049 produced an acute decrease of water intake, but 24 h intakes were not significantly changed. CONCLUSIONS: The results of this study strongly support the idea that an increase in the hypothalamic concentration of histamine produces a specific reduction of food intake and that this effect can be translated into a decrease in body weight.

Reduction of adiposity with prolonged growth hormone treatment in old obese rats: effects on glucose handling and early insulin signaling.

OBJECTIVES: Growth Hormone (GH) promotes loss of body fat and causes insulin resistance. It is debated whether reduction of body fat mass during long term growth hormone (GH) administration improves carbohydrate metabolism. To answer this question we assessed carbohydrate handling and tissue specific function of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) during prolonged GH treatment of obese rats. METHODS: Body fat % estimated by DEXA scanning, plasma IGF-I, glucose and insulin were studied in 17 months old dietary induced obese rats treated for 4, 21 or 41 days (GH: 4 mg/kg/d or saline total n=90). Adipose tissue, muscle and liver samples were obtained after 21 days and expression and tyrosine phosphorylation of IR and IRS-1 proteins and the degree of IRS-1-Janus Kinase-2 (JAK2) interaction were analyzed by immunoprecipitation and immunoblotting. RESULTS: Forty-one days GH treatment caused the body fat to decline significantly to 20+/-3% (Mean+/-SEM), whereas it remained steady on 51+/-4% in the pair fed group. Insulin levels in response to OGTT were significantly elevated throughout the experiment. IR amount was elevated in adipose tissue but decreased in liver after GH treatment while IR phosphorylation was increased in muscle only. IRS-1 amount was elevated in adipose tissue and muscle while IRS-1 phosphorylation was increased only in liver. The association of IRS-1 with JAK-2 was increased in liver and muscle. CONCLUSIONS: An extensive reduction of fat mass did not improved signs of insulin resistance in GH treated old obese rats. The molecular events associated with GH treatment included tissue specific changes in the function of IR and IRS-1 suggesting the liver to be the primary site of insulin resistance. Furthermore, the association of IRS-1with JAK-2 in the course of GH signaling could present a mechanism for GH to directly induce insulin resistance.

Growth hormone-mediated breakdown of body fat: insulin and leptin responses to GH are modulated by diet composition and caloric intake in old rats.

This work was performed to elucidate whether growth hormone (GH)-mediated loss of adipose tissue and responses in plasma insulin and leptin are modulated by diet composition. 12-month-old rats were first fed a high-fat (HF) diet or a low-fat (LF) diet for 14 weeks. After that, GH or saline was administered to rat groups that were maintained on either HF or LF diets or that were switched from the HF to the LF diet. All 6 groups had free access to food. One additional saline group was pair-fed with the GH group that was switched from the HF to the LF diet. The caloric consumption of this latter group was also translated to yet another GH group receiving restricted amounts of the HF diet. GH was given in a total dose of 4 mg/kg/d for three weeks. After sacrifice, blood was collected and tissues were excised. In groups injected with saline, the weight of excised adipose tissue was 60 +/- 4.7, 41 +/- 3.8 and 50 +/- 4.5 g in animals that continued with the HF diet, LF diet, or that were switched from HF to LF, respectively. Corresponding figures after GH treatment were significantly (p < 0.05) decreased to 38 +/- 2.7, 30 +/- 2.3, and 31 +/- 2.7 g, respectively. Pair-feeding had no effect, whereas only 26 +/- 3.0 g of adipose tissue was retrieved in rats fed restricted amounts of HF diet while receiving GH. In this group, plasma insulin and leptin were also significantly (p < 0.05) depressed compared with other GH groups, especially to the group fed the unrestricted HF diet (203 +/- 35 vs. 1345 +/- 160 pmol/l and 9.3 +/- 1.2 vs. 31 +/- 4.4 micro g/l). In conclusion, this study shows that GH mediates breakdown of adipose tissue under a variety of dietary conditions, and that induction of hyperinsulinemia can be prevented if GH treatment is combined with restricted feeding of a diet which is relatively low in carbohydrates and rich in fat. This will also promote a fall of plasma leptin.

