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364 Fukunaga, K., Noguchi, T., Takeda, H., Matozaki, T., Hayashi, Y., Itoh, H andKasuga, M (2000) Requirement for protein tyrosine phosphatase SHP-2 in insulin-

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365 Maegawa, H., Hasegawa, M., Sugai, S., Obata, T., Ugi, S., Morino, K., Egawa, K.,Fujita, T., Sakamoto, T., Nishio, Y., Kojima, H., Haneda, M., Yasuda, H., Kikkawa, R.and Kashiwagi, A (1999) Expression of a dominant negative SHP-2 in transgenic mice

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366 Neel, B G., Gu, H and Pao, L (2003) The ‘Shp’ing news: SH2 domain-containing

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367 Kayali, A G., Eichhorn, J., Haruta, T., Morris, A J., Nelson, J G., Vollenweider, P.,Olefsky, J M and Webster, N J G (1998) Association of the insulin receptor withphospholipase Cγ (PLCγ) in 3T3-L1 adipocytes suggests a role for PLCγ in metabolic

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368 Eichhorn, J., Kayali, A G., Austin, D A and Webster, N J (2001) Insulin activatesphospholipase C-gamma 1 via a PI 3-kinase dependent mechanism in 3T3-L1

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369 Lorenzo, M., Teruel, T., Hernandez, R., Kayali, A G and Webster, N J (2002)PLCgamma participates in insulin stimulation of glucose uptake through activation

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370 Lemmon, M A and Ferguson, K M (2000) Signal-dependent membrane targeting by

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371 Chan, T O., Rittenhouse, S E and Tsichlis, P N (1999) AKT/PKB and other D3phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent

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372 Li, H S., Shome, K., Rojas, R., Rizzo, M A., Vasudevan, C., Fluharty, F., Santy, L.C., Casanova, J E and Romero, G (2003) The guanine nucleotide exchange factor

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374 Krugmann, S., Anderson, K E., Ridley, S H., Risso, N., McGregor, A., well, J., Davidson, K., Eguinoa, A., Ellson, C D., Lipp, P., Manifava, M., Ktis-takis, N., Painter, G., Thuring, J W., Cooper, M A., Lim, Z Y., Holmes, A B.,Dove, S K., Michell, R H., Grewal, A., Nazarian, A., Erdjument-Bromage, H.,Tempst, P., Stephens, L R and Hawkins, P T (2002) Identification of ARAP3, anovel PI3K effector regulating both Arf and Rho GTPases, by selective capture on

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62 THE INSULIN RECEPTOR AND DOWNSTREAM SIGNALLING

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384 Romero, G and Larner, J (1993) Insulin mediators and the mechanism of insulin

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386 Muller, G., Jung, C., Frick, W., Bandlow, W and Kramer, W (2002) Interaction ofphosphatidylinositolglycan(-peptides) with plasma membrane lipid rafts triggers insulin-

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42 480 – 42 487

Direct and indirect regulation of glucose metabolism by insulin

in its classical target tissues

Insulin is more than an endocrine messenger for the transition from fasted- to fed-state metabolism; it is in fact a required regulator in all physiological states.

Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly

 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6

64 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

During fasting, circulating insulin levels are 5–20 per cent of those measured after a meal These low circulating concentrations of the hormone are required

to maintain the balance with counter-regulatory hormones to prevent sis The brain and cells such as erythrocytes that rely solely on glucose as their energy source consume glucose evenly in the fed and overnight fasted states.1Therefore, maintaining normoglycaemia despite fluctuations in the availability

ketoacido-of exogenous glucose relies on a coordinated regulation ketoacido-of glucose disposal and endogenous glucose production During fasting, the liver, and to a lesser degree the kidney, release glucose to the blood, matching its utilization by glucose- dependent tissues such as the brain Under these conditions, glucose disposal into skeletal muscle and adipocytes is low, where lipids are consumed as the major fuel Upon a meal, when circulating glucose and insulin levels rise, glu- cose is disposed of from the blood into muscle, fat and liver.1 These tissues therefore constitute the ‘classical target organs’ for insulin action through which the hypoglycaemic response of the hormone is directly achieved (Table 2.1).

