THE JOURNAL OF BPXOCEAL CHEM~~Y

Val 253, No I”, Issue of May 25, pp 3504-3508, 1978 Prvtfed m lJ S A

Ability of Insulin to Increase Calcium Uptake by Adipocyte Endoplasmic Reticulum*

(Received for publication, December 5, 1977)

JAY M. MCDONALD,DAVID E. BRUNS,SANDLEONARDJARETT

From the Division ofLaboratory Medicine, Departments ofPathology and Medicine, Washington University School ofMedicine, and Barnes Hospital, St. Louis, Missouri 63110

The endoplasmic reticulum of adipocytes possess an ATP- dependent calcium uptake system (Bruns, I). E., McDonald, J. M., and Jarett, L. (1976) J. Bid. Chem. 251, 7191-7197) which is shown in this report to be regulated by insulin treatment of intact adipocytes. Insulin (100 microunitslml) treatment of cells resulted in a 20% (p < 0.025 to 0.001 at various time points) increase in the steady state calcium filling capacity of endoplasmic reticulum. This insulin ef- fect was specific as determined by the insulin-concentration dependency between 5 and 100 microunits of insulin/ml and by the lack of effect by desoctapeptide insulin. This increase resulted from a 28% (p < 0.025) increase in the apparent V,,,,, of calcium uptake. Insulin treatment had no affect on the K,,, (1.4 PM) of the reaction nor on the rate of calcium efflux from the vesicles when efflux was initiated by either ethylene glycol bis(/3-aminoethyl ether)N,N’-tetraacetic acid or the calcium ionophore A23187. Consistent with these changes, insulin treatment of cells resulted in a 33% (p < 0.001) increase in the atomic absorption measurements of the total calcium content of the microsomal fraction. The total calcium content of the microsomal fraction consisted of a highly stable pool and a less stable or labile pool. Only the labile pool was altered by insulin treatment, and the insulin-induced change represented a 50% increase of this pool. Insulin treatment had no effect on the magnesium content of the microsomal fraction. These findings support the concept that the calcium uptake system of the endoplas- mic reticulum of adipocytes is likely to be physiologically important. Furthermore insulin regulation of intracellular calcium pools probably plays an essential role in the mech- anism of insulin action.

The role of calcium in the mechanism of insulin action remains unknown. This ion has been proposed as a second messenger for insulin (1, 2), however, no direct experimental evidence supports such a role. On the other hand, there is considerable indirect evidence supporting an important role

* This work was supported by United States Public Health Service Grants AM11892 and RR05389 and a grant from the Juvenile Diabetes Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department of Pathology, University of Vir- ginia, School of Medicine, Charlottesville, Va. 22901.

for calcium in the overall cellular mechanism of insulin action. This evidence includes the numerous intracellular enzymes and enzyme systems which are both insulin- and calcium-sensitive (3-5). Recently, more direct evidence has been reported, demonstrating that insulin treatment of adi- pocytes causes alterations in the subcellular distribution of calcium (6, 7). This includes an increase in calcium binding capacity by the plasma membranes (7) and a shift of stable calcium to a more labile form in mitochondria without altera- tion of the total calcium content of that organelle (6).

Another organelle, the endoplasmic reticulum, may play an important role in regulating intracellular calcium in non- contractile cells. ATP-dependent calcium uptake systems have been demonstrated in the endoplasmic reticulum of kidney cells (81, hepatocytes (91, adipocytes (lo), and cultured fibro- blasts (11). The endoplasmic reticulum of adipocytes has been shown to contain dense calcium deposits by electron micro- scopic x-ray microprobe analysis (12). In addition, the active ATP-dependent calcium uptake system in adipocyte endoplas- mic reticulum appears to be qualitatively analogous to the calcium uptake system in sarcoplasmic reticulum (10). If the calcium uptake system of the endoplasmic reticulum is hor- monally sensitive, then it could provide a potent modulator of cytosol calcium.

