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Endocrinology Vol. 139, No. 12 4953-4961
Copyright © 1998 by The Endocrine Society

ARTICLES

Insulin Produces a Growth Hormone-Like Increase in Intracellular Free Calcium Concentration in Okadaic Acid-Treated Adipocytes1

Shikha Gaur, Yael Schwartz, Lih-Ruey Tai, G. Peter Frick and H. Maurice Goodman

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Address all correspondence and requests for reprints to: H. Maurice Goodman, Ph.D., University of Massachusetts Medical School, Department of Physiology, 55 Lake Avenue, North, Worcester, Massachusetts 01655-0127. E-mail: Maurice.Goodman{at}banyan.ummed.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µM 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 µU/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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RAT ADIPOCYTES express receptors for both GH and insulin, and in vivo these hormones usually produce different, and often opposing effects on carbohydrate and lipid metabolism in these cells. However, in rat adipocytes that have been made GH deficient by hypophysectomy (1) or incubation in vitro for at least 3 h in the absence of GH (2, 3, 4), GH produces responses that are qualitatively indistinguishable from those of insulin. After 1–2 h, this acute insulin-like response to GH dissipates and gives way to the delayed action of GH that makes adipocytes refractory to acute stimulation of glucose metabolism by renewed exposure to GH (1). Adipocytes that are freshly isolated from normal rats, and hence recently exposed to GH, are also refractory to such insulin-like stimulation by GH, and their refractory state can be maintained in vitro for at least 4 h by brief treatment with GH at the onset of the incubation period (5).

The acute insulin-like effects of GH can probably be attributed to overlap in the signaling pathways for GH and insulin. The signal transduction pathways entrained by both GH (6) and insulin (7, 8) are initiated by phosphorylation of proteins on tyrosine residues, which results in the subsequent recruitment and activation of cytosolic proteins that regulate phosphorylation and dephosphorylation reactions and gene transcription. GH, via JAK 2, stimulates tyrosine phosphorylation of the insulin receptor substrates (9, 10, 11, 12) and SHC (13), and activates phosphatidylinositol-3 kinase (10), and the MAP kinase pathway (14, 15, 16). More recently, overlap of insulin signaling with the GH transduction pathway was reported when it was found that the insulin receptor kinase may activate stat 5B (17). Furthermore, insulin may also activate JAK 1 and JAK 2 (18). Despite these potential cross-overs between the GH and insulin transduction pathways, adipocytes usually express discrete responses that are appropriate for each hormone. Specificity of the hormonal response therefore is likely to be achieved through the activities of unshared elements in the transduction pathways that may block or reinforce particular routes of signal expression probably at branch points of the overlapping insulin and GH signaling pathways and hence restrict the response to a subset of possible actions. Refractoriness to insulin-like stimulation, which appears to require GH-dependent gene transcription for its expression (1), can thus be regarded as an effect of GH that provides some specificity to its long term actions.

Okadaic acid is an inhibitor of type I and IIa phosphoprotein phosphatases (19, 20) and affects glucose metabolism in fat cells in a manner that is reminiscent of GH. Like GH, okadaic acid initially mimics the actions of insulin (21, 22, 23), but subsequently, even after okadaic acid is removed by thorough washing, the ability of adipocytes to accelerate glucose metabolism in response to insulin is severely reduced (22, 23, 24). Okadaic acid produces such refractoriness to insulin-like stimulation without affecting the ligand affinity or tyrosine kinase activity of the insulin receptor (23, 24). Similarly, refractoriness to the insulin-like effect of GH is also produced without a change in the affinity or abundance of GH binding sites (5). These parallels suggested that the refractoriness produced by okadaic acid with respect to the actions of insulin might provide some insight into the refractoriness produced by GH with respect to its own insulin-like actions. Refractoriness to the insulin-like effect of GH is associated with, and may result from, GH-induced increases in intracellular free calcium concentrations ([Ca2+]i) (25, 26, 27), although the precise role of increased [Ca2+]i has not been established. Hence, in examining the effects of insulin in okadaic acid-treated adipocytes, we examined the effects of insulin on [Ca2+]i. We describe here experiments that demonstrate that, in addition to making adipocytes refractory to the effects of insulin on glucose metabolism, treatment with okadaic acid unmasks a remarkable GH-like effect of insulin on [Ca2+]i.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cells
Male rats of the CD strain were obtained from the Charles River Laboratories, Inc. (Kingston, NY) and studied when they attained body weights of 160–180 g. The animals were maintained in the vivarium under conditions of constant temperature (18 C) and lighting (lights on from 0600–1800 h) and had free access to food (Purina 5008, Ralston Purina Co., St. Louis, MO) and water. Procedures and protocols for animal handling were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC). For each experiment epididymal fat from 2–8 rats was pooled and minced for preparation of isolated adipocytes according to the procedure of Rodbell (28) as modified in this laboratory (5). After digestion for 20 min with 1 mg/ml collagenase (lot 143710, type A, Boehringer Mannheim Biochemicals, Indianapolis, IN) in KRBG (Krebs Ringer bicarbonate buffer) that contained 5.5 mM glucose and 40 mg/ml bovine serum albumin (BSA; Metrix, fraction IV, Reheis Chemical Co., Phoenix, AZ) the cells were washed four times in KRBG containing 10 mg/ml BSA, resuspended 1:3 (vol/vol) in the same buffer and incubated at 37 C under an atmosphere of 95% O2, 5% CO2.

