Nutrition and somatomedin. XXI. Insulin-like growth factor-I and somatomedin inhibitor in streptozotocin-diabetic rats: relation to ketogenesis and gluconeogenesis

Diabetes is associated with a fall in serum insulin-like growth factor-I (IGF-1) and a rise in somatomedin inhibitor, a circulating factor(s) of approximately 30,000 MW that is released by the liver and can antagonize both somatomedin and insulin action. Levels of inhibitor correlate with levels of glucose, beta-hydroxybutyrate, and weight loss. To study pathways that could underlie the fall in IGF-1 and rise in inhibitor, the effects of metabolic inhibitors on circulating metabolic fuels, serum IGF-1, and serum somatomedin inhibitor activity were studied. Rats given streptozotocin exhibited weight loss of 14% +/- 0.1%, glucose 457 +/- 26 mg/dL, and beta-hydroxybutyrate (BOHB) 6.3 +/- 0.5 mmol/L. Somatomedin inhibitor was separated from IGF-1 by size exclusion HPLC at pH 3; IGF-1 was measured by RIA, and somatomedin inhibitor by cartilage bioassay. Diabetic animals exhibited a fall in IGF-1 to 76% of normal (P less than .02) and a rise in inhibitor to 270% of normal (P less than .01). 3-Mercaptopicolinic (3-MPA) acid, an inhibitor of gluconeogenesis, lowered glucose to 68 +/- 2 mg/dL and BOHB to 0.96 +/- 0.09 mmol/L (both P less than .01 v diabetic), but levels of inhibitor did not fall. Nicotinic acid, an inhibitor of lipolysis, did not affect glucose but reduced BOHB to 0.42 +/- 0.02 mmol/L; somatomedin inhibitor fell 19% below diabetic levels (NS) but remained above normal (P less than .01). In contrast, inhibition of fatty acid oxidation with methyl-2-tetradecylglycidate reduced glucose to 191 +/- 18 mg/dL but lowered BOHB to normal, 0.16 +/- 0.02 mmol/L, accompanied by normalization of somatomedin inhibitor levels (152% +/- 33% of normal, NS). Below 1.0 mmol/L BOHB, somatomedin inhibitor and BOHB were strongly correlated (r = .67, P less than .001); no comparable relation was found with glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrition and somatomedin. XVI: Somatomedins and somatomedin inhibitors in fasted and refed rats

Nutritional deprivation is associated with poor growth and decreased levels of net circulating somatomedin activity, as measured by bioassay. Since somatomedin activity reflects the contributions both of somatomedins (which stimulate cartilage) and of somatomedin inhibitors (which antagonize the ability of the somatomedins to stimulate cartilage), we asked if changes in net somatomedin activity could involve progressive underlying alterations in levels of both somatomedins and somatomedin inhibitors. Groups of rats were killed during three days of fasting and 24 hours of refeeding. Fasting was associated with a rise in serum beta-hydroxybutyrate from 1.6 to 12.6 mmol/L after one day, followed by a decline to 4 mmol/L at three days. Somatomedins (low-MW) were separated from somatomedin inhibitors (high-MW) by gel permeation chromatography at acid pH on Sephadex G-50 and TSK-2000 HPLC. Somatomedins fell 35% after one day of fasting, and decreased to 59% below control levels after three days (P less than .05). Somatomedins did not change with six hours of refeeding, but then rose rapidly, reaching control levels after 24 hours. Somatomedins were correlated with change in weight (r = .41, P less than .05), but not with glucose or beta-hydroxybutyrate. Inhibitors rose to 195% above control-levels after one day of fasting, and continued to rise to 375% above control after three days (P less than .01). In contrast to the delayed change in somatomedins with refeeding, there was an abrupt fall in inhibitors (41% below three-day fasted levels after six hours), returning to control levels after 24 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrition and somatomedin: nutritionally regulated release of somatomedins and somatomedin inhibitors from perfused livers in rats