The present study investigates the effects of insulin on the active calcium uptake system of the endoplasmic reticulum. The hormonal treatment of the adipocyte causes an increased calcium content of the endoplasmic reticulum resulting from a stimulation of active calcium uptake.

EXPERIMENTAL PROCEDURES

Materials-Male Wistar rats (100 to 200 g) were purchased from National Laboratory Animal Co., O’Fallon, MO. Collagenase (type I) from Clostridium histolvticum. bovine serum albumin (fraction V). and ATP (disodium Sal6 were ‘purchased from Sigma Chemical Co.; St. Louis, MO. Lots of collagenase were selected which yielded a fat cell preparation maximally responsive to insulin as determined by bioassay (stimulation of glucose oxidation) and the albumin was determined to be free of insulin-like activity by immune- and bioassay. Calcium carbonate and magnesium acetate used in the preparation of standards for atomic absorption analysis were from the National Bureau of Standards (reference material #915) and J. T. Baker Chemical Co., respectively. Omnifluor and WaCl, (ap- proximately 1 mCi/pmol) were purchased from New England Nu- clear, Boston, Mass. All other materials were of reagent grade quality and were purchased from standard sources. All reagents were prepared with water deionized by a double-chambered mixed bed ion exchange resin system (Culligan, Inc., Northbrook, Ill.) which was filtered prior to use with a 0.25-pm pore size filter

3504

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Effect of Insulin on Calcium Uptake by Endoplasmic Reticulum 3505

(Ultipor, PTM Corp., Cortland, N. Y.). Resistance was constantly monitored at the outlet. Calcium contents of all reagents were measured by atomic absorption spectrophotometry and the concen- trations found were included in all calculation. Multiple-sample filtration manifolds and 0.45pm pore size filters (type HAWP) were purchased from Millipore Corp., Bedford, Mass. Porcine insulin and desoctapeptide insulin were gifts of Dr. R. Chance, Eli Lilly. Stock insulins were dissolved in 0.1 M HCl and diluted with 0.1% bovine serum albumin to the desired concentration. The calcium ionophore A23187 was a gift from Eli Lilly, Indianapolis, Ind. and was diluted in 100% ethyl alcohol. The final concentration of ethanol was less than 1% in the assay buffer and was present in controls.

Adipocyte S I and Microsomal Preparations- Fat cells were iso- lated from the epididymal fat pads by a modification (13) of the method of Rodbell (14) using 1.0 mg of collagenase/ml of modified Krebs-Ringer phosphate buffer, pH 7.4 at 37”C, containing 11 rnM D- glucose, 1.3 mM calcium, and 30 mg of bovine serum albumin/ml. The cells were washed three times, resuspended, and preincubated for 10 min at 37°C in two equal aliquots in modified Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 95% O,, 5% CO,, containing the same concentrations of glucose, bovine serum albu- min, and calcium as the Krebs-Ringer phosphate buffer. Following preincubation, one aliquot of cells received insulin (100 microunits/ ml unless otherwise stated) diluted in 0.1% bovine serum albumin (insulin cells) and the other received an equal volume of 0.1% bovine serum albumin (control cells). After 5 to 20 min at 37°C both aliquots were washed two times and homogenized in 0.25 M sucrose buffered with 10 mM Tris-HCl, pH 7.4, again adding insulin or bovine serum albumin to the appropriate cells at each step. The cells were homogenized in parallel as previously described (13, 15). EDTA was omitted from the homogenization buffers (7, 10) except as indicated. The supernatant of the initial 15min 20,000 x g centrifugation of the fat cell homogenate was termed S,. The microsomal fraction, which is highly enriched with endoplasmic reticulum (151, was prepared from S, by centrifugation at 160,000 x g for 1 h and the pellet was resuspended in 10 mM Tris-HCl, pH 7.4. S, preparations were assayed for calcium uptake immediately and microsomal pellets were frozen at -70°C until assayed for calcium, magnesium, and cytochrome c reductase activity.

