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Insulin-like growth factor-binding protein-1 (IGFBP-1) is one of six soluble binding proteins that regulate the actions of the insulin-like growth factors (IGFs). Liver is the major source of IGFBP-1 in non-pregnant humans. In normal physiology, IGFBP-1 transcription is potently inhibited by insulin and serum levels are limited by a rapid clearance rate. Elevated levels of IGFBP-1 in liver disease have been attributed to insulin resistance; however, the relationships between these analytes have not been defined. We studied insulin, proinsulin and IGFBP-1 in normal subjects (NL, N=47, 43+/-12 yr), cirrhosis (CIR, N=29, 54+/-14 yr), hepatocellular carcinoma (HCC, N=42, 61+/-11 yr), and other liver tumors (TUM, N=8, 60+/-17 yr). All three analytes were significantly increased in liver disease (mean+/-SEM; p-values relative to normals): IGFBP-1 (NL 24+/-4 ng/ml; CIR 235+/-53, p<0.0001; HCC 505+/-105, p<0.0001; TUM 118+/-36, p<0.0001), insulin (NL 72+/-4 pM; CIR 261+/-62, p<0.0002; HCC 180+/-25, p<0.0001; TUM 189+/-58, p<0.0001), proinsulin (NL 6.5+/-0.7 pM; CIR 36.8+/-7.7, p<0.0001; HCC 26.2+/-3.8, p<0.0001; TUM 32.1+/-9.7, p<0.0001). The ratio of proinsulin to insulin was also significantly elevated in liver disease. A typical curvilinear inverse relationship of insulin and IGFBP-1 was observed, but was shifted several fold higher for the liver disease groups. Our results demonstrate that insulin and proinsulin are elevated in liver disease. However, these elevations are paradoxically accompanied by elevated IGFBP-1 levels, indicating disruption of normal regulatory mechanisms. IGFBP-1 is postulated to play a dynamic role in metabolic substrate utilization via regulation of free IGF. Therefore, inappropriate elevation of IGFBP-1 could play an important role in the metabolic disturbances associated with liver disease.

Evidence suggests that magnesium (Mg) deficiency may play a key role in cardiovascular disease. In particular, Mg deficiency may lead to a potentiation of platelet aggregation. However, the factor(s) regulating intracellular-free Mg concentration ([Mg2+]i) in platelets is not known. We studied the effects of insulin on the changes of [Mg2+]i in human platelets. Preincubation of hirudinized platelet-rich plasma with insulin had a dose- and time-dependent effect on the increase of [Mg2+]i measured in Mag-fura-2-loaded cells with a fluorescence spectrophotometer. The maximal effect was achieved by incubation with 200 microU/mL insulin for 30 min. [Mg2+]i increased from the basal value of 266 +/- 23 mumol to 355 +/- 46 (SD, P < 0.001). In the presence of an antiinsulin receptor monoclonal antibody the effect of insulin was abolished suggesting that the Mg transport mechanism was an insulin-receptor mediated process. Furthermore, the insulin-stimulated Mg transport was inhibited by the addition of chelating agent ethylenediaminete-traacetate while the receptor binding was not altered. These findings suggest that insulin can translocate Mg from the extracellular space. Insulin alone had no effect on the changes of intracellular calcium (Ca) concentration using Ca-Fura-2 as a probe. In addition, glucose (5 mg/mL) was not effective in altering either the Mg or Ca concentration. Insulin (100 microU/mL) decreased thrombin-induced platelet aggregation (washed platelets resuspended in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-Tyrode buffer). Similarly, the production of the proaggregatory eicosanoids thromboxane B2 was inhibited by insulin from 16 +/- 1 ng/10(8) platelets to 13 +/- 2 (P < 0.05). The results suggest that insulin, through the interactions with its receptors may be a key factor regulating, Mg transport in human platelets.

In rats, a moderately hepatotoxic single dose of diethylnitrosamine (DEN) 100 mg/kg causing depletion of liver glycogen, elevation of aspartate aminotransferase and decreased liver uptake of 3-O-methylglucose, resulted in substantial changes in insulin and glucagon balance. Two days after DEN, insulin binding to liver membranes and insulin removal by the liver were sharply reduced whereas its binding to muscle and adipocyte membranes remained unaltered. Serum insulin (random and after an overnight fast) remained normal. Intravenous (I.V.) insulin (10 U/kg) caused the usual degree of hypoglycemia that, however, lasted longer than in the control animals. Removal of glucagon by liver was also depressed in spite of its normal binding to hepatocytes, and peripheral serum glucagon was increased three-fold. I.V. glucagon (40 micrograms/kg) resulted in a blunted response of plasma glucose. I.V. glucose tolerance test (1 g/kg) remained normal in spite of the insulin increase to a level twice as high as in the controls, and in spite of nonsuppressed glucagon. These changes were still present after 1-3 months, but disappeared by 6 months. The results demonstrate remarkable ability of homeostatic mechanisms to preserve normal plasma glucose and glucose tolerance in spite of dramatic changes in insulin and glucagon.