In addition to increasing glucose uptake into skeletal muscle and adipose sue, insulin promotes glucose storage as either glycogen (mainly in muscle and liver) or lipids (mainly in fat and liver) To avoid futile metabolic cycles, insulin simultaneously inhibits the breakdown of these macromolecules through glycogenolysis and lipolysis, respectively Similarly, in the liver and kidney endogenous glucose production is curbed by insulin through the inhibition of glycogenolysis and gluconeogenesis.3, 4

tis-Research of recent years utilizing tissue-specific gene deletions of the insulin receptor in animal models largely confirmed the direct effects of insulin on glucose metabolism in its classical target organs Mice lacking the insulin recep- tor in the liver (liver insulin receptor knock-out (LIRKO) mice) fail to sup- press endogenous glucose production during hyperinsulinaemic clamps.5 Like- wise, ablation of the insulin receptor gene in skeletal muscle (MIRKO mice) blunts insulin-stimulated glucose transport and glycogen synthesis in this tissue.6Remarkably, glucose uptake into adipose tissue is elevated in MIRKO mice, suggesting that insulin’s direct effects on its classical target tissues are coordi- nated, allowing for complex adaptive responses and balances in the regulation

of whole body glucose metabolism, as discussed further below Hence, insulin

can be viewed as a glucose flux regulator, promoting peripheral glucose uptake

and hepatic glucose storage during a meal, and allowing hepatic glucose output while preventing ketoacidosis between meals.

On top of the major direct effects of insulin on glucose fluxes in its classical target organs (Table 2.1), the hormone also engages indirect mechanisms in the

regulation of glucose metabolism These include insulin-mediated alterations in lipid and protein metabolism (described in detail in Chapters 3 and 4 of this book) that in turn impact on glucose metabolism, coordination of glucose fluxes between its various target tissues, and regulation of circulating factors involved

in cross-talk between skeletal muscle, adipose tissue and liver.

INSULIN AS A MASTER REGULATOR OF WHOLE BODY GLUCOSE DISPOSAL 65 Table 2.1 ‘Classical’ and ‘non-classical’ target organs for insulin-regulated glucosemetabolism

Main effect of insulin on glucose metabolism

↑ hexose monophosphate shunt∗

↓ NEFA availability andoxidation

Adipose tissue ↑ glucose uptake

↑ hexose monophosphate shunt∗

↑ lipogenesis

Regulation of adipokinessynthesis and/or secretion

↓ lipolysis

‘Non-classical targets’

Pancreatic beta cells ? Permissive effect on

glucose-stimulated insulinsecretion (phase 1 release)

↑ capillary recruitment

↑ NO secretion

A ‘direct effect’ of insulin on glucose metabolism is defined as an insulin-stimulated change in the flux

of glucose through a specific metabolic pathway that is initiated by the insulin receptor in the sametissue An ‘indirect effect’ is the regulation of glucose metabolism in one organ resulting from the effect

of insulin on other macronutrients (such as lipids) or in other organs

∗Insulin-stimulated lipogenesis consumes NADPH, and the resulting drop in NADPH/NADP+increasesthe activity of G6PD, i.e G6P flux through the shunt In addition, insulin regulates the mRNA levels

of G6PD – the rate-limiting enzyme in this pathway.2

Insulin is a key regulator of the interplay between glucose and fatty acids metabolism, as follows Insulin-mediated inhibition of lipolysis, i.e the release

of non-esterified fatty acids (NEFAs) from adipose tissue, contributes to the acute inhibition of hepatic glucose production induced by the hormone.7, 8

This is brought about by lowering NEFA oxidation, since in this process high intracellular ATP/ADP, NADH/NAD+and acetyl-CoA/CoA ratios are achieved, providing the metabolite milieu required for gluconeogenesis In addition, NEFA availability to skeletal muscle also influences insulin-regulated glucose utiliza- tion by this tissue The original hypothesis of Randle9 suggested that increased NEFA availability as a fuel source to the muscle (as occurs physiologically dur- ing fasting) blocks glycolysis through the elevated generation of acetyl-CoA and

66 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

citrate, resulting in allosteric inhibition of pyruvate dehydrogenase and fructokinase-1, respectively The ensuing accumulation of glucose-6-phosphate (G6P) would then secondarily diminish glucose uptake However, while a glu- cose–NEFA cycle probably exists, recent studies using nuclear magnetic res- onance (NMR) spectroscopy demonstrate that, in skeletal muscle, increased NEFA availability reduces, rather than elevates, intracellular G6P and free glu- cose levels.10, 11 These findings suggest that insulin-stimulated glucose uptake

phospho-is itself a primary site of inhibition by NEFA in skeletal muscle.

