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Biochem. J. (2009) 417, 791801 (Printed in Great Britain) doi:10.1042/BJ20081149 791

Modulating serine palmitoyl transferase (SPT) expression and activityunveils a crucial role in lipid-induced insulin resistance in rat skeletalmuscle cellsMaria L. WATSON*, Matthew COGHLAN and Harinder S. HUNDAL*1*Division of Molecular Physiology, Sir James Black Centre, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, U.K., and AstraZeneca, CVGI, Alderley Park,Macclesfield, Cheshire SK10 4TG, U.K.

Saturated fatty acids, such as palmitate, promote accumulationof ceramide, which impairs activation and signalling of PKB(protein kinase B; also known as Akt) to important end pointssuch as glucose transport. SPT (serine palmitoyl transferase) isa key enzyme regulating ceramide synthesis from palmitate andrepresents a potential molecular target in curbing lipid-inducedinsulin resistance. In the present study we explore the effects ofpalmitate upon insulin action in L6 muscle cells in which SPTexpression/activity has been decreased by shRNA (small-hairpinRNA) or sustained incubation with myriocin, an SPT inhibitor.Incubation of L6 myotubes with palmitate (for 16 h) increasesintramyocellular ceramide and reduces insulin-stimulated PKBactivation and glucose uptake. PKB inhibition was not associatedwith impaired IRS (insulin receptor substrate) signalling and wasameliorated by short-term treatment with myriocin. SilencingSPT expression (90%) by shRNA or chronic cell incubationwith myriocin (for 7 days) markedly suppressed SPT activityand palmitate-driven ceramide synthesis; however, challengingthese muscle cells with palmitate still inhibited the hormonal

activation of PKB. This inhibition was associated with reducedIRS1/p85-PI3K (phosphoinositide 3-kinase) coupling that arisesfrom diverting palmitate towards greater DAG (diacylglycerol)synthesis, which elevates IRS1 serine phosphorylation viaactivation of DAG-sensitive PKCs (protein kinase Cs). Treatmentof SPT-shRNA cells or those treated chronically with myriocinwith PKC inhibitors antagonized palmitate-induced loss ininsulin signalling. The findings of the present study indicatethat SPT plays a crucial role in desensitizing muscle cells toinsulin in response to incubation with palmitate. While short-term inhibition of SPT ameliorates palmitate/ceramide-inducedinsulin resistance, sustained loss/reduction in SPT expression/activity promotes greater partitioning of palmitate towards DAGsynthesis, which impacts negatively upon IRS1-directed insulinsignalling.

Key words: ceramide, diacylglycerol, insulin receptor substrate1 (IRS1), myriocin, palmitate, protein kinase B (PKB), proteinkinase C (PKC).

INTRODUCTION

Hyperlipidaemia is one of a cluster of abnormalities associatedwith the metabolic syndrome and one that is considered importantfor both development and progression of insulin resistance inskeletal muscle, a tissue that makes a major contribution to whole-body glucose homoeostasis. Sustained over supply of NEFAs(non-esterified fatty acids; free fatty acids) to skeletal muscle hasbeen shown to result in the accumulation of IMTGs (intramusculartriacylglycerols) and that of fatty-acid-derived metabolites, suchas DAG (diacylglycerol) and ceramide. Although accumulationof IMTG has been positively correlated with a loss in skeletalmuscle insulin sensitivity, the finding that muscle of endurance-trained athletes exhibit improved insulin sensitivity in the faceof an increase in IMTG implies that increases in intramuscularlipid alone may not in itself be the primary driver of lipid-inducedinsulin resistance [1]. Recent work from our laboratory and workof others has shown that increased availability of palmitate, themost prevalent circulating saturated fatty acid, impairs the insulin-dependent regulation of glucose transport and glycogen synthesisin skeletal muscle cells [2,3]. This impairment is due, in large part,to an attendant accumulation in muscle cells of the sphingolipidceramide, for which the fatty acid is a key precursor. Increasesin intramyocellular ceramide have previously been linked to loss

of insulin sensitivity in cultured muscle cells [4,5] and in skeletalmuscle of both rodents [6,7] and humans [8]. However, it is onlyrecently that the mechanistic basis of ceramide-induced insulinresistance is beginning to be understood.

