Stimulation Of Glucose Oxidation Research Articles

Recovery from Thyroid-Stimulating Hormone-Induced Refractoriness in Thyroid Slices: Effect of Removal of Hormone and New Protein Synthesis*

An initial incubation of bovine thyroid slices with TSH causes decreased responsiveness to the subsequent addition of the hormone when the adenylate cyclase -cAMP system and other metabolic parameters are measured. After the initial incubation with TSH, refractoriness persists despite incubation of thyroid slices for 24 h in the absence of added TSH. Removal of persistently bound TSH by trypsin or antibody to TSH did not reverse the refractoriness during a subsequent 2 h incubation without added TSH. However, normal TSH responsivity was restored by the removal of TSH bound during the first incubation by the addition of either trypsin or antibody to TSH at the beginning of a 24-h second incubation. Restitution of TSH responsiveness after treatment with trypsin or antibody to TSH requires new protein synthesis. While TSH-induced refractoriness does not modify stimulation of cAMP by cholera toxin, its effect on glucose oxidation is significantly diminished. Menadiol stimulation of glucose oxidation is not inhibited in thyroid slices refractory to TSH. Thus, the effect of menadiol is subsequent to the block induced by TSH, whereas that of cholera toxin is proximal to it.

Lipolytic effects of insulin in adipocytes isolated from hypophysectomized rats.

Adipocytes isolated from the epididymal fat of hypophysectomized rats by digestion with collagenase failed to respond to insulin with an increase in glucose utilization. These cells also exhibited an anomalous response to insulin when added in the presence of epinephrine. While insulin antagonized the lipolytic actions of epinephrine in normal adipocytes or in segments of epididymal fat from normal or hypophysectomized rats, it potentiated lipolysis in adipocytes isolated from hypophysectomized rats. This anomalous effect was also evident when isoproterenol, ACTH, or glucagon was used as the lipolytic agent, but the expected antilipolytic response was obtained when theophylline served as the lipolytic agonist. The lipolytic effects of insulin were not seen until 4-7 days after hypophysectomy. Treatment of hypophysectomized rats with a combination of GH (100 micrograms/rat . day), cortisone acetate (1 mg/rat . day), and T3 (1 micrograms/rat . day) for 5 days restored the antilipolytic response to insulin in cells of hypophysectomized rats, but no one hormone alone was effective. The data indicate that adipocytes of hypophysectomized rats retain their ability to recognize and response to insulin, but the ability of insulin to stimulate glucose oxidation or antagonize epinephrine-induced lipolysis is abolished by the cell isolation procedure. The findings underscore the need to consider the impact of hormonal status on the ability of cells to retain normal responsiveness during the rigors of the cell isolation procedure and suggest that failure to do so might lead to erroneous interpretations of the physiological actions of hormones.

Stimulation of glucose oxidation and transport in isolated rat adipocytes by riboflavin and visible light

Riboflavin, which is known to cause photooxidative damage in biological systems, is now shown to stimulate glucose transport and oxidation in isolated rat adipocytes in the presence of visible light. At low riboflavin concentrations, well within normal blood levels, there is a small but reproducible stimulation of C 1-labeled glucose oxidation to labeled CO 2 (30% stimulation at 10 −6 m), which does not require light. However, at higher concentrations (10 −5 m and above), light greatly potentiates this effect on C 1-glucose oxidation as well as stimulates C 6-glucose oxidation (two- to three-fold over controls). These apparent effects on the hexose monophosphate shunt and glycolytic-tricarboxylic acid pathways are blocked by 10 μ m cytochalasin B, a glucose transport inhibitor. Riboflavin in light is further shown to stimulate uptake of 3- O-methylglucose, a nonmetabolizable glucose analog. These light-dependent effects are not affected by catalase or superoxide dismutase, but they are inhibited by dimethylfuran, a singlet oxygen scavenger. This latter agent has no effect on glucose metabolism in untreated or insulin-treated cells. The results suggest a physiologically important potential effect of riboflavin and visible light.