Further examples of the indirect actions of insulin emerge from out animal models In particular, tissue-specific gene deletions have allowed investigators to manipulate glucose flux in a single tissue and then assess the ensuing changes in the non-targeted organs As mentioned above, MIRKO mice exhibited decreased insulin-stimulated glucose flux into the skeletal muscle, accompanied by an increased glucose flux in adipose tissue.6 Similarly, mice lacking the insulin-responsive glucose transporter GLUT4 selectively in the adi- pose tissue show not only reduced glucose uptake in fat cells but also blunted insulin regulation of glucose metabolism in muscle and liver.12 Such depen- dency of glucose metabolism among the different organs suggests the existence

knock-of mechanisms that mediate inter-organ cross-talk An exciting development in this regard has been the identification of adipose-derived factors (adipokines) that modulate glucose metabolism and insulin responsiveness in muscle and the liver.13, 14 While factors such as tumour necrosis factor α and interleukin 6 are not uniquely expressed in adipocytes, adiponectin and leptin are largely consid- ered as adipose-specific gene products Adiponectin (ACRP30) increases whole body insulin sensitivity largely by suppressing glucose production in the liver, as well as by increasing glucose uptake into skeletal muscle.15, 16 Leptin, the prod-

uct of the obesity (ob) gene, signals through receptors in the hypothalamus to decrease food intake and increase energy expenditure,17and may also act periph- erally to regulate whole body insulin sensitivity.18 Insulin positively regulates both the gene expression and the secretion of leptin from adipose tissue,19, 20and may also regulate the gene expression of adiponectin.21, 22Affecting the cir-

culating concentrations of these regulatory factors provides a newly recognized

potent indirect mechanism through which insulin regulates glucose metabolism

in its classical target organs.

Non-classical targets of insulin in the regulation of total body glucose metabolism

Although the major effect of insulin in controlling glucose metabolism is on its classical target organs (muscle, liver and fat), virtually every cell type expresses the insulin receptors This raises the possibility that additional sites exist for insulin action on carbohydrate metabolism Tissue-specific ablation of the insulin receptor has provided new insights about ‘non-classical’ target tissues for insulin action (Table 2.1) Target tissues include pancreatic β-cells, the central nervous

INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLIC PATHWAYS 67

system and vascular cells The findings suggest that these tissues may exert important effects on glucose metabolism at the whole body level For example, mice lacking insulin receptors in β-cells manifest impaired glucose-mediated insulin secretion Conversely, mice in which the insulin receptor was ablated in neuronal cells exhibit elevated food intake and diet-induced obesity, suggesting that insulin delivers an anorexogenic input to the central nervous system.23 Such

a role of insulin in the brain was suggested in early studies where the hormone was administered intra-cerebroventricularly to monkeys.24Finally, vascular cells are a target for insulin-induced vasodilatation and capillary recruitment that, by enhancing glucose delivery, may complement the hormone’s direct stimulatory effect on glucose uptake in muscle.25 Given that quantitatively β-cells, neuronal and vascular cells have only a minor contribution to insulin-stimulated whole body glucose disposal, these studies suggest that the actions of insulin in ‘non-

classical insulin targets’ are indirectly involved in the regulation of total body

glucose metabolism It will be interesting to see whether in addition insulin regulates glucose metabolism in these sites.

In summary, insulin engages both ‘classical’ and ‘non-classical’ target organs

in orchestrating the control of glucose metabolism (Table 2.1) In its classical target organs insulin directly modulates glucose uptake, metabolism and pro- duction In addition, the hormone affects glucose metabolism secondarily to alterations in the metabolism of other macronutrients, and by utilizing complex inter-organ cross-talk mechanisms.

pathways

Entry of the hydrophilic glucose molecule into the cell through a lipid brane requires a ‘gateway’ offered by glucose transporters (discussed later in this chapter) Once in the cytosol, glucose is phosphorylated into glucose-6- phosphate, and from this initial step its biochemical fate is diverse Glucose-6- phosphate is catabolized through glycolysis, the hexose monophosphate shunt and mitochondrial oxidation, yielding high-energy compounds such as ATP and NAD(P)H Alternatively, glucose can be stored in polymer form (glycogen) or converted to triglycerides In addition, glucose can be metabolized through sev-

mem-eral quantitatively minor pathways: it is a precursor for de novo synthesis of

nucleotides and certain amino acids, it can be converted to other sugars and hols (e.g sorbitol) and it is required for the generation of complex compounds such as glycoproteins and glycolipids.

alco-At the cellular level, insulin regulates the flow of glucose through these biochemical pathways by two basic mechanisms:

(a) by increasing uptake from the blood, as described in Section 2.4;

(b) by affecting regulatory enzymes in the various pathways of glucose metabolism, as outlined next.

