Similarities between the insulin-like stimulatory effect of human IgG and NSILP.

Since the insulin-like stimulatory effect of human IgG on adipocyte lipogenesis, exerted through its Fc moiety, is not neutralized by anti-insulin antisera, IgG may contribute significantly to the non-suppressible insulin-like activity (NSILA) of plasma. 91% of NSILA has been shown previously to be associated with a high mol. wt. protein (NSILP). The purpose of this investigation was to assess whether IgG and NSILP have similar stimulatory effects on adipocyte lipogenesis and whether this effect can be neutralized by preincubation with gamma-chain specific anti-IgG antiserum; whether IgG stimulates 35S-sulphate uptake by porcine cartilage, known to be stimulated by insulin-like growth factors but not NSILP; and whether gamma-chain specific anti-IgG antisera precipitate IgG in a fashion similar to that with IgG preparations. Our investigations show that both IgG and NSILP have similar dose response relationships with respect to the stimulation of adipocyte lipogenesis and that both lose their adipocyte stimulating effect following preincubation with anti-IgG antiserum; neither IgG nor NSILP stimulate 35S-sulphate uptake by porcine cartilage, unlike serum somatomedin and crude NSILA-s preparations; and that gamma-chain specific anti-IgG antisera form precipitin lines with NSILP. Therefore, NSILP and IgG molecules have immunological and biological similarities; there may occur a homology between the Fc fragment of IgG and the NSILP molecule.

New powerful insulin-like protein from human promyelocytic leukemia cells.

Potent insulin-like activity was found in the conditioned medium of human promyelocytic leukemia (HL-60) cells. The conditioned medium of HL-60 cells at high density stimulated [3H]glucose incorporation into lipids in rat adipocytes in a time- and dose-dependent manner. The dose-response curve for this factor was not parallel to that for insulin, and the maximal effect achieved was much greater than reached by insulin or multiplication-stimulating activity. Moreover, the maximal effect reached by either insulin or the conditioned medium was additive. The insulin-like activity was not suppressed in the presence of antiinsulin antibody. Insulin-like activity was not detectable by radioreceptor assay for insulin, suggesting that the factor does not act through the insulin receptor. The factor in the conditioned medium of HL-60 cells was heat stable and sensitive to trypsin. When the conditioned medium was subjected to gel filtration on a Sephadex G-100 column, the major part of insulin-like activity eluted in the position corresponding to an apparent molecular weight between RNAase and insulin markers. The remaining activity, approximately 10% of the total, appeared with a larger molecular weight species. On isoelectric focusing of the smaller molecular species, insulin-like activity was largely focused in the position corresponding to pI 7.8-8.2.

The effect of insulin-like growth factor (IGF) and of human serum on steps in proteoglycan synthesis.

Embryonic chick pelvic cartilages were incubated in the presence of insulin like growth factor (IGF) (1 - 100 microunits/ml), as well as normal human serum (5%), with radiolabelled precursors of proteoglycan (PG) synthesis: L-[3-3H]serine, D-[6-3H]glucosamine and [35S]Na2SO4. IGF alone (1 - 15 microunits/ml), stimulated in a dose-dependent manner D-[6-3H]glucosamine incorporation into tissue-bound and soluble isolated glycosaminoglycan (GAG) chains. L-[3-3H]serine incorporation into PG molecules was not stimulated by IGF (1 - 100 microunits/ml), despite the increase in the uptake of this precursor into intact cartilage. [35S]Na2SO4 incorporation was unaffected by IGF. Serum promoted the uptake of all three precursors into tissue-bound glycosaminoglycans. It was postulated that IGF could stimulate proteoglycan synthesis not only by elongating existing chondroitin sulphate chains but also by increased synthesis of other sugar chains e.g. keratan sulphate and oligosaccharides.

Responses of rat and chick chondrocytes and rate hepatoma cells to plasma fractions with insulin-like and growth-promoting activities.

