Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis,on inhibition of lipolysis by insulin,and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca²⁺ and Mg²⁺ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca²⁺ + Mg²⁺)-free incubation medium incorporation of L-[¹⁴C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[¹⁴C]-leucine incorporation only in the presence of added CaCl₂ or MgCl₂. Incubation of the cells in the (Ca²⁺ + Mg²⁺)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of ¹²⁵I-labelled insulin to the fat cells was reduced in the absence of Ca²⁺ and Mg²⁺ but was not abolished, even in the presence of EDTA. Ca²⁺ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg²⁺, Sr²⁺ and Ba²⁺.Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes

The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline-stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP.     

Role of extracellular magnesium in insulin secretion from rat insulinoma cells.

Magnesium (Mg2+) is an abundant intracellular cation that participates in the regulation of the intracellular concentration of ATP. In this study, we examined the relationship between insulin secretion and intracellular free Mg2+ ([Mg2+]i) in a rat-insulinoma cell line (RIN m5F), using a fluorescent dye (Mag-fura-2). KCI, forskolin, and D-glyceraldehyde increased [Mg2+]i and insulin secretion from RIN m5F cells in a dose-dependent fashion. Verapamil, a voltage-dependent Ca2+ channel blocker, inhibited the increase of [Mg2+]i that was evoked by KCI, forskolin, and D-glyceraldehyde. In a Mg(2+)-free buffer, these agents failed to cause an elevation in [Mg2+]i; however, the insulin response to KCI and forskolin was enhanced, compared with that in the presence of Mg2+ (1.25 mM). Our findings suggest that [Mg2+]i is dependent upon extracellular Mg2+, and the influx through the voltage-dependent Ca2+ channel. Mg2+ may competitively inhibit the voltage-dependent Ca2+ channel, which is known to play a role in insulin secretion. An absence of Mg2+ in the extracellular space may result in enhanced insulin secretion. [Mg2+]i may play a role in insulin secretion from RIN m5F cells.     

Insulin-stimulated phosphoinositide metabolism in isolated fat cells

Treatment of isolated fat cells with insulin produced increases of up to 4.8-fold in the incorporation of [3H]inositol into phosphatidylinositol. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 30 microunits/ml of insulin. Insulin increased the labeling of phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate but not phosphatidylinositol 4-monophosphate in cells which had been preincubated with [3H]inositol for 90 min. Incubation of the cells in a Ca2+-free buffer increased the basal level of phosphatidylinositol labeling and enhanced the effect of insulin. Glucagon and isoprenaline, both of which stimulate lipolysis, had no effect on phosphatidylinositol labeling but did potentiate insulin-stimulated incorporation of [3H]inositol into phosphatidylinositol. Phosphoinositide breakdown was measured by the accumulation of inositol phosphates. Insulin did not increase the level of the inositol phosphates at all concentrations of the hormone tested. By comparison, phenylephrine and vasopressin were able to stimulate phosphoinositide breakdown. Pretreatment of the cells with insulin enhanced the effect of phenylephrine on inositol phosphates' accumulation, suggesting that insulin may potentiate phenylephrine-mediated phosphoinositide turnover. From these data we conclude that insulin stimulates the de novo synthesis of phosphatidylinositol and phosphatidylinositol 4,5-biphosphate, but has no effect on phosphoinositide breakdown.     

The effect of digitonin of the stimulation by insulin of glucose uptake by isolated fat cells.

The effect of digitonin on glucose uptake by isolated fat cells in the presence and absence of insulin has been studied. At low concentrations of digitonin, the stimulation of glucose uptake by insulin was inhibited without severe cell damage as estimated by the leakage of lactate dehydrogenase from the cells. The inhibition of the insulin effect was not reversed by washing the cells or by the addition of cholesterol or lecithin-cholesterol liposomes to the incubation medium of the cells after treatment with digitonin. Cholesterol was shown to be present in the fat cells and it is suggested that the inhibition of the insulin effect is a consequence of the formation of digitonin-cholesterol complexes in the fat cell plasma membrane. Possible ways in which this may results in inhibition of the effect of insulin are discussed.     

The effect of extracellular Ca and Mg on mitochondrial ultrastructure in isolated intact neurons

The ultrastructural transformations of mitochondria in isolated crayfish neurons were studied after incubation of the cells in saline media containing different Ca2+ and Mg2+ concentrations. Incubation in a 5-fold higher Ca concentration resulted in the swelling of mitochondria that was prevented by the addition of the calcium channel blocker, verapamil. Exposure of the cells to Mg2+-depleted medium induced swelling of all the mitochondria, followed by substantial shrinkage of most of them. The absence of Ca as well as the presence of verapamil in Mg2+-free medium led to the inhibition of mitochondrial swelling and to a strong contraction of the mitochondria after 1 h incubation. The omission of Ca2+ from the saline medium or the addition of Ca2+-ionophore A23187 in the presence of Ca2+ resulted in strong mitochondrial shrinkage. These structural alterations of mitochondria are interpreted as an osmotic response of the inner mitochondrial membranes to changes in their potassium transport, induced by a disturbance in the cellular and mitochondrial Ca2+-Mg2+ homeostasis.     

