What signals are involved in the stimulation of glucose transport by insulin in muscle cells?

CellularSignallingVol. 5, No. 5, pp. 519-529, 1993. Printed in Great Britain.

0898-6568/93 $6.110+ .00 © 1993PergamonPressLtd

MINI REVIEW WHAT SIGNALS ARE INVOLVED IN THE STIMULATION OF GLUCOSE TRANSPORT BY INSULIN IN MUSCLE CELLS? AMIRA KLIP, TOOLSIE RAMLAL, PHILIP J. BILAN, ANDRI~ MARETTE, ZHI L m a n d YASUI-IIDE MITSUMOTO

Division of Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8 (Received 30 March 1993; and accepted 24 April 1993)

1. INTRODUCTION

potential messengers involved in the stimulation of glucose transport, i.e. "the response".

INSULIN signalling is one of the most tenaciously investigated and most controversial topics in the field of hormone action. Perhaps the fallacy lies in trying to find a unifying signalling pathway for a hormone which triggers a diversity of responses varying in their time of appearance, the metabolic pathways involved and the cellular targets. This Mini Review focuses on the possible signalling pathways involved in the stimulation of glucose transport by insulin in L6 cells, a line of rat skeletal muscle cells which re-enact myogenesis in vitro and respond to the hormone at the differentiated, multinucleated myotube stage [1, 2]. The choice of a cell line to study a hormonal response typical of muscle tissue and fat cells is based on the ease of analysing signalling pathways through a systematic experimental approach. In general, this consists of measuring changes in the levels of the purported signal, mimicking insulin action by artificially generating the signal, and interfering with insulin action by exogenous tampering with the generation of the proposed signal. This strategy can be readily implemented in cells in culture but neither easily nor unambiguously in intact tissues or organisms. The reader should not expect an unequivocal answer at the end of this review, as the signal(s) mediating stimulation of glucose transport by insulin remain outstanding. This chapter merely analyses the pathways that have been ruled out and suggests

2. NATURE OF THE RESPONSE The L6 cell line originates from leg skeletal muscle of day-old rats [1], and in the differentiated stage expresses many biochemical, morphological, electrical, contractile and metabolic characteristics of mature skeletal muscle [2]. The multinucleated L6 myotubes display the ability to respond to insulin or IGF-I with an elevation in glucose uptake [2, 3]. Receptors for each growth factor have been described in these cells [4-8], and at relatively low concentrations ( < 3nM for IGF-1, < 30nM for insulin), metabolic responses are elicited selectively through the IGF-I or insulin receptors by their corresponding ligand [7, 8]. At higher concentrations, IGF-I and insulin cross-activate each others' receptors. It has been observed that the mechanism of stimulation of glucose transport by each ligand is indistinguishable [3] (see below), and is perhaps mediated by hybrid insulin/IGF-I receptors as well [3]. Insulin rapidly stimulates the uptake of nonmetabolizable sugars into L6 myotubes, with maximal effects observed within 15 min of addition of the hormone. Typically the response elicited by 100 nM insulin achieves a 1.6- to 2fold rise in glucose uptake above basal levels [2]. This rate of uptake is sustained for up to 519

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A. KLIPet al.

120 min, when a second phase of stimulation starts if the hormone continues to be present in the medium [2, 3]. The first or acute phase of stimulation is independent of protein synthesis, whereas the further increase, which peaks in about 10 h and reaches a 3- to 4-fold increase in transport, is prevented by the inhibitor of protein synthesis, cycloheximide [2, 3, 9]. Glucose transport in L6 cells is mediated by transporters of the facilitative diffusion type, ' members of a multifamily of gene products. L6 cells express the ubiquitous GLUT1 transporter throughout muscle cell differentiation, although a less glycosylated form of the transporter appears during myogenesis and the overall content of the doublet progressively diminishes [10, 11]. In contrast, the muscle/fat specific glucose transporter isoform, GLUT4, appears only upon differentiation and is linked to the muscle-specific phenotype of these cells [10-12]. L6 myotubes constitute the only muscle cell line expressing this isoform in culture [13]. In addition, L6 cells have significant levels of the foetal muscle glucose transporter isoform, GLUT3 [14], which is present at fairly steady levels throughout L6 cell differentiation. Based on subceUular fractionation studies coupled to immunoblotting with isoform-specific antibodies, GLUT1 and GLUT3 polypeptides are detected largely in the plasma membrane and to a lesser extent in an intracellular light microsomal pool, whereas the GLUT4 polypeptide is found mostly in the latter, with only a minor part co-purifying with the plasma membranes [11]. In response to insulin or IGF-1, the content of all three transporter isoforms diminishes in the intracellular light microsomes and increases in the plasma membranes [11, 13, 14] (Fig. 1). The largest relative change occurs for GLUT4, as is the case in mature skeletal muscle [15]. The overall gain in glucose transporters regardless of isoform (measured as D-glucoseprotectable cytochalasin B binding sites) in the plasma membranes is matched by the loss of transporters from the light microsomes [16]. Moreover, the fold-increase in cytochalasin B binding sites in the plasma membrane is virtually identical to the fold stimulation of

