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Glycogen synthetase in the sea mussel, mytilus edulis L.—III. Regulation by glucose in a mantle tissue slice preparation
Comp. Biochem. Physiol. Vol. 83B, No. 1, pp. 209-214, 1986
0305-0491/86 $3.00+0.00 Pergamon Press Ltd
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GLYCOGEN SYNTHETASE IN THE SEA MUSSEL, M Y T I L U S EDULIS L.--III. REGULATION BY GLUCOSE IN A MANTLE TISSUE SLICE PREPARATION M. A. WHITTLE* a n d P. A. GABBOTTt N.E.R.C. Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd, UK (Tel: 0248-351151)
(Received 31 May 1985) A~tract--1. Treatment of mantle tissue slices with glucose (2-10 mM) increased the I-activity of glycogen synthetase but had no effect on the total (I + D)-activity. The glucose effect was time- and concentrationdependent with maximum stimulation by 5 mM glucose. 2. In the summer the tissue slices had a lower threshold for glucose activation of glycogen synthetase than in the winter. 3.2-Deoxyglucose but not galactose substituted for glucose. The glucose transport inhibitor phloridzin decreased both the I- and (I + D)-activities in the control slices but had no effect on the activation of glycogen synthetase by added 5 mM glucose. 4. Neither mammalian insulin alone or insulin in combination with 2 mM glucose had any effect on glycogen synthetase activity. Our findings are in agreement with the results obtained by injecting insulin (or anti-insulin serum) into whole mussels but are at variance with those of other workers (see Discussion).
INTRODUCTION In a c o m p a n i o n p a p e r we have shown that the activity of glycogen synthetase in the m a n t l e tissue o f Mytilus edulis varies seasonally ( G a b b o t t a n d Whittle, 1985). In the s u m m e r the I-activity (independent of G6P) increased up to 10-fold a n d was higher in mussels o n the shore t h a n for animals starved in the laboratory. Starved mussels r e t u r n e d to the shore showed a n increase in the I-activity of glycogen synthetase. Injection of glucose into the a d d u c t o r muscle resulted in a n increase in the c o n c e n t r a t i o n of glucose in the m a n t l e fluid a n d a t i m e - d e p e n d e n t activation o f glycogen syntbetase. It was further s h o w n that, on the shore, the glucose level in the m a n t l e fluid increased o n submersion, presumably as a result o f feeding. The implication o f these findings is t h a t glucose acts as a physiological regulator o f glycogen synthetase activity. The purpose o f the present investigation was to further investigate the effect o f added glucose o n the activity o f glycogen synthetase using the m a n t l e tissue slice p r e p a r a t i o n described by Z a b a a n d Davies (1980). A preliminary report o f o u r findings has been published elsewhere ( G a b b o t t et al., 1979). MATERIALS AND METHODS Mussels of uniform size (6.5 cm in length) were collected along the mid-spring tide level at Tay-y-Foel in the Menai Strait, Anglesey, UK. For the winter experiments the mussels were kept without food in laboratory tanks, at ambient *Present address: Department of Biochemistry, University of Manchester, Oxford Road, Manchester, UK. tCorrespondence should be addressed to P. A. Gabbott, Department of Biochemistry and Soil Science, University College of North Wales, Bangor, Gwynedd, UK.
temperature, for up to seven days before treatment. For the summer experiments the mussels were collected from the shore and used within 1 hr of collection.
Preparation of mantle tissue slices The method used was essentially the same as described by Zaba and Davies (I 980). Mantles were excised from approx. 30mussels for each experiment and sliced by applying downward pressure to a block of 10 razor blades set I mm apart. The slices were placed in a small plastic sieve, suspended in ice-cold artificial saline (450 mM NaC1, i0 mM KC1, 10mM CaCI2, 55mM MgSO4, 25mM NaHCO 3 gassed with 5% CO2 in air, to pH 7.2) and gently agitated with a magnetic stirrer. After washing for 30 min the slices were rinsed twice with fresh saline and placed on filter paper. Clean-cut, intact slices were weighed on a torsion balance (ca 500 mg per treatment) and added to 4.5 ml saline. The slices were then pre-incubated for a further 30 min to allow temperature equilibration before further treatment. In some instances the pre-incubation medium also contained additives used in the experimental treatments (see Results).
Incubations and methods of analysis Incubations were carried out in squat, 10 ml glass vials in a shaking incubator. For experiments at 7.5°C the incubator was placed in a cold-room with an air temperature of 44i°C. Over a 3 hr incubation period the water bath temperature rose from 5°C to a maximum of 10°C (mean 7.5°C) due to heating from the shaker motor. The experiments were started by adding 0.5 ml saline, with or without additions, to the pre-incubation mixture. At the end of the incubations the slices were strained through 0.05 mm nylon mesh, rinsed twice with saline and transferred to 2.5 ml ice-cold 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM EDTA, 5 mM DTT and 25 mM KF (homogenization buffer). Glycogen synthetase activity was assayed at 25°C by the two-step fluorimetric assay of Passoneau and Rottenberg (1973), as described in the companion paper (Gabbott and Whittle, 1985). The total (I + D)- and I-activities were measured in the presence and absence of 5 mM G6P, respectively. 209
CBP 83/11~-N
210
M.A. WHITTLEand P. A. GABBOTT Total (140) activity
RESULTS AND DISCUSSION
Mantle tissue sfices The mantle tissue of M. edulis contains a number of different cell types, the relative proportions of which vary seasonally (Lowe et al., 1982). The storage tissue is composed of vesicular cells storing large amounts of glycogen and adipogranular cells containing lipid droplets and protein granules (see photomicrographs in Lubet et al., 1976 and Gabbott, 1983). A tissue slice preparation was selected for the experiments because it reflects closely the composition and physiological behaviour of the mantle. With respect to the utilization of added glucose, mantle slices show metabolic activities that respond to effectors which are known to influence the behaviour of the mantle tissue in the intact mussel (Zaba and Davies, 1980; Zaba et al., 1981), and there is a good correlation between the rate of incorporation of [14C]glucose into glycogen, in mantle tissue slices, and the specific activity of glycogen synthetase (see Gabbott, 1983). We can confirm that the mantle tissue slice preparation described by Zaba and Davies (1980) is robust and convenient to use. In the summer, however, it proved difficult to maintain the integrity of the mantle slices until such time (usually in late July-August) as the number of adipogranular cells had increased sufficiently to provide mechanical support for the much larger vesicular cells. Loss of glycogen occurs from the mantle slices during the incubations (Zaba and Davies, 1980) and glycogen released into the medium was expected to be at least partially degraded by hydrolytic enzymes (see Alemany and Rossell-Perez, 1973). The level of free glucose determined in 80% v/v ethanol extracts of the incubation medium, after 1 hr at 20°C was ca 0.5 mM (unpublished results). Experiments with winter mussels Activation o f glycogen synthetase by added glucose. Figure 1 summarizes the results obtained with mantle slices incubated for l - 3 h r in the presence of 2-10 mM glucose. The added glucose concentrations were approx. 10 times higher than the range of seasonal values measured in the mantle tissue and haemolymph by Livingstone and Clarke (1983). In the former case the level of 3-4 #moles/g wet wt determined for female mussels in January-February 1979 was an exceptionally high value. In all three tissue slice experiments the same pattern of results was obtained with maximal I-activities associated with the 5 mM glucose treatment. By contrast, the total (I + D)-activities remained the same for all treatments showing that the activation of glycogen synthetase occurs by conversion of the D (phosphorylated) to I (unphosphorylated) form of the enzyme (for a review of the effects of phosphorylation on the properties of glycogen synthetase see Roach, 1981). The activation of glycogen synthetase by 5 mM glucose was temperature- and time-dependent (Tables 1 and 2) with maximal stimulation after 3 hr treatment at 20°C. The I/(I + D) activity ratio increased to a value of 0.24, the same as the maximum level observed in summer mussels (see Cook and Gabbott, 1978). Although the 7.5°C conditions re-
I _~]_= 2 IO ¢qlm
O
3h 7 . 5 o c
,c
lh 2 0 ° C
3h 2 0 ° C
I-activity
g
T
T
==
=; c
3h 7 . 5 o c
O o
lh 2 0 ° C
3h 2 0 ° C
Activity ratio I/(I+D)
.2
0 3h 7 . 5 ° C
lh 2 0 o c
3h 2 0 o c
Fig. 1. The effect of added glucose on the activity of glycogen synthetase in mantle tissue slices prepared from winter mussels. The total (I + D)- and I-activities were assayed in the presence and absence of 5 mM G6P, respectively. Values are the means of replicated experiments and error-bars represent the SD.