Growth hormone-mediated breakdown of body fat: effects of GH on lipases in adipose tissue and skeletal muscle of old rats fed different diets.

Lipid storage and breakdown is mainly controlled by lipoprotein lipase and hormone-sensitive lipase. The aim of this work was to elucidate whether growth hormone mediated loss of adipose tissue involves a concerted action on tissue lipases, and to what degree such events are modulated by dietary regimen. Twelve-month-old rats fed first a high-fat diet or a low-fat diet for 14 weeks were injected with saline or growth hormone (4 mg/kg/d) for four days or three weeks in different combinations with either high- or low-fat diets. In adipose tissue, growth hormone generally inhibited lipoprotein lipase and also attenuated the inhibiting effect of insulin on hormone-sensitive lipase activity. Growth hormone treatment combined with restricted high-fat feeding reduced the activity of both lipases in adipose tissue and stimulated hormone-sensitive lipase in muscle. Generally, plasma levels of free fatty acids, glycerol and cholesterol were reduced by growth hormone, and in combination with restricted high-fat feeding, triglyceride levels improved too. We conclude that growth hormone inhibits lipid storage in adipose tissue by reducing both lipoprotein lipase activity and insulin's inhibitory action on hormone-sensitive lipase. We also propose that growth hormone's effects on tissue lipases and blood lipids are modulated by dietary regimen.

The growth hormone secretagogue ipamorelin counteracts glucocorticoid-induced decrease in bone formation of adult rats.

The ability of the growth hormone secretagogue (GHS) Ipamorelin to counteract the catabolic effects of glucocorticoid (GC) on skeletal muscles and bone was investigated in vivo in an adult rat model. Groups of 8-month-old female rats were injected subcutaneously for 3 months with GC (methylprednisolone) 9 mg/kg/day or GHS (Ipamorelin) 100 microg/kg three times daily, or both GC and GHS in combination. The maximum tetanic tension of the calf muscles was determined in vivo in a materials testing machine. The maximum tetanic tension was increased significantly, and the periosteal bone formation rate increased four-fold in animals injected with GC and GHS in combination, compared with the group injected with GC alone. In conclusion, the decrease in muscle strength and bone formation found in GC-injected rats was counteracted by simultaneous administration of the growth hormone secretagogue.

Daily low-dose administration of growth hormone secretagogue stimulates pulsatile growth hormone secretion and elevates plasma insulin-like growth factor-1 levels in pigs.

Repeated administration of growth hormone secretagogues (GHSs) has proven to be a delicate matter owing to development of tolerance. The aim of the present study was to define conditions during which the responsiveness to the orally active NN703 was maintained over several days. Growing pigs were fitted with stomach and vascular catheters, permitting unstressed intragastric administrations and blood sampling. NN703 or vehicle was administered once daily. When NN703 was given at a dose of 18 mg/kg, there was a massive acute increase in plasma growth hormone (GH) levels, but this was only seen on the first day of administration. A dose of 1.8 mg/kg did not cause a significant acute increase in plasma GH concentrations, whereas stimulation of pulsatile GH release was sustained over a 4-d period. During the first 7 h following injection of vehicle, the area under the curve of plasma GH was 1211+/-144 (microg/[L x 7 h]), but increased to 1770+/-269 and 1824+/-198 (microg/[L x 7 h]) on the first and fourth day of NN703 administration, respectively. Deconvolution analysis of the 7-h profiles revealed that the GH mass per burst as well as the GH burst amplitude were significantly (p < 0.001) increased during treatment with NN703, which led to an increase in pulsatile GH secretion rate (p < 0.001). Insulin-like growth factor-1 plasma concentrations increased steadily during NN703 administration (p < 0.01) and decreased after termination of treatment. The sustained increase in GH pulsatility observed with low-dose NN703 treatment suggests that development of tolerance to this GHS may be obviated by minimization of dose.

Comparison of methods of analysis of body composition in hypophysectomized rats treated with rat growth hormone.