Calcium Uptake and Effluz Assays-Calcium accumulation by endoplasmic reticulum was assayed using the S, preparation since the calcium uptake system is labile and therefore cannot be studied in the microsomal fraction (10). Assays were performed by filtration as described previously (10). The standard incubation medium contained 0.1 M KCl, 5 mM MgCl,, 5 mM ATP, 0 to 10 mM potassium oxalate, 50 rnM Tris-HCl, pH 7.0 at 24”C, 1 to 75 PM CaCl,, 0.25 to 0.50 &i of “C!aCl*, and 30 to 60 pg of protein of the S, fraction in a total volume of 500 ~1. The endoplasmic reticulum vesicles have been shown to be responsible for the calcium uptake in this assay system utilizing the S, fraction (10). Therefore, results are expressed as calcium transported/mg of endoplasmic reticulum protein as previously reported (10). Incubations were carried out at 24°C for 2 to 16 min as indicated in the legends.

Calcium efflux was measured after calcium uptake had been allowed to proceed for 6 to 10 min in the standard buffer with 25 FM

calcium in the absence of oxalate. Calcium eBIux was initiated by the addition of 2.5 rnM EGTA’ or 1 FM calcium ionophore (A213871 and the calcium efIIux rates followed by filtering aliquots at the indicated times thereafter.

Other Assays-The atomic absorption analysis of the calcium and magnesium contents and measurements of cytochrome c reductase activity of the microsomal fraction were determined as previously described (15, 16). Protein was determined by the method of Lowry et al. (17) using bovine serum albumin as standard.

Calculations - Free calcium concentrations in the presence of 5 mM MgZ+ and ATP were calculated as described by Katz (18). Linear regressions of Eadie-Hofstee plots performed by computer assisted least squares fit were used to obtain values for K,, values and maximum velocities (I’,,,,,) for the uptake reactions. Significance was determined by the Student paired t test. Data are expressed as mean t standard error.

RESULTS

Purity and Recovery of Endoplasmic Reticulum from Insu- lin-treated and Control Preparations - The studies reported

1 The abbreviation used is: EGTA, ethylene glycol bis(p-amino- ethyl etherlN,N’-tetraacetic acid.

here utilized both the first supernatant of the fat cell homoge- nate (S,) and the microsomal preparations from control and insulin-treated (100 microunits/ml) preparations, necessitat- ing the characterization of the effect of insulin on the compo- sition of these fractions. Insulin treatment of intact adipocytes for either 10 or 20 min had no effect on the purity or protein content of the S, and microsomal fractions (Table I). Further- more, the cytochrome c reductase levels of 324 2 37 (controls) and 331 + 33 (insulin-treated) were similar to the values of 338 i 46 (n = 11) originally reported by McKee1 and Jarett (15). These values further confirmed that the present modifi- cation of the original fractionation scheme by the omission of EDTA from the fractionation media did not affect the purity of the microsomal fraction. Furthermore the microsomal frac- tions prepared in the presence or absence of EDTA did not differ by electron microscopic examination (not shown). Cyto- chrome c reductase levels could not be accurately assessed in the first supernatant of the fat cell homogenate (S,) used for the calcium uptake studies because of the dilute nature of the

ST. Effect of Insulin on Calcium and Magnesium Content of

Endoplasmic Reticulum - Insulin (100 microunits/ml) treat- ment of cells for 10 min resulted in a 32% increase in total calcium content of the subsequently isolated microsomal frac- tion as determined by atomic absorption spectrophotometry (Table II). The magnesium content was unaffected by insulin treatment. Inclusion of EDTA in the fractionation and homog- enization media altered the above results. Both the calcium and magnesium content of the endoplasmic reticulum were

TABLE I

Purity and recovery of endoplasmic reticulum from insulin-treated and control cells

The S, and microsomal fractions were isolated in parallel from equal volumes of insulin-treated (100 microunitslml for 10 or 20 min) and control cells and total protein content and cytochrome c reduc- tase (cyto c reductase) assayed as described under “Experimental Procedures.” The values represent the mean 5 S.E. of five paired preparations. The values for control and insulin-treated prepara- tions were not significantly different as assessed by the paired t test.