Circulating somatomedin activity reflects the presence of both somatomedins and somatomedin inhibitors, factors which antagonize the growth-promoting actions of somatomedins. Although both are regulated by nutrition, somatomedin inhibitors respond more rapidly than somatomedins to refeeding in fasted animals. To explore the role of the liver in such responses, release of somatomedin activity and somatomedin inhibitor activity was assessed during perfusion of livers from normal, fasted, and fasted-refed rats. Size-exclusion high-performance liquid chromatography (HPLC) revealed that liver perfusates contain both somatomedin and somatomedin inhibitor activity of apparent molecular weight (mol wt) comparable to that found in the circulation (approximately 7,000 and approximately 30,000, respectively), as well as activity of apparently higher wt. In subsequent studies, responses to nutrition were evaluated as fluctuations in bioactivity only of mol wt comparable to that found in the circulation. Release of both somatomedin and somatomedin inhibitor activity was progressive over at least two hours of recirculating perfusion. Perfusates of livers from normal fed rats had somatomedin activity (stimulation of cartilage SO4 uptake) 94 +/- 19% above buffer (P less than .01), which fell to undetectable levels after three days of fasting. With refeeding, perfusate somatomedin activity rose within three hours to approximately 25% of levels in fed rats, but did not become significant until after 12 hours (29 +/- 7%, P less than .02). Perfusates of livers of fed rats also contained somatomedin inhibitor activity (42 +/- 10% inhibition of cartilage stimulation by normal serum), which rose after three days of fasting to 114 +/- 22% (P less than .02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Nutrition and somatomedin. XIV. Altered levels of somatomedins and somatomedin inhibitors in rats with streptozotocin-induced diabetes

Diabetes is associated with poor growth despite elevated levels of growth hormone (GH). Skeletal GH effects are mediated by somatomedins; in diabetes, somatomedins measured by radioassay are normal, yet somatomedin activity measured by bioassay is low. Since bioassay measurements reflect the presence of both somatomedins and somatomedin inhibitors, we asked if diabetes might be associated with discordant regulation of these circulating factors. Graded severity of diabetes was induced in rats by injection of streptozotocin at 37, 73, 146, and 293 mg/kg. After two days, metabolic derangement varied from normal serum beta-hydroxybutyrate with slight increase in glucose and minimal weight loss at 37 mg/kg streptozotocin to beta-hydroxybutyrate 10.6 mmol/L, glucose 447 mg/dL, and 33 g weight loss at 293 mg/kg streptozotocin. After fractionation of serum on Sephacryl S-300 pH 7.0, somatomedins and somatomedin inhibitors were measured by rat cartilage bioassay. Somatomedins (Kav 0.25 to 0.50) were comparable to control levels despite beta-hydroxybutyrate 2 mmol/L, glucose 534 mg/dL, and weight loss 11 g at 73 mg/kg streptozotocin and fell only at higher streptozotocin dosage. In contrast, somatomedin inhibitors (Kav 0.62 to 0.88) began to rise at 37 mg/kg streptozotocin and increased with higher dosage. Levels of somatomedins were correlated weakly only with beta-hydroxybutyrate (r = 0.48, P less than 0.05), while somatomedin inhibitors were correlated significantly with all indices, particularly beta-hydroxybutyrate (r = 0.78, P less than 0.0001). The early rise in somatomedin inhibitors but late fall in somatomedins could explain low somatomedin activity (and poor growth) despite normal levels of somatomedins measured by radioassay; measurement of somatomedin inhibitors may provide an index of growth potential in diabetes mellitus.     

Nutrition and somatomedin. X. Comparison of insulin-like activity of somatomedins extracted from liver and serum

Uncontrolled diabetes in rats is associated with reduced levels of both serum somatomedins and hepatic somatomedins. Hepatic somatomedins are recognized after extraction with 5 mol/L acetic acid, have a higher molecular weight (about 30,000) than serum somatomedins (about 8,000), despite acid conditions that dissociate somatomedins from circulating carrier proteins, and stimulate cartilage when given in vivo. To determine if hepatic somatomedins—as potential prohormones—have insulin-like activity comparable with serum somatomedins, their effects on rat epididymal adipose tissue were examined. Somatomedins were prepared from serum and liver extracts by gel filtration on Sephadex G-75, pH 2.4. In initial studies with fat pad segments, extracted hepatic somatomedins increased glucose oxidation only 64 ± 11% above buffer (mean ± SEM), while stimulation of 372 ± 48% was provided by extracted serum somatomedins of comparable cartilage-stimulating potency (P < 0.01, liver v serum). Further examination was performed with isolated adipocytes in a system sensitive to insulin at a concentration of 10 μU/mL (stimulation 100% above buffer). In dose-response studies measuring glucose oxidation, hepatic somatomedins had insulin-like activity of 16 μU/mL versus 55 μU/mL for serum somatomedins equipotent on cartilage (P < 0.05); measuring glucose incorporation into total lipids, hepatic somatomedins had undetectable activity while serum somatomedins had activity of 28 μU/mL. It is concluded that hepatic somatomedins with potent cartilage-stimulating activity have greatly reduced insulin-like activity. The apparent dissociation in biologic activity of hepatic somatomedins suggests that while they may be prohormones, they may also represent a class of growth factors separate from the circulating somatomedins.     