The present study compared estimates of body composition derived from dual-emission X-ray absorptiometry (DEXA) and from chemical analyses. The primary aim was to compare the two methods because growth hormone (GH) may cause fluid retention, and DEXA does not distinguish water from lean mass. Hypophysectomized rats were fed ad libitum and were treated with continuous infusions of rat GH in doses of 0, 10, 30, and 100 microg/day for 14 days. By chemical analysis, a decrease in percentage fat from 12.9% in the control group to 11.3%, 11.0%, and 10.2% in the low, medium, and high dose groups was observed (P < 0.0001). The fat percentages were about 3-4% higher by DEXA, but showed the same decline (P < 0.03). Lean mass increased from 74.4% in the control group to 75.8%, 78.0%, and 78.6% in the treatment groups (P < 0.001). A significant increase in the wet weight of the quadriceps muscle, but no difference in dry weight was observed in all four treatment groups, indicating that the increase in muscle weight was exclusively caused by water. This accumulation of water was reflected in the total water content of the carcasses, which increased from 62.0% in the control group to 64.9%, 66.1%, and 66.8% in the GH groups (P < 0.0001). The protein content decreased from 19.8% in the control group to 19.4%, 19.1%, and 18.9% in the GH groups (P < 0.001). Regardless of the decrease in protein, the GH treated groups contained more water in relation to protein as the g water/g protein ratio was increased by 13% from 3.14 in the control group to 3.55 in the group treated with the highest GH dose (P < 0.0001). Also, a close relationship between feed intake and body weight were found, together with increases in epiphyseal growth plate width, insulin-like growth factor I (IGF-I), and insulin-like growth factor binding protein 3 (IGFBP-3). In conclusion, the study shows that estimation of lean mass by DEXA should be carefully evaluated when used in connection with treatment of drugs that cause water retention.

Insulin-like growth factor-I, but not growth hormone, is dependent on a high protein intake to increase nitrogen balance in the rat.

The aim of the present study was to investigate the influence of dietary protein level on the protein anabolic effects of growth hormone (GH) and insulin-like growth factor-I (IGF-I). Female growing rats were fed on either a high- or a low-protein diet with crude protein contents of 222 and 83 g/kg respectively. The diets contained the same amount of metabolizable energy (15.1 MJ/kg) and were given during a 14 d period. During the same time, three groups of rats (n 8) on each diet received subcutaneous infusions of either saline, recombinant human GH (rhGH) or recombinant human IGF-I (rhIGF-I). rhGH and rhIGF-I were given in doses of 360 and 500 micrograms/d respectively. The low-protein diet alone reduced significantly (P < 0.05) IGF-I concentrations in serum and in tissue taken from the gastrocnemius muscle as well as IGF-I mRNA from the same muscle. The responses to rhGH and rhIGF-I in terms of muscle IGF-I and its mRNA were variable. However, when rhIGF-I was infused into rats on the high-protein diet, significantly elevated levels of IGF-I in muscle tissues could be observed. This was associated with a significantly (P < 0.05) increased N balance, whereas rhGH significantly (P < 0.05) enhanced the N balance in rats on the low-protein diet. Thus, it can be concluded that the level of dietary protein ingested regulates not only the effect of IGF-I on whole-body N economy but also the regulation of IGF-I gene expression in muscles. The exact mechanism by which GH exerts its protein anabolic effect, however, remains to be elucidated.

The role of growth hormone and glucocorticoid in glucose handling in vivo.