68 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

Table 2.2 Major target proteins of insulin in the regulation of glucose metabolism, and themain mechanism(s) of regulation

Biochemical

Major mechanism ofregulation by insulin Reference

Stimulatory effect by insulin

Glucose uptake GLUT4 Translocation from intracellular

pools to the plasma membrane

26

Expressional regulation? 28Glucose

phosphorylation

Glucokinase Transcriptional activation 29Hexokinase II Transcriptional activation 30Glycolysis Phospho-fructokinase 2 Reversible phosphorylation? 31

Allosteric activationPhosphofructokinase 1 Allosteric activation

Phosphorylation and actin binding 32Pyruvate kinase Transcriptional/post-transcriptional

activation

33Dephosphorylation

Glycogenesis Glycogen synthase Dephosphorylation:

inhibited GS kinase (GSK3) 34stimulated dephosphorylation

Glucose oxidation Pyruvate dehydrogenase Dephosphorylation by inhibition

of PDK-4/2 expression 36Allosteric activation

Lipogenesis Acetyl CoA carboxylase Transcriptional activation 37

Allosteric activationReversible phosphorylation?

Inhibitory effect by insulin

Glycogenolysis Glycogen phosphorylase Dephosphorylation 38, 39

GP-kinase DephosphorylationGluconeogenesis G6Pase Transcriptional repression 40

Acute inhibition by 3-PIPs?

PEPCK Transcriptional repressionPyruvate carboxylase Transcriptional repression 41

GLUT4 – glucose transporter 4, GP-kinase – glycogen phosphorylase kinase, G6Pase – phosphatase, GSK3 – glycogen synthase kinase 3, PDK – pyruvate dehydrogenase (PDH) kinase,

pro-tein phosphatase 1

? – ambiguity exists regarding the precise effect of insulin or its physiological relevance

Enzyme regulation is achieved by changes in the phosphorylation state and/or

in expression levels The result is a change in Km and/or Vmax in the first case, or only Vmax in the second In addition, the rise in glucose flux alters the intracellular concentration of metabolites that in turn act as allosteric modulators, and may regulate the expression levels of specific enzymes Frequently, these

GLUCOSE UPTAKE INTO SKELETAL MUSCLE – THE RATE-LIMITING STEP 69

different mechanisms operate in a concerted fashion, translating metabolic and hormonal information into both short and long term regulation of enzymatic activity Short term (seconds to minutes) regulation is frequently achieved by allosteric modulation and reversible phosphorylation, whereas long term (hours

to days) regulation largely employs alterations in gene and protein expression Table 2.2 summarizes the major molecular mechanism(s) engaged by insulin in regulating its major protein targets.

An example of combined allosteric modulation, covalent modifications and expressional regulation is offered by the pyruvate dehydrogenase complex (PDH) This large enzymatic complex catalyses the irreversible oxidative decarboxylation of pyruvate to form acetyl CoA, linking glycolysis to the mitochondrial citric acid cycle PDH is negatively regulated allosterically mainly

by its products acetyl CoA and NADH, curbing energy production from glucose when NEFA are abundant as a fuel source In addition, reversible phosphorylation is achieved by at least four PDH kinases and two phosphatases, which respectively decrease or increase the overall catalytic activity of the complex Insulin rapidly decreases the expression level of PDH kinases 4 and

2, reducing the phosphorylation input on PDH and ultimately elevating its catalytic activity.

The above sections discussed the complex mechanisms utilized by insulin to regulate glucose metabolism and its homeostasis in the context of total body fuel metabolism Among these, the stimulation of glucose uptake by insulin into skeletal muscle remains quantitatively at the centre of the hypoglycaemic actions of this hormone under normal physiological conditions Impairment of this process calls into action extreme adaptive responses to maintain glucose homeostasis, in the form of compensatory hyperinsulinaemia and/or glucose funnelling into alternative sites (as demonstrated by the MIRKO mice) De- compensation of these mechanisms results in pathological manifestations (e.g diabetes) We next focus on the cellular and molecular mechanisms by which insulin stimulates the uptake of glucose into skeletal muscle.

step in glucose metabolism

As discussed above, glucose entering muscle cells encounters various fates, but

it rarely accumulates as free glucose in the sarcoplasm.42 – 44 This observation has led to the concept that, under both fasting and fed conditions, glucose transport across the membrane of the muscle fibre is rate limiting for glucose utilization This notion was further confirmed by the use of magnetic resonance spectroscopy, which allows detection of intracellular glucose-6-phosphate43 as well as intracellular glucose levels.10 However, under certain physiological conditions (e.g exercise)45 or in other tissues (e.g cardiomyocytes)46 steps

70 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

beyond glucose transport such as glucose phosphorylation might become rate limiting.