Nutrition and somatomedin. XIII. Usefulness of somatomedin-C in nutritional assessment

Malnutrition is common in hospitalized patients, but current measures of nutritional status are limited. Because levels of somatomedins are regulated by nutrition, the utility of somatomedin-C measurement in nutritional assessment was studied. Thirty-seven malnourished patients had measurement of somatomedin-C and conventional nutritional indexes. In 28 patients seen before therapy, the somatomedin-C level was reduced (38 percent of normal) and was lower than the albumin level (66 percent, p less than 0.01), transferrin level (59 percent, p less than 0.05), and lymphocyte count (43 percent, p = NS). Somatomedin-C level was lowest with combined "kwashiorkor-marasmus" (25 percent of normal) and also reduced with "kwashiorkor" (51 percent) or "marasmus" (57 percent) alone. Somatomedin-C was correlated with albumin, transferrin, and lymphocyte count (p less than 0.02 for each). In six patients given nutritional therapy, somatomedin-C levels rose by more than 70 percent in each (mean increase 181 percent), whereas lymphocyte counts rose in four (increase 78 percent for all patients), transferrin levels rose in four (increase 33 percent), and albumin levels rose in one (-6 percent). In 20 patients with detailed dietary analysis, only somatomedin-C was correlated with intake of protein and calories (p less than 0.005 for each). Somatomedin-C may be a sensitive marker of malnutrition and the response to nutritional therapy.     

Kidney tissue somatomedin C and initial renal growth in diabetic and uninephrectomised rats

Summary Kidney growth after induction of experimental diabetes in rats was compared to compensatory renal growth in response to unilateral nephrectomy. After 4 days of diabetes, kidney weight had increased from 816±21 mg (SEM) to 940±42 mg (15%). In insulin-treated diabetic rats kidney weight was unchanged at the end of the study, namely 828±15 mg.In unilaterally nephrectomised rats kidney weight increased from 840±20 mg (SEM) to 1050±60 mg during 4 days (24%). We observed increased kidney content of somatomedin C in both diabetic and uninephrectomised rats. In untreated diabetic rats it was maximal after 48 h, with an increase of 77% (3469±312 ng/g (SEM) versus 1961±173 ng/g). After 4 days the somatomedin C content had returned to initial levels. In insulin-treated rats somatomedin C content did not increase during the observation period. The somatomedin C content of the remaining kidney after unilateral nephrectomy was maximal after 24 h with an increase of 58% (from 1340±203 ng/g (SEM) to 2122±214 ng/g). The somatomedin C content returned to normal at day 4. Serum somatomedin C declined insignificantly in diabetic animals during the experimental period, but a significant decrease (p<0.02) was found in uninephrectomised rats. This study demonstrates that kidney somatomedin C peaks during the first or second day after uninephrectomy or induction of diabetes, respectively, and that insulin treatment sufficient to prevent kidney growth abolishes the increase. These similar rapid initial hypertrophies/hyperplasies may thus be dependent on local somatomedin C formation.     