Experimental design
Adipocytes that were incubated for 3 h in the absence of GH (GH-deprived cells) became sensitive to insulin-like stimulation by GH (2, 3, 4). The refractoriness of freshly isolated cells to the insulin-like effects of GH was sustained in replicate aliquots of cells by treating them with 100 ng/ml of hGH (GH-treated cells) for 1 h followed by washing, resuspension in KRBG, and continued incubation for 2 or 3 h in the absence of GH (25). Similarly, okadaic acid-induced refractoriness was produced by incubation of the cells for 1 h with 0.25 µM okadaic acid (LC Laboratories, Woburn, MA), followed by washing three times with KRBG and an additional incubation for 2 h in KRBG in the absence of okadaic acid. In order to draw reliable conclusions about responsiveness to GH and insulin in okadaic acid-treated cells, it was necessary to maintain a stable baseline rate of glucose incorporation into triglycerides. Although insulin-like stimulation of glucose metabolism was reported to disappear upon removal of okadaic acid (22), the basal rate of glucose conversion to triglycerides was often considerably elevated in adipocyte suspensions 2 h after okadaic acid was removed. This problem was alleviated by increasing BSA content of the KRBG to 40 mg/ml during the second and third hours of incubation of okadaic acid-treated adipocytes. After 3 h of incubation, GH-treated, GH-deprived and okadaic acid-treated adipocytes were collected by centrifugation and washed before testing for their ability to respond to 500 ng/ml of hGH or 100 µU/ml of insulin. The hGH used in these experiments was generously provided by Genentech, Inc., (South San Francisco, CA). Crystalline insulin was obtained from Eli Lilly & Co. (Indianapolis, IN).

Determination of lipogenesis
Incorporation of 3H from D-[3-3H]-glucose (New England Nuclear Corp., Boston, MA) into lipids as described by Moody et al. (29) and modified in this laboratory (25), served as an index of the insulin-like response. Briefly, GH-deprived, GH-treated, or okadaic acid-treated adipocytes diluted 1:30 (vol/vol) were incubated for an additional hour in the absence or presence of GH or insulin in Krebs Ringer bicarbonate buffer that contained 10 mg/ml BSA and 0.25 mM D-[3-3H]-glucose (specific activity 0.8 µCi/µM). The incubation was terminated by the addition of 5 ml/tube of scintillation fluid that contained 0.3 g of 1,4-bis[2(4-methoxy-5-phenyloxazolyl)]benzene and 5.0 g of 2,5-diphenyloxazole/liter of toluene. After vigorous mixing, the tubes were allowed to stand for 1 h to permit extraction of lipids into the toluene phase before counting in a Packard Tri-Carb 4530 scintillation counter.