Growth hormone (GH) can oppose the catabolic effects of glucocorticoids. However, both hormones have adverse effects on carbohydrate metabolism. Here we examined the interactive effects of GH and the glucocorticoid methylprednisolone (MP) on glucose tolerance, insulin resistance and [3H]2,6-deoxyglucose uptake of peripheral tissues in rats. Female Wistar rats received either saline, GH (2.7 mg/kg), MP (5.0 mg/kg) or GH+MP. After 7 days treatment, animals were subjected to an i.v. glucose tolerance test. In a second experiment, animals treated as above were anesthetized and injected with human insulin (0.5 U/kg), [3H]2,6-deoxyglucose (500 microCi/kg), and [14C]mannitol (25 microCi/kg), to estimate insulin resistance and [3H]2,6-deoxyglucose uptake in fat and muscle. Weight gain in controls was 7.6+/-1.7 g, while GH treatment increased the mean body weight by 18.7+/-2.2 g (P<0.0002) and MP inhibited weight gain down to 0.0+/-1.0 g (P<0.004). This drop in weight gain was reversed back to normal when GH was given in combination with MP. After a glucose tolerance test no significant differences in glucose area under the curve were detected when comparing individual groups with the control group, but samples taken just before this test revealed that basal insulin was significantly elevated in the group treated with GH (174+/-27 pM, P<0.008), or GH+MP (209+/-21 pM, P<0.004), when compared with controls (107+/-17 pM). MP alone had no effect (122+/-19, P<0.3). After an i.v. bolus of insulin the group receiving GH+MP had a significantly (P<0.007) higher level of circulating glucose compared with controls (6.5+/-0.3 mM vs 4.4+/-0.7 mM). Despite this, there were no differences in peripheral glucose uptake between the two groups. In conclusion this study shows that a combined administration of GH and MP decreases the potency by which insulin decreases circulating glucose levels, but that peripheral tissues are not primarily involved in this insulin resistance.

Effects of human growth hormone on the porto-arterial concentration differences of glucose and amino acids in the newborn piglet.

It is known that growth hormone (GH) increases the mitotic index of duodenal crypt cells. In early life, such an effect could be of particular importance for the functional development of the intestine in terms of absorptive capacity. In this study, osmotic mini pumps were introduced into the abdominal cavity of newborn piglets. The pumps permitted a continuous infusion of either recombinant human growth hormone (rhGH) at a rate of 0.1 IU GH x kg(-1) x 24 h(-1) or of vehicle. After 7 days of treatment, a bolus of amino acids and glucose was infused into the duodenum. Following this bolus, there was a prompt rise in the plasma concentration of both amino acids and glucose, especially in blood withdrawn from the portal vein. Thus, when the differences in concentrations of both amino acids and glucose between portal and arterial blood plasma were calculated, these differences reached maximum values between 30 and 60 minutes after the bolus. In animals treated with GH, maximum values occurred at a lower level than in control animals. These reductions were in the order of 60% (P > 0.01) if calculated over the first hour of absorption. From this study, it might be concluded that GH does not improve the absorptive capacity of the small intestine in newborn piglets. Instead, GH seems to reduce the absorption dynamics of glucose and amino acids. The reason for this is obscure, but could imply a specific effect of GH on enterocyte function.

Methylprednisolone does not inhibit the release of growth hormone after intravenous injection of a novel growth hormone secretagogue in rats.

The present study was undertaken to study the growth hormone-releasing properties and growth-promoting effect of a GH secretagogue ipamorelin (IPA) in rats given the synthetic glucocorticoid methylprednisolone (MP). In a first experiment, rats received either saline or MP (5.0 mg/kg) for 8 days. Treatment with MP significantly (P< 0.001) decreased body weight gain, but the acute response to either IPA or growth hormone releasing hormone (GHRH) in terms of plasma GH was not changed. In a second experiment, venous catheters were surgically implanted. On the next day, rats were randomly allocated to receive saline alone, MP alone (5.0 mg/kg) or MP plus IPA in doses of 0.4 or 1.6 mg/kg/day for 10 days. IPA was administered intravenously four times a day.MP treatment significantly (P< 0.05) retarded recovery from surgery in terms of body weight. Thus, saline treated animals lost 4.0 +/- 3.5 g over the entire experimental period, whereas animals receiving MP lost 13. 6 +/- 2.9 g. When IPA was given together with MP, losses in body weight were significantly (P< 0.05) reduced to 2.3 +/- 2.0 and 1.6 +/- 2.0 g in animals given the high and low dose of IPA, respectively. In parallel with this IGF-I levels increased. In conclusion, this work shows that MP does not disrupt the response of the GH-IGF-I axis to an exogenous stimulus like IPA, and repeated stimulation leads to increases in IGF-I and of body weight gain.