Total protein recovered Cytochrome c reductase in

S, Microsomes microsomes

m&T nmolimgprotein

Control 21.6 k 2.3 1.92 t 0.22 324 2 37

Insulin-treated 22.1 -+ 2.0 1.92 f 0.18 331 * 33

TABLE II

Effect of insulin on calcium and magnesium content of endoplasmic reticulum

Paired microsomal fractions were obtained from insulin-treated (100 microunits/ml for 10 min) and control cells in the presence or absence of EDTA throughout the fractionation and calcium and magnesium content analyzed by atomic absorption spectrophotome- try. Data is expressed as the mean 2 S.E. of the number of paired preparations indicated in parentheses. Significance was determined using the paired t test.

Calcium Magnesium

-EDTA +EDTA -EDTA +EDTA

nmollmg protein Control 17.4 i- 2.6 10.1 t 1.8 42.7 k 3.4 8.4 k 1.5

Insulin-treated 22.9 ? 3.8” 11.4 ? 1.8 44.8 t 2.3 7.2 i 1.1

(10) (7) (10) (7)

a p < 0.01 compared to control; all other numbers not significantly different from controls.

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3506 Effect of Insulin on Calcium Uptake by Endoplasmic Reticulum

significantly lowered when the fractionation media included EDTA. Insulin had no effect on either the calcium or magne- sium remaining in the microsomal fractions isolated under these conditions (with EDTA). The values obtained from microsomes isolated in the presence of EDTA represent a highly stable ion pool, whereas the values obtained from the fractions isolated in the absence of EDTA represent both the stable and a less stable pool. Expressing the insulin-induced changes on the basis of a change only in this less stable pool demonstrate that the hormone treatment caused a 50% in- crease in this pool.

Effect of Insulin on Calcium Uptake and Efflux by Endo- plasmic Reticulum-The effect of insulin-treatment of adipo- cytes on calcium uptake by endoplasmic reticulum was first measured in the absence of a permeant anion such as oxalate in the calcium uptake assay. Under these conditions the endoplasmic reticulum (suspended in S,) obtained from insu- lin-treated cells had an increased capacity to accumulate calcium (Fig. 1). This increase was approximately 20% above control levels at all time points of the calcium uptake assay. Furthermore the percentage stimulation was insulin concen- tration-dependent (Fig. 2) and began to plateau at approxi- mately 50 microunits of insulin/ml. This concentration de- pendence is similar to the insulin sensitivity of metabolic processes such as glucose oxidation and antilipolysis measured in isolated fat cells (19).

v I 5 25 50 1c

INSULIN (M~romts /ml)

1 10

FIG. 2. Insulin concentration dependency of calcium uptake mea- sured in the absence of oxalate. Assays were performed as outlined in Fig. 1 except that the concentration of insulin was varied from 5 to 100 microunits/ml. The data represents the combined mean per cent stimulation by insulin ? S.E. of individual time points between 2 to 16 min in three to eight experiments at each concentration. The numbers in parentheses represent the number of individual assay points, each performed in triplicate.

TABLE III

Effect of insulin on rate of calcium uptake

Calcium uptake measured in the absence of oxalate repre- sents the net accumulation of calcium, reflecting both active calcium uptake and efflux, and has been termed the steady state filling capacity (20). Therefore the insulin stimulation of calcium uptake by endoplasmic reticulum from insulin-treated adipocytes found in the above studies could reflect 1) stimula- tion of the rate of calcium uptake, 2) a decrease in the rate of

uaired t test.

calcium eRlux or 3) a combination of both of these processes. The following studies were performed to investigate these possibilities.