We [2,4,9], and others [5,10,11], have shown that increasesin intracellular ceramide promote a targeted loss in the insulin-dependent activation of the serine/threonine kinase PKB (proteinkinase B; also known as Akt). This kinase plays a pivotal rolein the hormonal activation of glucose transport and glycogensynthesis, and consequently its dysregulation is likely to impactsignificantly upon the homoeostatic control of blood glucose. Theloss of PKB activation by ceramide has been proposed to occurby one of two mechanisms involving either dephosphorylationof its two regulatory sites (Thr308 and Ser473) by a type 2A-likephosphatase activity [10] or alternatively, as we have shown,inhibition in the cell-surface recruitment and phosphorylationof PKB [4,12] by a process dependent upon activation ofatypical PKC (protein kinase C) isoforms (/ ) [9,11]. Increasingintracellular ceramide [by provision of short-chain (C2) ceramideor stimulating endogenous palmitic (C16)-ceramide synthesis fromfatty acids such as palmitate] results in potent activation ofPKC (for a review, see [13]) and, as such, this has importantimplications for PKB activation. In unstimulated cells, PKC hasbeen shown to directly interact with and repress PKB in numerous

Abbreviations used: DAG, diacylglycerol; DTT, dithiothreitol; FBS, foetal bovine serum; IMTG, intramuscular triacylglycerol; IRS, insulin receptor substrate;LCB, long-chain base; -MEM, -minimal essential medium; NEFA, non-esterified fatty acid; NF-B, nuclear factor B; PI3K, phosphoinositide 3-kinase;PKB, protein kinase B; PKC, protein kinase C; shRNA, small-hairpin RNA; SPT, serine palmitoyl transferase; TLR, Toll-like receptor.

1 To whom correspondence should be addressed (email h.s.hundal@dundee.ac.uk).

c The Authors Journal compilation c 2009 Biochemical Society

792 M. L. Watson, M. Coghlan and H. S. Hundal

cell types [9,11,1417], but dissociates upon cell treatment withgrowth factors [14] and insulin [9], thus allowing PKB to becomeactivated. In contrast, ceramide stabilizes the interaction betweenthe two kinases and, moreover, ablates the ability of insulin to notonly dissociate the kinase complex, but to also activate PKB [9].It follows that limiting intramuscular ceramide production and/oraccumulation would confer an insulin-sensitizing effect and thatenzymes participating in ceramide synthesis may potentially serveas therapeutic targets for promoting insulin sensitivity of tissuessuch as skeletal muscle. Indeed, recent in vivo work suggeststhat attenuating endogenous ceramide synthesis ameliorates theloss in skeletal muscle insulin sensitivity induced in response tosaturated fat, glucocorticoids and obesity [18].

The synthesis of ceramide from saturated fatty acids, suchas palmitate, is crucially dependent upon the activity of SPT(serine palmitoyl transferase), which catalyses the first and rate-limiting step of sphingolipid biosynthesis involving the condens-ation of the amino acid L-serine with palmitoyl-CoA to form3-oxodihydrosphingosine [19]. Mammalian SPT belongs to thefamily of pyridoxal 5-phosphate-dependent enzymes and is com-posed of two distinct protein subunits known as LCB1 (long-chainbase-1; 53 kDa) and LCB2 (long-chain base-2; 63 kDa), withstability of the latter being critically dependent upon expressionof the former. More recently, a third SPT subunit (LCB3) hadbeen identified, but given that its tissue expression is highlyvariable, being expressed predominantly in the placenta andcells of trophoblastic origin [20], it is likely to be dispensablewith regard to SPT function in other cell and tissue types. SPTactivity is susceptible to inhibition by serine analogues such ascycloserine and by molecules that structurally resemble transition-reactive intermediates such as the anti-fungal agent myriocin [19].In the present study we have modulated SPT expression andactivity in cultured rat skeletal muscle cells by either silencingthe expression of the LCB1 gene or subjecting muscle cellsto sustained incubation with myriocin in an attempt to assessthe impact of these interventions on palmitate-induced insulinresistance. We have shown previously that ceramide generatedde novo from palmitate is a major factor promoting insulinresistance in muscle cells and that short-term inhibition of SPThas an ameliorative effect on insulin signalling [2]. However,under circumstances when expression of SPT has been repressedor is chronically inhibited, we show that utilization of palmitate isdiverted towards greater synthesis of DAG, which subsequentlyactivates DAG-sensitive PKCs that in turn promote a reduction inIRS (insulin receptor substrate)-directed insulin signalling.

EXPERIMENTAL

Materials

-MEM (-minimal essential medium), FBS (foetal bovineserum) and antibiotic/antimycotic solution were from Life Tech-nologies. Most other reagent grade chemicals, myriocin,insulin, palmitate, palmitoyl-CoA, DAG, sn-1,2-DAG kinase andantibodies against actin were purchased from SigmaAldrich.Ro 31.8220 and GF109203X were from CalbiochemMerck.C2-ceramide was from Tocris and C16-ceramide was purchasedfrom Avanti. Radioactive chemicals, 2-deoxy [3H]glucoseand [3H]serine were purchased from PerkinElmer. [ -32P]ATP and protein-GSepharose beads were from AmershamBiosciences. Antibodies to PKB/Akt, phospho-PKB(Ser473),PKC and phospho-IRS1(Ser1101) were purchased from NewEngland Biolabs. Antibodies against the p85 subunit of PI3K(phosphoinositide 3-kinase) and anti-IRS1 were obtained fromUpsta

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