Measurement of [Ca2+]i
[Ca2+]i was measured in individual adipocytes using the hexakis(acetoxymethyl) ester (AM) of the calcium sensitive dye fura-2 (30) (Molecular Probes, Inc., Eugene, OR) as previously described (26). Briefly, after loading for 30 min with 20 µM fura-2, adipocytes were pipetted into a temperature controlled plexiglass perfusion chamber mounted on the stage of an inverted microscope. The cells were illuminated from below with 3 nsec pulses of light (337 or 380 nm) delivered every 33 msec through a bifurcated quartz fiber extending from a nitrogen laser and a tunable dye laser (Laser Science, Cambridge, MA). Emitted light was recorded with a video camera and processed (Recognition Technology, Westborough, MA) with a NEC Powermate I computer. The instrument was programmed to allow data collection over a 9-min interval, during which a single cell could be monitored for the entire time, or multiple cells selected at random could each be scanned for a period of 10–15 sec. Fluorescence ratios were converted to [Ca2+]i with a standard calibration curve from fluorescence ratios measured in the cytosol of refractory and sensitive adipocytes as adapted from the procedure described by Williams et al. (31).

Materials
D609 (Tricyclodecan-9-yl-xanthogenate), KT5720, actinomycin D, verapamil, cycloheximide, and chelerythrine chloride were obtained from Calbiochem (San Diego, CA). Calphostin C was obtained from Sigma Chemical Co. (St. Louis, MO), and staurosporine was obtained from Boehringer Mannheim, and sn 1,2-dioctanoyl glycerol (DOG), and nimodopine were obtained from Research Biochemicals International (Natick, MA). BAY K 5552 was obtained through the courtesy of Dr. Cheryl Scheid of this department from material generously provided by Dr. Alexander Scriabine of Bayer Laboratories (New Haven, CT). All other reagents were of the highest purity obtainable.

Statistical analyses
Adipocytes pooled from 4–8 rats were used in each metabolic experiment. Data obtained in at least five replicate experiments were analyzed by analysis of variance for repeated measures (32, 33). Statistical significance was assessed by multiple pairwise t tests using the Bonferonni adjustment to correct for additive type I errors due to multiple comparisons (34). [Ca2+]i was measured in 25–50 adipocytes in each of the eight or nine treatment groups studied in each experiment using adipocytes pooled from two to four rats. Data for every treatment variable were obtained in at least three independent experiments. Statistical significance was determined for each variable both within individual experiments and for the means of replicate experiments. The data are presented as the means and standard errors calculated using each experiment as a single observation, and evaluated by Student’s t test for paired or unpaired samples again using the Bonferonni adjustment to correct for additive type I errors due to multiple comparisons (34). Statistical significance (P < 0.05) of any treatment variable demonstrated in this way invariably reflected statistical significance of that same variable within individual experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The refractory phenomenon
The incorporation of 3H from 3[3H]-glucose into triglycerides was increased 2- to 3-fold when either GH or insulin was added in the fourth hour to GH-deprived adipocytes (Fig. 1Go). In contrast, addition of GH in the fourth hour to adipocytes that were treated with GH during the first hour of the 3-h preincubation period (GH-treated cells) failed to stimulate lipogenesis, although the response to insulin was unchanged. Adipocytes that were treated with 0.25 µM okadaic acid in the first hour of the preincubation period (oka-treated cells) were refractory to insulin-like stimulation when either GH or insulin was added 2 h later (Fig. 1Go). Because earlier studies indicated that the failure of GH to stimulate glucose metabolism in GH-treated adipocytes was associated with increased uptake of calcium (26, 27), we determined if increased [Ca2+]i uptake might be similarly associated with the refractoriness to the actions of insulin on glucose metabolism in oka-treated cells. When added in the fourth hour, GH increased [Ca2+]i in GH-treated, but not GH-deprived adipocytes (Fig. 2Go). Insulin had no effect on [Ca2+]i in either GH-treated or GH-deprived cells, and when added along with GH in the fourth hour, blocked GH-induced increase in [Ca2+]i. In contrast, both GH and insulin produced large increases in [Ca2+]i when added to oka-treated cells in the fourth hour (Fig. 2Go). Furthermore, insulin failed to block the GH-dependent increase in [Ca2+]i in oka-treated cells, and in at least some experiments, submaximal effects of GH and insulin on [Ca2+]i were additive.