sembled field temperatures in the winter, the activation of the enzyme was lower than at 20°C. Therefore both factors, an increased blood glucose level (due to feeding) and higher temperatures, may contribute to the activation of glycogen synthetase in the summer (see Gabbott and Whittle, 1985). The winter experiments were carried out in December-January. The I-activity of the control slices was always markedly above the value for field mussels, whereas the total (I + D)-activity was always below the corresponding field value (see later, Fig. 2). These activity changes are almost certainly the result of washing and pre-incubation of the slices. The temperature sensitivity of the D form of glycogen synthetase (Cook and Gabbott, 1978) could account for the loss of the total (I + D)-activity. The increased I-activity in washed tissue slices could be explained by removal of an inhibitory factor suppressing glycogen synthesis during gametogenesis (see Whittle et al., 1983) or by stimulation of glucose transport, due to
Regulation of glycogen synthetase by glucose Table 1. Temperature dependence of glucose activation of glycogen synthetase (U/10g wet wt) Treatment Control 5 mM glucose Control 5 mM glucose
Temperature (°C)
I
(I + D)
I/I + D ratio
7.5 7.5
0.42 0.59
4.60 4.87
0.09 (N = 5) 0.12 (N = 5)
0.63 1.05
4.29 4.39
0.15 (N = 8) 0.24 (N = 8)
0.007
0.379
20 20
Error mean square* (df = 22)
All treatments were for 3 hr at the temperatures indicated. *The use of the error mean square statistic is discussed in the companion paper (Gabbott and Whittle, 1985).
mechanical agitation of the slices (see Vega and Kono, 1979), and subsequent glucose activation of glycogen synthetase. Notwithstanding more complex interpretations it is interesting to note that Vega and Kono (1979) initially suspected that an unknown transport inhibitor had been removed by extensive washing of rat epididymal fat cells, but no such activity was found in the washings. After 1 hr incubation at 20°C the free glucose concentration determined in the medium was 0.5mM (see previous section). This is within the physiological range for activation of glycogen synthetase but is 10 times lower than the concentration required for maximal stimulation (Fig. 1). The 50-fold dilution of the tissue during extraction and enzyme assay precludes the possibility that increased G6P levels in the control slices resulted in allosteric activation of glycogen synthetase D. Furthermore, by removing the tissue slices from the medium before homogenization (cf. Zaba and Davis, 1980) the measured changes in glycogen synthetase activity must have occurred within the cells and not in the cell-free medium. Effect of different hexoses. To discover whether other hexoses could substitute for glucose in the activation of glycogen synthetase, mantle slices were incubated at 20°C with 2-deoxyglucose and galactose at a concentration of 5 raM. The results of three experiments are shown in Table 3. Treatment with 2-deoxyglucose but not with galactose increased the I-activity to give 5 mM hexose/control ratios similar to those found with 5 mM glucose. This result is the same as that described for the activation of glycogen synthetase in rat adipocytes by Lawrence and Lamer (1978). In animals the major proportion of 2-deoxyglucose that enters the cell is found as 2-deoxyglucose 6-phosphate which accumulates as a stable metabolite. Again, however, dilution of the
211
tissue extract before enzyme assay precluded the possibility of allosteric activation of glycogen synthetase. According to Lawrence and Lamer (1978) activation of glycogen synthetase in adipocytes by glucose and 2-deoxyglucose takes place via a transport-coupled mechanism involving phosphorylation of the hexose.
Effect of the glucose transport inhibitor phloridzin. If, as suggested above, the activation of glycogen synthetase by added glucose involves the hexose transport system then such activation should be abolished by treatment with glucose transport inhibitors (see Lawrence and Lamer, 1978). The limited solubility of phloridzin meant that the maximum concentration in the incubation medium was 3 mM at 20°C. When present, phloridzin was included in the pre-incubation step as well as during incubation of the mantle slices in the presence of 5 mM glucose (Table 4). The result of the phloridzin treatment
Total (1+0) activity 6
_e U.
o
Summer
Winter
I-activity Ol 0
"I-
T
T
@
c
"1-
0
Winter
0 0
nl
Summer
Activity ratio I1(I,,O) i
.2
Table 2. Time dependence of glucose activation of glycogen synthetase (U/10g wet wt) Time (hr)
I
(I + D)
I/I + D ratio
Control 5 mM glucose
1 1
0.53 0.83
3.74 3.88
0.14 (N = 9) 0.21 (N = 9)
Control 5 mM glucose
3 3
0.63 1.05
4.29 4.39
0.15 (N = 8) 0.24 (N = 8)
Treatment
Error mean square* 0.008 0.423 (df = 30) All treatments were at 20°C for the times indicated. *See Table 1.
Winter
Summer
Fig. 2. The effect of a d d e d glucose on the activity o f glycogen s y n t h e t a s e in m a n t l e tissue slices from w i n t e r a n d s u m m e r mussels. The i n c u b a t i o n s were carried out for 3 hr at 20°C. O t h e r details are given in the legend to Fig. I. Field values were t a k e n from d a t a in the c o m p a n i o n paper, expressed on a wet w t basis (see f o o t n o t e in G a b b o t t a n d Whittle, 1985 referring to Whittle, 1982).
