The 3-Phosphorylated Phosphoinositide Response of 3T3-L1 Preadipose Cells Exposed to Insulin,Insulin-Like Growth Factor-1, or Platelet-Derived Growth Factor

We compared the pattern of 3-phosphorylated phosphoinositides produced by confluent 3T3-L1 preadipose cells upon exposure to growth factors that either induce differentiation (insulin, insulin-like growth factor-1) or do not (platelet-derived growth factor). Following addition of insulin or insulin-like growth factor-1, PI(3,4,5)P3 rapidly rose, on average, to levels tenfold over basal. PI(3,4)P₂ either did not change (after insulin) or slightly increased (1.5 fold). Time course studies with insulin demonstrated that the rise in PI(3,4,5)P₃ peaked by 1 minute, and levels then remained steady over 30 minutes. Dose-response experiments showed that insulin at a concentration of 1 nM was sufficient for the PI(3,4,5)P₃ response. Insulin failed to increase PI(3,4)P₂ at any of the time points or at any of the doses used. In contrast, after addition of platelet-derived growth factor, both PI(3,4)P₂ and PI(3,4,5)P₃ rose concurrently and to comparable extents. These data suggest that one possible mechanism contributing to insulin/insulin-like growth factor-1-induced 3T3-L1 preadipose cell differentiation is a distinct pattern of 3-phosphorylated phosphoinositide accumulation, defined by a prominent increase in PI(3,4,5)P₃ with no (in the case of insulin), or a minimal (in the case of IGF-1), rise in PI(3,4)P₂.     

Antihypertensive effects of captopril without adverse effects on glucose tolerance in hyperinsulinemic rhesus monkeys

The rhesus monkey (Macaca mulatta), which has been found to develop spontaneous obesity, non-insulin dependent diabetes mellitus (NIDDM; Type 2), and hypertension, was used to evaluate the potential blood pressure-lowering effects of captopril as well as the specific effects, if any, on the prediabetic state. Intravenous and oral glucose tolerance testing was carried out with oral captopril dosing. Results showed that captopril significantly decreased both systolic and diastolic blood pressure in all monkeys and significantly decreased fasting plasma glucose levels. Based on these preliminary studies in monkeys, we conclude that captopril exerted antihypertensive effects without adverse effects on glucose metabolism.     

Immunoneutralization of insulin partially modifies dorsoventral patterning in developing frog embryos

Determination of anteroposterior and dorsoventral axes is an important early event in the development of vertebrates involving extensive cellular interactions including inductive events. Recently we showed that insulin plays an essential role in prepancreatic development of the frogMicrohyla ornata. In the present study we have investigated the effects of immunoneutralization of endogenous insulin on the process of pattern formation. Treatment of neurulating embryos with antiserum to insulin caused abnormal pattern formation. The defects included loss of normal architecture of the neural tube, reduction in the size of the neural tube and, most conspicuously, rotation of the dorsoventral axis of the neural tube, notochord and adjoining mesodermal elements. The effects could be alleviated partially by pretreatment of embryos with exogenous insulin. This supports our belief that insulin plays an important role in induction and pattern formation of the amphibian nervous system. In addition, using 2-deoxy-α-D-glucose, an inhibitor of glucose metabolism, it is shown that the stimulatory effects of exogenous insulin on developing frog embryos are, at least partially, through the glucose metabolism pathway. Preliminary results of this study were presented at the National Symposium on Genes and Human Environment, held at Hyderabad, February 1994 and DAE Symposium on Stress and Adaptive Responses in Biological Systems, held at Vadodara, March 1994.     

Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Adenosine effects upon insulin action on lipolysis and glucose transport in human adipocytes

The dose response effect of a new adenosine analogue, GR 79236 (N-[1S trans-2-hydroxycyclopentyl] adenosine) upon insulin sensitivity was examined in human adipocytes. The influence of adenosine upon insulin sensitivity for suppression of lipolysis and stimulation of glucose transport was examined. Removal of adenosine by use of adenosine deaminase stimulated lipolysis to the same extent as did 10^–9 M noradrenaline. GR79236 brought about dose dependent inhibition of lipolysis with half-maximal effect at 11.3±7.8×10^–9 M. When lipolysis was stimulated by noradrenaline alone the subsequent inhibition of lipolysis brought about by GR79236 was significantly greater than that of insulin. To examine adenosine effects on the insulin signalling pathway separately from those on lipolysis, the insulin sensitivity of glucose transport was examined. Removal of adenosine brought about a small but significant increase in the concentration of insulin required for half-maximal stimulation of glucose transport. Adenosine agonists offer promise as new agents for the modulation of metabolism in diabetes and other states of insulin resistance.     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Modulation of insulin action by vanadate: evidence of a role for phosphotyrosine phosphatase activity to alter cellular signaling