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  		Figure 1. Refractoriness produced by GH or okadaic acid. Freshly isolated adipocytes were incubated for 1 h in KRBG (GH-deprived), KRBG that contained 100 ng/ml GH (GH-treated) or KRBG that contained 0.25 µM okadaic acid (Oka-treated). The cells were then washed and resuspended in KRBG enriched with 4% bovine serum albumin and reincubated for 2 h, after which the cells were again washed and resuspended in KRB containing 1% BSA and 0.1 mM 3-[3H]-glucose and reincubated for 1 h without or with GH (500 ng/ml) or insulin (100 µU/ml). Each bar represents the mean ± SEM for 14 independent cell populations, each including cells pooled from 4–8 rats. *, P < 0.01 compared with corresponding control value.

 


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  		Figure 2. Effects of GH and insulin on [Ca2+]i. Adipocytes were incubated for the first 3 h as described in Fig. 1Go. Aliquots of cells were then incubated for 30 min in KRBG that contained 1% BSA and 20 µM fura 2-AM. The cell suspensions were then transferred to the perfusion chamber for measurement of [Ca2+]i. Throughout the 9-min period in which [Ca2+]i was monitored, the cells were perfused with KRBG that contained 0.1% BSA and either GH (500 ng/ml) or insulin (100 µU/ml). Each bar represents the mean ± SEM for three independent populations, each including measurement of [Ca2+]i in 30–50 cells selected at random. *, P < 0.05 compared with control values for that group. [Ca2+]i was significantly higher (P < 0.05) in GH-treated controls than in GH-deprived or Oka-treated controls.

 
Characterization of the calcium response to insulin
The ability of oka-treated adipocytes to increase [Ca2+]i in response to insulin developed slowly with a latency of 1–2 h (Fig. 3Go). When added immediately after okadaic acid was washed out, or even 1 h later, insulin produced no significant change in [Ca2+]i, but the response was fully developed by 2 h after removal of okadaic acid and persisted for at least two additional hours (data not shown). During this time basal [Ca2+]i declined to about half the concentration seen in freshly isolated cells (Fig. 3Go) as reported previously for GH-deprived cells (26, 27). The minimal concentration of insulin needed to increase [Ca2+]i was about 30 µU/ml, and the response was maximum at about 100 µU/ml (Fig. 4Go). Curiously, at 500 µU/ml, insulin produced a significantly smaller increase in [Ca2+]i than at 100 µU/ml.



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  		Figure 3. Time course for the development of the increased [Ca2+]i response to insulin. Adipocytes were incubated for 1 h with 0.25 µM okadaic acid. The cells were then washed and reincubated for 1, 2, or 3 h before measurement of [Ca2+]i in the absence or presence of 100 µU/ml of insulin. In each case, the cells were incubated with 20 µM fura2-AM for the final 30 min before measurement of [Ca2+]i. Each bar is the mean ± SEM of three independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random. *, P < 0.01 compared with the corresponding control value. **, P < 0.05 compared with 0 h control value.

 


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  		Figure 4. Dose-response relationship for the increased [Ca2+]i response to insulin. Adipocytes were incubated with 0.25 µM okadaic in the first hour, and the response to insulin was measured in the fourth h as described in the legend for Fig. 2Go. Each point is the mean of four independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random. The vertical brackets indicate the SEM. *, P < 0.01, **, P < 0.05 vs. control.

 
The data presented in Figs. 2–4GoGoGo and in the following figures are the average values of [Ca2+]i measured in many individual cells over 10–15 sec intervals during the 9 min scanning period beginning approximately 1 min after addition of insulin, and therefore may not accurately reflect the peak or plateau values for [Ca2+]i. The responses of individual adipocytes were also monitored for longer periods by focusing on a single cell before, during, and up to 8 min after insulin was introduced into the perfusion chamber. An increase in [Ca2+]i usually began within 2 min, became maximal over the next 1 to 4 min, and was usually, but not always, sustained for as long as the hormone was present. In the 20 cells examined in this way, there was considerable variability in the intervals between addition of hormone and onset of response, between the onset and attainment of the maximum response, and the time over which the maximum response was sustained. The data for insulin-treated cells in the preceding and following figures probably underestimate [Ca2+]i somewhat, because it is unlikely that each of the 1,015 oka-treated cells reported here was scanned during its peak response to insulin.