The comparative effects of insulin and ethanolamine on 14CO2 production and lipid synthesis from [U-14C]-D-glucose in isolated rat adipocytes were studied. Ethanolamine (10 mM) increased 14CO2 production (glucose oxidation) about 5-fold and lipogenesis about 3-fold as compared to the control. Ethanolamine was more efficient than 25 microU/ml insulin regarding both parameters, but it was less efficient than 200 microU/ml insulin in glucose oxidation, and equally potent in lipogenesis. The combination of ethanolamine and insulin was more active than insulin alone. The mechanisms of ethanolamine action include facilitation of glucose transport and increase of pyruvate dehydrogenase activity.

51CrCl3 was added to the incubation medium of Saccharomyces cerevisiae for up to 48 hr. After repeated freezing and thawing, lysing in 9 M urea with 1% NP-40 detergent, and dialysis against water, the lower molecular weight (Mr less than 3500) dialysate was retained on a SE53 cationic exchange column, eluted with 0.25 M NH4OH and fractionated on a Bio-gel P-2 column. The insulin-like biological activity of the fractions was measured by the 14C-glucose oxidation in isolated rat adipocytes. The biological activity that was found in two of nine fractions did not correspond to their chromium content. Moreover, identical findings were obtained when chromium was added not to the live yeast but to the yeast extract, which showed that its binding was a chemical process not requiring cellular activity. No fraction demonstrated insulin-potentiating activity on rat adipocytes.

Experimental chemical hepatocellular carcinomas that were induced in male F344 rats using three different regimens of limited exposure to the carcinogens 2-acetylaminofluorene or diethylnitrosamine were characterized by very low (as compared to peritumorous or normal tissues) binding of epidermal growth factor and decreased autophosphorylation of the epidermal growth factor receptors. Similar changes were also found in the insulin receptors. We suggest that the carcinogens 2-acetylaminofluorene and diethylnitrosamine cause an initial chemical effect on the great majority of cells. Most of them with time recover their receptor function, and only a small minority become truly initiated and retain these changed characteristics up to the tumor stage. The observed changes appear to be associated with the altered growth state induced by chemical carcinogens. Simultaneous changes observed in the two receptors raise the possibility of a common underlying mechanism.

MCF-7 cells were cultured either in RPMI-1640 medium containing 10% fetal calf serum (FCS) or in a serum-free (SF) medium supplemented with insulin, epidermal growth factor (EGF) and transferrin. Binding studies were performed with 125I-insulin or 125I-EGF. In the FCS containing culture, down-regulation was seen for insulin receptors (47%), and for the EGF receptors (75%). Using cells grown in the serum-free medium we could not demonstrate down-regulation of the insulin receptors, while the EGF receptors were down-regulated to the same extent (74%). The number of binding sites per cell was about twice as much in cells cultured in FCS as that in SF medium. No significant differences were observed for receptor affinity of insulin or EGF in cells grown in both media. The rate of internalization of insulin or EGF into cells was similar in both culture conditions. The mechanism in which only EGF but not insulin demonstrated receptor down-regulation in SF medium remains unknown.

The effects of ethanolamine on insulin secretion by the perfused rat pancreas were examined. During the second phase of glucose-induced insulin secretion 5-minute perfusions of ethanolamine at final concentrations of 0.1, 1 and 10 mM inhibited insulin release in a dose-related manner. When given throughout the experiment the highest dose of ethanolamine markedly suppressed both phases of glucose-induced insulin release. The inhibitory effect of ethanolamine was blunted in the presence of phentolamine. It is concluded that ethanolamine inhibits glucose-induced insulin secretion by the perfused rat pancreas and that alpha-adrenergic receptors play a role in its actions on insulin output.

Using the isolated pancreas of male Sprague-Dawley rats, three different concentrations of glucose were tested in the perfusion medium: 4.0, 6.7, and 9.4 mM. The insulinotropic effects of several potent secretagogues were examined with and without a 10(-7)-M corticosterone-21-acetate (CS) background. When the perfusion medium contained 4.0 mM glucose, there was no insulin secretion in the basal state, but 10(-6)M carbamylcholine chloride, 5 mM L-leucine, and 5 mM calcium chloride elicited moderate but sharp responses of insulin output. When the secretagogues were superimposed on CS infusion, their stimulatory effects were abolished. When the perfusion medium contained 6.7 mM glucose, a steady insulin secretion was observed. A 4- to 5-fold stimulation of insulin release was elicited by carbamylcholine, leucine, and calcium. With CS in the perfusate, the secretagogues were ineffective. However, at a higher glucose level (9.4 mM), the stimulants could partially escape the inhibitory effects of CS. It is concluded that CS may play a role in the regulation of insulin output by directly modulating the activities of some potent secretagogues.

Polyethylene glycol (PEG) and killed Staphylococcus aureus cells (S. aureus) were used as agents to separate free insulin from antibody-bound insulin in diabetic sera. Insulin was determined by conventional double antibody radioimmunoassay. The free insulin values after PEG treatment were almost half of those after S. aureus treatment. The free insulin levels in high-antibody containing sera preincubated at 37 degrees C, 2 h were double the value of fresh sera. PEG treatment caused about 40% loss of total serum protein. The addition of bovine serum albumin (BSA) to the PEG-treated serum greatly increased the immuno-reactive insulin values. This may suggest that protein concentration plays a role in insulin radioimmunoassay. PEG treatment may also enhance the interaction between free insulin and free antibodies resulting in underestimation of free insulin level.