212
M.A. WHITTLEand P. A. GABaOTT
Table 3. The effect of different hexoses on the activity of glycogen synthetase Treatment
Activity
Ratio = experimental/control 1 hr at 20°C
5 mM glucose 5 mM 2-deoxyglucose 5 mM galactose
I I+D I I+ D 1 I+ D
1.67 0.96 1.71 0.97 1.04 0.89
2.26 1.11 1.86 1.09 1.24 1.17
3 hr at 20°C 1.94 1.01 2.04 0.84 1.00 0.92
Table 5. The effect of gluconeogenic precursors on the activity of glycogen synthetase Treatment
Activity
Ratio = experimental/control
5 mM glucose
I I+D
1.59 + 0.23 0.97_+0.15
5 mM pyruvate
I I+D
0.93 + 0.08 1.17_+0.05
5 mM aspartate
1 I+ D
0.88 + 0.05 1.20 _%0.08
Values are means + SD for three experiments in each group. Treatments were for 1 hr at 20°C.
alone was to decrease both the I- and (I + D)-activities of glycogen synthetase below the level of the controls. The decreased experimental/control ratio for total (I + D)-activity (Table 4) suggests that glucose from the incubation medium stabilizes or protects the G6P-dependent activity. The effect of phloridzin may have been to inhibit the re-entry of endogenous glucose into the tissue slices. Competition for transport binding sites between 3 mM phloridzin and the estimated 0.5 mM glucose released from the slices during pre-incubation would probably favour phloridzin. When the Iactivities of the combined (glucose+phloridzin) treatment were expressed relative to the phloridzin treatment alone, the ratios were 1.37 and 1.41 in the two experiments, showing that phloridzin had no effect on the activation of glycogen synthetase by added 5 mM glucose. Similarly, in summer mussels, phloridzin (or phloretin) at a concentration of 2 mM reduced the I-activity to below that of the controls (experimental/control ratio 0.88 + 0.07; N = 5) but had no effect on the activation of glycogen synthetase by added 2 mM glucose. These results are consistent with the idea that the increased 1-activity in the control slices (compared to field mussels) is due to activation of glycogen synthetase by glucose released into the incubation medium. In summer mussels, the transport inhibitors abolished the activation of glycogen synthetase by added 2 mM 2-deoxyglucose (results not shown). The latter result is, however, preliminary and requires further verification. Gluconeogenic precursors. The number of potential gluconeogenic precursors for M. edulis is extensive, and includes the carbon skeletons associated with gluconeogenesis in mammalian systems, the endproducts of anaerobic metabolism such as succinate and propionate (De Zwaan and Wijsmann, 1976; De Zwann, 1977) and the amino acid pool used in osmoregulation (Hoyaux et al., 1976). As examples, pyruvate and aspartate were included in the incubations at 5 mM (Table 5). Neither substrate resulted in a measureable change in the I-activity of glycogen Table 4. The effect of phloridzin on the activation of glycogen synthetase Treatment
Activity
Ratio = experimental/control
5 mM glucose
I I+ D
1.64 1.03
1.47 0.92
3 mM phloridzin
I I+ D
0.81 0.82
0.74 0.75
5 mM glucose+ 3 mM phloridzin
I I+ D
1.11 0.84
1.04 0.79
Treatments were for 3 hr at 20°C.
synthetase. However, both treatments gave experimental/control ratios for the total (I + D)-activity which were significantly greater than one. This was an unexpected result for which no explanation can be offered based on the glucose treatments. We have noted, however, that for rat hepatocytes Katz et al. (1979) have shown that in response to treatment with glucose + gluconeogenic precursors, the activity of the G6P-dependent form of glycogen synthetase increases in parallel with changes in the I-activity (contrary to the 'either I or else D' concept for the regulation of glycogen synthetase; see Katz et al., 1979). Experiments with summer mussels
In mantle slice preparations made in August, the total (I + D)-activities were markedly lower than the activity in field mussels (Fig. 2). As in the winter experiments, however, the I-activity in the control slices was twice the activity in the field animals. Glucose treatment had no effect on the total (I + D)-activity but the I-activity increased above the controls at all three glucose concentrations (Fig. 2). The mantle tissue appears to have a lower threshold for glucose activation of glycogen synthetase in the summer than in the winter. Thus, the level of Iactivity after the 2 mM glucose treatment was the same as after the 5 mM glucose treatment in the winter, giving I/(I + D) activity ratios of 0.26 (N = 9) and 0.24 (N = 8), respectively. The thickness of the mantle tissue is clearly important when considering the availability of exogenous substrates and modulators. For example, Zaba and Davies (1980) have shown that the utilisation of added glucose is more rapid in tissue slices than in the unsliced, excised mantle. Since the mantle is generally thicker in the winter than in the summer, the difference in the effectiveness of 2 mM glucose as an activator of glycogen synthetase is consistent with a permeability effect or apparent lowering of the threshold for glucose activation. Glucose utilization by mantle slices exhibits saturation kinetics with a Km for glucose of approx. 1 mM (Zaba and Davies, 1980). Although hyperbolic kinetics are consistent with a carrier-mediated transport mechanism for glucose uptake, measurement of 3H20 release from [2-3H]glucose can only identify a ratelimiting step prior to the formation of fructose 6-phosphate. Thus glucose transport or hexokinase activity, or both could be limiting. Zaba (1981) has shown that in the mantle tissue of M. edulis the hexokinase reaction is displaced far from equilibrium
Regulation of glycogen s y n t h e t a s e in agreement with its known regulatory function in other organisms. The activation of glycogen synthetase by added glucose seems to reflect closely, the kinetics of glucose utilization in agreement with the hypothesis that activation occurs subsequent to the transport and phosphorylation of the hexose (see Lawrence and Lamer, 1978). The effect of other modulators (2 mM concentrations of 2-deoxyglucose, phloridzin and phloretin) were the same as described previously for mantle slices from winter mussels (results not shown). Absence o f an effect o f mammalian insulin on the activity o f glycogen synthetase The aim of this series of experiments was to investigate the direct action of insulin, and the possible synergistic effect with added glucose, on the activation of glycogen synthetase (see, for example, Lawrence et al., 1977). Since in winter mussels the activation of the enzyme appears to be saturated by 5 mM glucose, the insulin treatments were combined with a lower glucose concentration of 2 mM. Insulin, when present, was included in the pre-incubation medium as well as the experimental incubations. Crystalline bovine insulin was dissolved in a minimum volume of 1 M NaOH, neutralized with HC1 and diluted with physiological saline to a concentration equivalent to 1 U/ml. In the winter experiments the results obtained for each treatment after 1 and 3 hr at 20°C were not significantly different from each other and were combined to give the data presented in Table 6. The results show that neither mammalian insulin alone or insulin in combination with 2 mM added glucose had any effect on the activity of glycogen synthetase. The absence of an insulin effect in summer mussels is particularly important since it was suspected that the marked increase in I-activity observed seasonally in May-June (Gabbott and Whittle, 1985) might be due to an insulin-like substance (see Plisetskaya et al., 1978, 1979). The experiments were then repeated with concanavalin A (lectin from jack bean, Sigma Grade IV) which shares with insulin the ability to activate glycogen synthetase in rat adipocytes (Lawrence and Lamer, 1978). As with the insulin treatments concanavalin A was also included in the pre-incubation medium, at a final concentration of 0.1 mg/ml. None of the treatments, however, had any effect on the activity of glycogen synthetase (results not shown). Apparently, in mantle
Table 6. Absence of an effect of insulin on the activation of glycogen synthetase Ratio = experimental/control Winter mussels* Summer musselst
Treatment
Activity
2 mM glucose
I I+ D
1.49 + 0.12 1.08 + 0.02
1.48 ___0.05 1.00 ___0.05
1 U/ml insulin
I I+D
1.14 + 0.09 1.02+0.12
1.07 ___0.05 1.10±0.07
2 mM glucose+
I
1.56 ± 0.19
1.41 ± 0.18
1U/ml insulin
I+D
1.10 ± 0 . 1 8
1.04±0.08
Values are means -t- SD for three experiments in each group. *For winter mussels data were combined for treatments of 1 and 3 hr at 20°C (see text). t F o r summer mussels treatments were for 3 hr at 20°C.