Insulin activates L-type calcium channels in okadaic acid-treated adipocytes
The increase in [Ca2+]i produced by insulin in oka-treated cells was blocked by the dihydropyridine channel antagonist, nimodipine (100 nM), and was mimicked by incubation of the cells with 30 mM KCl to partially depolarize the plasma membrane, or with the L-type calcium channel agonist Bay K 5552 (Fig. 5Go). These results strongly suggest that insulin activates voltage-sensitive L-type calcium channels in oka-treated cells. Virtually identical findings were obtained in an earlier study of the effects of GH on [Ca2+]i in freshly isolated or GH-pretreated adipocytes (35). As reported for the effects of GH (35), the increase in [Ca2+]i produced by insulin was also mimicked by sn- 1,2-dioctanoyl glycerol (DOG), an activator of protein kinase C, and blocked by inhibiting protein kinase C with chelerythrine chloride (1 µM) added just before insulin, suggesting a crucial role for protein kinase C in the transduction pathway of this response to insulin (Fig. 6Go). Consistent with this premise, D609, a specific inhibitor of phosphatidylcholine-specific phospholipase C (36), also blocked the response to insulin. These agents also blocked the increase in [Ca2+]i produced by GH (35). In contrast, the ineffectiveness of KT5720, a specific inhibitor of protein kinase A (37) suggests that this kinase may not have a role in expression of the insulin signal. The importance of protein phosphorylation for the activity of calcium channels in these cells is underscored by the findings that a variety of channel-activating signals, depolarization, DOG, BayK 5552 as well as GH and insulin produced greater increases in [Ca2+]i in oka-treated adipocytes than in untreated control cells (Fig. 7Go).



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  		Figure 5. A, Effects of 100 nM nimodipine on the increase in [Ca2+]i produced by 100 µU/ml of insulin, or partial depolarization produced by 30 mM KCl in oka-treated adipocytes. B, The effects of Bay K 5222 on [Ca2+]i in oka-treated adipocytes. The cells were treated with 0.25 µM okadaic acid for 1 h as described in Fig. 2Go, and [Ca2+]i was measured in the fourth h. Each bar is the mean ± SEM of three independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random. *, P < 0.01 compared with the corresponding control value.

 


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  		Figure 6. Effects of insulin, sn-1,2 dioctanoylglycerol (DOG) and enzyme inhibitors on [Ca2+]i (100 µU/ml) and DOG (50 µM) were added immediately before measurement of [Ca2+]i. Chelerythrine chloride (1 µM), D609 (50 µM), or KT5720 (100 nM) were added 1 min before insulin or DOG. Each bar represents the mean ± SEM for three independent cell populations, each including measurements in 30–50 cells selected at random for each experimental condition. *, P < 0.01 compared with the corresponding control value.

 


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  		Figure 7. Enhanced responsiveness to calcium channel activating agents in oka-treated adipocytes. Adipocytes were treated as described in the legend to Fig. 2Go, and [Ca2+]i was measured 2.5–4 h after okadaic acid was washed away. Insulin (100 µU/ml), KCl (30 mM), DOG (50 µM) and Bay K 5222 (10 µM) were added immediately before measuring [Ca2+]i. Each bar is the mean ± SEM of the indicated number of independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random for each experimental condition. The effects of KCl, DOG, and Bay K were significant (P < 0.01) compared to the corresponding control values in each group, and were significantly greater (P < 0.05) in the oka-treated than control cells. The effects of insulin were significant (P < 0.01) only in the oka-treated cells.

 
Okadaic acid alters gene expression
The approximately 2 h lag period required for the acquisition of responsiveness to the [Ca2+]i-raising effect of insulin is similar to the lag time required for GH to produce responsiveness to its own [Ca2+]i-raising effects (25). This delay in the expression of the GH effect appears to be required for transcriptional and translational events and suggested that okadaic acid might also influence gene expression. To test this possibility, freshly isolated adipocytes were incubated with 0.25 µM okadaic acid added simultaneously with either 5 µg/ml of actinomycin D or 20 µg/ml of cycloheximide. The ability of these cells to increase [Ca2+]i in response to insulin was measured in the fourth hour as in previous experiments (Fig. 8Go). Once again, insulin dramatically increased [Ca2+]i in oka-treated cells, but blockade of RNA or protein synthesis abolished the ability of these cells to respond subsequently to insulin.