Glucocorticoid effects on IGF-1/somatomedin-C and somatomedin inhibitor in streptozotocin-diabetic rats

Diabetes is associated with a fall in serum levels of insulin-like growth factor-1/somatomedin-C (IGF-1/Sm-C) and a rise in somatomedin inhibitor, a factor which antagonizes somatomedin action. We attempted to determine if the presence of glucocorticoids was required for diabetes-related alterations in these circulating growth factors. Diabetes was induced with streptozotocin in intact or adrenalectomized rats. Adrenalectomized-nondiabetic and adrenalectomized-diabetic rats were given either no glucocorticoids or daily hydrocortisone acetate at 0.5 or 50 mg/kg body weight, and killed 48 hours after streptozotocin treatment. After serum fractionation via size exclusion high performance liquid chromatography (HPLC), IGF-1/Sm-C was determined by radioimmunoassay, and somatomedin inhibitor by bioassay according to the ability of serum fractions to blunt cartilage stimulation by normal serum. Intact-diabetic rats had 22% weight loss, glucose 427 mg/dL, and beta-hydroxybutyrate 7.2 mmol/L (all P less than .001 v control). Serum IGF-1/Sm-C levels in intact-diabetic rats were decreased 71%, while somatomedin inhibitor rose to 470% of the control values (both P less than .004). Adrenalectomized-diabetic rats displayed comparable hyperglycemia (greater than 400 mg/dL) and decline in IGF-1/SmC, with or without glucocorticoid replacement. However, adrenalectomized-diabetic rats had greatly reduced weight loss (10%), beta-hydroxybutyrate (1.5 mmol/L), and somatomedin inhibitor (59% of control), all P less than .01 v intact-diabetic. Hydrocortisone 0.5 mg/kg in these animals increased weight loss but had no significant effect on beta-hydroxybutyrate or somatomedin inhibitor levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of somatomedin from sheep serum

A rapid, high yield preparative technique for the isolation of sheep somatomedin is reported. Purification of biologically active somatomedin from the 60% ammonium sulfate precipitate of sheep serum was accomplished using three gentle fractionation steps. Biological activity during purification was monitored using the rat adipocyte nonsuppressible insulin-like activity (NSILA) assay. A stepwise pH elution (pH 2.85, 3.5, 4.5, and 6.0) from SP-Sephadex resulted in the elimination of more than 99% of the serum proteins and a 500-fold enhancement of biological activity. The active fraction eluted at pH 6.0 and was further fractionated on Sephadex G-50 (fine) chromatography at pH 2.85. This resulted in about a 10,000-fold enhancement of activity over serum activity. The most active fractions from Sephadex chromatography were further separated on reverse phase HPLC in 0.1% trifluoroacetic acid using a linear gradient of 24-60% acetonitrile. The biological activity of the final preparation was enhanced 61,000- to 182,000-fold over that of serum (mean, 93,000-fold) when assayed in the NSILA assay. Protein yield was estimated to be 467 micrograms/liter serum. In addition to the NSILA activity, the final preparation demonstrated dose-dependent sulfation factor activity in the embryonic chick pelvic leaflet bioassay. Sheep somatomedin was active at physiological levels in both bioassays. Analysis of the somatomedin preparation by sodium dodecyl sulfate-electrophoresis at pH 8.8 showed that it was homogeneous by this criterion. The activity eluted from Sephadex G-50 was estimated to have a molecular size of 6900. Two Coomassie blue-stained bands were present in the final sheep somatomedin preparation after polyacrylamide gel electrophoresis at pH 3.2. Our purification process is a rapid, high yield technique which yields a polypeptide fraction enriched in NSILA and somatomedin-like activity. The molecular size and biological activity in the NSILA and sulfation factor assays suggest that our sheep NSILA is analogous to somatomedins purified from other species of animals.     

Nutrition and somatomedin. XV. Growth plate, growth factor and biologically active somatomedins in rats with streptozotocin-induced diabetes