glucose uptake in parallel cultures [16]. These observations strongly suggest that translocation of glucose transporters suffices to produce the observed increase in transport activity, and is the essence of the acute hormonal response of glucose transporters. In contrast, the long-term response to insulin involves a net increase in GLUT1 glucose transporter protein preceded and matched by an increase in GLUT1 mRNA levels [9]. These are produced by both enhanced transcription and improved stability of the transcript [17, 18]. IGF-I causes similar increases in GLUTI and GLUT3 protein (Fig. 2A) and mRNA (Fig. 2B), contrasting with a diminution in GLUT4 protein and mRNA in the course of several hours. The elevated levels of GLUTI and GLUT3 protein are reflected in a gain in these proteins in the plasma membrane as well as in the intracellular pool. Conversely, GLUT4 transporters are particularly reduced in the latter pool. Questions arising are: what signals emanate from the activated receptor which impinge on the intracellular vesicles? Do all transporters reside in the same vesicles and respond to the same signal? Which specific intracellular components aid in vesicular traffic? What mediates vesicle fusion with the plasma membrane? Do different signals mediate the acute and longterm responses? What is the mechanism for the insulin-dependent induction of genes encoding specific glucose transporter isoforms?

3. POTENTIAL SIGNALS INVOLVED IN THE ACUTE RESPONSE TO INSULIN OF L6 CELLS Insulin receptor substrate- 1 ( I R S - 1 )

The earliest response to insulin is probably the phosphorylation of intracellular products by direct association with the activated insulin receptor endowed with tyrosine kinase activity. IRS-1, also known as ppl60/pp185 based on its molecular size in SDS-PAGE, is thought to be the first substrate of the insulin receptor kinase in rat liver cells [19]. We have detected insulin-

PM --

+

IM --

+

GLUT 1

GLUT 3

GLUT 4 FIG. 1. Acute effect of insulin on the subcellular distribution of glucose transporters in L6 myotubes. Cell monolayers were deprived of serum for 5h and exposed to 100nM insulin for I h in a-minimum essential medium (~-MEM). Purified plasma membranes (PM) and intracellular membranes (IM) were prepared and the different glucose transporter isoforms were detected by SDS-PAGE and western blot analysis using isoformspecific antibodies (dilutions: 1:1000 East Acres anti-GLUTl antiserum; 1:300 anti-GLUT3 antiserum, kind gilt of Dr Ian Simpson, NIH, Bethesda; 1:500 East Acres anti-GLUT4 antiserum) and ~25I-labelled protein A. Virtually identical results were observed using 3 nM insulin-like growth factor-I instead of insulin. Due to the different antibody potencies, the results cannot be compared quantitatively in the vertical direction, only in the horizontal direction.

521

A GLUT 1

GLUT3

GLUT 4 B 1B 2 0.5 1 1.5 2 4 6 8 1 6 2 4

HOURS

B

GLUT 1

GLUT3

GLUT4 B 0.5

1

1.5

2

4

6

8

16

24

HOURS FIG. 2. Long-term effect of insulin-like growth factor-1 on total content of glucose transporter proteins (A) and m R N A (B) in L6 myotubes. Cell monolayers were incubated in ~-MEM containing 2% foetal bovine serum and 5 mg/ml BSA, in the presence of 10 nM IGF-1 for the indicated time (in hours). (A) Total membranes were prepared from post-nuclear supernatants by sedimentation at 177,000g for 1 h. Fifteen micrograms of protein were applied to each lane of S D S - P A G E gels followed by analysis of glucose transporter polypeptides by western blots as described in Fig. 1. (B) Total R N A was isolated and 15/tg were applied to 0.9% formaldehyde agarose gels for analysis of glucose transporter m R N A by northern blots, using specific cDNA probes (kind gifts of Drs M. Birnbaum, C. Burant and D. James for GLUT1, GLUT3 and GLUT4, respectively), 32p-labelled by the random priming procedure. Controls showed no change with time in the total amount of 28S and 18S ribosomal R N A or on the labelling of c-Hras m R N A (results not shown).