Insulin-like growth factor-I and growth hormone administration in intestinal ischemia shock in the rat.

The effect of exogenous insulin-like growth factor (IGF)-I and growth hormone (GH) was examined in a rat model of intestinal ischemia-reperfusion (I/R). Animals were anesthetized, vascular catheters were placed, and intestinal ischemia was induced for 60 min. Thereafter, the intestine was reperfused, and rats received a primed, constant infusion of either IGF-I or GH (500 microg/rat + 500 microg/day) for the remainder of the study; control rats received an equal volume of vehicle. The plasma IGF-I concentration gradually declined after I/R in the vehicle-shock group and was reduced 30% at 48 h. GH infusion completely prevented this reduction, whereas the effect of IGF-I was intermediate. The IGF-I content in liver was increased by IGF-I (78%) and further enhanced in the GH-treated group (140%). Comparable increases were seen for the abundance of IGF-I mRNA in liver in these two treatment groups, compared to the vehicle control. In contrast, while both IGF-I and GH elevated the IGF-I content in skeletal muscle similarly (80%), no increase in IGF-I mRNA expression was observed in this tissue. Neither treatment altered the IGF-I content in small intestine. At the time tissues were sampled (48 h), the plasma concentration of glucose and corticosterone was not different among the three groups. However, plasma insulin was reduced 50% in the IGF-I-infused animals, compared to values in either the shock-GH or shock-vehicle group. These data demonstrate that chronic administration of GH and, to a lesser extent, IGF-I, after intestinal I/R maintains levels of IGF-I in the blood, liver, and muscle. Thus, adjunct treatment with these anabolic agents may help blunt the increased catabolism observed in individuals following intestinal I/R.

The role of insulin, insulin-like growth factor-I and growth hormone in counteracting dexamethasone induced nitrogen wasting in rats.

Administration of glucocorticoids is associated with decreased nitrogen balance. The main aim of the present study was to elucidate whether this could be counteracted by subcutaneous infusions of rhGH (360 micrograms/d), rhIGF-1 (500 micrograms/d) or insulin (39 micrograms/d). During a study period of 8 days one out of 10 groups of rats received dexamethasone (dex, 26 micrograms/d) alone, whereas 7 groups were given one or more of the three peptides in combination with dex. One group was given saline alone and one was food restricted to match the average food intake among dex groups. Food restriction and saline alone produced relative nitrogen balances, retained N/ingested N (x100) of 44 (SE 3) and 36% (SE 4), respectively. This was decreased (p < 0.001) to 7% (SE 3) with dex alone. However, if either insulin/rhGH/rhIGF-1, rhIGF-1 alone or insulin/rhIGF-1 were given together with dex, the relative nitrogen balances increased significantly (p < 0.03) up to 16, 22 and 24% (SE 3), respectively. Insulin or rhGH alone were without effect as was rhGH combined with either rhIGF-I or insulin. Although the relative nitrogen balances associated with insulin/rhIGF-1 and rhIGF-1 alone were not found to be significantly different, the former infusion regimen produced a significantly (p < 0.02) lower plasma urea. It is concluded that in the rat, rhIGF-1 has a potential to counteract the decrease in nitrogen balance induced by potent glucocorticoids, whereas rhGH as administered in this experiment does not have this effect.

Effects of insulin-like growth factor I (IGF-I) on the small intestine: a comparison between oral and subcutaneous administration in the weaned rat.

Several studies have suggested that growth factors play an important role in the development and the maintenance of the gastrointestinal tract. Colostrum and normal breast milk as well as intestinal secretion are reported to contain IGF-I. Thus, the objective of this study was to investigate whether the route of administration of IGF-I, subcutaneously and orally, was important for the way of influencing the gastrointestinal tract in the weaned rat and further to observe if this effect was affected by different feed regimens. Well nourished weaned rats subcutaneously administered IGF-I (2 mg/kg body weight and day during 14 days) were found to have significantly increased relative weights of the small intestine, increased duodenal crypt depths and villi heights compared with control rats. The same dose given orally in bovine milk had no effect. Furthermore, a study was performed with the same dose of IGF-I given for 7 days together with a restricted feed regimen. Similar observations, although of a slightly smaller magnitude, were found. These observations indicate that the effect of IGF-I on gastrointestinal renewal in the weaned rat is not depending on an IGF-I supply through the gastrointestinal fluids but rather through a supply via the circulation or by a local intestinal production. The effects were statistically significant both in well fed and malnourished conditions.

