Glucose Metabolism In Fat Cells Research Articles

Insulin produces a growth hormone-like increase in intracellular free calcium concentration in okadaic acid-treated adipocytes.

In vivo, GH and insulin usually produce opposing effects on carbohydrate and lipid metabolism in adipocytes, even though their signal transduction pathways overlap. However, when added to rat adipocytes that have been made GH deficient, GH briefly produces responses that are qualitatively like those of insulin. Subsequently, GH induces refractoriness to this acute insulin-like response, in a sense restricting its effects to a unique subset of possible physiological actions. Okadaic acid is an inhibitor of type I and IIa phosphoprotein phosphatases and affects glucose metabolism in fat cells in a manner that is reminiscent of GH. Okadaic acid initially mimics the actions of insulin, and subsequently, even after it has been removed by thorough washing, blunts the ability of adipocytes to accelerate glucose metabolism in response to insulin or GH. Because refractoriness to the insulin-like effect of GH is associated with GH-induced increases in intracellular free calcium concentrations ([Ca2+]i), we examined the effects of insulin on [Ca2+]i in okadaic acid-treated adipocytes. Adipocytes were incubated with 0.25 microM okadaic acid for 1 h, washed, and reincubated without okadaic acid for 2 h before measurement of [Ca2+]i using fura-2 as a calcium indicator. Neither GH (500 ng/ml) nor insulin (100 microU/ml) affected [Ca2+]i in cells in which glucose metabolism was stimulated, but both hormones rapidly increased [Ca2+]i in adipocytes that were refractory to insulin-like stimulation. The characteristics of the increase in [Ca2+]i produced by insulin were identical to those previously reported for GH. The effect of insulin was mimicked by the dihydropyridine calcium channel activator BayK 5552 or depolarization of the cell membrane with 30 mM KCl and was blocked by the dihydropyridine calcium channel blocker, nimodipine (100 nM), implicating activation of voltage-sensitive L-type Ca2+ channels. The increase in [Ca2+]i was also mimicked by sn-1,2-dioctanoylglycerol and blocked by inhibitors of protein kinase C (staurosporine, chelerythrine chloride, and calphostin), and D609, an inhibitor of phospholipase C, as reported for GH. Acquisition of the ability to increase [Ca2+]i in response to insulin required a lag period of at least 2 h after removal of okadaic acid and was prevented by inhibitors of RNA and protein synthesis. Adipocytes that were incubated with inhibitors of protein kinase A (KT-5720), or protein kinase C (staurosporine) along with okadaic acid also failed to increase [Ca2+]i in response to insulin. Conversely, adipocytes that were incubated with dibutyryl cAMP, methylisobutyl xanthine, or phorbol ester instead of okadaic acid increased [Ca2+]i when treated with insulin 2 h later. These results suggest that phosphorylated substrates of protein kinases A and C may mediate the transcriptional event(s) that enable adipocytes to activate L-type Ca2+ channels in response to insulin. Blockade of protein kinases A or C or removal of calcium from the incubation medium did not restore the ability of okadaic acid-treated adipocytes to increase glucose metabolism in response to insulin, nor did pretreatment of adipocytes with dibutyryl cAMP or phorbol ester decrease insulin-induced stimulation of glucose metabolism. The failure of insulin to increase glucose metabolism in okadaic acid-treated adipocytes thus cannot be ascribed to the increase in [Ca2+]i. These findings indicate that just as GH can produce an insulin-like response, so too can insulin produce a GH-like response, and highlight the need to understand how specificity of hormone action is achieved in cells that respond to different hormones that share elements of their transduction pathways.

The effect of in vivo thyroxine treatment on insulin receptors, glucose transport and GLUT4 in rat adipocytes.

Daily i.p. administration of thyroxine (T4; 50 micrograms/100 g BW) to adult male rats for one week doubled the thyroxinemia and induced significant augmentation of glycemia and insulinemia. Hyperthyroxinemia was also manifested by significant elevation of liver malic enzyme activity and by the presence of smaller fat cells. Adipocytes of T4 treated rats displayed significantly lower density of insulin receptors, decreased insulin stimulatory effect on glucose transport and less glucose transporter (GLUT4) protein in plasma membranes after insulin stimulation as those of controls. These results indicate a deleterious effect of hyperthyroxinemia on insulin controlled glucose metabolism in fat cells.