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  		Figure 8. Inhibitors of transcription and translation block the effect of okadaic acid. Adipocytes were treated as described in Fig. 2Go except 20 µg/ml of cycloheximide or 5 µg/ml of actinomycin d were also added along with okadaic acid at the start of the incubation, and were present throughout the incubation period. Each bar is the mean ± SEM of three independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random. *, P < 0.001 compared with the corresponding control value.

 
On the assumption that the effects of okadaic acid are secondary to its inhibition of phosphatase activity, adipocytes were incubated with inhibitors of protein kinases C and A before, and during incubation with okadaic acid (Fig. 9Go) to gain insight into which protein kinase activity or activities might account for this phenomenon. When added before okadaic acid, KT-5720 was as effective as actinomycin D or cycloheximide in preventing the induction of responsiveness to the effects of insulin on [Ca2+]i, suggesting a role for protein kinase A. Similarly, the protein kinase C inhibitors, staurosporine, chelerythrine chloride, or calphostin C were also effective in preventing the subsequent response to insulin.



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  		Figure 9. Effects of protein kinase inhibitors added immediately before and along with okadaic acid on the subsequent response to 100 µU/ml of insulin. Adipocytes were treated with okadaic acid as described in Fig. 2Go except that chelerythrine chloride (1 µM), calphostin C (100 nM), staurosporine (500 nM), or KT5720 (100 nM) were added to some cells 15 min before okadaic acid and were present throughout the incubation period. Each bar is the mean ± SEM of three independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random for each experimental condition. *, P < 0.01 compared with the corresponding control value.

 
To determine if indeed protein kinases A and C might catalyze reactions that result in the induction of sensitivity to the effect of insulin on [Ca2+]i, freshly isolated adipocytes were incubated with activators of these enzymes instead of okadaic acid, and their response to insulin was tested in the fourth hour. To evaluate the importance of protein kinase A, adipocytes were incubated for 1 h with the membrane permeant analog, dibutyryl cAMP. The cells were washed and reincubated for 2.5 h without dibutyryl cAMP. Addition of insulin in the fourth hour resulted in a nearly 3-fold increase in [Ca2+]i (Fig. 10Go). Similar results were obtained when endogenous cAMP levels were increased by inhibition of cAMP phosphodiesterase with methylisobutyl-xanthine (MIX) (data not shown). Intracellular calcium concentrations were also increased by insulin in adipocytes that were preincubated with phorbol myristic acid (PMA) to stimulate protein kinase C (Fig. 10Go). Insulin produced no greater increase in [Ca2+]i when added to adipocytes that had been preincubated with the combination of DBC and PMA to activate both protein kinase A and protein kinase C, than in adipocytes that had been incubated with either agent alone.



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  		Figure 10. Effects of protein kinase activators on the subsequent response to 100 µU/ml of insulin. Freshly isolated adipocytes were incubated for 1 h with 1 mM dibutyryl cyclic AMP (DBC), 10 µM phorbol myristic acid (PMA), or both. Insulin (100 µU/ml) added 2 h later significantly (P < 0.05) increased [Ca2+]i in adipocytes that had been treated with DBC, PMA, or DBC+PMA. Each bar is the mean ± SEM of three independent cell populations, each including measurements of [Ca2+]i in 30–50 cells selected at random for each experimental condition. *, P < 0.05 compared with the corresponding control value.

 
Is acquisition of the ability to increase [Ca2+]i linked to refractoriness?
To determine if responsiveness to the effects of insulin on glucose metabolism is linked to the increase in [Ca2+]i we examined the effects of inhibitors of protein kinases A and C on the ability of oka-treated adipocytes to increase glucose metabolism in response to insulin (Fig. 11AGo). Despite their effectiveness in preventing an increase in [Ca2+]i in response to insulin, these agents failed to restore sensitivity to the effects of insulin on glucose metabolism. A small, but statistically significant stimulation of glucose metabolism was seen in the cells that were pretreated with staurosporine in agreement with the findings of Shisheva and Schecter (38), but the response was severely blunted. Conversely, treatment of adipocytes with dibutyryl cAMP or phorbol ester at concentrations that induced responsiveness to the effects of insulin on [Ca2+]i failed to make the cells refractory to the effects of insulin on glucose metabolism (Fig. 11BGo). Similarly, preventing the influx of calcium by incubating oka-treated cells in calcium-free medium was ineffective in restoring increased glucose metabolism in response to insulin although a small, but statistically significant stimulation was evident (Fig. 11CGo). Taken together these results suggest that the increase in [Ca2+]i produced by insulin in oka-treated adipocytes does not account for the failure of insulin to increase glucose metabolism.