Reliability of the western ligand blot method for the simultaneous relative estimations of serum insulin-like growth factor binding proteins (IGFBPs).

It is well established that nutrition is an important regulator of both serum insulin-like growth factor-I (IGF-1) and its binding proteins (IGFBPs). The Western ligand blot method (WLB) for simultaneous determinations of IGFBPs in serum or plasma samples was evaluated and validated with emphasis on its reproducible capabilities. After electrophoretic separation and transfer, the membranes were incubated with a mixture of recombinant labeled human (GF-I/IGF-II(rhIGF-I/rhlGF-II) and band intensities measured by autoradiography. The typical electrophoretic profile for pig serum, as determined with molecular weight markers, showed four mainbands of approximately 42-39, 32, 30-28 and 24 kDa which seemed to correspond to IGFBP-3, IGFBP-2, IGFBP-1 and IGFBP-4 respectively. Likewise, a triplet of approximately 42-39 kDa (IGFBP-3), a broad area called IGFBP-30 region (most probably IGFBP-1, -2 and -3 variants) and a third band of approximately 24 kDa (IGFBP-4) were seen in rat samples. Determination of IGFBP-2 and -1 in rat serum samples, as two separate bands on 12% gels was difficult due to their close electrophoretic migration and possibly to the reported lower levels of IGFBP-2 in adult rat serum. Dilutions tested on 0.2 micron nitrocellulose membranes with samples volumes between 0.25 to 1.5 microliters (1:10-1:60 dilutions), showed IGFBPs curves with good linearity which suggest first, that there exist a quantitative relation between the amount of each protein and the densitometric response and second, that the transfer of the proteins was linear across the range of 0.25 to 1.5 microliters (1:10-1:60 dilutions). Moreover, the results also suggest that losses were equally spread and that the proteins retained their binding properties after the transfer process. Reproducibility showed intraassay coefficients of variation (CVs) of 15% or lower using either a transfer device without cooling system or a combination of a transfer device with cooling system and manually defined band boundaries. In summary, it was shown that the optimized experimental conditions here described for the WLB method, allow reliable simultaneous measurements of the main pig and rat serum IGFBPs and therefore, could be utilised to detect changes in the serum profile after dietary manipulations.

Effects of insulin-like growth factor I (IGF-I) on the porto-arterial concentration differences of amino acids and glucose: a comparison between oral and intraperitoneal administration in the newborn piglet.

It is established that insulin-like growth factor I (IGF-I) influences cell differentiation and proliferation. Less is known, however, if such changes also influence the flow of nutrients from the intestinal lumen to the portal circulation. In the present study, we tested if IGF-I treatment (0.3 mg IGF-I/24 hr and kg) during 7 days affects the porto-arterial concentration differences of amino acids and glucose in piglets. Two routes of administration, oral and intraperitoneal, were compared. Following the IGF-I treatment, a bolus of nutrients was administered to the proximal duodenum and the porto-arterial concentration differences of amino acids and glucose were determined. We found that intraperitoneal administration of IGF-I significantly increased the difference in concentration between portal and arterial plasma for amino acids, whereas no such effect was seen with glucose. This might suggest that IGF-I has a specific effect on amino acid transporters in the intestinal wall. The same dose of IGF-I given orally did not exhibit the same effect on the absorption of amino acid as the animals which were given the peptide intraperitoneally.

Effects of insulin-like growth factor-I and growth hormone on the net flux of amino acids across the hind limbs in the surgically traumatized pig.