by glucose
213
slices, concanavalin A does not overcome the specificity problems associated with mammalian insulin assayed in the molluscan system. On the other hand the absence of an insulin effect is consistent with the results obtained by injecting insulin or anti-insulin serum into whole mussels but is at variance with the findings of Plisetskaya and co-workers who have also used the mammalian mone in molluscan studies (see Discussion in companion paper and review by Gabbott, 1983). In seeking to reconcile this disagreement we have suggested that there may be tissue-specific differences between the mantle and the muscle tissues (used by Plisetskaya et al., 1978, 1979). Another possible explanation is that, in summer mussels, insulin produces a stable change in the glucose transport system or its microenvironment by increasing membrane fluidity (see Pilck et al., 1980). In this way 'priming' of the mantle tissue by endogenous molluscan insulin could result in the observed summer-winter differences in the activation of glycogen synthetase by low concentrations of added glucose (Fig. 2). The insulin effect might be stable throughout the slice preparation and further treatment with mammalian insulin would therefore be without effect. In view of the complex cellular composition of the mantle tissue it is also possible that insulin stimulation of glucose uptake in one cell type (presumably sensitive to inhibition by phloridzin) could be masked by an insulinindependent effect in other cells.
REFERENCES
Alemany M. and Rossell-Perez M. (1973) Two different amylase activities in the sea mussel, Mytilus edulis L. Revtg esp. Fisiol. 29, 217-222. Cook P. A. and Gabbott P. A. (1978) Glycogen synthetase in the sea mussel Mytilus edulis L. I. Purification, interconversion and kinetic properties of the I and D forms. Comp. Biochem. Physiol. 60B, 419~t21. De Zwaan A. (1977) Anaerobic energy metabolism in bivalve molluscs. Oceanogr. Mar. Biol. Ann. Rev. 15, 103-187. De Zwaan A. and Wijsman T. C. M. (1976) Anaerobic metabolism in bivalvia (Mollusca). Characteristics of anaerobic metabolism. Comp. Biochem. Physiol. 54B, 313-324. Gabbott P. A. (1983) Developmental and seasonal metabolic activities in marine molluscs. In The Mollusca (Edited by Hochachka P. W.), Vol. 2, pp. 165-217. Academic Press, New York. Gabbott P. A. and Whittle M. A. (1985) Glycogen synthetase in the sea mussel Mytilus edulis L.--II. Seasonal changes in glycogen content and glycogen synthetase activity in the mantle tissue. Comp. Biochem. Physiol. 8313, 197-207. Gabbott P. A., Cook P. A. and Whittle M. A. (1979) Seasonal changes in glycogen synthase activity in the mantle tissue of Mytilus edulis L.: regulation by tissue glucose. Biochem. Soc. Trans. 7, 895-896. Hoyaux J., Gilles R. and Jeuniaux C. (1976). Osmoregulation in molluscs of the intertidal zone. Comp. Biochem. Physiol. 5 3 A , 3 6 1 - 3 6 5 , K a t z J., G o l d e n S. a n d W a l s P. A. (1979) G l y c o g e n s y n thesis b y r a t h e p a t o c y t e s . Biochem. 3". 180, 3 8 9 - 4 0 2 .
Lawrence J. C. and Lamer J. (1978) Activation of glycogen synthase in rat adipocytes by insulin and glucose involves increased glucose transport and phosphorylation. J. biol. Chem. 253, 2104-2113.
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Lawrence J. C., Guinovart J. J. and Lamer J. (1977) Activation of rat adipocyte glycogen synthase by insulin. J. biol. Chem. 252, 444-450. Livingstone D. R. and Clarke K. R. (1983) Seasonal changes of hexokinase from the mantle tissue of the common mussel Mytilus edulis L. Comp. Biochem. Physiol. 74B, 691 702. Lowe D. M., Moore M. N. and Bayne B. L. (1982) Aspects of gametogenesis in the marine mussle Mytilus edulis. J. Mar. Biol. Assoc. U.K. 62, 133-145. Lubet P., Herlin P., Mathieu M. and Collin F. (1976) Tissue de r6serve et cycle sexuel chez les lamellibranches. Haliotis 7, 59~52. Passoneau J. V. and Rottenberg D. A. (1973) An assessment of the methods for the measurement of glycogen synthetase activity including a new direct one-step assay. Analyt. Biochem. 51, 528-541. Pilch P. F., Thompson P. A. and Czech M. P. (1980) Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin treated adipocytes. Proc. natn. Acad. Sci. U.S.A. 77, 915-918. Plisetskaya E., Kazakov V. K., Soltitskaya L. and Leibson L. G. (1978) Insulin-producing cells in the gut of freshwater bivalve molluscs Anodonta cygnea and Unio pictorum and the role of insulin in the regulation of their carbohydrate metabolism. Gen. comp. Endocrinol. 35, 133-145.