Control

Calcium uptake into endoplasmic reticulum from seven paired insulin-treated (100 microunits/ml for 20 min) and control prepara- tions was assayed in the presence of 10 rnM potassium oxalate and varying calcium concentrations. The V,,,,, values and K,,, values were determined by Eadie-Hofstee plots using linear regression performed by least squares best iit of at least live data points assayed at calcium concentrations between 1.5 to 75 FM. The means IT S.E. are represented. Significance was determined using the

V max K!,

nmollmgll0 min !JJ.f

42.6 k 6.8 1.4 f 0.2

An estimate of the rate of calcium uptake by the endoplas- mic reticulum may be obtained by including 10 mM potassium oxalate in the calcium uptake buffer which was found to linearize the reaction. Under these conditions, endoplasmic reticulum from insulin-treated cells had a 28% increase in the

Insulin-treated 54.7 f 1.7” 1.4 k 0.2

a p i 0.025 compared to control.

r I , I

rate of calcium uptake without an alteration of the apparent K,,, (Table III). Substitution of 10 mM inorganic phosphate for oxalate also linearized the calcium uptake reaction and re- sulted in a similar percentage stimulation by insulin; however

1 I / I 4 8 12 16

TIME (Mln)

FIG. 1. The effect of insulin on calcium uptake measured in the absence of oxalate. S, was obtained from insulin-treated (100 mi- crounitslml for 20 min) and control cells as described under “Exper- imental Procedures.” Calcium uptake was assayed at the time points indicated in the standard buffer containing 25 FLM calcium and no oxalate. The means tr S.E. are indicated. The number of paired experiments are indicated in parentheses and the p values were obtained using the paired t test.

the V,,, for control and insulin-treated preparations were approximately 50% of those found with oxalate (data not shown).

Insulin treatment of cells had no effect on the rate of calcium efflux from endoplasmic reticulum vesicles in contrast to its effect on the rate of calcium uptake. Calcium eRlux was initiated by the addition 2.5 mM EGTA to S, obtained from parallel insulin-treated and control cells after calcium uptake had progressed in the presence of 25 pM calcium for 6 to 10 min without oxalate. The mean rate of efflux was 0.0014 nmol/ mg/s at 24°C and there was no significant difference between the S, preparations from control and insulin-treated cells (Fig. 3). Release of calcium from the vesicles also was analyzed using 1 PM calcium ionophore A21387 after uptake had pro- gressed under the same conditions as the above experiments. Calcium ionophore caused an immediate (<lo s) release of greater than 95% of the calcium from the vesicles (Fig. 3). The remaining calcium associated with the endoplasmic reticulum could be entirely accounted for by passive binding to endoplas- mic reticulum (21). There was no difference between prepara- tions from insulin-treated and control cells in the speed or the

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Effect of Insulin on Calcium Uptake by Endoplasmic Reticulum 3507

i:I/ i:;, , ;t , , / , 1 $?..-------~4i------------!;’ I 2 3 4 5 6 7 8

TIME (MIN)

FIG. 3. The effect of insulin on the rate of calcium ef?lux from the endoplasmic reticulum. Calcium uptake was carried out under standard conditions in the presence of 25 ~CLM calcium and no oxalate for 6 to 10 min from control (0) and insulin-treated cells (0). EGTA (2.5 mM) or calcium ionophore (A23187) were added (time 0) and the remaining calcium in the endoplasmic reticulum was assayed by filtration at the times indicated. The values represent the mean values of the number of experiments indicated in parentheses.

amount of the calcium released. Thus the effect of insulin upon the steady state filling capacity appeared to be due entirely to an effect of insulin upon the rate of calcium uptake.

The effect of insulin upon the rate of calcium transport was also demonstrable after short exposure of the cells to insulin. Insulin treatment (100 microunits/ml) of cells for 10 min resulted in only a 10.5% (n = 4) stimulation of the rate of calcium uptake, which was less than that seen with the 20- min incubation usually employed. However, treatment of cells with higher concentrations of insulin (500 to 1000 microunitsi ml) for 5 min showed an 18.2% (n = 4) stimulation of endoplasmic reticulum calcium uptake which was more com- parable to that found with 20-min treatment with 100 mi- crounits of insulin/ml.