1. After surgery three groups of six female pigs weighing on average 52.2 kg (SD 3.5) received vehicle, recombinant insulin-like factor-1 (364.4 micrograms day-1 kg-1) or recombinant human growth hormone (467.7 m-i.u. day-1 kg-1) for two post-operative days. Vehicle and peptides were infused intravenously together with total parenteral nutrition providing 129 kJ day-1 kg-1 non-protein calories and 0.35 g N day-1 kg-1. 2. On both post-operative days the mean concentration of insulin-like growth factor-1 in arterial blood samples was clearly below presurgical levels in animals receiving vehicle or recombinant human growth hormone, whereas recombinant human insulin-like growth factor-1 infusions more than restored insulin-like growth factor-1 concentrations. These last samples, however, contained significantly (P < 0.05) less insulin than those from other animals. 3. Infusion of recombinant human growth factor was often associated with higher circulating levels of amino acids compared with recombinant human insulin-like growth factor-1 infusions. Despite this, both hormones significantly (P < 0.05) increased the hind limb net balance of total amino acids on post-operative day 1. Net balances of -44.2, +69.5 and +100.9 mumol/min (pooled SE 35.3) were associated with infusion of vehicle, recombinant human insulin-like growth factor-1 and recombinant human growth hormone respectively. This response was also closely reflected in the group of non-essential amino acids. 4. The net efflux of alanine from the hind limbs was also significantly (P < 0.002) reduced, whereas glutamine was less affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Human growth hormone does not cross the placenta of the pregnant rat.

The purpose of this study was to investigate if human growth hormone (hGH) crosses the placenta to the fetus of the pregnant rat. Pregnant rats were injected i.v. with 125I-hGH alone or co-injected with unlabelled hormone on gestational day 20 or 21. The rats were sacrificed 10 min after injection, and the distribution of the radioactivity was determined by direct measurement in brain, thymus, liver, kidney, rectus femoris muscle, placenta and fetus. Free iodine was determined in samples from maternal liver, placental and fetal tissue homogenates after precipitation of proteins by trichloroacetic acid. Two animals were injected with a higher dose of radioactivity. One of them was co-injected with a high dose of unlabelled hormone. They were sacrificed after 10 min, frozen, and sections of 20 microns were cut sagittally for autoradiography. A small amount of radioactivity was detected in the fetal tissue. However, this activity was shown to be free iodine, and no inhibition of that uptake was found by unlabelled hGH. No radioactivity was detected in the fetuses of either rat exposed to autoradiography. Therefore, we conclude that there is no transfer of hGH from the mother to the fetus of the pregnant rat. Our result is in agreement with some early observations in humans and rabbits but disagrees to some extent with recent results found in rats, where a small passage of 125I-hGH from mother to fetus was reported.

Effects of guar gum and cellulose on glucose absorption, hormonal release and hepatic metabolism in the pig.

Six Large White pigs (mean body-weight 59 (SE 1.7) kg) were surgically fitted with permanent catheters in the portal vein, the brachiocephalic artery and the right hepatic vein, as well as with electromagnetic flow probes around the portal vein and the hepatic artery, and allowed to recover. The non-anaesthetized animals were given a basal non-fibre diet (diet A) alone or together with 60 g guar gum/kg (diet B) or 150 g purified cellulose/kg (diet C) by substitution for mica. The diets were given for weekly periods and according to a replicated 3 x 3 Latin square design. On the last day of each such adaptation period, test meals of 800 g were given before blood sampling. Sampling was continued for 8 h. Guar gum strongly reduced glucose apparent absorption without changing the absorption and the hepatic uptake profiles. Production rates of insulin, gastric inhibitory polypeptide and insulin-like growth factor-1 (IGF-1) were lowest after guar gum ingestion. However, the reductions in peripheral blood insulin levels caused by guar gum were not associated with a change in hepatic insulin extraction. IGF-1 appeared to be strongly secreted by the gut, whereas the liver had a net uptake of the peptide. Ingestion of guar gum increased the hepatic extraction coefficient of gut-produced IGF-1. Guar gum ingestion appeared also to decrease glucagon secretion. Cellulose at the level consumed had very few effects on the variables considered. It is suggested that the modulation of intestinal mechanisms by guar gum was sufficient to mediate the metabolic effects described.