Glucose oxidation, glucose transport and insulin binding in isolated rat epididymal adipocytes in the presence of anesthetics

Insulin binding and glucose oxidation were measured in isolated rat adipocytes in the presence of several anesthetics; ethanol, n-octanol, pentobarbital, chlorpromazine and tetracaine. Ethanol and chlorpromazine, at anesthetic and pentobarbitol at sub-anesthetic concentrations are inhibitory to both basal and insulin stimulated rates of glucose oxidation. At all concentrations of ethanol, pentobarbital or chlorpromazine tested binding of insulin is not affected. Since anesthetics may alter membrane fluidity, it is suggested that an anesthetic-induced increase in membrane fluidity beyond that which occurs at 37°C is detrimental to glucose oxidation. Of the 5 anesthetics examined, only chlorpromazine (10 μM or less) and tetracaine (500 μM) stimulate glucose oxidation. These two agents are known to bind to a cell's cytoskeletal system; the binding of chlorpromazine to microtubules is entropy driven. The temperature and concentration dependence of chlorpromazine stimulation of glucose oxidation (transport) are consistent with this form of binding. It is proposed that chlorpromazine binds to the cytoskeletal system of the adipocyte and that this system is normally restrictive to the motion of membrane proteins. Disruption of the cytoskeletal system by chlorpromazine or tetracaine would increase the frequency of insulin-receptor and glucose-carrier contact. Activation of glucose transport could ensue.

Decreased Stimulated Glucose Oxidation and lodination by Polymorphonuclear Leukocytes from Insulin-Treated Diabetic Subjects

To investigate the mechanism of the reported abnormality of polymorphonuclear leukocyte (neutrophil) function in diabetes, the burst of oxidative activity accompanying the killing reaction and the neutrophil iodination reaction were measured in 21 stable, insulintreated diabetic subjects and 32 nondiabetic subjects of similar age range. Responses to standard killed preparations of Candida albicans (C. albicans) (glucose oxidation and neutrophil iodination) and Staphylococcus aureus (S. aureus) (neutrophil iodination) were studied. The iodination reaction was studied using the subjects' neutrophils suspended both in autologous serum and in pooled normal human serum, and the glucose oxidation test was performed using the neutrophils in pooled normal human serum. There was a significant decrease in the conversion of 14C-1-glucose to 14CO2 in the neutrophils from diabetics [0.47 ± 0.04 (SE) cf. 0.64 ± 0.04 nmol/2 × 106 neutrophils/60 min, P < 0.005] and in neutrophil iodination in response to S. aureus in both autologous serum (diabetics .089 ± 0.008 cf. 0.114 ± 0.008 nmol iodine/106 neutrophils/90 min, P < 0.025) and in pooled human serum (diabetics 0.086 ± 0.009 cf. 0.105 ± 0.006, P < 0.05). There was no significant difference in neutrophil iodination in response to C. albicans. There was a significant negative correlation between the diabetics' fasting plasma glucose level and neutrophil iodination in response to S. aureus in pooled human serum (r = - 0.54, P < 0.05), but not between fasting plasma glucose and glucose oxidation. It is concluded that there are significant decreases in stimulated glucose oxidation and in the neutrophil iodination reaction (reflecting decreased function of the myeloperoxidase-hydrogen peroxide-halide microbicidal system) in neutrophils from insulin-treated dia-betic subjects. These abnormalities may contribute to the defective bactericidal activity of diabetic neutrophils.

Changes in prostaglandin E 1 stimulation of glucose oxidation in rat adipocytes during maturation and aging

Prostaglandin E 1 stimulates glucose oxidation in isolated rat adipocytes in a time and concentration dependent manner. Maximal stimulation requires 2 hours exposure to prostaglandin, although effects can be detected by 0.5 hours or earlier. In contrast to prostaglandin E 1, prostaglandin F 2 α has essentially no effect on glucose oxidation. Maximal stimulation by prostaglandin E 1 at all ages tested occurs at concentrations of 10 −5 − 10 −4M. Stimulation is greatest in cells of mature (10–12 month old) animals at 81 ± 9% above basal levels of glucose oxidation. This is to reduced to 48 ± 8% in cells of senescent (23–26 month old) animals, and at 23 ± 18% in cells of young (2–3 month old) rats is not significantly different from basal oxidation in most animals. These results are consistent with data for adipocytes and other cell types indicating that responsiveness to certain hormones is altered during maturation and aging.

12-asparagine-B] human insulin. An analogue with modification in the hydrophobic core of insulin.

