Robert S. Bar

Insulin, insulin-like growth factors, and vascular endothelium

Endothelial cells form the intimal lining of the entire vascular system. The vascular endothelium is continuously and directly bathed by components of the bloodstream and represents the initial fixed anatomical surface with which these components come in contact. In the past decade, the methodologies for studying endothelial cell functions have markedly advanced, enabling direct and detailed study of the vascular endothelium. From such studies, it is now apparent that the vascular endothelium represents an extraordinarily complex network of cells demonstrating a multitude of distinct anatomic, metabolic, and immunologic properties critical to such processes as angiogenesis, atherosclerosis, thrombosis, neoplasia, and a variety of metabolic disorders including homocystinuria and diabetes mellitus. This report will focus on the interactions of insulin and the insulin-like growth factors (IGFs) with vascular endothelium, based on studies with cultured endothelial cells, isolated microvessels, and perfused organ systems. Data will be presented relevant to the following concepts: (1) endothelial cells, in culture and in vivo, have specific receptors for insulin, IGF-I, and IGF-II; (2) insulin, IGF-I, and IGF-II have both distinct and overlapping functions in cultured endothelial cells; (3) cultured endothelial cells process receptor-bound insulin, IGF-I, and IGF-II, by distinct processes; (4) in vivo, capillary endothelial receptors are integrally involved in the transport of intact insulin to subendothelial sites of insulin action; and (5) vascular endothelium has specialized cellular features that are likely to contribute to the unique interactions of endothelial cells with insulin and the IGFs.

Linoleic acid metabolism and prostaglandin production by cultured bovine pulmonary artery endothelial cells.

When bovine pulmonary artery endothelial cells are cultured in a medium supplemented with linoleic acid, their capacity to produce prostacyclln (PGI2) is reduced by about 60%. This reduction occurs when PGI2 formation Is stimulated by the addition of either the calcium lonophore A23187 or arachidonic acid. In addition, supplementation with linoleic acid reduced the production of prostaglandin E2 and F2n from 1-14 C-arachidonic acid by more than 50%. The capacity of cultured bovine pulmonary vein and aortic endothelial cells to convert extracellular arachidonic acid Into PGI2 also was reduced by about 50% when the growth medium was supplemented with linoleic acid. Although bovine pulmonary artery endothelial cells Incorporated large amounts of 1-14 C-linolelc acid into cellular phospholiplds and triglycerldes, a maximum of only 2.3% of the radioactivity was converted to arachidonic acid In 24 hours. The most prevalent radioactive metabolite was elcosadlenoic acid, the elongation product of linoleic acid. As compared with linoleic acid, the bovine endothelial cells Incorporated 30% more 1-14 C-arachldonlc acid Into phospholiplds and 60% more Into triglycerldes. When the growth medium was supplemented with linoleic acid, the percentage of this fatty acid In cellular liplds Increased 3-to 4.5-fold and eicosadlenoic acid accumulated, accounting for up to 9% of the cellular fatty acids. This Increase was accompanied by a 30% to 45% reduction in arachidonic acid. These findings, together with our previous results with human umbilical vein endothellum, suggest that an inability to convert large amounts of linoleic to arachidonic acid and a suppresslve effect of linoleic acid enrichment on prostaglandin production may be general properties of endothelial cells.

Production of IGF-binding proteins by vascular endothelial cells.

Conditioned serum-free media from cultured human, bovine and rodent endothelial cells contained binding proteins with high affinity for the insulin-like growth factors (IGFs). After partial purifications on heparin or Multiplication Stimulating Activity (MSA)-affinity columns, 2 species of binding protein were identified, a major protein having Mr approximately 35,000 and a minor 22-28,000 protein. The binding proteins had greater affinity for IGF-I than IGF-II with no affinity for insulin or proinsulin. Substantial amounts of the binding proteins remained cell-associated, loosely bound to the outer cell surface of the endothelial cell. Binding protein(s) from human endothelial cells cross-reacted with antibodies to the 53,000 dalton acid-stable human serum binding protein. Production of endothelial binding proteins was not stimulated by growth hormone or insulin. We conclude that endothelial cells in culture produce large quantities of specific IGF binding proteins. Such binding proteins should be relevant in understanding the complex metabolism and function of the IGFs in the intact host.