Plisetskaya E. M., Soltitskaya L. P. and Leibson L. G. (1979) Participation of insulin in metabolic regulation in marine bivalve molluscs. J. Evol. Biochem. Physiol. (English translation) 15, 243-248. Roach P. J. (1981) Glycogen synthase and glycogen synthase kinases. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. K.), Vol. 20, pp. 45 105. Academic Press, New York. Vega F. V. and Kono T. (1979) Sugar transport in fat cells: effects of mechanical agitation, cell-bound insulin and temperature. Archs Biochem. Biophys. 192, 12(~ 127. Whittle M. A., Mattieu M., Gabbott P. A. and Lubet P. (1983) The effect of glucose and neuro-endocrine factors on the activity ratio of glycogen synthetase in organ cultures of the mantle of Mytilus edulis. In Molluscan Neuroendocrinology (Edited by Lever J. and Boer H. H.), p. 183. North-Holland, Amsterdam. Zaba B. N. (1981) Glycogenolytic pathways in the mantle tissue of Mytilus edulis L. Mar. Biol. Lett. 2, 67-74. Zaba B. N. and Davies J. I. (1980) Glucose metabolism in an in vitro preparation of the mantle tissue from Mytilus edulis L. Mar. Biol. Lett. 1, 235 243. Zaba B. N. Gabbott P. A. and Davies J. I. (1981) Seasonal changes in the utilization of 14C- and 3H-labelled glucose in a mantle tissue slice preparation of Mytilus edulis L. Comp. Biochem. Physiol. 70B, 689-695.
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GLYCOGEN SYNTHETASE IN THE SEA MUSSEL, M Y T I L U S EDULIS L.--III. REGULATION BY GLUCOSE IN A MANTLE TISSUE SLICE PREPARATION M. A. WHITTLE* a n d P. A. GABBOTTt N.E.R.C. Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd, UK (Tel: 0248-351151)
(Received 31 May 1985) A~tract--1. Treatment of mantle tissue slices with glucose (2-10 mM) increased the I-activity of glycogen synthetase but had no effect on the total (I + D)-activity. The glucose effect was time- and concentrationdependent with maximum stimulation by 5 mM glucose. 2. In the summer the tissue slices had a lower threshold for glucose activation of glycogen synthetase than in the winter. 3.2-Deoxyglucose but not galactose substituted for glucose. The glucose transport inhibitor phloridzin decreased both the I- and (I + D)-activities in the control slices but had no effect on the activation of glycogen synthetase by added 5 mM glucose. 4. Neither mammalian insulin alone or insulin in combination with 2 mM glucose had any effect on glycogen synthetase activity. Our findings are in agreement with the results obtained by injecting insulin (or anti-insulin serum) into whole mussels but are at variance with those of other workers (see Discussion).
INTRODUCTION In a c o m p a n i o n p a p e r we have shown that the activity of glycogen synthetase in the m a n t l e tissue o f Mytilus edulis varies seasonally ( G a b b o t t a n d Whittle, 1985). In the s u m m e r the I-activity (independent of G6P) increased up to 10-fold a n d was higher in mussels o n the shore t h a n for animals starved in the laboratory. Starved mussels r e t u r n e d to the shore showed a n increase in the I-activity of glycogen synthetase. Injection of glucose into the a d d u c t o r muscle resulted in a n increase in the c o n c e n t r a t i o n of glucose in the m a n t l e fluid a n d a t i m e - d e p e n d e n t activation o f glycogen syntbetase. It was further s h o w n that, on the shore, the glucose level in the m a n t l e fluid increased o n submersion, presumably as a result o f feeding. The implication o f these findings is t h a t glucose acts as a physiological regulator o f glycogen synthetase activity. The purpose o f the present investigation was to further investigate the effect o f added glucose o n the activity o f glycogen synthetase using the m a n t l e tissue slice p r e p a r a t i o n described by Z a b a a n d Davies (1980). A preliminary report o f o u r findings has been published elsewhere ( G a b b o t t et al., 1979). MATERIALS AND METHODS Mussels of uniform size (6.5 cm in length) were collected along the mid-spring tide level at Tay-y-Foel in the Menai Strait, Anglesey, UK. For the winter experiments the mussels were kept without food in laboratory tanks, at ambient *Present address: Department of Biochemistry, University of Manchester, Oxford Road, Manchester, UK. tCorrespondence should be addressed to P. A. Gabbott, Department of Biochemistry and Soil Science, University College of North Wales, Bangor, Gwynedd, UK.
temperature, for up to seven days before treatment. For the summer experiments the mussels were collected from the shore and used within 1 hr of collection.
Preparation of mantle tissue slices The method used was essentially the same as described by Zaba and Davies (I 980). Mantles were excised from approx. 30mussels for each experiment and sliced by applying downward pressure to a block of 10 razor blades set I mm apart. The slices were placed in a small plastic sieve, suspended in ice-cold artificial saline (450 mM NaC1, i0 mM KC1, 10mM CaCI2, 55mM MgSO4, 25mM NaHCO 3 gassed with 5% CO2 in air, to pH 7.2) and gently agitated with a magnetic stirrer. After washing for 30 min the slices were rinsed twice with fresh saline and placed on filter paper. Clean-cut, intact slices were weighed on a torsion balance (ca 500 mg per treatment) and added to 4.5 ml saline. The slices were then pre-incubated for a further 30 min to allow temperature equilibration before further treatment. In some instances the pre-incubation medium also contained additives used in the experimental treatments (see Results).