522

I m m u n o p p t n : cx I R S - 1 p o l y ab

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10 -3

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116

C

C

I

I

G

G

I m m u n o b l o t : a P Y m o n o ab FIG. 3. Effect of acute treatment with insulin and insulin-like growth factor-1 on the phosphorylation of the insulin receptor substrate 1 (IRS-I) in L6 myotubes. Cell monolayers were deprived of serum for 5 h, followed by exposure to control buffer (C), 100nM insulin (I), or l nM 1GF-1 (G) for 15 min. The cells were immediately frozen in liquid nitrogen, thawed in vanadate-containing lysis buffer, and the lysate was incubated overnight at 4°C with a 1:200 dilution of a polyclonal antibody raised to IRS-I (kind gift of Dr A. Snltiel, Parke Davis Laboratories) followed by precipitation with protein A-Sepharose beads for 2h. Immunoprecipitated protein was dissolved in sample buffer and analysed by S D S - P A G E and western blots using a monoclonal anti-phosphotyrosine antibody (1:3000 dilution) and labelling was visualized by the enhanced chemilluminiscence method. Samples from two assays of each condition are illustrated. The position of molecular weight standards is shown on the left margin.

M r x 10 -3

116

alNa+/K+-ATPase --I 97

-,131 v-ras (H) bH

CPM

1M PM

FIG. 4. Presence of v-ras (H)-like proteins in subcellular fractions of L6 myotubes. Subcellular fractions from L6 myotubes were prepared as in Fig. 1, and samples were analysed of homogenates (H), crude plasma membranes (CPM), purified plasma membranes (PM) and light intracellular microsomes (IM) by S D S - P A G E (10 #g protein/lane) and western blotting, using a monoclonal antibody (1:10,000 dilution) raised to a fusion protein consisting of a v-ras (H) synthetic peptide (residues 96-118) coupled to keyhole limpet haemocyanin (lower panel) or a monoclonal antibody (McK1, kind gift of Dr K. Sweadner, Harvard University) raised to the ~1 subunit of the Na+/K+-ATPase and used at a 1:100 dilution. The bottom panel stems from 10% polyacrylamide gels, the top panel from 7.5% polyacrylamide gels, each calibrated with pre-stained molecular weight standards.

523

Total membranes

GLUT 1 r C XFF/xl

GLUT 4 m C X F F/X I FIG. 6. Effect of long-term exposure to control conditions (C), forskolin (F), isobutyl methyl xanthine (X), forskolin and isobutyl methylxanthine (F/X) or insulin (I) on the net cellular content of glucose transporters in L6 myotubes. All incubations were as described in Fig. 5. Total membrane polypeptides were prepared as described in Fig. 2A, followed by S D S - P A G E as described in Fig. 1. Thirty micrograms of protein were applied to each lane. Top panel: total cellular content of G L U T I polypeptides, (r) indicates red blood cell standard (0.1 #g protein). Bottom panel: total cellular content of GLUT4 polypeptides, m, skeletal muscle membrane standard (3 #g protein).

PM

IM

CF

CF

GLUT 1

GLUT 4 C F F/X FIG. 7. Effect of long-term exposure to control conditions (C), forskolin (F) or forskolin plus IBMX (F/X) on the subcellular distribution of glucose transporters. Incubations were as described in Fig. 5. Purified plasma membranes (PM) and intracellular light microsomes (IM) were isolated as described in Fig. 1. Fifteen micrograms of protein were loaded per lane and analysed by S D S - P A G E as described in Fig. 1. Top panel: distribution of G L U T I polypeptides. Bottom panel: distribution of GLUT4 polypeptides in the PM.

524

Glucose transport in musclecells dependent tyrosine phosphorylation on polypeptides of the size of IRS-1 in L6 muscle cells [20] and have immunoprecipitated IRS-I from L6 myotubes with a specific polyclonal antibody. The immunoprecipitate contains a polypeptide of 170,000-180,000 M r phosphorylated in tyrosine residues based on its detection with a monoclonal anti-phosphotyrosine antibody on western blots. The phosphotyrosine content of this band dramatically rises when the cells are pre-treated with insulin or IGF-I (Fig. 3). The migration of the polypeptide is further retarded as a function of increased phosphorylation. The role of IRS-1 in stimulation of glucose transport has not been tested. However, the levels of IRS-I, detected from immunoblots of total cell extracts, do not seem to vary during L6 cell myogenesis [21], whereas the response of glucose transport develops after maturation into myotubes [2, 10].