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.     

Enhancement (by ATP, insulin, and lack of divalent cations) of ouabain inhibition of cation transport and ouabain binding in frog skeletal muscle; effect of insulin and ouabain on sarcolemmal (Na + K)MgATPase.

Using small, intact frog muscles, the basic properties of Na+ and K+ transport were shown to resemble those of the (Na+ + K+)Mg2+ATPase (EC 3.6.1.3) isolated from skeletal muscle. (a) External K+ is essential for Na+ exit and K+ entry after the muscles are Na+-loaded and K+-depleted; (b) the ouabain concentration causing maximum inhibition of recovery is the same for transport as for the inhibition of the isolated enzyme. Ouabain causes a decrease in the sorbitol space and causes muscle fibre swelling. Absence of Ca2+ and Mg2+ inhibits recovery of normal Na+ and K+ concentrations and increases the sorbitol space. Insulin stimulates K+ uptake and Na+ loss in intact muscles but has no effect on the isolated sarcolemmal (Na+ + K+)Mg2+ATPase. Absence of divalent cations, addition of external ATP and of insulin enhance the ouabain inhibition of recovery. Bound ouabain was measured using [3H]ouabain and [14C]sorbitol (to measure the extracellular space). The process of binding was slowly reversible and was saturable within a range of ouabain concentrations from 1.48 X 10(-7) to 5.96 X 10(-7) M. From the nonexchangeable ouabain bound, the density of glycoside receptors was estimated to be 650 molecules per square micrometre of membrane surface. The absence of divalent cations, addition of external ATP and of insulin significantly enhanced the amount of ouabain bound. Substitution of Na+ and K+ by choline greatly reduced the bound ouabain.     

Translation of poly U by ribosomes from rel+ and rel- E. coli strains]

A translation of polyU in a cell-free system from CP78 (relA+) and CP79 (relA-) E. coli strains is investigated. The strains studied no not differ in misreading at Mg2+ concentration of 20 mM and concentrations of 16 mM (optimal for phenylalanine incorporation) and 18-22 mM (optimal for leucine incorporation) respectively. It is found that leucine incorporation increases similarly in both strains under conditions simulating an amino acid deficience (in phenylalanine-free incubation medium): the ratio leucine(-phenylalanine)/leucine (+phenylalanine) is 3.5-4 at a concentration of enzymatic fraction protein being 15-30 mkg per example, and it is 2 st a concentration of enzymatic fraction of 60-180 mkg of protein. It is suggested that differences in activities of a number of enzymes under amino acid starvation in Rel+ and Rel- cells are not due to differences in the precision of mRNA translation, but depend on the activity of respective genes.     

The relationship between extracellular K+ and Ca2+ on aminopyrine accumulation in rat parietal cells.

The effects of extracellular K+ in relation to extracellular Ca2+ on acid production were studied. Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by 14C-aminopyrine (AP) accumulation. In the absence of K+ in the incubation medium histamine-stimulated AP accumulation ratios were significantly decreased independently in the presence or absence of extracellular Ca2+. Under basal conditions, in the absence of extracellular Ca2+, increasing concentrations of extracellular K+ enhanced AP accumulation ratios to significantly higher than those found in the presence of Ca2+. In histamine-, cAMP-, and carbachol-stimulated parietal cells, high K+ concentrations increased AP accumulation significantly less in Ca(2+)-free than in Ca(2+)-containing media. High K+ also induced significantly both an increase in cytosolic free Ca2+ concentration and 45Ca2+ uptake. The present results confirmed the importance of K+ in gastric acid production and suggested a role for Ca2+ as a modulator of mechanisms of parietal cell stimulation.     

Significance of regional difference in ion concentrations in lizard, Hemidactylus flaviviridis (Rüppell): assessment of ionic influence on sperm motility in vitro

A gradual increase in the concentration of Ca2+ from anterior to the posterior region was observed when mono- and divalent cations were estimated in different segments of the epididymis in wall lizard. Na+ and K+ levels increased from anterior to middle segment but declined significantly in the posterior segment. However, no significant difference in the levels of Mg2+ was observed in various segments. To study the influence of mono- and divalent cations on sperm motility in vitro, the spermatozoa from posterior region of the epididymis were incubated in medium with varying concentrations of Na+, K+, Ca2+ and Mg2+. Spermatozoa were non-motile when suspended in Na+-free medium. Addition of NaCl induced the acquisition of sperm motility in a concentration-dependent manner. Further, amiloride, a Na+-influx blocker, markedly reduced the Na+-induced forward progressive motility. Unlike Na+, the presence of K+ or Ca2+ in the incubation medium reduced the motility of spermatozoa even at very low concentrations. The inhibitory effect of Ca2+ decreased when nifedipine, a Ca2+-influx blocker, was added to the medium. Mg2+ at high concentrations only was able to reduce the forward progressive motility.     