Incubations and methods of analysis Incubations were carried out in squat, 10 ml glass vials in a shaking incubator. For experiments at 7.5°C the incubator was placed in a cold-room with an air temperature of 44i°C. Over a 3 hr incubation period the water bath temperature rose from 5°C to a maximum of 10°C (mean 7.5°C) due to heating from the shaker motor. The experiments were started by adding 0.5 ml saline, with or without additions, to the pre-incubation mixture. At the end of the incubations the slices were strained through 0.05 mm nylon mesh, rinsed twice with saline and transferred to 2.5 ml ice-cold 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM EDTA, 5 mM DTT and 25 mM KF (homogenization buffer). Glycogen synthetase activity was assayed at 25°C by the two-step fluorimetric assay of Passoneau and Rottenberg (1973), as described in the companion paper (Gabbott and Whittle, 1985). The total (I + D)- and I-activities were measured in the presence and absence of 5 mM G6P, respectively. 209
CBP 83/11~-N
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M.A. WHITTLEand P. A. GABBOTT Total (140) activity
RESULTS AND DISCUSSION
Mantle tissue sfices The mantle tissue of M. edulis contains a number of different cell types, the relative proportions of which vary seasonally (Lowe et al., 1982). The storage tissue is composed of vesicular cells storing large amounts of glycogen and adipogranular cells containing lipid droplets and protein granules (see photomicrographs in Lubet et al., 1976 and Gabbott, 1983). A tissue slice preparation was selected for the experiments because it reflects closely the composition and physiological behaviour of the mantle. With respect to the utilization of added glucose, mantle slices show metabolic activities that respond to effectors which are known to influence the behaviour of the mantle tissue in the intact mussel (Zaba and Davies, 1980; Zaba et al., 1981), and there is a good correlation between the rate of incorporation of [14C]glucose into glycogen, in mantle tissue slices, and the specific activity of glycogen synthetase (see Gabbott, 1983). We can confirm that the mantle tissue slice preparation described by Zaba and Davies (1980) is robust and convenient to use. In the summer, however, it proved difficult to maintain the integrity of the mantle slices until such time (usually in late July-August) as the number of adipogranular cells had increased sufficiently to provide mechanical support for the much larger vesicular cells. Loss of glycogen occurs from the mantle slices during the incubations (Zaba and Davies, 1980) and glycogen released into the medium was expected to be at least partially degraded by hydrolytic enzymes (see Alemany and Rossell-Perez, 1973). The level of free glucose determined in 80% v/v ethanol extracts of the incubation medium, after 1 hr at 20°C was ca 0.5 mM (unpublished results). Experiments with winter mussels Activation o f glycogen synthetase by added glucose. Figure 1 summarizes the results obtained with mantle slices incubated for l - 3 h r in the presence of 2-10 mM glucose. The added glucose concentrations were approx. 10 times higher than the range of seasonal values measured in the mantle tissue and haemolymph by Livingstone and Clarke (1983). In the former case the level of 3-4 #moles/g wet wt determined for female mussels in January-February 1979 was an exceptionally high value. In all three tissue slice experiments the same pattern of results was obtained with maximal I-activities associated with the 5 mM glucose treatment. By contrast, the total (I + D)-activities remained the same for all treatments showing that the activation of glycogen synthetase occurs by conversion of the D (phosphorylated) to I (unphosphorylated) form of the enzyme (for a review of the effects of phosphorylation on the properties of glycogen synthetase see Roach, 1981). The activation of glycogen synthetase by 5 mM glucose was temperature- and time-dependent (Tables 1 and 2) with maximal stimulation after 3 hr treatment at 20°C. The I/(I + D) activity ratio increased to a value of 0.24, the same as the maximum level observed in summer mussels (see Cook and Gabbott, 1978). Although the 7.5°C conditions re-
I _~]_= 2 IO ¢qlm
O
3h 7 . 5 o c
,c
lh 2 0 ° C
3h 2 0 ° C
I-activity
g
T
T
==
=; c
3h 7 . 5 o c
O o
lh 2 0 ° C
3h 2 0 ° C
Activity ratio I/(I+D)
.2
0 3h 7 . 5 ° C
lh 2 0 o c
3h 2 0 o c
Fig. 1. The effect of added glucose on the activity of glycogen synthetase in mantle tissue slices prepared from winter mussels. The total (I + D)- and I-activities were assayed in the presence and absence of 5 mM G6P, respectively. Values are the means of replicated experiments and error-bars represent the SD.
sembled field temperatures in the winter, the activation of the enzyme was lower than at 20°C. Therefore both factors, an increased blood glucose level (due to feeding) and higher temperatures, may contribute to the activation of glycogen synthetase in the summer (see Gabbott and Whittle, 1985). The winter experiments were carried out in December-January. The I-activity of the control slices was always markedly above the value for field mussels, whereas the total (I + D)-activity was always below the corresponding field value (see later, Fig. 2). These activity changes are almost certainly the result of washing and pre-incubation of the slices. The temperature sensitivity of the D form of glycogen synthetase (Cook and Gabbott, 1978) could account for the loss of the total (I + D)-activity. The increased I-activity in washed tissue slices could be explained by removal of an inhibitory factor suppressing glycogen synthesis during gametogenesis (see Whittle et al., 1983) or by stimulation of glucose transport, due to
Regulation of glycogen synthetase by glucose Table 1. Temperature dependence of glucose activation of glycogen synthetase (U/10g wet wt) Treatment Control 5 mM glucose Control 5 mM glucose
Temperature (°C)
I
(I + D)
I/I + D ratio
7.5 7.5
0.42 0.59
4.60 4.87
0.09 (N = 5) 0.12 (N = 5)
0.63 1.05
4.29 4.39
0.15 (N = 8) 0.24 (N = 8)
0.007
0.379
20 20
Error mean square* (df = 22)
All treatments were for 3 hr at the temperatures indicated. *The use of the error mean square statistic is discussed in the companion paper (Gabbott and Whittle, 1985).
mechanical agitation of the slices (see Vega and Kono, 1979), and subsequent glucose activation of glycogen synthetase. Notwithstanding more complex interpretations it is interesting to note that Vega and Kono (1979) initially suspected that an unknown transport inhibitor had been removed by extensive washing of rat epididymal fat cells, but no such activity was found in the washings. After 1 hr incubation at 20°C the free glucose concentration determined in the medium was 0.5mM (see previous section). This is within the physiological range for activation of glycogen synthetase but is 10 times lower than the concentration required for maximal stimulation (Fig. 1). The 50-fold dilution of the tissue during extraction and enzyme assay precludes the possibility that increased G6P levels in the control slices resulted in allosteric activation of glycogen synthetase D. Furthermore, by removing the tissue slices from the medium before homogenization (cf. Zaba and Davis, 1980) the measured changes in glycogen synthetase activity must have occurred within the cells and not in the cell-free medium. Effect of different hexoses. To discover whether other hexoses could substitute for glucose in the activation of glycogen synthetase, mantle slices were incubated at 20°C with 2-deoxyglucose and galactose at a concentration of 5 raM. The results of three experiments are shown in Table 3. Treatment with 2-deoxyglucose but not with galactose increased the I-activity to give 5 mM hexose/control ratios similar to those found with 5 mM glucose. This result is the same as that described for the activation of glycogen synthetase in rat adipocytes by Lawrence and Lamer (1978). In animals the major proportion of 2-deoxyglucose that enters the cell is found as 2-deoxyglucose 6-phosphate which accumulates as a stable metabolite. Again, however, dilution of the
211
tissue extract before enzyme assay precluded the possibility of allosteric activation of glycogen synthetase. According to Lawrence and Lamer (1978) activation of glycogen synthetase in adipocytes by glucose and 2-deoxyglucose takes place via a transport-coupled mechanism involving phosphorylation of the hexose.
Effect of the glucose transport inhibitor phloridzin. If, as suggested above, the activation of glycogen synthetase by added glucose involves the hexose transport system then such activation should be abolished by treatment with glucose transport inhibitors (see Lawrence and Lamer, 1978). The limited solubility of phloridzin meant that the maximum concentration in the incubation medium was 3 mM at 20°C. When present, phloridzin was included in the pre-incubation step as well as during incubation of the mantle slices in the presence of 5 mM glucose (Table 4). The result of the phloridzin treatment
Total (1+0) activity 6
_e U.
o
Summer
Winter
I-activity Ol 0
"I-
T
T
@
c
"1-
0
Winter
0 0
nl
Summer
Activity ratio I1(I,,O) i
.2
Table 2. Time dependence of glucose activation of glycogen synthetase (U/10g wet wt) Time (hr)
I
(I + D)
I/I + D ratio
Control 5 mM glucose
1 1
0.53 0.83
3.74 3.88
0.14 (N = 9) 0.21 (N = 9)
Control 5 mM glucose
3 3
0.63 1.05
4.29 4.39
0.15 (N = 8) 0.24 (N = 8)
Treatment
Error mean square* 0.008 0.423 (df = 30) All treatments were at 20°C for the times indicated. *See Table 1.