Role of G proteins Insulin-dependent phosphorylation of IRS-1 is thought to be reduced by action of GTP-binding proteins, based on the sharp reduction in phosphotyrosine content of IRS-I upon incorporation of GTPyS into permeabilized L6 cells [20]. This effect of GTP?S is effectively blocked by GDPflS. The nature of the purported G proteins involved and their possible role in further down-stream signalling is yet to be investigated. In search for smallmolecular weight G proteins that might participate in the process of recruitment of glucose transporters, we probed the donor and recipient subcellular fractions involved in glucose transporter traffic with antibodies specific for v-ras (H)-like proteins on western blots. A polypeptide doublet of 23,000 M r is present in several subfractions of these cells, each fraction applied at equal loading (10/~g protein/lane; Fig. 4). The amount of these polypeptides was faint in homogenates, increased in isolated crude plasma membranes (CPM) and peaked in purified plasma membranes (PM). The 23,000 M, immunoreactive bands were several-fold more abundant in the PM, i.e. the glucose

525

transporter recipient fraction, than in the purified intracellular light microsomes (IM; i.e. the donor fraction). For comparison, the distribution of the plasma membrane marker ~tl subunit of the Na÷/K÷-ATPase is shown in the upper panel. A similar enrichment of the smallmolecular weight G proteins was found in isolated plasma membranes from mature rat skeletal muscle compared to intracellular membrane fraction (results not shown), as well as when L6 myotube or muscle membrane fractions were tested for polypeptides binding GTPTS on a GTP?S overlay assay (Van der Meulen J. et al:, unpublished observation). Collectively, these results suggest that if smallmolecular weight G proteins immunologically related to v-ras (H) participate in insulin action, they may act at the point of fusion of vesicles with the recipient plasma membrane rather than at the point of sensing an insulin signal on the donor vesicles. One cannot rule out at this stage, however, that the low level of smallmolecular weight G proteins co-purifying with the light microsomal pool may play a role in sensing insulin action. Ca 2+ signals Signalling through changes in cytosolic calcium levels, [Ca2+]~, is central in the action of growth factors such as EGF and hormones such as vasopressin. [Ca2+]i plays its role as a signal by activating Ca2+-calmodulin dependent kinase, inducing membrane changes (including fusion) and activating proteases. These activities could potentially participate in insulinmediated intracellular traffic. However, the [Ca2+]i signalling pathway does not appear to be essential for insulin action. In fact, [Ca2÷]~ does not vary in response to insulin in L6 muscle cells [22], 3T3-LI adipocytes [23], 3T3 Swiss fibroblasts [24] or rat cardiocytes [25], measured with diverse intracellular fluorescent Ca 2+ indicators. Consistent with these findings, insulin action is not associated with changes in inositol phosphates [26, 27]. Moreover, stimulation of glucose transport by insulin occurs in the absence of extracellular Ca 2+ in both L6

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TABLE I. EFFECT OF BAFILOMYCIN A I ON BASAL AND ACUTE INSULIN-STIMULATED HEXOSE UPTAKE IN L6 MYOTUBES

Insulin stimulation Condition Control Bafilomycin A1

Basal pmol/min •mg

pmol/min -mg

%

A

P

17.25_+0.58 11.61 _+0.23

24.06_+0.43 15.24_+0.81

140 131

6.81 3.63

< 0.005 < 0.005

L6 myotubes grown in ct-MEM containing 2% foetal bovine serum were exposed to control buffer or 200 nM Bafilomycin A1 for 1 h, followed by a second l-h incubation with or without 100 nM insulin in the presence of the drug. Control cells were only exposed to insulin for 1 h. Hexose uptake was measured for 5 min using 10 #M [3H]2-deoxy-D-glucose. Results are the mean ___S.E.M. of four independent experiments, each performed at least in triplicate. P values derived from Student's t-test are for insulin-stimulated vs basal uptake. muscle cells and 3T3-L1 adipocytes loaded with Ca 2+ buffers [22, 23]. However, specific [Ca2+]i levels may play a permissive role in insulin action, as substantiated by observations of decreased insulin stimulation of glucose transport in adipocytes with elevated [Ca2+]i [28].