Increased insulin mediated glucose metabolism in fat cells from I versus C57BL mice.

The purpose of this study was to determine whether adipocytes from I strain mice, which are characterized by a greater in vivo glucose tolerance than most other strains, had a higher capacity to utilize glucose in response to physiological concentrations of insulin. Using C57BL mice as a control strain, we examined the effect of insulin on glucose metabolism in epididymal and inguinal adipocytes from 2-month-old male mice. Body weight was only slightly less (7%) for the I mice than for the C57BL mice, but fat pad sizes were 60 and 20% less for epididymal and inguinal depots, respectively, in the I mice. Fat cell size was also smaller in epididymal adipocytes from the I mice than from the C57BL mice. Fat cell size of inguinal adipocytes was similar in the two strains. Without insulin the rates of [U-14C]glucose incorporation into CO2 or lipids were twofold higher in cells from the I mice than in those from the C57BL mice. Maximal insulin concentration (2.5 nM) increased glucose metabolism by 140 and 500% in epididymal and inguinal adipose cells, respectively, in the I mice versus 30 and 50% in the C57BL mice. The maximal effect of insulin was reached at a much higher insulin concentration in the I mice than in the C57BL mice. The activity of fatty acid synthetase was four- to sixfold higher in fat cells from I than in those from C57BL mice. These results demonstrate an increased insulin responsiveness of glucose metabolism in fat cells from the I mice related to an increased lipogenic capacity. Furthermore, they show that adipose tissue in mice exhibits significant regional differences in terms of insulin responsiveness of glucose metabolism.

Insulin stimulation of glucose metabolism in rat adipocytes: possible implication of protein kinase C.

The hypothesis that insulin stimulation of glucose and lipid metabolism in rat adipocytes may involve the activation of protein kinase C was evaluated. 4 beta-Phorbol 12 beta-myristate, 13 alpha-acetate (PMA, 0.1-1000 ng/ml), a potent tumor promoter acting as a substitute for diacylglycerol to activate protein kinase C, stimulated 2-deoxyglucose transport in a time- and dose-dependent manner, without affecting passive glucose diffusion. PMA (0.1-1000 ng/ml) also elicited a dose-dependent activation of lipogenesis from [3-3H] glucose. Maximal PMA effects (100 ng/ml) on both processes were 60% of insulin maximal effects. In contrast, PMA (1-1000 ng/ml) failed to mimic the ability of insulin to stimulate lipogenesis from [3H]acetate. 4 beta-Phorbol 12,13 dibutyrate, mezerein, 1-oleyl-2-acetyl-glycerol, 1,2 diolein, known as protein kinase C activators, also markedly stimulated glucose metabolism whereas 4 alpha-phorbol 12,13 didecanoate and 4 beta-phorbol 13-monoacetate, shown not to activate protein kinase C, were ineffective. Mellitin, a cytotoxin-inhibiting protein kinase C, markedly decreased both PMA and insulin stimulation of glucose metabolism. PMA decreased insulin submaximal effects on 2-deoxyglucose transport without inhibiting insulin binding. Maximal PMA and insulin effects on 2-deoxyglucose transport and lipogenesis from [3-3H]glucose were not additive. Both PMA and insulin activated each metabolic process in a calcium-dependent manner. PMA, like insulin, no longer stimulated 2-deoxyglucose transport in fat cells treated with 2,4-dinitrophenol. These data show that PMA exhibited specific insulin-like properties on glucose metabolism in fat cells, without any effect on lipid synthesis from acetate. They indicate that PMA and insulin bioeffects may share a common step implicating a calcium- and energy-dependent process, distal to the initial insulin binding event. Our results suggest that protein kinase C may play a role in insulin regulation of glucose metabolism.