In contrast to the effect of native insulin on the rate of calcium uptake by endoplasmic reticulum, there was no effect on calcium transport when cells were treated with equimolar concentrations of desoctapeptide insulin which has approxi- mately 1% of the biologic activity of insulin. Similarly, direct addition of up to 500 microunits of insulin/ml to S, had no effect on calcium uptake.

DISCUSSION

These studies demonstrate that insulin treatment of adipo- cytes increased the ability of endoplasmic reticulum to ac- tively accumulate calcium. The net effect of insulin treatment was to increase the steady state filling capacity of the endo- plasmic reticulum. This insulin-induced increase was found to be due entirely to an increase in the apparent V,,, of calcium uptake without an alteration in the K,,, Insulin had no effect on the rate of calcium efIlux from the endoplasmic reticulum. These findings from in vitro ‘WaZ+ uptake studies were confirmed by an independent atomic absorption method which showed that the total calcium content of the endoplasmic reticulum isolated from insulin-treated adipocytes was greater than the calcium content of endoplasmic reticulum from controls.

Recent evidence has suggested that the endoplasmic reticu- lum of a variety of cells may play an important role in the regulation of cytosol calcium concentration. The importance of sarcoplasmic reticulum in the control of cytosol calcium and thus in the regulation of the muscle contraction-relaxation cycle is well documented (22). More recently the identification of ATP-dependent calcium uptake systems in a variety of

noncontractile cells which are qualitatively analogous to that in sarcoplasmic reticulum (8-11) has suggested an important role of this organelle in the regulation of intracellular calcium distribution. This concept is strengthened by the finding that the K,n values of the endoplasmic reticulum calcium uptake systems range between 1.0 and 4.0 PM which is similar to the estimated cytosol calcium concentration in most cells (0.1 to 1.0 pM) (23). Further support for the importance of the endoplasmic reticulum calcium uptake system in adipocytes are the present observations that the calcium uptake and the calcium content of the endoplasmic reticulum are responsive to insulin.

The ability of insulin treatment of adipocytes to increase the steady state filling capacity and the V,,,,, of calcium transport by endoplasmic reticulum by 20 and 28%, respec- tively, could markedly alter the cytosol calcium concentration. Calculations based upon an intracellular water space of 2% of the cell volume of the adipocyte (24) and a 6% yield of homogenate protein as endoplasmic reticulum (15) indicate that, at the K,,, of the reaction, the rate of calcium uptake in control preparations could alter the cytosol calcium concentra- tion by approximately 1 pmol/liter/s at 37°C. The 28% increase in uptake rate after insulin treatment would change this rate by approximately 0.3 pmol/liter/s. This change could clearly represent a significant factor in the rapid regulation of cytosol calcium and thus the activities of calcium-dependent cytosol and endoplasmic reticulum-associated enzymes.

The insulin-induced increase in the atomic absorption meas- urements of the total calcium content of the isolated endoplas- mic reticulum (Table II) has been taken as confirmatory evidence that an increase in the V,,,,, and the filling capacity of the calcium uptake system occurs. The atomic absorption studies indicate that there are two pools of calcium in the endoplasmic reticulum, a stable pool, and a less stable or labile pool. This is indicated by the values obtained for endoplasmic reticulum isolated in the presence and absence of EDTA. The values for endoplasmic reticulum isolated in the presence of EDTA represent a highly stable calcium pool since EDTA chelates calcium in the media, preventing postho- mogenization calcium uptake and also dissociating the more loosely complexed calcium from the organelle. On the other hand the values obtained for endoplasmic reticulum isolated in the absence of EDTA are almost twice as large as the stable pool and represent the total calcium content which includes the stable pool in addition to a more labile pool. It is only this labile pool of calcium which is increased by insulin treatment and the magnitude of increase is approximately 50% of this pool or 5.5 nmol/mg of endoplasmic reticulum. It must be emphasized that this estimate of the labile pool may not reflect the actual in uiuo size of this pool because of postho- mogenization changes. This is further supported by the mag- nitude of the insulin-induced increase in this pool since a change of such magnitude in vivo would be many times greater than the estimated cytosol concentration. Such a change would seem excessive for an intracellular regulatory process. Nevertheless, whether the measured changes in the labile pool represent primarily an in vitro or an in uivo change they are consistent with the major findings that insulin induces an increase in the steady state calcium capacity of the endoplasmic reticulum.