Increasing effect of vanadate on lipoprotein lipase activity in isolated rat fat pads

Vanadate increased lipoprotein lipase (LPL) activity in the isolated fat pads in a time- and dose-dependent manner. The increasing effect of vanadate was inhibited by amiloride, similar to that of insulin, and it also was not additive to that of insulin. Although the increasing effects of vanadate and insulin were preserved in K(+)-free medium, appreciable decreases in both effects were observed by replacement of Na+ with choline ion or omission of Ca2+ in the medium. Vanadate showed the full effect in the presence of cycloheximide at concentrations that inhibited protein synthesis of the fat pads, suggesting that the action of vanadate is not due to the increase in protein synthesis. Tetrakis (acetoxymethyl) ester of quin 2 at 50 microM concentration never inhibited the action of vanadate though it showed a little inhibition at a concentration of 300 microM. No inhibition of the action of vanadate was observed with ruthenium red. These results suggest that vanadate increases the LPL activity via a process less sensitive to the intracellular Ca2+ concentration. Adrenaline, dibutyryl cyclic AMP, and 3-isobutyl-1-methylxanthine all inhibited the action of vanadate, suggesting that the action is inhibited with increase in the intracellular concentration of cyclic AMP. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited the action of vanadate. In contrast, the action of insulin was never inhibited by monensin. Tunicamycin and 2-deoxyglucose, at rather high concentrations, inhibited both actions. These findings suggest that vanadate increases the LPL activity through mechanisms of action involving amiloride- and monensin-sensitive pathways dependent on energy.     

Insulin-stimulated phosphorylation of calmodulin by rat liver insulin receptor preparations

Insulin stimulates autophosphorylation of the beta subunit of its receptor and activates the associated tyrosine kinase. This kinase, in turn, phosphorylates a number of specific protein substrates; however, the functional and structural identity of these substrates is largely unknown. In this study, we demonstrate that insulin also stimulates the phosphorylation of calmodulin by rat hepatocyte insulin receptors partially purified by wheat germ agglutinin affinity chromatography. Phosphorylation occurred predominantly on tyrosine residues and had an absolute requirement for insulin receptors, divalent cations, and certain basic proteins. Maximal 32P incorporation was observed at an insulin concentration of 5 X 10(-9) M, and the K0.5 for insulin was approximately 4 X 10(-10) M. Phosphorylation of calmodulin was dependent upon ATP, saturating at 100 microM ATP with a K0.5 of 30 microM. Insulin-stimulated phosphorylation of calmodulin was also dependent upon Mg2+ or Mn2+, but was approximately 12-fold greater in the presence of Mg2+. Maximal phosphorylation was observed in the absence of Ca2+ and was inhibited at Ca2+:EGTA ratios greater than 0.8 (0.16 microM free Ca2+). Certain basic proteins, such as polylysine, histone Hf2b, and protamine sulfate, were necessary to observe insulin-stimulated phosphorylation of calmodulin. The relative amount of insulin-stimulated phosphorylation of calmodulin observed in the presence of each of these proteins differed. Maximal insulin-stimulated phosphorylation was observed in the presence of polylysine. These data suggest that both Ca2+ and calmodulin may participate in the early post-receptor events in the cellular mechanism of insulin action in hepatocytes.     

The effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

Membrane potential modulates divalent cation entry in rat parotid acini

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)     

A significant increase of lysophosphatidylinositol 4-phosphate with insulin in isolated rat fat cells

We studied the effects of insulin on the incorporation of 32Pi into phospholipids in rat fat cells. When the cells were treated with insulin, a new radioactive phospholipid was detected on thin layer chromatography. The substance migrated slower than phosphatidylinositol 4,5-bisphosphate and was hardly detectable in the absence of insulin. This effect of insulin was both time- and dose-dependent with half-maximal stimulation at 120 microU/ml. Pretreatment of insulin with anti-insulin antibody or the cells with anti-insulin receptor antibody inhibited the effect of insulin. The product of phosphatidylinositol 4-phosphate hydrolyzed by phospholipase A2 was coincided with the substance on thin layer chromatography. Quinacrine inhibited the formation of the substance in a dose-dependent manner. These results suggested that insulin stimulates the generation of lysophosphatidylinositol 4-phosphate through the insulin-receptor interaction.