Does GLUT4 dictate glucose uptake in muscle and fat cells?

The GLUT family comprises 13 members, but only seven of these (GLUTs 1–4, 6, 8 and 11) have demonstrated glucose transport activity.47 Muscle and fat cells express predominantly GLUT4 From this expression pattern as well as functional studies showing that GLUT4 translocates to the plasma membrane in response to insulin, GLUT4 is generally believed to mediate insulin-stimulated glucose uptake This notion was further confirmed recently by experiments using GLUT4 gene ablation or a rather selective inhibitor of GLUT4 (the HIV protease inhibitor indinavir), as outlined below Mice lacking GLUT4 have diminished glucose uptake in response to insulin or exercise,48, 49and mice lacking GLUT4 selectively in skeletal muscle50 or in adipose tissues12 have impaired glucose and insulin tolerance Similarly, heterozygous GLUT4-null mice are severely insulin resistant and develop diabetes.51

Overall, these findings underline the importance of GLUT4 for and contraction-dependent glucose uptake In addition, muscle-specific GLUT4 knockout mice demonstrate the important regulatory role of skeletal muscle

insulin-in glucose homeostasis.50 Surprisingly, homozygous GLUT4-null mice had normal glucose tolerance even though they showed impaired insulin tolerance, suggesting insulin resistance The mechanism by which homozygous GLUT4- null mice are protected from diabetes is still unclear The most likely explanation

is that compensatory mechanisms are induced.52, 48 For example, other GLUT

isoforms may be expressed at higher levels, which then compensate for GLUT4 Although no up-regulation of GLUT1, GLUT3 or GLUT5 could be detected,52, 48 glucose uptake into isolated soleus muscle was mediated by a saturable glucose-transport process and blocked by the classical inhibitor of GLUTs, cytochalasin B.48Thus glucose uptake into soleus muscle from GLUT4- null mice appears to be mediated by some other member of the GLUT family (potentially GLUT8).

Genetic ablation of GLUT4 has confirmed its importance for normal insulin sensitivity; however, it does not reveal the quantitative contribution of GLUT4

to glucose influx into normal tissues, since compensatory mechanisms in the affected tissue are induced Until recently, glucose uptake through a specific GLUT isoform could not be directly assessed given the lack of inhibitors with sufficient selectivity for any given GLUT The HIV protease inhibitor indinavir was recently found to selectively block GLUT4-mediated glucose uptake into

of indinavir on glucose uptake was much lower in oocytes expressing GLUT4 compared with those expressing GLUT2, GLUT1, GLUT3 or a GLUT8 mutant directed to the cell surface.54 Using this approach, we studied the contribution

GLUCOSE UPTAKE INTO SKELETAL MUSCLE – THE RATE-LIMITING STEP 71

Table 2.3 Percentage inhibition of glucose uptake by the HIV

protease inhibitor indinavir (100µM) in skeletal muscle and

Cells or tissues were stimulated for 20–30 min without (basal) or with

100 nM insulin, followed by the measurement of 2-deoxyglucose uptake

in the absence or presence of 100µM indinavir Values are the

percent-age inhibition by indinavir

of GLUT4 to insulin-stimulated glucose uptake in mammalian cell lines as well

as in primary adipocytes and isolated skeletal muscles.55We found that GLUT4

is the major contributor to insulin-stimulated glucose uptake into skeletal cle, white and brown adipocyte and L6 wild type muscle cells as well as L6 cells overexpressing a myc-tagged GLUT4 (Table 2.3) However, in 3T3-L1 adipocytes, the effect of indinavir on glucose uptake was more variable, averag- ing a 67 per cent inhibition of insulin-stimulated glucose uptake These results confirm the high contribution of GLUT4 to insulin-stimulated glucose uptake in mature muscle and cells; however, the contribution of GLUT4 to basal glucose uptake is less clear Among cell lines representing these tissues, only in L6 cells (over)expressing GLUT4myc, but not wild-type L6 or 3T3-L1 adipocytes, GLUT4 accounted functionally for the majority of basal glucose uptake.