Diabetic children may exhibit poor growth, yet levels of growth hormone and somatomedins measured by specific radioligand assays usually are normal. In the present studies, biological assays based on costal cartilage from hypophysectomized rats were used to test the possibility that diabetes is associated with decreases in circulating growth-related factors. 'Growth plate growth factor' activity was evaluated with tissue from the osteochondral junction (which resembles epiphyseal cartilage), and 'somatomedin' activity was measured with resting cartilage distant from the growth plate. Diabetes was induced in rats by administration of streptozotocin (STZ) at 40, 80, 160 and 310 mg/kg. Two days later, animals receiving STZ 40 mg/kg exhibited slight hyperglycemia but normal beta-hydroxybutyrate and weight gain; glucose and beta-hydroxybutyrate rose, and weight fell progressively with higher dosage. Gel filtration on Sephadex G-75 at pH 2.4 was used to separate rat serum into somatomedins (KAv 0.50-0.75) and growth plate growth factor (KAv 0.38-0.50). Somatomedins were 102, 92, 83 and 68% of control in STZ-treated animals, i.e. unchanged from control at 40 mg/kg STZ and falling only with higher dosage. In contrast, the growth plate growth factor declined at all doses of STZ to 93, 84, 69 and 57% of control. Thus, the growth plate growth factor began to fall with mild hyperglycemia alone (glucose 190 mg/dl), while a comparable fall in somatomedins was not seen until glucose was greater than 400 mg/dl and beta-hydroxybutyrate was three times normal. Only the growth plate growth factor was correlated with changes in body weight (r = 0.44, p less than 0.025). We conclude that decreases in levels of a circulating growth plate growth factor may contribute to growth impairment in diabetes. Measurements of this factor may be useful in examining underlying mechanisms.     

Comparison of somatomedin activity in rat serum and lymph.

A chick embryo fibroblast (CEF) assay has been used to measure the somatomedin activity in serum and lymph from normal rats. Lymph contains about one-half the SM activity found in serum. The somatomedin activites in serum and lymph have identical elution volumes on Sephadex G-50 in 1N acetic acid. Chromatography at neutral pH demonstrates that somatomedin peptides are bound to large proteins in both normal rat serum and lymph. The relative restriction of somatomedin to the intravascular space may be due to this protein binding.     

The effects of a new aldose reductase inhibitor (tolrestat) in galactosemic and diabetic rats

Tolrestat(N-[[5-(trifluoromethyl)-6-methoxy-1-naphthalenyl] thioxomethyl]-N-methylglycine; AY-27,773; Alredase) is a potent, structurally novel inhibitor of aldose reductase (AR). In vitro, tolrestat inhibited in dose-dependent fashion the AR from bovine lenses (IC50, 3.5 X 10(-8) mol/L) and the formation of sorbitol in human RBC incubated with glucose (IC50, 3 X 10(-8) mol/L). Upon administration with the diet to rats made galactosemic or diabetic, tolrestat decreased, in a dose-related fashion, the accumulation of galactitol or sorbitol in the sciatic nerve and lens. The effectiveness of tolrestat depended upon the experimental conditions and tended to be higher in less severe galactosemia and after suitable pretreatment, particularly in galactosemic rats, resulting in ID50 of 5 mg/kg/d in the sciatic nerve and 12-15 mg/kg/d in the lens. Tolrestat also decreased, in dose-related manner, the RBC sorbitol levels in normal and in streptozotocin diabetic rats; in the latter, at less than 2 mg/kg/d, the RBC sorbitol was reduced to control levels.     

Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats

We have studied potential mechanisms by which zinc deficiency (ZD) may result in growth impairment in young animals. Dietary-induced ZD in young rats resulted in diminished skeletal growth as measured by tibial epiphyseal width. Treatment with bovine GH (bGH) did not increase skeletal growth suggesting GH resistance rather than GH deficiency in zinc-deficient rats. Serum levels of basic somatomedin (SM) were lower in zinc-deficient rats than in control rats receiving a zinc adequate diet, either ad libitum or in pair matched amounts, and were restored to normal by zinc repletion but not by bGH treatment, suggesting that SM production is impaired by ZD. There was a high correlation between tibial epiphyseal widths and serum or femur zinc concentrations. These findings, along with observations that despite similar levels of serum basic SM the bGH-treated zinc-deficient rats had smaller tibial epiphyseal widths than pair fed control rats, additionally suggest that the action of SM on skeletal growth is impaired by ZD.     

