Lawrence S. Phillips

Nutrition and Somatomedin: II. Serum Somatomedin Activity and Cartilage Growth Activity in Streptozotocin-diabetic Rats

Since diabetes mellitus is a condition in which poor growth occurs despite elevation of plasma GH, we have attempted to determine if poor growth in diabetes, as in malnutrition, could be associated with a decrease in somatomedin activity. Young male rats were rendered diabetic with intravenous streptozotocin (STZ). The growth activity of their cartilage was estimated by 35SO4 incorporation in vitro, and somatomedin (SM) activity in their serum was determined by the stimulation of SO4 incorporation by cartilage from hypophysectomized rats or normal young pigs. Cartilage growth activity was significantly decreased 24 hours after STZ and fell to hypopituitary levels after 48 hours. The decreased growth activity could not be attributed to decreased cartilage responsiveness to SM, since incubation of diabetic cartilage with normal rat serum (normal SM) resulted in significant stimulation of cartilage SO4 incorporation. SM in diabetic serum decreased to hypopituitary levels 24 hours after STZ, and decreased further after 48 hours. The decrease in SM and cartilage growth activity was not prevented by the administration of high doses of bovine GH. The fall in bioassayable SM appeared to be due in part to the presence of an SM inhibitor in the diabetic serum, since addition of diabetic serum to normal serum decreased to measurable SM in the normal serum. Administration of insulin to diabetic rats 48 hours after STZ led to significant increases in SM and cartilage growth activity, and insulin therapy 24 hours after STZ prevented the decreases in SM and cartilage growth activity which occurred without insulin. Thus, acute STZ-induced diabetes in rats was associated with a significant decrease in both serum SM and cartilage growth activity; these changes were not ameliorated by administration of GH, and insulin therapy could both prevent and reverse the fall in SM and cartilage growth activity. From these observations, we conclude that (1) that fall in somatomedin activity and cartilage growth activity associated with STZ-induced diabetes appears to be due to insulin deficiency and (2) growth failure in diabetes, as in malnutrition, may be due to decreased somatomedin activity.

Nutrition and somatomedin. XI. Studies of somatomedin inhibitors in rats with streptozotocin-induced diabetes.

Diabetics may have normal somatomedins by radioimmunoassay yet decreased somatomedin activity by bioassay. The discrepancy appears due to circulating inhibitory factors; inhibitors in whole diabetic serum antagonize the action of both somatomedins and insulin via noncompetitive interactions. Since little is known about the nature of the inhibitory activity, serum from streptozotocin-diabetic rats was fractionated, and inhibitory activity measured as the ability of fractions to blunt stimulation of SO4 uptake by hypophysectomized rat costal cartilage exposed in vitro to somatomedins in normal rat serum. After establishing that inhibitory activity was stable to pH and lyophilization, diabetic rat serum was gel filtered at pH 7 (somatomedins bound to carrier proteins) and 2.4 (somatomedins dissociated, mol wt ∼8000). Using Sephadex and Sephacryl columns at neutral pH, inhibitors were detected at mol wt ∼250,000, ∼24,000, and ∼940. Predominant activity was at ∼24,000 and ∼940. In contrast, Sephadex columns at acid pH revealed inhibitors only at mol wt ∼21,000. Diabetic rat serum was also subjected to ion-exchange chromatog-raphy on CM-Sepharose. A single band of activity at pH 5–7 was found on elution with increasing pH, suggesting an isoelectric point(s) lower than that of the somatomedins. However, three areas of activity were seen on elution with increasing ionic strength at pH 5— at 0.02 M and 0.14–1.4 M and at 2.0 M pH 5.0–5.5. These studies indicate that reduced anabolism in diabetes may be due in part to three species of circulating somatomedin inhibitors, largely of mol wt ∼ 21–24,000 and∼940, and also of mol wt ∼250,000. The inhibitors have generally acidic properties, and molecular weights both larger and smaller than the somatomedins.