Impaired insulin receptor binding and postbinding defects of adipocytes from normal and diabetic pregnant women.

To evaluate the relative contribution of insulin binding and postbinding defects of glucose utilization in peripheral tissue during normal and diabetic pregnancy, we have studied the in vitro insulin action of isolated adipocytes from eight nondiabetic pregnant women and nine pregnant women with insulin-dependent diabetes mellitus who were undergoing cesarian section. The pregnant women were compared with a matched group of normal nonpregnant women undergoing gynecologic surgery. Insulin binding to adipocytes measured at tracer insulin concentration was reduced by 45% (P less than 0.01) in normal pregnant women and by 30% (P less than 0.02) in pregnant women with diabetes. In contrast, no changes were found between the three groups in insulin binding to pure monocytes and erythrocytes. The glucose transport system in fat cells from both groups of pregnant women was characterized by impaired maximal (P less than 0.05) and half-maximal (P less than 0.05) response to insulin. When fat cell glucose metabolism was studied, pregnant diabetic women exhibited decreased basal lipogenesis (P less than 0.05) and decreased maximal responses of lipogenesis and glucose oxidation to insulin stimulation (P less than 0.05). Similar but less pronounced abnormalities were seen in glucose metabolism of adipocytes from nondiabetic pregnant women. In conclusion, both in late normal and diabetic pregnancy, insulin binding to adipocytes is significantly reduced and accompanied by decreased insulin sensitivity and reduced maximal insulin responsiveness of glucose transport and by impaired basal and maximally insulin-stimulated glucose metabolism.

Trifluoperazine inhibits insulin action on glucose metabolism in fat cells without affecting inhibition of lipolysis.

One of the specific inhibitors of calmodulin action, trifluoperazine, blocked the stimulating action of insulin on 2-deoxyglucose uptake and glucose metabolism. The inhibitory effect of insulin on lipolysis was not altered by the drug. The active (insulin-stimulated) state and the basal state of lipogenesis were inhibited half-maximally at 80 and 550 microM trifluoperazine, respectively. 2-Deoxyglucose uptake was inhibited half-maximally at a trifluoperazine concentration of 70 microM. Other less potent calmodulin inhibitors also inhibited glucose metabolism in fat cells but in a nonspecific manner. The inhibition was noncompetitive and was not altered in Ca2+- free medium. The stimulating activity of wheat germ agglutinin and of sodium vanadate were also inhibited by trifluoperazine. The dose-dependent inhibitions were indistinguishable whether the active (stimulated) state was produced by insulin, wheat germ agglutinin, or vanadate. The data indicate that a late event in the sequence that ultimately leads to enhanced glucose transport activity in fat cells is specifically inhibited by trifluoperazine. The possible involvement of calmodulin or another related Ca2+-dependent regulatory protein in the exocytic (fusion) reaction that recruits glucose-transport activity from storage sites to the plasma membranes is discussed.

Variations in insulin responsiveness in rat fat cells are due to metabolic differences rather than insulin binding.

Insulin resistance was studied by comparing insulin response and insulin binding in four groups of rats. Glucose metabolism in isolated fat cells from male Wistar rats weighing 340 g was less responsive to a supramaximal dose of insulin than glucose metabolism in fat cells from rats weighing 200 g. Induction of streptozotocin-diabetes in rats weighing 200 g resulted in a marked decrease in the insulin responsiveness of fat cells. Ventromedial hypothalamic lesions of 340 g rats had the opposite effect and restored the insulin responsiveness of fat cells. The responsiveness in the four groups was correlated to the rate of glucose conversion to fatty acids in fat cells. The binding of 125I-insulin was the same in both 340 and 200 g rats. The ventromedial hypothalamic lesioned rats and the diabetic rats showed, in spite of their great difference in insulin responsiveness, the highest binding of 125I-insulin to fat cells. Insulin binding was not correlated to the plasma insulin level which however was reflected in the lipoprotein lipase activity in the adipose tissue. In conclusion, these results indicate that variations in insulin responsiveness in fat cells are due to alterations in cellular metabolism rather than in insulin binding.