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  		Figure 11. Effects of insulin on incorporation of 3H from 3-[3H]-glucose into triglycerides. Adipocytes were treated with okadaic acid as described in Fig. 1Go and were incubated for a final hour with 0.1 mM 3-[3H]-glucose in the presence or absence of 100 µU/ml of insulin. Each bar represents the mean ± SEM increase attributable to insulin. *, P < 0.01, **, P < 0.05 compared with zero. A, KT5720 (100 nM), chelerythrine chloride (1 µM), or staurosporine (500 nM) were added as described in Fig. 10Go. The basal rate of incorporation for six independent cell populations was 709 ± 39 cpm/mg triglyceride x h. B, Adipocytes were incubated for 1 h with 1 mM dibutyryl cAMP (DBC) or 10 µM phorbol myristic acid (PMA) instead of okadaic acid, but otherwise treated as in Fig. 1Go. The basal rate of incorporation for five independent cell populations was 1306 ± 100 cpm/mg triglyceride·h. C, Control and oka-treated adipocytes were incubated for a final hour with 0.1 mM 3-[3H]-glucose in the presence or absence of 100 µU/ml of insulin in KRB, or KRB from which calcium had been omitted. The basal rates of incorporation for nine independent cell populations were 863 ± 127 and 715 ± 55 CPM/mg triglyceride·h for cells incubated in KRB or calcium-free KRB.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present findings highlight the remarkable similarity between the effects of insulin on [Ca2+]i in oka-treated adipocytes and the previously reported effects of GH on [Ca2+]i in freshly isolated or GH-pretreated adipocytes (35). After a delay of a minute or more, both hormones appear to produce a sustained activation of voltage-sensitive L-type calcium channels. The patterns, magnitude, and duration of the changes in [Ca2+]i produced by both hormones are virtually identical. Furthermore, both hormones appear to initiate the same biochemical sequence of reactions that lead to activation of calcium channels. Acute addition of inhibitors of phospholipase C (D609) and protein kinase C (staursporine, calphostin C, and chelerythrine chloride) blocked the increase in [Ca2+]i stimulated by either hormone (Fig. 6Go and Ref. 35). D609, however, failed to block the effects of DOG on [Ca2+]i, consistent with inhibition of the formation rather than the action of diacylglycerol (35). The manner by which either hormone initiates this reaction sequence is unknown. Both GH (6) and insulin (18) can activate the JAK family of kinases, but stimulation of calcium uptake by GH appears to occur by a mechanism that is independent of JAK2 (39), at least in Chinese hamster ovary (CHO) cells engineered to express GH receptors. In these cells, deletion of the proline-rich docking site for JAK2 (box 1) on the GH receptor prevented other responses to GH but failed to diminish the GH-induced increase in [Ca2+]i (39).

The lingering effects of okadaic acid treatment resemble some of the delayed effects of GH on adipocytes. In less than 3 h after treatment with GH, adipocytes lose the ability to increase glucose metabolism in response to GH, but instead respond to GH with an increase in [Ca2+]i (26, 27). Similarly, in less than 3 h after treatment with okadaic acid, adipocytes lose their ability to increase glucose metabolism in response to insulin, and instead respond with an increase in [Ca2+]i. These delayed consequences of treatment with either okadaic acid or GH do not require the continuous presence of either okadaic acid or GH (27) and were blocked by inhibitors of transcription or translation (27), suggesting that both agents induced a genomically directed change in the transduction pathways that caused both GH and insulin to activate L-type channels.