Role of cytosolic and intravesicular pH A well-recognized function of insulin in muscle is the alkalinization of the cytoplasm, which facilitates activation of phosphofructokinase with a consequent augmentation of glycolytic flux [29, 30]. In L6 myotubes, insulin rapidly elevates cytosolic pH (pHi) through activation of Na+/H + exchange [31]. This response peaks within 10 min and hence precedes the full stimulation of glucose transport. However, prevention of the alkalinization with the inhibitor of the Na+/H + antiport, amiloride, does not preclude stimulation of hexose uptake. Moreover, cytoplasmic alkalinization in the absence of insulin, through the use of the ionophore monensin or by elevating extracellular pH, does not augment glucose uptake [30, 31]. These observations make it unlikely that the alkalinization of the cytoplasm plays other than a permissive role for the hormonal stimulation of glucose transport. By analogy to secretory granules and endosomes, it is possible that the intracellular vesicles endowed with glucose transporters

maintain an acidic intravesicular pH. Intravesicular acidity is required for exocytosis in several cell systems. We therefore investigated whether the light microsomal pool of L6 myotubes contains H÷-ATPases of the v (vacuolar) type common to endomembranes and explored the relevance of intravesicular pH gradients in insulin action. Two lines of evidence suggest that the donor intracellular pool either does not have an acidic intravesicular space or that such an acidic milieu is not essential for insulin response in L6 cells. Firstly, collapsing the intravesicular pH gradient with monensin [31, 32] or low chloroquine concentrations [33] does not prevent stimulation of glucose transport by the hormone [31-33]. Secondly, the inhibitor of v-type H÷-ATPases, Bafilomycin A 1, does not impede stimulation of glucose transport by insulin (Table 1). This drug was tested in the presence of serum where insulin stimulated glucose uptake by about 40% above basal levels. Although Bafilomycin A1 diminished somewhat the basal uptake, insulin still caused a 31% increase in glucose uptake above the diminished basal value in the presence of the drug. It is intriguing, however, that if the data are expressed as the portion of transport that is stimulated by insulin (i.e. insulin-stimulated "minus" basal transport) the effect of insulin is halved, from 6.8 in the absence to 3.6 pmol/min.mg protein in the presence of Bafilomycin A I. These results

Glucose transport in musclecells suggest that the full stimulation of glucose transport, perhaps the full effectiveness of recruitment of glucose transporters, may utilize a pathway involving proton pumps. The extent of such participation remains controversial given the monensin experiments described above.

Activation of protein kinase C ( PKC) The role of PKC in the stimulation of glucose uptake by insulin action is complex and controversial [27]. In L6 cells, PKC-activating phorbol esters stimulate glucose uptake, but down-regulation of about 90% of the total cellular PKC activity by 24 h pre-exposure to high levels of PMA does not abort the hormonal response, yet blocks the acute response to phorbol dibutyrate [34]. This would suggest that insulin does not require the major part of PKC to elicit the response of glucose uptake. However, it is possible that the remaining 10% of PKC activity, mediated by specific isoforms of the enzyme, suffices to mediate the response to insulin. This is indeed the case in the smooth muscle-like cell line BC3H-1, where the PKC fl isoform appears to be important for insulin action, while the ~ isoform appears to be the responsive one to phorbol esters [35]. If the hormone activates PKC, this is not achieved through the same mechanism as activation by diacylglycerol or exogenous phorbol esters, which promote anchoring of cytosolic PKC on to the cell membrane that subsequently activates the enzyme. Insulin does not cause translocation of PKC from the cytoplasm to membranes in L6 cells, but it does cause a 9% rise in PKC activity in the cytosol [34]. The consequence of this increase in glucose transport has not been assessed. However, inhibition of PKC with the specific inhibitor H7 does not prevent insulin action in L6 myotubes [36]. With prolonged incubation, this compound diminishes both basal and insulin-mediated uptake, making it difficult to assess the role of PKC in the stimulation of transport by this means [36]. The effect of phorboi esters on the subcellular localization of glucose transporters