The human [12-Asparagine-B] insulin [Asn12-B] insulin) which differs from the parent molecule in that the valine residue at position B12 is substituted by an asparagine residue, has been synthesized by the procedures developed in this laboratory. In stimulating glucose oxidation and lipogenesis the analogue exhibited potencies of 0.19% and 0.14%, respectively, as compared to insulin. In insulin receptor binding [Asn12-B] insulin was found to possess a potency of ca. 0.29% compared to the natural hormone. At high concentrations this analogue is shown to have the same maximal activity in the in vitro assays as the natural hormone. This indicates that despite the low affinity for of the analogue for the insulin receptor, the analogue-receptor complex is fully capable of initiating the series of chemical events that leads to the biological response. It is concluded that the B12 valine contributes greatly in the maintenance of a structure possessing the proper receptor-binding characteristics, but does not have any role in modulating the activity of the hormone-receptor complex. The potency of this analogue by radioimmunoassay is at least 100-fold higher than the in vitro biological assays, indicating that the immunological determinants of insulin can be essentially unrelated to the biological activity of the molecule.

Insulin Binding and Glucose Transport

This chapter discusses the insulin binding and glucose transport. The insulin binding is explained with the help of recent data on cellular localization, cooperativity, regulation of receptors and receptors in normal and transformed cells. The action of insulin starts with the binding of the hormone to specific receptors on the plasma membrane of target cells. Studies of insulin receptors require a satisfactory reagent probe, one with the same biological properties as the native insulin. The monoiodinated insulin has a biological activity similar to native insulin, based on stimulation of glucose oxidation by fat cells. For glucose transport, the chapter reviews various regulatory aspects, such as cell growth, adaptation, and hormonal influences in normal and neoplastic cells. The chapter focuses on the mechanism and regulation of glucose transport in cells where glucose is transported by facilitated diffusion. This process is mediated by a specific glucose carrier but does not require energy; therefore, the direction of glucose flux is in accordance with its concentration gradient. The most common experimental design for measuring rates of substrate transport is the zero-trans influx procedure.

A method for the iodination of insulin and its binding to dissociated mouse mammary cells.

The iodination of insulin was accomplished by a modification of the lactoperoxidase method. The use of a low concentration of hydrogen peroxide (1.5 ng/ul) followed by Sephadex gel filtration and purification on a cellulose column yielded iodoinsulin with an activity equal to that of native insulin in stimulation of glucose oxidation in rat epididymal fat cells and with high specific binding to collagenase-dissociated mouse mammary cells from pregnant and lactating mice. Other hormones tested did not displace the binding. Analysis of displacement curves and scatchard plots suggests that both the affinity and the number of sites for insulin binding differ between pregnant and lactating mammary cells.

Adrenergic and cholinergic mediated glucose oxidation by rat parotid gland acinar cells during aging

Adrenergic (both α and β) and cholinergic agonists stimulated glucose oxidation in dispersed parotid acinar cells obtained from various aged rats. Basal levels of glucose oxidation were constant over the adult rat life span. Epinephrine and norepinephrine effects on parotid cell glucose oxidation were reduced about 50% in cells from 24 mo rats compared to results with 3 and 12 mo rats. The epinephrine effect was mediated primarily via the α-adrenergic system. In addition, an α-adrenergic agonist (phenylephrine) stimulated glucose oxidation significantly less in parotid cells from 24 mo rats than in those from younger rats. β-Adrenergic (isoproterenol) and cholinergic (carbachol) stimulation did not change with age. These results suggest that an age-related impairment occurs specifically in the α-adrenergic system of rat parotid acinar cells.

Growth hormone stimulation of glucose transport in isolated rat hepatocyte suspensions and primary cultures.

[14C]-3-O-Methyl-D-glucose transport in response to GH stimulation was measured in freshly prepared and cultured hepatocytes from hypophysectomized (hypox) adult female rats. Sugar transport in freshly isolated hepatocytes from hypox animals was stimulated by 10(-9) M human GH (hGH) and 10(-9) M rat GH (rGH) during 0-30 and 60-90 min of incubation, respectively. Monosaccharide transport in freshly isolated hepatocytes from normal female rats was unaffected by either hGH or rGH addition. Specific [125I]iodo-hGH binding to freshly isolated hepatocytes from hypox rats was detectable as early as 9 min of incubation and reached maximum binding levels by approximately 30-60 min. Specific [125I]iodo-rGH binding was not detectable until 30 min of incubation and reached a binding plateau after 60 min of incubation. Porcine insulin at 10(-9) and 10(-7) M was ineffective in stimulating sugar transport in either freshly isolated or cultured hepatocytes from hypox animals. Sugar transport in cultured hepatocytes was stimulated by 10(-7) M hGH ad 10(-7) rGH, with 30-60 and 60-90 min of incubation being the most frequent stimulatory intervals observed. Maximum specific [125I]iodo-hGH binding was observed at 60 min of incubation and decreased by 72% and 77% at 90 and 120 min, respectively. Specific [125I]iodo-rGH binding, however, was not detectable in cultured hepatocytes. Both hGH at 10(-9) M stimulated glucose oxidation to CO2 in freshly isolated hepatocytes from hypox animals. Stimulation was observed after 30 min of incubation with GH and indicated a regulatory function for GH in maintaining basal levels of glucose oxidation. The results presented here suggest a possible role for GH in glucose uptake and metabolism in the liver and indicate that differences may exist between the effects of hGH and rGH on glucose uptake.