Nutrition and Somatomedin. VIM. The “Somatomedin Inhibitor” in Diabetic Rat Serum is a General Inhibitor of Growing Cartilage

Serum somatomedin activity appears to reflect the presence of both somatomedins and “somatomedin inhibitor,” a factor(s) that blunts cartilage stimulation by somatomedins. Although poor growth in uncontrolled diabetes is associated with an increase in inhibitor, suggesting a possible physiologic role, little is known of the interactions between this factor and somatomedins. We examined such interactions by measuring the effects of serum from normal rats (NRS, enriched in somatomedins) and serum from streptozo-tocin-diabetic rats (DRS, enriched in inhibitor) on costal cartilage from hypophysectomized rats. A 48-h exposure of cartilage to NRS resulted in an uptake of sulfate and uridine 125 ± 36% and 59 ± 14% (m ± SEM) above buffer levels, respectively, but addition of DRS to NRS produced sulfate and uridine uptake of 66 ± 3% and 95 ± 2% below buffer, respectively, indicating that chondroitin sulfate and RNA synthesis were both affected. Significant inhibition of uridine uptake (an early cartilage process) occurred as little as 4 h after exposure to DRS, while inhibition of sulfate uptake (a later process) was not found until 20 h. The decrease in isotope uptake following 24 h of exposure to DRS persisted over the next 24 h despite transfer of cartilage to fresh DRS-free medium and addition of NRS during the second 24-h period. When cartilage was incubated with a fixed concentration of DRS and increasing concentrations of NRS, Lineweaver-Burk analysis of both sulfate and uridine uptake indicated that NRS/DRS interactions were noncompetitive. These results indicate that the inhibitory factor in DRS affects different cartilage processes, produces profound, long-lasting decreases in cartilage activity, and has noncompetitive interactions with somatomedins in NRS. We conclude that somatomedin inhibitor in the serum of diabetic rats is a general inhibitor of growing cartilage that not only blocks stimulation by somatomedins, but also inhibits cartilage metabolism directly.

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—XII. Fractionation of somatomedins and somatomedin inhibitors in normal and diabetic rats

Bioassayable somatomedins and somatomedin inhibitors were examined after chromatographic separation, using serum from normal rats (enriched in somatomedins) and diabetic rats (enriched in somatomedin inhibitors). At neutral pH, gel filtration on Sephacryl S-300 revealed somatomedins at mol. wt approximately 140,000 (presumably carrier-bound) and inhibitors at mol. wts approximately 250,000, approximately 24,000 and approximately 1,000. At acid pH, gel filtration on Sephadex G-50 revealed somatomedins at mol. wt approximately 8,000 (presumably carrier-free) and a single inhibitor at mol. wt approximately 21,000. Ion exchange chromatography revealed that the inhibitor(s) may be more acidic than the somatomedins, but only low quantities of somatomedins were recovered. Sephadex G-50 fractionation was applied to pathophysiologic models in rats: 3 days of fasting were associated with a 62% fall in somatomedins and a 159% rise in inhibitors; 2 days of diabetes were associated with a 60% fall in somatomedins and a 344% rise in inhibitors. Since chromatography on Sephadex G-50 at pH 2.4 appears to provide adequate separation of somatomedins and somatomedin inhibitors with good estimated recovery of biological activity, this simple approach may be a probe useful in examining the regulation of somatomedins and somatomedin inhibitors in vivo.

Homocysteic acid: An examination of its possible growth hormone-like activity☆

Hypophysectomized rats were injected intraperitoneally for 4 days with various doses of homocysteic acid or growth hormone. The effects of these compounds on epiphyseal cartilage thickness and circulating somatomedin activity levels were evaluated in an attempt to repeat the results of Clopath, Smith, and McCully, who reported that this compound had growth hormone-like activity. DNA polymerase activity in livers of animals treated with growth hormone or with 10 mg/day of homocysteic acid was also measured. Using larger number of animals and including higher doses of homocysteic acid than those previously employed, we did not observe an increase of epiphyseal cartilage thickness in homocysteic acid treated hypophysectomized rats. Growth hormone significantly increased cartilage thickness. DNA polymerase levels in homocysteic acid treated hypophysectomized rats were not substantially increased although a larger, dose-dependent increase was observed with pGH and hGH. Neither homocysteic acid nor GH increased circulating somatomedin activity under the conditions used in this investigation. These observations demonstrate that homocysteic acid was not a substance with growth hormone-like activity in our hands and cast doubt on its possible future usefullness as a substitute for GH in clinical situations.