Plasma protein Cohn fraction IV-1 was extracted and fractionated according to the method of Van Wyk et al.[1], with the omission of the Sephadex G-75 step. The fractionated polypeptides were investigated for insulin-like activity (lipid synthesis and lactate production) and growth-stimulatory activity (DNA synthesis) in three bioassay systems: rat hepatoma cells, and rat and chick chondrocyte suspensions. Binding of each fraction to insulin receptors in isolated plasma membranes was also determined. Three peptide subfractions, with molecular weights ranging from 400 to 20 000, stimulated lipid synthesis, lactate production and DNA synthesis in all three bioassay systems. There were distinct species- and cell-type-specific variations in the quantitative patterns produced by each subfraction. In contrast, authentic insulin (0.005-0.1 ng/ml) had no effect on DNA and lipid synthesis anad lactate production in hepatoma cells and chondrocytes. One of the three active subfractions and two relatively inactive fractions displaced insulin from its receptors. These observations indicate that acid-ethanol extracts of plasma contain a variety of peptide factors with insulin-like and growth-promoting effects whose expression in different cell types is modulated by inherent properties of each cell type which are determined genetically presumably. Surface receptors for authentic insulin do not appear to play an essential role in the insulin-like activities of the isolated plasma fractions.

Inhibition of the action of nonsuppressible insulin-like activity on isolated rat fat cells by binding to its carrier protein.

Nonsuppressible insulin-like activity extracted and purified from human serum (NSILA-S) mimics all insulin-like effects in vitro and, after injection, in vivo in the presence of excess insulin antibodies. However, there is no evidence that it exerts acute insulin-like effects in its native form in the circulation, where it is almost completely bound to a specific large molecular weight carrier protein. In this paper we show that partially purified NSILA-S-carrier protein, devoid of endogenous insulin-like activity, inhibits the stimulatory effect of NSILA-S, but not of insulin, on 3-0-methylglucose transport and on lipogenesis from [U-(14)C]glucose in isolated rat fat cells. Concomitantly, it prevents binding of (125)I-labeled NSILA-S to the insulin receptor and to the NSILA-S-binding site. The following explanation is, therefore, offered for the absence of acute insulin-like effects of native NSILA-S in vivo: In native serum NSILA-S occurs almost exclusively as NSILA-S-carrier complex. According to recent findings the passage of this complex through blood capillaries is restricted. The present results indicate that, in addition, it is metabolically inactive, or, at least, possesses reduced metabolic activity. The well-known phenomenon that whole serum, nevertheless, exerts pronounced nonsuppressible insulin-like effects on adipose tissue in vitro seems, therefore, to be mainly caused by the presence of a large molecular weight insulin-like protein not identical to the NSILA-S-carrier complex.

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.

Effects of insulin and NSILA on adipocytes of normal and diabetic rats: receptor binding, glucose transport and glucose metabolism.

Isolated fat cells from normal and streptozotocin-diabetic rats were compared with respect to metabolic indices (glucose-uptake, 3-0-methyl-glucose efflux) with and without stimulation by insulin and nonsuppressible insulin-like activity (NSI-LA). In addition, binding studies were carried out with these two hormones. Basal 14C-glucose oxidation and incorporation into lipids was decreased in diabetic cells and their response to insulin and NSILA was greatly reduced. Basal efflux of 3-0-methylglucose from diabetic cells was somewhat faster than from normal cells. The response to insulin and NSILA was less than in normal cells and it was delayed. The apparent number of insulin binding sites as well as their affinity for insulin was increased in diabetic cells. In contrast, the apparent number of binding sites for NSILA was decreased in diabetic cells and their affinity for NSILA was increased. In normal cells insulin enhanced binding of 125I-NSILA more markedly than in diabetic cells. These findings show that the rate-limiting step of impaired glucose metabolism (oxidation and lipogenesis) in diabetic fat cells is beyond the interaction of the hormone with the receptor. They suggest that the apparent number of hormone receptors (insulin, NSILA) on the cell membrane is regulated individually for each binding site.