mus-Insulin-mediated GLUT4 traffic

Glucose transport into skeletal muscle fibres probably occurs along the two domains of the sarcolemma: the plasma membrane and the transverse tubules This assumption is based on the detection of GLUT4 glucose transporters in both domains when isolated by subcellular fractionation,56 or imaged upon GLUT4 photolabelling,57 immunoelectron microscopy58, 59 or fluorescence microscopy

of GLUT4-GFP.60 GLUT1 can also be detected on isolated plasma membranes

of rodent and human muscle (but not on isolated transverse tubules),56 and by immunocytochemistry on muscle sections.61, 56 However, contributions from

endothelial cells to the isolated fractions, or ambiguity in the cence detection, cast doubt on the significance of GLUT1 presence at the muscle surface Moreover, as discussed above, it is GLUT4 that has preponderance in dictating to glucose influx into skeletal muscle in the absence and presence

immunofluores-of insulin.

72 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

Insulin increases the rate of glucose influx into skeletal muscle measured

in vivo, ex vivo (in isolated muscles, teased fibres) or in muscle cells in culture.

The magnitude of this response ranges from two- to eightfold, with a mean around threefold when 20 studies were analysed.62 This is in contrast to the larger response observed in rodent (but not in human) adipose cells.

How does GLUT4 mediate the increase in glucose uptake caused by insulin?

In 1981 it was first reported that isolated plasma membrane from treated rat diaphragm had a higher number of cytochalasin B-binding sites than membranes from control muscles.63 Cytochalasin B is a rather specific ligand of GLUT-family proteins, and hence the results suggested that, as in rat adipocytes, there is a gain in the number of glucose transporters in response

insulin-to the hormone Shortly thereafter, we and others reported that purified plasma membranes and transverse tubules of hindlimb skeletal muscles show a gain in cytochalasin B-binding sites,64, 65and these were then identified as GLUT4 upon immunoblotting with specific antibodies.66, 56 The gain in GLUT4 at the surface

of muscles was further established by a variety of techniques including electron microscopy of ultrathin muscle slices,58 surface affinity photolabelling with bis-mannose derivates followed by either GLUT4 immunoprecipitation57

immuno-or avidin pull-down of the photolabel followed by GLUT4 immunoblotting,67

and more recently detection of electrotransfected GLUT4-GFP by fluorescence microscopy.60 Interestingly, when analysed, there was a qualitatively parallel reduction in the GLUT4 in intracellular membranes None of the techniques listed above provide the opportunity to quantitatively recover the surface mem- branes and the intracellular membranes Some, surface affinity photolabelling for example, do not afford detection of the intracellular pool Hence, to date

it has not been possible to quantitatively account for the gain in surface

glu-cose transporters vis-`a-vis their loss in intracellular stores Moreover, the gain in

surface GLUT4 remains a semiquantitative measurement, as all the approaches listed have confounding factors such as contamination with intracellular mem- branes, incomplete immunoprecipitation or limited sampling of the cell surface

in ultrathin sections The limitations of these approaches are discussed in a recent publication.68

Muscle cells in culture offer the possibility to examine in more detail the mechanism of GLUT4 translocation to the cell surface, the signals involved and the distribution of the intracellular pools Although primary cultures of mus- cle have been of limited use to answer these questions (largely due to their low levels of GLUT4, their low response to insulin and the variability from culture to culture69, 70) muscle cell lines have been more yielding The L6

muscle cell line, originally derived from satellite cells of thigh muscle from day-old rats, expresses GLUT4 upon cell differentiation from myoblasts into myotubes.a Moreover, these cells have the capacity to express large levels of

aAs with most cell lines, there is currently diversity in the clones available Not all clones express

GLUT4, and not all clones show myoblast fusion into myotubes

GLUCOSE UPTAKE INTO SKELETAL MUSCLE – THE RATE-LIMITING STEP 73

exogenous GLUT4 without mistargetting it.71 We have established and terized an L6 muscle cell line expressing myc-tagged GLUT4 at levels about fivefold higher than those of endogenous GLUT4 in skeletal muscle The myc tag is present in the first exofacial loop, allowing one to detect surface GLUT4 immunologically without cell lysis This feature provides a means to measure GLUT4 translocation to the cell surface without interference from intracellular GLUT4 either stored or docked below the plasma membrane, and in parallel to measure glucose uptake.71, 72 The exofacial tag is also instrumental in tracing the intracellular route of GLUT4 as it internalizes from the cell surface and re- emerges in response to insulin Using this tool, we have established the following features of GLUT4myc stably expressed in L6 muscle cells (L6GLUT4myc) (a) GLUT4myc continuously cycles to and from the cell surface The half-time

charac-of this cycling is 2 h in the basal state and 40 min in the presence charac-of insulin.