Immunosuppressive serum factors in viral hepatitis. I. Characterization of serum inhibition factor(s) as lymphocyte antiactivator(s)

Sera from patients with acute viral hepatitis B were found to inhibit the in vitro proliferation of normal lymphocytes induced by different mitogens and antigens. In addition, an effect on concanavalin A-induced T suppressor cell activity and pokeweed mitogen-stimulated IgG and IgM synthesis was demonstrated. Studies concerning the kinetics of serum immunosuppressive effects indicated that serum immunosuppressive factor (SIF) interfered with the intermediate phase of mitogen-induced lymphocyte activation which was defined by protein and RNA synthesis. Thus, when SIF-positive sera were added to lymphocytes, which were already activated by phytohemagglutinin, for 8, 12, or 18 hr, the inhibitory effect decreased in relation to the duration of lymphocyte activation. No inhibition could be demonstrated when SIF-positive sera were added 24 hr after initiation of mitogen stimulation. Furthermore, similar inhibitory effects were found measuring either uptake of [3H]uridine (RNA synthesis) or [3H]leucine (protein synthesis) in a 24 hr culture of phytohemagglutinin-stimulated lymphocytes or [3H]thymidine uptake (DNA synthesis) after 48 hr. These results indicate that SIF act(s) like an antiactivator and may belong to immunoregulatory physiologic serum factors.     

Insulin increases plasma somatomedin C (IGF-1) concentrations in adult type 1 diabetic patients

The role of insulin in the control of somatomedin release has been investigated in people with Type 1 diabetes. Six patients underwent insulin-induced hypoglycaemia of 20 min duration and 8 patients were studied using the hyperinsulinaemic euglycaemic clamp technique at insulin infusion rates of 0.25, 1.25, 2.5, and 0.25 mU kg-1 min-1 for 1 h at each rate. In the first study plasma insulin concentrations rose from (median, range) 23 (10-36) to 114(60-200) mU l-1 at the onset of hypoglycaemia, and fell to 53 (23-100) mU l-1 after 20 min hypoglycaemia and 30 (15-73) mU l-1 on return to normoglycaemia. Plasma IGF-1 rose from 140 (96-292) to 179 (127-352) micrograms l-1 (p less than 0.05) at the onset of hypoglycaemia and fell to 131 (125-173) micrograms l-1 after 20 min and to 154 (121-287) micrograms l-1 at the end of the study. During hyperinsulinaemia plasma insulin rose from 23 (19-40) mU l-1 at the lowest infusion rate to 61 (33-84) and 148 (68-200) mU l-1 at the two higher infusion rates. Over the same period, plasma IGF-1 increased from 91 (13-203) to 123 (98-182) micrograms l-1 (p less than 0.05) and 109 (84-160) micrograms l-1. There was no correlation between growth hormone levels and IGF-1 in either study. These results suggest that insulin produces short-term increases in IGF-1 levels in man in the absence of a growth hormone response.     

Isolation of a somatomedin from plasma of rats bearing growth hormone secreting tumors.

We have purified a protein which has somatomedin-like properties from the serum of Wistar-Furth rats bearing a growth hormone producing pituitary tumor (MStT/W15). Activity was measured by a placental insulin and/or somatomedin C radioreceptor assay (SmC-RRA). The serum was initially filtered through Sephadex G-150 equilibrated with 0.1 M NH4HCO3 and 0.02% NaN3. On the G-150 column, radioreceptor insulin (RRI) and radioreceptor somatomedin C (RRSm-C) activities coincided and appeared predominantly in the 160,000 mol wt range with a minor proportion in the 50,000 mol wt range. The pooled active fractions were boiled for 30 min at pH 5.5. After removing denatured protein by centrifugation, the extract was passed through G-50 Sephadex equilibrated with 1% formic acid and 0.15 M NaCl. Sixty to 90% of the SmC-RRA activity in the effluent appeared in the 9000 mol wt range. This material has an isoelectric focusing range of 8.4--9.6, similar to that described for human somatomedin C. On SDS-urea polyacrylamide gel electrophoresis only one protein band was seen. The isolated peptide (rSm) stimulated sulfate uptake in hypophysectomized rat cartilage. The potency of two preparations was variously assayed from 14.0 to 54.7 units/mg. Rat somatomedin was iodinated and purified by absorption on and elution from placental membranes. Eight to 12% of rat [125I]Sm was specifically bound by human placental membranes. Rat [125I]Sm was displaced by hSmC and rSm and human NSILA-S, partially displaced by procine proinsulin and poorly displaced by rat insulin. In preliminary studies, rat [125I]Sm was displaced from receptors on human placental membranes by sera from pituitary tumor bearing rats greater than normal rat sera greater than hypophysectomized rat sera.