Insulin-Like Growth Factor (IGF)-Binding Protein-4 Proteolytic Degradation in Bovine, Equine, and Porcine Preovulatory Follicles: Regulation by IGFs and Heparin-Binding Domain-Containing Peptides

Abstract We recently showed that insulin-like growth factor-binding protein-4 (IGFBP-4) proteolytic degradation in ovine preovulatory ovarian follicles is IGF-dependent and regulated by the heparin-binding domain (HBD) from IGFBP-3 and from connective tissue growth factor (CTGF), heparan/heparin-interacting protein (HIP), and vitronectin. The present study investigated regulation of IGFBP-4 proteolytic degradation in porcine, bovine, and equine ovarian preovulatory follicles. Follicular fluid from such preovulatory follicles contains proteolytic activity, degrading exogenous IGFBP-4. An excess of IGF-I enhanced IGFBP-4 degradation. In contrast, IGFBP-2 or -3 or monoclonal antibodies against IGF-I or -II dose-dependently inhibited IGFBP-4 degradation, and IGF-I or -II reversed this inhibition in a dose-dependent manner. Heparin-binding peptides derived from the C-terminal domain of IGFBP-3 or -5 inhibited IGFBP-4 degradation. Other heparin-binding peptides derived from CTGF, HIP, and vitronectin also inhibited IGFBP-4 degradation, except in porcine follicles. Finally, IGFBP-3 that was mutated in its HBD was less effective at inhibiting IGFBP-4 degradation. Thus, in bovine, porcine, and equine preovulatory follicles, IGFBP-4 proteolytic degradation both depends on IGFs and is inhibited by peptides containing HBD. Overall, these results suggest that during terminal development of follicles to the preovulatory stage in domestic animal species, the increase in IGF bioavailability might enhance IGFBP-4 degradation. In contrast, in atretic follicles, the decrease in IGF bioavailability, resulting partly from the increase in IGFBP-2 (sow, heifer, mare) and IGFBP-5 (heifer) expression would participate in the decrease of IGFBP-4 degradation. In bovine atretic follicles, IGFBP-5 would also strengthen the inhibition of IGFBP-4 degradation by direct interaction of its HBD with the protease. The involvement of other HBD-containing proteins in the modulation of intrafollicular proteases degrading IGFBP-4 remains to be investigated.

Cultured capillary endothelial cells from bovine adipose tissue: A model for insulin binding and action in microvascular endothelium☆

Capillary endothelial cells were cultured from bovine adipose tissue. The endothelial nature of the cultures was documented by characteristic morphology, uniform presence of factor VIII antigen, and uptake of the endothelial cell marker Dil-Ac-LDL. The capillary cell cultures had specific, high affinity binding sites for insulin, demonstrating time and temperature dependence of binding, pH optimum, analog specificity, and inhibition of insulin binding by anti-insulin receptor antibodies. In both subconfluent and confluent cultures, insulin stimulated thymidine incorporation into DNA; significant stimulatory effects of insulin were observed at insulin concentrations of 1 ng/ml with maximal 8- to 10-fold increases at hormone concentrations of 1,000 to 10,000 ng/ml. Because of the ease of routine preparation, cell purity, presence of high affinity insulin binding sites, and insulin-sensitive metabolic responses, we suggest that the bovine capillary endothelial cultures could serve as a model cell system for the detailed study of insulin interactions with capillary endothelial cells.

Interactions of cultured endothelial cells with TGF-β, bFGF, PDGF and IGF-I

Abstract Endothelial cells in culture synthesize the growth factors transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF) and, perhaps, insulin like growth factor I (IGF-I). We have previously demonstrated that IGF-I and PDGF have both high affinity receptors and stimulate glucose and AIB uptake in the microvessel cells under study and that IGF-I, but not PDGF, has similar high affinity receptors in cultured large vessel endothelial cells. In the present study, cultured bovine endothelial cells were exposed to these four growth factors to determine a) their effects on the acute metabolic processes of neutral amino acid (AIB) and glucose uptake and b) their interactions at the endothelial cell surface. In microvessel endothelial cells, each growth factor stimulated AIB and glucose uptake 2–4 fold whereas in large vessel endothelial cells only bFGF stimulated glucose uptake. Each growth factor had specific high affinity binding to the microvessel cells that was not influenced by the presence of the other growth factors. In large vessel endothelial cells, similar high affinity binding was present only for IGF-I and to a lesser degree TGF-β. When cells were exposed to a given growth factor for 18 hours, homologous receptor downregulation was observed, with a maximal 60–95% decrease in surface binding. These findings suggest several potential levels of interaction of the growth factors TGF-β, bFGF, PDGF and IGF-I in cultured vascular endothelial cells.