Winter
Summer
Fig. 2. The effect of a d d e d glucose on the activity o f glycogen s y n t h e t a s e in m a n t l e tissue slices from w i n t e r a n d s u m m e r mussels. The i n c u b a t i o n s were carried out for 3 hr at 20°C. O t h e r details are given in the legend to Fig. I. Field values were t a k e n from d a t a in the c o m p a n i o n paper, expressed on a wet w t basis (see f o o t n o t e in G a b b o t t a n d Whittle, 1985 referring to Whittle, 1982).
212
M.A. WHITTLEand P. A. GABaOTT
Table 3. The effect of different hexoses on the activity of glycogen synthetase Treatment
Activity
Ratio = experimental/control 1 hr at 20°C
5 mM glucose 5 mM 2-deoxyglucose 5 mM galactose
I I+D I I+ D 1 I+ D
1.67 0.96 1.71 0.97 1.04 0.89
2.26 1.11 1.86 1.09 1.24 1.17
3 hr at 20°C 1.94 1.01 2.04 0.84 1.00 0.92
Table 5. The effect of gluconeogenic precursors on the activity of glycogen synthetase Treatment
Activity
Ratio = experimental/control
5 mM glucose
I I+D
1.59 + 0.23 0.97_+0.15
5 mM pyruvate
I I+D
0.93 + 0.08 1.17_+0.05
5 mM aspartate
1 I+ D
0.88 + 0.05 1.20 _%0.08
Values are means + SD for three experiments in each group. Treatments were for 1 hr at 20°C.
alone was to decrease both the I- and (I + D)-activities of glycogen synthetase below the level of the controls. The decreased experimental/control ratio for total (I + D)-activity (Table 4) suggests that glucose from the incubation medium stabilizes or protects the G6P-dependent activity. The effect of phloridzin may have been to inhibit the re-entry of endogenous glucose into the tissue slices. Competition for transport binding sites between 3 mM phloridzin and the estimated 0.5 mM glucose released from the slices during pre-incubation would probably favour phloridzin. When the Iactivities of the combined (glucose+phloridzin) treatment were expressed relative to the phloridzin treatment alone, the ratios were 1.37 and 1.41 in the two experiments, showing that phloridzin had no effect on the activation of glycogen synthetase by added 5 mM glucose. Similarly, in summer mussels, phloridzin (or phloretin) at a concentration of 2 mM reduced the I-activity to below that of the controls (experimental/control ratio 0.88 + 0.07; N = 5) but had no effect on the activation of glycogen synthetase by added 2 mM glucose. These results are consistent with the idea that the increased 1-activity in the control slices (compared to field mussels) is due to activation of glycogen synthetase by glucose released into the incubation medium. In summer mussels, the transport inhibitors abolished the activation of glycogen synthetase by added 2 mM 2-deoxyglucose (results not shown). The latter result is, however, preliminary and requires further verification. Gluconeogenic precursors. The number of potential gluconeogenic precursors for M. edulis is extensive, and includes the carbon skeletons associated with gluconeogenesis in mammalian systems, the endproducts of anaerobic metabolism such as succinate and propionate (De Zwaan and Wijsmann, 1976; De Zwann, 1977) and the amino acid pool used in osmoregulation (Hoyaux et al., 1976). As examples, pyruvate and aspartate were included in the incubations at 5 mM (Table 5). Neither substrate resulted in a measureable change in the I-activity of glycogen Table 4. The effect of phloridzin on the activation of glycogen synthetase Treatment
Activity
Ratio = experimental/control
5 mM glucose
I I+ D
1.64 1.03
1.47 0.92
3 mM phloridzin
I I+ D
0.81 0.82
0.74 0.75
5 mM glucose+ 3 mM phloridzin
I I+ D
1.11 0.84
1.04 0.79
Treatments were for 3 hr at 20°C.
synthetase. However, both treatments gave experimental/control ratios for the total (I + D)-activity which were significantly greater than one. This was an unexpected result for which no explanation can be offered based on the glucose treatments. We have noted, however, that for rat hepatocytes Katz et al. (1979) have shown that in response to treatment with glucose + gluconeogenic precursors, the activity of the G6P-dependent form of glycogen synthetase increases in parallel with changes in the I-activity (contrary to the 'either I or else D' concept for the regulation of glycogen synthetase; see Katz et al., 1979). Experiments with summer mussels
In mantle slice preparations made in August, the total (I + D)-activities were markedly lower than the activity in field mussels (Fig. 2). As in the winter experiments, however, the I-activity in the control slices was twice the activity in the field animals. Glucose treatment had no effect on the total (I + D)-activity but the I-activity increased above the controls at all three glucose concentrations (Fig. 2). The mantle tissue appears to have a lower threshold for glucose activation of glycogen synthetase in the summer than in the winter. Thus, the level of Iactivity after the 2 mM glucose treatment was the same as after the 5 mM glucose treatment in the winter, giving I/(I + D) activity ratios of 0.26 (N = 9) and 0.24 (N = 8), respectively. The thickness of the mantle tissue is clearly important when considering the availability of exogenous substrates and modulators. For example, Zaba and Davies (1980) have shown that the utilisation of added glucose is more rapid in tissue slices than in the unsliced, excised mantle. Since the mantle is generally thicker in the winter than in the summer, the difference in the effectiveness of 2 mM glucose as an activator of glycogen synthetase is consistent with a permeability effect or apparent lowering of the threshold for glucose activation. Glucose utilization by mantle slices exhibits saturation kinetics with a Km for glucose of approx. 1 mM (Zaba and Davies, 1980). Although hyperbolic kinetics are consistent with a carrier-mediated transport mechanism for glucose uptake, measurement of 3H20 release from [2-3H]glucose can only identify a ratelimiting step prior to the formation of fructose 6-phosphate. Thus glucose transport or hexokinase activity, or both could be limiting. Zaba (1981) has shown that in the mantle tissue of M. edulis the hexokinase reaction is displaced far from equilibrium