There are also important differences between the consequences of treatment with GH or okadaic acid. GH produces refractoriness only to the effects GH on glucose metabolism, and produces responsiveness to calcium channel activation by GH but not by insulin. Okadaic acid, on the other hand, alters these responses to both insulin and GH. Differences in the manner by which GH and okadaic acid desensitize cells to the stimulatory effects of GH and insulin on glucose metabolism may account for differences in the importance of increased [Ca2+]i for expression of the refractory phenomenon. Little or no relationship between [Ca2+]i and the refractory phenomenon produced by okadaic acid was evident in these experiments, but increased [Ca2+]i was clearly implicated in the refractoriness produced by GH (25, 26, 27). Okadaic acid apparently interferes with stimulation of glucose metabolism by interfering with tyrosine phosphorylation of IRS 1 and 2, perhaps secondary to causing hyperphosphorylation of serine and threonine residues on these proteins (40, 41). Refractoriness produced by GH, however, was seen despite increased tyrosine phosphorylation of IRS, at least in one laboratory (9), although not in another (42).

The finding that okadaic acid failed to sensitize adipocytes to the [Ca2+]i-raising effect of insulin when RNA and protein synthesis were blocked implies some alteration in gene expression may be required. Okadaic acid is known to promote widespread protein phosphorylation (20) and affects transcription of many genes in various tissues (43, 44, 45, 46, 47, 48, 49, 50), probably by increasing phosphorylation of nuclear regulatory proteins. The findings that inhibition of protein kinase A blocks and that dibutyryl cAMP mimics these effects of okadaic acid suggest that the one or more members of the CREB (the cAMP response element binding) family of transactivating proteins (51) might be involved. Transcriptional activation associated with activation of protein kinase C and CREB has also been reported (52, 53). No evidence yet relates GH activity to these transcription factors. The nature and activity of the gene product(s) induced by okadaic acid in adipocytes and their possible relationships, if any, to GH-sensitive genes is unknown.

Although insulin alone did not increase [Ca2+]i in otherwise untreated rat adipocytes, it produced transient increases in [Ca2+]i in rat hepatocytes (54) and in CHO cells that were stably transfected with the cDNA for the human insulin receptor (55). These actions of insulin are probably not related to the latent potential to activate L-type calcium channels in adipocytes that was unmasked by okadaic acid. In both the CHO cells and the hepatocytes, the insulin-stimulated influx of calcium appeared to be through cation channels that were insensitive to the L-type calcium channel blockers, nifedipine (55) or verapamil (54), and at least in the CHO cells, activation of calcium entry appeared to be voltage independent.

Insulin and GH normally activate very different, even opposite, processes in adipocytes. However, under certain circumstances, particularly after a period of GH deprivation (1, 2, 3, 4), GH can activate some elements of the insulin signaling pathway and produce the familiar insulin-like effects that qualitatively, at least, are indistinguishable from the effects of insulin. The present results suggest that conversely, some aspect of the insulin signaling pathway intersects with the GH signaling pathway with the potential for activating calcium channels in a manner that is indistinguishable from the activation produced by GH. That potential is realized in oka-treated adipocytes, but no physiologically relevant circumstances that allow expression of this response to insulin have yet been reported. Adipocytes (and other cells) that are responsive to multiple hormones have the remarkable ability to respond to each hormone in a specific way, even though the signaling pathways arising from different activated receptors may intersect at one or more points. How cells limit the transmission of signals along only prescribed pathways is an important aspect of hormone action that has received little attention. It is likely that, in addition to activating a particular cascade of biochemical reactions or initiating assembly of a reaction complex that leads to expression of a unique pattern of cellular responses, the activated hormone receptor also initiates a cascade of biochemical changes that shuts down alternative pathways and thus directs the signal to a specific outcome. One interpretation of the present findings is that okadaic acid somehow crippled that mechanism so that the signal that arose from activating the insulin receptor went astray.


   Acknowledgments
 
The authors are grateful to Dr. Hiroshi Yamaguchi for technical support, helpful discussions, and encouragement.


   Footnotes
 
1 This publication was made possible by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-19392. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. Preliminary reports of some of these findings were made at the 75th and 80th Meetings of The Endocrine Society, Las Vegas, NE, (Abstract 35) 1993, and New Orleans, LA (Abstract P1-111), respectively. Back

Received March 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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