It takes 6 h for all the intracellular GLUT4myc to cycle to the surface in the basal state, and this time is reduced to 3 h in the presence of insulin.73

(b) GLUT4myc largely resides intracellularly, with only 10 per cent of the total content being present at the surface of unstimulated muscle cells Insulin causes a two- to threefold gain in surface GLUT4myc.74

(c) The large intracellular depot of GLUT4myc segregates away from GLUT1 but coincides with the insulin-responsive aminopeptidase (IRAP).71 The latter protein has been used to characterize the insulin-sensitive GLUT4 compartment in fat cells and skeletal muscle.75

(d) GLUT4myc dictates glucose uptake, given its almost 100-fold excess over endogenous GLUT4, GLUT1 or GLUT3.76 This was functionally demon- strated by the nearly complete inhibition of basal and insulin-stimulated glucose uptake by indinavir.

(e) GLUT4myc responds to insulin, hyperosmolarity, mitochondrial uncouplers and anti-diabetic drugs.77, 74, 78, 72

(f) Surface-labelled GLUT4myc traverses the recycling endosomes, in both the absence and presence of insulin Strikingly, the hormone accelerates the transit time through these endosomes, presumably as part of the mechanism

of speeding up GLUT4myc recycling to the cell surface.73

(g) A large proportion (70 per cent) of the insulin-dependent gain in surface GLUT4myc requires intact VAMP2,79 a vesicular SNARE that mediates fusion of vesicles in the regulated exocytic pathway in differentiated cells.80

This property is shared by the endogenous GLUT4 in adipose cells81 and muscle cells.82

(h) A subset of GLUT4myc segregates into the actin mesh that forms below the cell surface in response to insulin This location also includes vesicles

74 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

containing VAMP2 and IRAP Actin remodelling is required for effective GLUT4myc translocation to the cell surface.83 – 85

All of these features lead us to propose a model for GLUT4myc cycling

in muscle cells, as outlined next and depicted in Figure 2.1 Specific isons are made with GLUT4 traffic in adipose cells in culture (3T3-L1) The model offers paradigms that should now be tested in mature muscle fibres In the basal state, GLUT4 continuously cycles to and from the plasma membrane, re-entering the endosomal system and reaching the perinuclear endosomes con- taining transferrin receptor Here, GLUT4 appears to be sorted into a storage or specialized compartment that however is not static, since all GLUT4 molecules eventually reach the muscle plasma membrane.73 (In 3T3-L1 adipocytes about half of the GLUT4 molecules are also segregated away from the transferrin receptor.86) This specialized compartment is marked by the presence of VAMP2 and the lack of transferrin receptor In fact, imaging of the perinuclear GLUT4

compar-Transferrin receptor

VAMP2

GLUT4myc

Recyclingvesicle

Specializedvesicle

Endocytosis

Exocytosis

Insulin

Sorting (recycling)endosome

Figure 2.1 The GLUT4 cycle and its regulation by insulin: proposed model for GLUT4translocation in muscle cells (see the text for details)

GLUCOSE UPTAKE INTO SKELETAL MUSCLE – THE RATE-LIMITING STEP 75

compartment shows a tight perinuclear ring containing both GLUT4 and ferrin receptor, and a pointed ‘cone’ emanating from it devoid of the latter receptor.87 (A recent model also proposes the existence of a storage compart- ment in 3T3-L1 adipocytes that is in equilibrium with the endosomal/transGolgi network.26) Recycling GLUT4 molecules are envisaged to exit the recycling endosome directly en route to the plasma membrane Such basal traffic is not affected by ablating VAMP2 or VAMP3 with tetanus toxin.79

trans-Insulin stimulation causes the loss of the GLUT4 ‘cone’, presumably due to budding of GLUT4 containing vesicles These vesicles find their way to the plasma membrane presumably via microtubules88, 89 and eventually are trapped

in a submembranous, insulin-dependent actin mesh.83, 84 As well, the transit of GLUT4 through the endosomal system is accelerated,73 presumably to continu- ously feed the storage compartment and vesicles emanating from it (sorting of GLUT4 at a post-recycling endosome step also occurs in 3T3-L1 adipocytes90and primary fat cells91) Vesicles gathered by the actin mesh contain VAMP2 and IRAP–a marker of GLUT4 compartments GLUT4 eventually fuses with the plasma membrane via VAMP2 binding to plasma membrane target-SNAREs syntaxin4 and SNAP23.80 GLUT4 emerges all over the muscle cell surface but with certain predominance above actin-remodelled sites, in regions resembling membrane ruffles.84 Whether GLUT4 vesicles contribute to ruffle formation or the ruffle membrane curvature facilitates their fusion remains to be determined.