Enhanced sensitivity to insulin in rats treated with antibodies to rat growth hormone.

Although prolonged treatment with GH can result in insulin resistance and hyperglycemia, it is not known whether endogenous GH influences sensitivity to insulin in normal animals. To investigate this possibility, sensitivity to insulin was assessed in chronically cannulated normal rats treated acutely with antiserum to rat GH (ArGH) produced in a rhesus monkey. Controls received equivalent treatment with normal monkey serum. In rats which received a regimen of six pulses of ArGH over a 24-h period, plasma glucose and insulin levels were not different from those in controls. When sensitivity to administered insulin was tested at the end of the 24-h regimen, the fall in plasma glucose was only slightly greater and more prolonged in ArGH-treated rats than in controls. However, a significant increase in sensitivity to insulin was evident in adipose tissue from the ArGH-treated rats as a greater stimulation of glucose oxidation by insulin (50 microU/ml). In addition, GH (0.5 micrograms/ml) stimulated glucose oxidation in adipose tissue from the ArGH-treated rats, while tissues from normal monkey serum-treated controls were unresponsive (refractory) to GH. The loss of the typical refractory effect of GH with ArGH treatment indicates that the biological activity of endogenous GH was neutralized. Similar results were obtained in rats treated for 6 or even 3 h with ArGH. These observations indicate that acute treatment with ArGH can neutralize GH activity and enhance sensitivity to insulin in adult rats. Thus, endogenous GH appears to participate in the regulation of carbohydrate metabolism in normal rats on an hour to hour basis.

Divergence of the in vitro biological activity and receptor binding affinity of a synthetic insulin analogue, [21-asparaginamide-A]insulin.

The [21-asparaginamide-A]insulin ([Asn-(NH2)21-A]insulin) was synthesized by the procedures developed in this laboratory to investigate the contribution of the C-terminal residue, asparagine, of the A chain to the biological activity and receptor binding affinity of insulin. Its secondary structure was investigated by circular dichroism studies. The biological behavior of this analogue was compared with that of insulin in in vitro and in vivo tests and in receptor binding assays. In contrast to other naturally occurring insulins and to all insulin analogues synthesized thus far, [Asn-(NH2)21-A]insulin displays a disparity between receptor binding affinity and in vitro biological potency. In stimulating glucose oxidation and lipogenesis the analogue exhibited potencies of 12 and 14.8%, respectively, compared to insulin. In insulin receptor binding assays, however, this analogue was found to possess a relative potency at least fourfold higher than the in vitro biological activities. In rat liver membranes and in isolated fat cells the analogue exhibited affinities of ca. 63.9 and 51.4%, respectively, compared to the natural insulin. Both the synthetic analogue and the natural hormone have the same maximal activity in the in vitro assays and their dose-response curves are parallel. When assayed in vivo by the mouse convulsion test, [Asn(NH2)21-A]insulin displays a potency of ca. 72% that of the native insulin. This might indicate partial amidolysis of the analogue in vivo, resulting in conversion to the natural hormone. The implications of these observations are considered with regard to insulin-receptor interactions and the generation of the physiological response to the hormone.

Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulinlike growth factor) using antibodies to the insulin receptor.

Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.

Metabolic, cationic and secretory effects of hypoglycemic sulfonylureas in pancreatic islets.

Tolbutamide, gliclazide and glibenclamide failed to stimulate glucose oxidation in rat pancreatic islets. Tolbutamide also failed to stimulate pyruvate and palmitate oxidation and decreased the islet content of NAD(P)H and ATP. Tolbutamide stimulated 45Ca net uptake, inhibited 86Rb net uptake and tended to increase 22Na net uptake by the islets. The effect of theophylline upon islet function differed from that of tolbutamide by the magnitude of its insulinotropic action as a function of the glucose concentration, by its stimulant action upon the utilization of endogenous nutrients in islets deprived of glucose and by the lack of gross alteration in 45Ca and 86Rb net uptake. It is concluded that the insulinotropic effect of hypoglycemic sulfonylureas cannot be merely equated to a facilitation of nutrient metabolism or inactivation of phosphodiesterase in the islet cells. The primary action of these drugs could be to affect cations transport across the B-cell membrane.