527

in L6 cells has not been explored, largely due to the smaller response they produce compared to that of insulin. By comparison, phorbol esters produce a larger response than insulin in BC3H-1 myocytes, suggesting that in those cells PKC may play a more significant role in the regulation of glucose transport. 4. POTENTIAL SIGNALS INVOLVED IN THE LONG-TERM STIMULATION OF GLUCOSE TRANSPORT BY INSULIN IN L6 CELLS As stated above, long-term exposure ( > 2 h) of L6 myotubes to insulin causes stimulation of glucose transport requiring new protein synthesis and accompanied by biosynthetic increases in GLUTI and GLUT3 glucose transporters. Conversely, GLUT4 glucose transporter protein and mRNA levels are depressed. Recent reports in the literature demonstrate that agents which raise intracellular cAMP in 3T3-L1 adipocytes such as cholera toxin and dibutyryl cAMP cause an elevation in GLUTI protein and a fall in GLUT4 protein [37, 38]. This led us to hypothesize that the long-term action of insulin in L6 myotubes might be mediated by an elevation in cAMP. Insulin typically depresses cAMP levels acutely, but no information is available on the status of cAMP upon sustained exposure to the hormone. To test this possibility, we incubated L6 cells with a potent activator of adenylyl cyclase, forskolin, or the inhibitor of cAMP phosphodiesterase, isobutyl methyl xanthine (IBMX). These compounds effectively elevate cAMP levels in L6 muscle cells [39]. Like insulin, both drugs stimulate glucose uptake measured after 16 h (Fig. 5) and their effect develops in several hours (results not shown). However, in contrast to the prolonged action of insulin, the stimulation caused by forskolin or IBMX is not prevented by inhibiting protein synthesis with cycloheximide (Fig. 5). Moreover, the stimulation caused by IBMX is additive to that elicited by insulin (Fig. 5). Forskolin (and IBMX, not shown) also provokes a rise in total levels of GLUTI protein (Fig. 6), and this action is further poten-

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F1G. 5. Effect of long-term exposure to insulin (Ins), isobutyl methyl xanthine (X) and forskolin (Forsk) in the absence (open bars) or presence (cross-hatched bars) of cycloheximide on glucose transport in L6 myotubes. Cells were exposed to 100nM insulin, I mM isobutyl methyl xanthine without or with insulin, or 20pM forskolin for 16h in a ct-MEM containing 2% foetal bovine serum in the presence or absence of 1 #g/ml cycloheximide, following which uptake of 2-deoxyglucose (5 rain, 10#M) was assayed.

tiated when both drugs are used in combination where a synergistic action on cAMP levels is expected. The forskolin-dependent elevation in GLUT1 protein was evident both in the plasma membrane and in the intracellular pool (Fig. 7). The increase at the cell surface of this transporter isoform may be responsible for the elevation in transport activity, since the amount of G L U T 4 polypeptides in the plasma membrane does not change. Unlike 3T3-LI adipocytes in which the stimulation of hexose transport by cholera toxin was higher than the gain in cell surface glucose transporters [37], the forskolinmediated increase in GLUT1 protein in the plasma membrane of L6 myotubes is substantial and commensurate with the increase in transport activity (Figs 5, 6). Interestingly, contrasting with the insulin effect, forskolin does not reduce the total cell content of G L U T 4 protein (Fig. 6) even in the combined presence of forskolin and IBMX. These results can be rationalized by the recent finding that different response elements mediate the actions

of cAMP and insulin on the G L U T 4 gene of 3T3-L1 adipocytes [40]. In conclusion, agents that elevate cAMP levels for several hours stimulate glucose transport activity, but the mechanism of this activation may differ from that of long-term exposure to insulin. Both the agents and the hormone raise GLUT1 protein levels, but whereas the hormone acts biosynthetically the cAMP-elevating agents appear to stabilize the protein against degradation. The net result in both cases is an elevated number of G L U T I glucose transporters in the plasma membrane. Future studies will likely reveal the interplay between the insulin signalling pathway and the cAMP cascade in the regulation of glucose transport. Acknowledgements--We thank ROBERT SARGEANT for help with some photographic material. We thank DRS K. SWEADNER, A. SALanEL. I. SIMPSONand S. GRINSTEINfor kindly providing antibodies to the ~tl subunit of the Na+/K+-ATPase, IRS-I, GLUT3 and v-ras (H) proteins, respectively, as well as DRs M. BIRNBAUM, D. JAMESand C. BURANTfor providing the cDNA probes for GLUTI, GLUT4 and GLUT3. The work summarized was supported by the Medical Research Council (M.R.C.) and the Muscular Dystrophy Association of Canada. P.J.B. held a studentship from the M.R.C.A.M. held a postdoctoral fellowship from the M.R.C., and Y.M. held a post-doctoral fellowship from the J.D.F.I.

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