A number of vanadium compounds (vanadate, vanadyl sulfate, metavanadate) have insulin-mimicking actions bothin vitro andin vivo. They have multiple biological effects in cultured cells and interact directly with various enzymes. The inhibitory action on phosphoprotein tyrosine phosphatases (PTPs) and enhancement of cellular tyrosine phosphorylation appear to be the most relevant to explain the ability to mimic insulin. We demonstrated that in rat adipocytes both acute insulin effects, e.g. stimulation of IGF-II and transferrin binding and a chronic effect, insulin receptor downregulation, were stimulated by vanadate. Vanadate also enhanced insulin binding, particularly at very low insulin concentrations, associated with increased receptor affinity. This resulted in increased adipocyte insulin sensitivity. Finally vanadate augmented the extent of activation of the insulin receptor kinase by submaximal insulin concentrations. This was associated with a prolongation of the insulin biological response, lipogenesis, after removal of hormone.In conclusion: in rat adipocytes vanadate promotes insulin action by three mechanisms, 1) a direct insulin-mimetic action, 2) an enhancement of insulin sensitivity and 3) a prolongation of insulin biological response. These data suggest that PTP inhibitors have potential as useful therapeutic agents in insulin-resistant and relatively insulin-deficient forms of diabetes mellitus.     

The relationship between insulin and vanadium metabolism in insulin target tissues

Vanadium (V) is an orally effective treatment for diabetes, but relatively little is known about the mechanisms controlling its normal metabolism nor the long term pharmacokinetics of oral administration. We have examined the accumulation of V in various organs from rats fed liquid diet for up to 18 days, containing no additional V, 1.6, 80, or 160 mole/kg/day as either sodium orthovanadate (SOV) or vanadyl sulfate (VS). V content was assayed using a sensitive neutron activation analysis method. The organs of the nonsupplemented animals contained widely varying concentrations (ng of V/g dry tissue weight) with brain < fat < blood < heart < muscle < lung < liver < testes < spleen < kidney. All organs accumulated V in a dose dependent manner. Not all organs showed steady state amount of V at 18 days, so additional rats were fed SOV or VS, switched to control diet, and assayed at 0, 4 and 8 days. From this data we calculated organ half lives of V. Insulin sensitive tissue tissues, such as liver and fat, had shorter half-lives than tissues that are relatively less insulin sensitive, such as spleen, brain and testes. SOV and VS fed rats showed similar patterns, but VS had somewhat shorter t1/2's. Additional studies of old and young rats fed control diet for 45 days show accumulation of V in spleen and testes. These results indicate that vanadium metabolism varies widely among different organs, and that insulin, either directly or indirectly has effects on the retention of vanadium. This may have impact on the therapeutic use of vanadium in Type I diabetics with no insulin, or Type II patients who may be relatively hyperinsulinemic.     

Evidence for selective effects of vanadium on adipose cell metabolism involving actions on cAMP-dependent protein kinase

The insulin-like effects of vanadiumin vivo are likely to be achieved at micromolar concentrations. Demonstrated effects of vanadium on adipose tissue of streptozotocin-diabetic rats include inhibition of basal and stimulated rates of lipolysis and effects on fat cell protein phosphorylation. The studies described below examined the effects of vanadium (to a maximum concentration of 0.5 mM) on adipose cells or tissuein vitro. Vanadium, added as a vanadyl-albumin complex or as sodium orthovanadate, produced a marked (greater than 50%) inhibition of isoproterenol-stimulated lipolysis. Inhibition of lipolysis equivalent to that seen with insulin, was achieved with 100 M vanadium. In contrast, no insulin-like stimulation ofde novo fatty acid biosynthesis was observed with vanadium below 0.5 mM. Surprisingly, the antilipolytic effects of vanadium persisted in the presence of cilostamide, an inhibitor of the insulin-sensitive isoform of cyclic nucleotide phosphodiesterase. Studies with purified preparations of the catalytic subunit of cyclic AMP-dependent protein kinase revealed dose-dependent inhibition with vanadyl-glutathione (to a maximum of 40% inhibition). Equivalent inhibition of cyclic AMP-dependent phosphorylation of Kemptide (50%) was observed upon incubation of freshly-prepared fat-pad supernatant fractions with vanadyl-glutathione. These results suggest that effects of low concentrations of vanadium may be mediated, at least in part, by actions on the catalytic subunit of cyclic AMP-dependent protein kinase.     

Expression of hepatic calcium-binding protein regucalcin mRNA is elevated by refeeding of fasted rats: Involvement of glucose,insulin and calcium as stimulating factors

The effect of refeeding on the expression of Ca²⁺-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.