Insulin-like stimulation of glucose oxidation in rat adipocytes by vanadyl (IV) ions

The mechanism of insulin action is still unknown1. One approach to this problem is to apply substances which mimic the action of the hormone to target cells. Ouabain and deprivation of extracellular K+ (refs 2,3), which inhibit the active transport of Na+ and K+ ions, are both known to activate glucose transport and oxidation in isolated adipocytes. Vanadate (V) ions have recently been shown to act as very efficient inhibitors of the sodium pump or (Na+ + K+)ATPase in in vitro preparations4. They have a natriuretic and diuretic effect in rats5 and a positive inotropic effect on cat heart muscle6. Many tissues contain vanadium at a concentration of about 0.1–1.0 μM (ref. 7) and so endogenous vanadate could be a physiological regulator of the sodium pump. But this is still open to debate, because the bulk of the vanadium is probably in the vanadyl (IV) form8,9 and VO2+ ions bind tightly to proteins8,10. VO2+ is a relatively ineffective inhibitor of (Na+ + K+) ATPase in vitro8,11. We report here that externally applied vanadate ions at low concentrations mimic fully the effect of insulin on glucose oxidation in rat adipocytes. However, this simulation seems to be due mainly to the effects of vandyl (IV) ions, probably produced within the cells, and not primarily to inhibition of the sodium pump. Also, externally applied vanadyl (IV) ions stimulate glucose oxidation substantially. Vanadyl ions are known to be powerful inhibitors of alkaline phosphatase12 and we therefore consider the possibility that they inhibit a cellular phosphatase activity. An early event in insulin action may involve alteration of the degree of phosphorylation of protein(s) involved in regulation of sugar transport.

Effects of wheat germ agglutinin on insulin binding and insulin sensitivity of fat cells.

The plant lectin (wheat germ agglutinin, WGA) produces several alterations in the ability of fat cells to bind and respond to insulin. Although WGA markedly stimulated glucose oxidation, it caused only a modest stimulation of glucose transport. WGA (0.25-20 micrograms/ml) increased the binding of insulin by adipocytes, apparently by increasing the binding affinity of the insulin receptor. With low WGA concentrations (0.25-2.5 micrograms/ml), the elevation in binding was accompanied by an increase in the sensitivity of the adipocytes to insulin stimulation of glucose transport. However, the sensitivity of these cells to vitamin K5 and H2O2 was not altered. With higher WGA concentrations (5-20 micrograms/ml), stimulation of the glucose transport system by insulin, vitamin K5, or H2O2 was markedly inhibited, an effect that is reversed by the addition of ovomucoid. These findings suggest that low WGA concentrations increase the affinity of the insulin receptor and the insulin sensitivity of the cells. At higher concentrations, the lectin appears to act at another site(s) to inhibit the activation of the transport system by insulin or other agents.

Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells.

SJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS-a, a thioguanine-resistant myeloma cell line derived from P3X63 Ag8 (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D-(1-14C) glucose to 14CO2. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D-(1-14C) glucose to 14CO2 in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin-fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: 1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; 2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D-(1-14C) glucose to 14CO2; and 3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.

Effects of bilirubin on fat cell metabolism and lipolysis.

Bilirubin (0.45 mM) inhibited lipolysis and stimulated [1-14C]glucose oxidation by rat fat cells in the presence of an equimolar concentration of bovine serum albumin. Bilirubin was an insulinlike agent with respect to inhibition of lipolysis and stimulation of glucose oxidation. There was a marked inhibition of adenylate cyclase activity of fat cell ghosts by 0.001--0.1 mM bilirubin in the absence of albumin, which was largely reversed by the addition of albumin. Although both bilirubin and free fatty acids bind to albumin, the primary binding sites appear to be separate. The effects of bilirubin at a molar ratio to albumin of 2 or less were not influenced by free fatty acid-to-albumin ratios up to 3. Triglyceride lipase activity of partially purified rat fat cell homogenates was inhibited by bilirubin in the presence of an equimolar concentration of bovine albumin. These data indicate that the antilipolytic action of bilirubin is probably due to direct inhibition of triglyceride lipase through a mechanism that does not involve competition with free fatty acids for binding to albumin.

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.