Effect of amino acids on insulin-stimulated glucose metabolism in fat cells.

The effect of amino acids on insulin responsiveness in epididymal adipose tissue was examined. It was found that insulin-stimulated glucose oxidation in fat cells was significantly inhibited by glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, lysine, phenylalanine, and proline. The effect of insulin on glucose incorporation into triglyceride is also severely diminished by these amino acids. In addition, alanine reduced the incorporation of precursors ([U-14C]glucose or [1-14C]palmitate) into triglyceride both in vitro and in vivo. The Ki values of alanine were 0.4 and 0.5 mM toward the precursors of glucose and palmitate, respectively. The mechanism of reduction of insulin responsiveness in rat adipose tissue is discussed on the basis of these results.

Cu++-dependent Thiol Stimulation of Glucose Metabolism in White Fat Cells

Abstract EDTA, added either alone or with equimolar concentrations of CaCl2 or MgCl2, was without effect on the increase in fat cell glucose oxidation due to insulin, vitamin K5, menadione, or sucrose hyperosmolarity. In contrast, EDTA markedly inhibited the action of cysteine, glutathione, and an insulin-like factor from serum on fat cell glucose metabolism. The inhibition by EDTA was greater on high concentrations of cysteine (1.5 or 2 mm) than lower concentrations (0.5 or 1 mm). In fat cells incubated in a 3% albumin medium which had been incubated for 30 min with 2 mm cysteine prior to addition of EDTA and cells, EDTA was ineffective in blocking the action of cysteine. The stimulation by cysteine, glutathione, and vitamin K5 of white fat cell glucose oxidation was inhibited in the absence of albumin while the response to insulin or sucrose hyperosmolarity was unaffected. The effect of albumin on the action of cysteine was biphasic. The stimulatory effect of 0.05 or 0.5 mm cysteine was about 3-fold greater than 1.5 mm cysteine in the presence of 0.75% albumin while with 3% albumin present the response of cells to 1.5 and 0.5 mm cysteine was maximal and that to 0.05 mm cysteine was nearly abolished. Dialysis of albumin reduced its ability to support the effect of cysteine as did passage over Sephadex G-50. The stimulation by cysteine and glutathione of glucose metabolism was inhibited by almost 90% if fat cells were incubated in albumin which had been exposed to 3.3 mm phenanthroline and then chromatographed on Sephadex G-50 while the increase due to insulin was diminished by only 25%. Cu++ at 3 x 10-6 and 10-5 m, but not 10-4 m, restored the stimulatory effect of cysteine on fat cell glucose metabolism in the presence of the phenanthroline, Sephadex-treated albumin. These results indicate that the stimulatory effects of cysteine on white fat cell glucose utilization involve a mechanism dependent on the presence of divalent copper.

Glucose metabolism in fat cells stimulated by insulin and dependent on sodium.

SPECIFIC transport of organic and inorganic substances through biological membranes has been well established1. Transport of organic substances, such as amino-acids and sugars, may be active and is, as a rule, linked to the transport of cations2, most frequently those of sodium and potassium3. Other transport mechanisms may agree rather with the definition of facilitated diffusion which seems to be independent of cations4. It is usually thought that in insulin tissues such as muscle and adipose tissue which respond to insulin, this hormone somehow favours the facilitated diffusion of glucose and of certain other sugars5,6. Insulin also facilitates, however, the entry of some substrates which pass through by active transport7, and, furthermore, it has been shown to increase the resting electrical potential of muscle8 and of adipose tissue9, while stimulating decreasing retention of potassium and sodium ions respectively in these tissues10,11. While Hagen12 has reported that insulin has less effect on glucose metabolism by adipose tissue in the absence of sodium or potassium ions, Rodbell13 has observed normal responsiveness to insulin of isolated fat cells in the absence of sodium ions. These conflicting views have encouraged us to investigate the effects of changing the concentration of cations in the medium glucose transport and metabolism by isolated fat cells, both in the presence and absence of insulin. A marked sodium dependency of insulin-stimulated glucose metabolism in fat cells on sodium has been found.