In adipose cells, the actin cytoskeleton also participates in GLUT4 translocation

to the plasma membrane, but actin remodelling appears to involve filaments pendicular to caveolae and parallel to the plasma membrane Other stimuli, such

per-as hyperosmolarity and potentially agents that lower ATP levels, cause GLUT4 translocation via vesicles not requiring VAMP2 nor involving the cytoskeleton and without taxing the GLUT4 ‘cone’.87The simplest interpretation is that such stimuli promote GLUT4 exit directly from the recycling endosome An attrac- tive hypothesis is that the endosome is the store of GLUT4 molecules recruited

to the plasma membrane in response to exercise in skeletal muscle, a scenario supported by immunofluorescence detection of GLUT4 in the intact tissue.59

Signals regulating GLUT4 traffic

The above segregation of GLUT4 in specialized compartments is likely brought about by so far unidentified proteins involved in retention and sorting of the transporter, presumably interacting via distinctive sequences at its amino- and carboxy-terminal cytosolic tails.92 The acceleration of GLUT4 interendosomal transit and distinct mobilization of GLUT4 from the specialized compartment require input from signals elicited by the occupied insulin receptor There is now little doubt that phosphorylation of insulin receptor substrates (IRS) is important for the insulin-dependent mobilization of GLUT4.93This participation

is manifest by binding and activation of phosphatidylinositol 3-kinase (PI3K), a

76 INSULIN-MEDIATED REGULATION OF GLUCOSE METABOLISM

lipid kinase that phosphorylates inositol phospholipids (PIPs) in the 3
position, mostly PI3,4,5-P3 This lipid product is membrane bound and is generated on both the plasma membranes and membranes gathered by the insulin-dependent actin mesh.94 PI3,4,5-P3 serves a dual function, activating phosphatidylinositol dependent kinase (PDK) and attracting PDK substrates such as protein kinase

B (PKB)/Akt There is increasing support for the concept that Akt is required for insulin-dependent translocation of GLUT495 – 97as is another PDK substrate, the atypical protein kinase C.98, 99 At which level in the cycle of GLUT4 traffic

these enzymes exert their input remains to be determined In muscle cells, the interendosomal acceleration of GLUT4 requires input of PI3K → PKB, and the formation of the actin mesh requires input of PI3K but not PKB.97 In this instance, PI3K leads to activation of the small GTPase Rac that determines actin remodelling Atypical PKC appears to phosphorylate VAMP2 and this may promote GLUT4 insertion into the plasma membrane.100Additional inputs may occur at the level of vesicle docking, leading to membrane fusion, involving these or other enzymes Regulation is also envisaged for additional functions such as GLUT4 budding from the specialized compartment and loading onto microtubules.

In 3T3-L1 adipocytes, a signalling pathway emanating from the receptor but distinct from the IRS → PI3K → PKB pathway has been recently described In this case, the receptor leads to tyrosine phosphorylation on Cbl aided by the pro- teins CAP and APS.101 – 103 The CAP–pCbl complex then migrates to caveolae, where it links to flotillin, and through a relay of binding events links the proteins CrkII and C3G to the small GTPase TC10, ultimately regulating cortical actin dynamics independently of PI3K Interplay between the TC10 pathway and the PI3K pathway appears to occur at the level of atypical PKC.104, 105 Whether the TC10 pathway also operates in skeletal muscle is currently unknown, but

it does not seem to be a major regulator of actin dynamics in muscle cells

in culture.106 With the exception of VAMP2, the pertinent phosphorylated strates of atypical PKC or PKB remain to be mapped Recent advances in the use of targeted elimination of specific genes, such as through small interference RNA, may bring us closer to this goal.

sub-Does GLUT4 translocation explain the full increase in glucose uptake?

From the above accounts, it is clear that insulin increases glucose uptake into skeletal muscle and adipose tissue via translocation of GLUT4 from intracellular compartments to the cell surface, and that GLUT4 is responsible for insulin- dependent glucose uptake Does translocation account for the full gain in glucose uptake in response to insulin? Several studies showed a discrepancy between the extent of GLUT4 translocation and the stimulation of glucose uptake in response to insulin in skeletal muscle,107, 108, 56 rat white adipocytes109 and brown adipocytes.110, 111 In addition, the percentage increase of glucose uptake