If the calcium uptake system of the adipocyte endoplasmic reticulum is analogous to the sarcoplasmic reticulum, then one would predict that the insulin-induced increase in the calcium uptake system would be associated with an increase

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3508 Effect of Insulin on Calcium Uptake by Endoplasmic Reticulum

in the activity of a calcium transport ATPase. There is considerable evidence to suggest that calcium transport in the adipocyte endoplasmic reticulum is associated with such a calcium ATPase. A calcium ATPase in adipocyte endoplaamic reticulum has been identified and correlated with 75-A intra- membranous particles similar to those seen in sarcoplasmic reticulum (25). Purthermore the ratio of calcium transported to ATP hydrolyzed has been shown to be at least l:l, which is similar to the values of 1:l to 2:l reported for sarcoplasmic reticulum (26). However, investigation of the effect of insulin upon the calcium ATPase is complicated by the fact that this enzyme represents only about 7% of the total ATPase in endoplasmic reticulum and therefore changes of 20 to 30% would be difficult to document. Recent studies have shown that the calcium ATPase in endoplasmic reticulum can be puriiled by at least 3-fold by extraction with deoxycholate which should permit its further investigation.2

In addition to the present findings, several recent reports have suggested that the endoplasmic reticulum may play an important role in at least part of the mechanism of insulin action. Coensyme A ligase, an endoplasmic reticulum-associ- ated enzyme, has been shown to be insulin sensitive (27). Avruch et al. (28) have reported that insulin treatment of adipocytes results in an altered phosphorylation of a protein localized to the endoplasmic reticulum and cytosol. Although the relationship of either of these insulin-sensitive processes to the present findings has not been investigated, there is a suggestion that the insulin-sensitive phosphoprotein may be related to calcium. Avruch et al. (29) showed that the subcel- lular localization of this insulin-sensitive phosphoprotein is ion-dependent since when calcium and magnesium were added back to the fractionation media, this protein became more prominent in the endoplasmic reticulum fraction as opposed to the cytosol. Furthermore it is interesting to note that the molecular weight of this insulin-sensitive phosphoprotein is 123,000 (28) which is similar to the molecular weight of the calcium transport ATPase of sarcoplasmic reticulum which is approximately 100,000 (26).

Calcium has been suggested as a second messenger for insulin (1, 2). The present findings in conjunction with our previous studies on insulin’s effects on calcium metabolism by adipocyte mitochondria and plasma membranes suggest that calcium is not the second messenger for insulin. However, the necessity for intact cells to be treated by insulin to cause the effects on calcium metabolism by these organelles supports the concept that a second messenger or messenger system is required for insulin’s action. The effects of insulin on calcium metabolism by these organelles are extensive and would not likely result from changes in calcium fluxes alone. However these changes could regulate the various intracellular calcium sensitive enzymes involved in the final metabolic responses to

insulin. Thus calcium could be envisioned as part of a tertiary messenger system responsible for the intracellular effects of insulin.

Acknowledgment-We wish to thank Mrs. Robert Smith for her excellent technical assistance.

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J M McDonald, D E Bruns and L Jarettreticulum.

Ability of insulin to increase calcium uptake by adipocyte endoplasmic

1978, 253:3504-3508.J. Biol. Chem. 

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