Reduction of embryotoxicity by protein in embryo culture media.

Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media. Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free. The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure. Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture. Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples. Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentration, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.     

Reduction of rapid eye movement (REM) sleep by glucose alone or glucose and insulin in rats

Administration of a high dose of glucose (2.5 g/kg, i.p.) that is known to produce severe hyperglycemia in euglycemic rats suppressed rapid eye movement (REM) sleep time significantly during the first three hours of 8 hr total electroencephalogram (EEG) recording period. Co-administration of glucose (2.5 g/kg, i.p.) and a non-convulsive dose of insulin (1.0 I.U./kg, i.p.) produced a significant reduction in REM sleep time during 1st through 5th hour and an increase in slow-wave sleep (NREM) time in the 3rd and 4th hour of 8 hr total EEG recording period. However, awake, NREM and REM sleep time in the 8 hr total EEG recording period were unaffected by either glucose alone or glucose plus insulin treatments. These results strongly suggest that the insulin's effects on the sleep-awake cycle i.e. reduction in REM and a slight increase in NREM sleep times of rats is not due to indirect effects of insulin on the central nervous system via hypoglycemia as reported by us previously, but could possibly be due to its direct effects on brain chemistry of neurotransmitters such as serotonin, catecholamines and acetylcholine which are believed to modulate the sleep-awake cycle pattern in rats.     

Influence of temperature on insulin secretion induced by different concentrations of glucose]

Lowering of the temperature from 37.5 degrees C to 28 degrees C provokes a decrease in the response of the beta cell to the stimulation by glucose (1.5 g/1, 3 g/1 and 5 g/1). The insulin secretion obtained at 28 degrees C, compared to that obtained at 37.5 degrees C, is weaker for strongly stimulating concentrations (3 g/1 and 5 g/1) than for a slightly stimulating concentration (1.5 g/1).     

Reduction of insulin-stimulated glucose uptake by peroxynitrite is concurrent with tyrosine nitration of insulin receptor substrate-1

Inducible nitric oxide synthetase plays an essential role in insulin resistance induced by a high-fat diet. The reaction of nitric oxide with superoxide leads to the formation of peroxynitrite (ONOO-), which can modify several proteins. In this study, we investigated whether peroxynitrite impairs insulin-signalling pathway. Our experiments showed that 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), a constitutive producer of peroxynitrite, dose-dependently inhibited insulin-stimulated glucose uptake. While SIN-1 did not affect the insulin receptor protein level and tyrosine phosphorylation, it reduced the insulin receptor substrate-1 (IRS-1) protein level, and IRS-1 associated phosphatidylinositol-3 kinase (PI-3 kinase) activity. Although SIN-1 did not induce Ser307 phosphorylation of IRS-1, tyrosine nitration of IRS-1 was detected in SIN-1-treated-Rat1 fibroblasts expressing human insulin receptors. Mass spectrometry showed that peroxynitrite induced at least four nitrated tyrosine residues in rat IRS-1, including Tyr939, which is critical for association of IRS-1 with the p85 subunit of PI-3 kinase. Our results suggest that peroxynitrite reduces the IRS-1 protein level and decreases phosphorylation of IRS-1 concurrent with nitration of its tyrosine residues.     

Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations

Glucose transport activity was found to increase over 5 h in rat epitrochlearis muscle in response to a moderate concentration (50-100 microunits/ml) of insulin. This process was examined using 3-methylglucose. The increase in permeability to 3-methylglucose was 2- to 4-fold greater after 5 h than after 1 h in muscles incubated with 50 microunits/ml of insulin and 1 or 8 mM glucose. The increase in permeability to 3-methylglucose during the period between 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mM glucose was due to an increase in the apparent Vmax of sugar transport. There were two components to this activation of glucose transport. One, which was not influenced by inhibition of protein synthesis, resulted in activation of sugar transport to the same extent by 50 microunits/ml as by 20,000 microunits/ml of insulin; however, this activation took approximately 20 times longer with 50 microunits/ml insulin. The other, which was blocked by cycloheximide, resulted in a further activation of sugar transport to a level higher than that attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar transport during the first hour, but a high concentration (20-36 mM) of glucose prevented the further activation of glucose transport during prolonged treatment with 50 microunits/ml of insulin. It appears from these results that prolonged exposure to a moderate concentration of insulin has previously unrecognized effects that include: a progressive activation of glucose transport over a long time that eventually results in as great a response as a "supramaximal" insulin concentration, and in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport.     