Interactions of insulin with bovine endothelium

Bovine endothelial cells have been isolated from pulmonary and systemic vessels and grown in culture as primary, passaged and passaged cloned-strains. The cultures were shown to be endothelial in nature on the basis of several endothelial-specific and endothelial-associated traits. Endothelial cells from all sources had specific receptors for insulin in primary culture and after serial passage. Endothelial cells derived from pulmonary arteries and aortas bound 2.5 times more insulin than cells derived from the pulmonary vein. Each endothelial cell type maintained a specific complement of receptors through at least 25 passages in vitro. Coupled with previous findings of insulin receptors on endothelial cells from human umbilical vessels, these data suggest that insulin receptors may be an intrinsic component of all vascular endothelium.

Insulin-Like Growth Factor Binding Protein-4 Proteolytic Degradation in Ovine Preovulatory Follicles: Studies of Underlying Mechanisms1

The regulation of insulin-like growth factor binding protein (IGFBP)-4 proteolytic degradation by insulin-like growth factors (IGFs) has been largely studied in vitro, but not in vivo. The aim of this study was to investigate the involvement of IGFs, IGFBP-2, IGFBP-3, and IGFBP-3 proteolytic fragments in the regulation of IGFBP-4 proteolytic activity in ovine ovarian follicles. Follicular fluid from preovulatory follicles contains proteolytic activity degrading exogenous IGFBP-4. The addition of an excess of IGF-I enhanced IGFBP-4 proteolytic degradation, whereas addition of IGFBP-2 or -3 or monoclonal antibodies against IGF-I and -II dose dependently inhibited IGFBP-4 proteolytic degradation. IGF-I and IGF-II, but not LongR3-IGF-I, reversed this inhibition in a dose-dependent manner. C-terminal, but not N-terminal, proteolytic fragments derived from IGFBP-3 (aa 161–264), as well as heparin-binding domain-containing peptides derived from the C-terminal domain of IGFBP-3 and -5 also induced the inhibition ...

Primary empty sella, galactorrhea, hyperprolactinemia and renal tubular acidosis

Discussed here is a 41 year old woman with galactorrhea associated with the empty sella syndrome and mild renal tubular acidosis. Basal serum prolactin (PRL) levels were normal, but a 24 hour serum PRL secretory profile demonstrated an increased mean PRL concentration. Serum PRL was appropriately suppressed by the administration of L-dopa; however, chlorpromazine stimulation resulted in a blunted serum PRL response. Pituitary luteinizing hormone, follicle stimulating hormone, ACTH and thyroid stimulating hormone levels were normal. Thus, galactorrhea associated with an enlarged sella does not establish the diagnosis of a pituitary tumor, and pneumoencephalography must be performed to exclude the empty sella syndrome.

Vascular Endothelium and Diabetes Mellitus

Diabetes mellitus is a disease complex defined by abnormalities of glucose homeostasis and characterized by a group of chronic complications that affect the function of the eyes (retinopathy), kidneys (nephropathy), peripheral nerves (neuropathy), large blood vessels (atherosclerosis), and microvessels (microangiopathy). Indeed, the vascular dysfunction(s) in diabetes may underlie all of the chronic complications of the disease. These complications account for the vast majority of morbidity and mortality that accompanies diabetes, with atherosclerosis underlying the susceptibility to myocardial infarction and stroke, and the microvascular disease underlying retinopathy, nephropathy, and, perhaps, neuropathy. Diabetes mellitus is divided into two general categories, type I [insulin-dependent diabetes mellitus (IDDM)] and type II [non-insulin-dependent diabetes mellitus (NIDDM)]. The general characteristics of type I and type II diabetes are outlined in Table 1. Both type I and type II patients have similar complications, although the frequency of each complication differs between the two groups, with type I patients having a much greater incidence of microvascular complications (nephropathy and retinopathy) and type II patients having a somewhat higher frequency of microvascular complications that are associated with atherosclerosis. Although the precise etiologies of type I and II diabetes, as well as the causes of the diabetic complications, are not known, abnormalities of the vascular endothelium have been shown to occur early in the disease and have been implicated in the pathogenesis of chronic complications in this complex illness.