Regulation of glycogen s y n t h e t a s e in agreement with its known regulatory function in other organisms. The activation of glycogen synthetase by added glucose seems to reflect closely, the kinetics of glucose utilization in agreement with the hypothesis that activation occurs subsequent to the transport and phosphorylation of the hexose (see Lawrence and Lamer, 1978). The effect of other modulators (2 mM concentrations of 2-deoxyglucose, phloridzin and phloretin) were the same as described previously for mantle slices from winter mussels (results not shown). Absence o f an effect o f mammalian insulin on the activity o f glycogen synthetase The aim of this series of experiments was to investigate the direct action of insulin, and the possible synergistic effect with added glucose, on the activation of glycogen synthetase (see, for example, Lawrence et al., 1977). Since in winter mussels the activation of the enzyme appears to be saturated by 5 mM glucose, the insulin treatments were combined with a lower glucose concentration of 2 mM. Insulin, when present, was included in the pre-incubation medium as well as the experimental incubations. Crystalline bovine insulin was dissolved in a minimum volume of 1 M NaOH, neutralized with HC1 and diluted with physiological saline to a concentration equivalent to 1 U/ml. In the winter experiments the results obtained for each treatment after 1 and 3 hr at 20°C were not significantly different from each other and were combined to give the data presented in Table 6. The results show that neither mammalian insulin alone or insulin in combination with 2 mM added glucose had any effect on the activity of glycogen synthetase. The absence of an insulin effect in summer mussels is particularly important since it was suspected that the marked increase in I-activity observed seasonally in May-June (Gabbott and Whittle, 1985) might be due to an insulin-like substance (see Plisetskaya et al., 1978, 1979). The experiments were then repeated with concanavalin A (lectin from jack bean, Sigma Grade IV) which shares with insulin the ability to activate glycogen synthetase in rat adipocytes (Lawrence and Lamer, 1978). As with the insulin treatments concanavalin A was also included in the pre-incubation medium, at a final concentration of 0.1 mg/ml. None of the treatments, however, had any effect on the activity of glycogen synthetase (results not shown). Apparently, in mantle
Table 6. Absence of an effect of insulin on the activation of glycogen synthetase Ratio = experimental/control Winter mussels* Summer musselst
Treatment
Activity
2 mM glucose
I I+ D
1.49 + 0.12 1.08 + 0.02
1.48 ___0.05 1.00 ___0.05
1 U/ml insulin
I I+D
1.14 + 0.09 1.02+0.12
1.07 ___0.05 1.10±0.07
2 mM glucose+
I
1.56 ± 0.19
1.41 ± 0.18
1U/ml insulin
I+D
1.10 ± 0 . 1 8
1.04±0.08
Values are means -t- SD for three experiments in each group. *For winter mussels data were combined for treatments of 1 and 3 hr at 20°C (see text). t F o r summer mussels treatments were for 3 hr at 20°C.
by glucose
213
slices, concanavalin A does not overcome the specificity problems associated with mammalian insulin assayed in the molluscan system. On the other hand the absence of an insulin effect is consistent with the results obtained by injecting insulin or anti-insulin serum into whole mussels but is at variance with the findings of Plisetskaya and co-workers who have also used the mammalian mone in molluscan studies (see Discussion in companion paper and review by Gabbott, 1983). In seeking to reconcile this disagreement we have suggested that there may be tissue-specific differences between the mantle and the muscle tissues (used by Plisetskaya et al., 1978, 1979). Another possible explanation is that, in summer mussels, insulin produces a stable change in the glucose transport system or its microenvironment by increasing membrane fluidity (see Pilck et al., 1980). In this way 'priming' of the mantle tissue by endogenous molluscan insulin could result in the observed summer-winter differences in the activation of glycogen synthetase by low concentrations of added glucose (Fig. 2). The insulin effect might be stable throughout the slice preparation and further treatment with mammalian insulin would therefore be without effect. In view of the complex cellular composition of the mantle tissue it is also possible that insulin stimulation of glucose uptake in one cell type (presumably sensitive to inhibition by phloridzin) could be masked by an insulinindependent effect in other cells.
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Lawrence J. C., Guinovart J. J. and Lamer J. (1977) Activation of rat adipocyte glycogen synthase by insulin. J. biol. Chem. 252, 444-450. Livingstone D. R. and Clarke K. R. (1983) Seasonal changes of hexokinase from the mantle tissue of the common mussel Mytilus edulis L. Comp. Biochem. Physiol. 74B, 691 702. Lowe D. M., Moore M. N. and Bayne B. L. (1982) Aspects of gametogenesis in the marine mussle Mytilus edulis. J. Mar. Biol. Assoc. U.K. 62, 133-145. Lubet P., Herlin P., Mathieu M. and Collin F. (1976) Tissue de r6serve et cycle sexuel chez les lamellibranches. Haliotis 7, 59~52. Passoneau J. V. and Rottenberg D. A. (1973) An assessment of the methods for the measurement of glycogen synthetase activity including a new direct one-step assay. Analyt. Biochem. 51, 528-541. Pilch P. F., Thompson P. A. and Czech M. P. (1980) Coordinate modulation of D-glucose transport activity and bilayer fluidity in plasma membranes derived from control and insulin treated adipocytes. Proc. natn. Acad. Sci. U.S.A. 77, 915-918. Plisetskaya E., Kazakov V. K., Soltitskaya L. and Leibson L. G. (1978) Insulin-producing cells in the gut of freshwater bivalve molluscs Anodonta cygnea and Unio pictorum and the role of insulin in the regulation of their carbohydrate metabolism. Gen. comp. Endocrinol. 35, 133-145.
Plisetskaya E. M., Soltitskaya L. P. and Leibson L. G. (1979) Participation of insulin in metabolic regulation in marine bivalve molluscs. J. Evol. Biochem. Physiol. (English translation) 15, 243-248. Roach P. J. (1981) Glycogen synthase and glycogen synthase kinases. In Current Topics in Cellular Regulation (Edited by Horecker B. L. and Stadtman E. K.), Vol. 20, pp. 45 105. Academic Press, New York. Vega F. V. and Kono T. (1979) Sugar transport in fat cells: effects of mechanical agitation, cell-bound insulin and temperature. Archs Biochem. Biophys. 192, 12(~ 127. Whittle M. A., Mattieu M., Gabbott P. A. and Lubet P. (1983) The effect of glucose and neuro-endocrine factors on the activity ratio of glycogen synthetase in organ cultures of the mantle of Mytilus edulis. In Molluscan Neuroendocrinology (Edited by Lever J. and Boer H. H.), p. 183. North-Holland, Amsterdam. Zaba B. N. (1981) Glycogenolytic pathways in the mantle tissue of Mytilus edulis L. Mar. Biol. Lett. 2, 67-74. Zaba B. N. and Davies J. I. (1980) Glucose metabolism in an in vitro preparation of the mantle tissue from Mytilus edulis L. Mar. Biol. Lett. 1, 235 243. Zaba B. N. Gabbott P. A. and Davies J. I. (1981) Seasonal changes in the utilization of 14C- and 3H-labelled glucose in a mantle tissue slice preparation of Mytilus edulis L. Comp. Biochem. Physiol. 70B, 689-695.