Effect of glucose and of insulin on the fast induced decrease of insulin response to glucose of the in vitro perfused rat pancreas]

A 24 hours fast decreases by 50% the first and second phases of insulin response to glucose by the isolated and perfused rat pancreas, while the response to tolbutamide remains unchanged. Intra peritoneal administration of low doses of glucose (0.3 g), four times during fasting restored the insulin response. Administration of insulin (0.25 U x 4). Tolbutamide (1.25 mg x 4) or L-leucine (0.1 g x 4), did not. These results show that an exogenous or endogenous insulin impregnation is not the factor responsible for the maintenance of the B cell gluco-receptor during fasting.     

Regulation of glucose transport by insulin and non-hormonal factors

Glucose uptake by nucleated cells is mediated by facilitated diffusion. In adipocytes, fibroblasts and muscle fibers uptake is regulated by a variety of hormones, environmental factors, and metabolic conditions. Glucose uptake by mammalian red cells also occurs by facilitated diffusion, but is not regulated by the same factors and conditions as in nucleated cells; yet the pharmacological and selectivity properties of this transport system resemble those of glucose uptake in regulated cells. The glucose transporter in the human red cell is a 55, 000 dalton protein, which has been purified to homogeneity and functionally reconstituted in artificial systems. Little is known about the molecular identity of the sugar carrier in other cell types. Glucose uptake is stimulated by insulin in muscle, fat and skin cells but not in bone, brain, placenta, erythrocytes nor probably lymphocytes. In responsive cells, stimulation occurs within seconds of exposure to the hormone; it requires cellular integrity but once elicited, it persists in isolated membranes; protein synthesis is not required for either the onset of the response or the return to basal conditions after hormone removal; on the other hand, intracellular energy is required for both steps; the cytoskeleton does not seem to be involved in the regulation of glucose uptake by insulin. In general, insulin increases V_t while K_t is unaffected. The hormone could affect the rate of turnover of the transporter in the membrane, and/or the number of transporters active at any time. An increase in the number of transport sites in the plasma membrane, due to incorporation of additional sites originating from intracellular membranes, has recently been proposed on the basis of both ³H-cytochalasin B binding and glucose transport determinations in isolated plasma and intracellular membranes. The feasibility and implications of a rapid and reversible translocation of glucose transport sites from specific intracellular pools to the plasma membrane are discussed.     

Activation of the glucose transporter GLUT4 by insulin.

The transport of glucose into cells and tissues is a highly regulated process, mediated by a family of facilitative glucose transporters (GLUTs). Insulin-stimulated glucose uptake is primarily mediated by the transporter isoform GLUT4, which is predominantly expressed in mature skeletal muscle and fat tissues. Our recent work suggests that two separate pathways are initiated in response to insulin: (i) to recruit transporters to the cell surface from intracellular pools and (ii) to increase the intrinsic activity of the transporters. These pathways are differentially inhibited by wortmannin, demonstrating that the two pathways do not operate in series. Conversely, inhibitors of p38 mitogen-activated protein kinase (MAPK) imply that p38 MAPK is involved only in the regulation of the pathway leading to the insulin-stimulated activation of GLUT4. This review discusses the evidence for the divergence of GLUT4 translocation and activity and proposed mechanisms for the regulation of GLUT4.     

Metal ion- and phosphate-mediated transport of glucose by insulin.

Insulin, in the presence of Mg²⁺ and Pi, can transport D-glucose across a bulk phase separating two aqueous phases. All three molecular species (Mg²⁺, Pi, D-glucose) are transported simulataneously in 1:1:1 stoichiometry. The same system will transport D-galactose and L-arabinose, but not L-glucose, D-arabinose, D-mannitol, D-fructose and 3-0-methyl glucose. Phloridzin completely suppresses transport, not only of glucose, but also of Mg²⁺ and Pi. Other divalent metal ions are less efficient (Mg²⁺ >Mn²⁺ >Ca²⁺ >Zn²⁺). The capability of insulin for transport of D-glucose is not duplicated by proinsulin or glucagon. Amino acids and citric cycle substrates are also transported, some as rapidly as D-glucose. Pi is replaceable by phosphate esters such as AMP, ADP and ATP, less efficiently with Mg²⁺, but more efficiently with Ca²⁺ as metal ion. The transport of D-glucose in the systems formed by insulin, Ca²⁺ and nucleotide is less sensitive to phloridzin than the standard Mg²⁺, Pi system.