A Role for Sphingolipids in Producing the Common Features of Type 2 Diabetes, Metabolic Syndrome X, and Cushing's Syndrome

Scott A. Summers; Don H. Nelson


Diabetes. 2005;53(3):591-602. 

In This Article

Role of Ceramides in Insulin Resistance

Since the majority of glucose disposal occurs in skeletal muscle, insulin resistance in this tissue is generally thought to contribute most significantly to the glucose intolerance associated with nutrient or glucocorticoid oversupply. Thus far, ceramide has been shown to accumulate in insulin-resistant muscles in both rodents[25,26] and humans.[27,28] Moreover, Straczkowski et al.[28] described a negative correlation between ceramide content of muscles and insulin sensitivity in 13 human subjects and further demonstrated that lipid infusion markedly elevated ceramide levels while decreasing insulin sensitivity. Exercise training, which improves insulin sensitivity, markedly decreases muscle ceramide levels in both rats and humans.[48,49,50] Although the increase in ceramide seen in these samples is modest, recent studies in cultured myotubes indicate that inducing a comparable increase (1.5- to 2-fold) in endogenous ceramide is sufficient to inhibit insulin signaling.[23,24]

Insulin accelerates glucose entry into skeletal muscle and adipose tissue by evoking the translocation of GLUT4 glucose transporters from intracellular stores to the plasma membrane. Simultaneously, the hormone regulates numerous metabolic enzymes (e.g., glycogen synthase or pp70 S6-kinase) to promote storage of the incoming glucose as glycogen, triglyceride, or protein. Insulin initiates these responses through its receptor, a tyrosine kinase, which subsequently phosphorylates a family of insulin receptor substrates (IRSs) (IRS-1, IRS-2, etc.). The phosphorylated IRS proteins activate a signaling pathway involving the sequential activation of phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase B (PKB), which are obligate intermediates in insulin's metabolic, antiapoptotic, and mitogenic effects.[51]

Ceramide acutely inhibits insulin-stimulated glucose uptake, GLUT4 translocation, and/or glycogen synthesis in cultured adipocytes and/or muscle.[52,53,54,55] These effects appear to result from the sphingolipid's ability to block activation of either IRS-1 or Akt/PKB (Figure 3). Specifically, in 1996, two independent laboratories found that treating cultured cells with short-chain ceramide analogs or bacterial sphingomyelinases, which hydrolyze sphingomyelin to form choline and ceramide (Figure 1), blocked insulin-stimulated tyrosine phosphorylation of IRS-1 and its subsequent recruitment and activation of PI3K.[56,57] A third group found that ceramide directly inhibited PI3K isolated from serum-stimulated cells.[58] However, the effect of ceramide on IRS and/or PI3K appears to be specific to certain cells or treatment conditions, as a number of different laboratories have shown that ceramide has no effect on PI3K or the production of its lipid products.[23,53,54,55,59,60] In all cell types tested, however, ceramide has been shown to block activation of Akt/PKB "downstream" of PI3K by either inhibiting its translocation to the cell membranes[61,62,63] and/or by promoting its dephosphorylation via protein phosphatase 2A.[23,60,62,64,65,66] While acute treatment with ceramides has these effects on insulin signaling, prolonged treatment of 3T3-L1 adipocytes with the sphingolipid was shown to downregulate GLUT4 expression.[67]

Figure 3.

Schematic diagram depicting the pathways linking ceramides to the inhibition of IRS-1 and Akt/PKB. As described in the text, ceramides have been shown to independently promote the serine/threonine phosphorylation and inhibition of IRS-1, block the translocation of Akt/PKB to the plasma membrane, and catalyze the dephosphorylation of Akt/PKB by protein phosphatase 2A.

Skeletal muscles exposed to excess lipid demonstrate decreased sensitivity to insulin. For example, incubating isolated muscle strips or cultured muscle cells with FFAs,[22,68,69,70,71] infusing lipid emulsions into rodents or humans,[72,73,74,75] or expressing lipoprotein lipase in skeletal muscle of transgenic mice[76,77] promotes intramyocellular lipid accumulation and compromises insulin-stimulated glucose uptake. To evaluate the role of ceramides in the insulin resistance associated with lipid oversupply, scientists have investigated the lipid's role in FFA-induced insulin resistance using cultured myotubes. Schmitz-Pfeiffer et al.[22] first observed that treating C2C12 myotubes with concentrations of saturated FFAs within the physiological serum range increased the intracellular pool of ceramide, while simultaneously inhibiting activation of Akt/PKB, but not PI3K. In this cell type, short-chain ceramide analogs recapitulated this pattern of effects on insulin signal transduction. The authors thus speculated that ceramide was the primary intermediate linking saturated fats to the inhibition of insulin signaling. To definitively identify a role for ceramide as an intermediate in these effects of saturated FFAs in C2C12 myotubes, Chavez et al.[23] demonstrated that inhibitors of the biosynthetic enzymes SPT or dihydroceramide synthase prevented the effects of palmitate on both ceramide accumulation and Akt/PKB. Moreover, inhibitors of ceramide degradation (i.e., its glycosylation or deacylation) were shown to both mimic and exacerbate the palmitate effect on ceramide accrual and insulin signaling.[23] Collectively these studies strongly indicate that ceramide is required for the inhibitory effects of saturated FFAs in cultured myotubes.

Of note, researchers recently demonstrated that infusing a lipid mixture enriched in unsaturated fatty acids into rodents may induce insulin resistance through a ceramide-independent mechanism. Specifically, infusion of Liposyn II (Abbott, North Chicago, IL), a triglyceride emulsion that is predominantly comprised of the fatty acid linoleate (18:2), induced insulin resistance by inhibiting insulin signaling to IRS-1 and PI3K.[78] This lipid cocktail did not affect Akt2/PKB-ß or glycogen synthase kinase 3-ß,[79] nor did it induce ceramide accumulation.[75,78] In a subsequent study, a lipid emulsion of comparable composition was shown to increase muscle ceramide levels in humans, and the authors speculated that different methods of muscle preparation may account for the opposite findings.[28] Nonetheless, the relative absence of saturated fats in this cocktail makes it unlikely to markedly induce ceramide synthesis, which is dependent on the availability of palmitate. Quite possibly, different types of fat induce insulin resistance through distinct mechanisms, with saturated FFAs inducing insulin resistance through a ceramide-dependent pathway and unsaturated ones doing so through another lipid intermediate (e.g., diacylglycerol). Indeed, some evidence supports the hypothesis that diacylglycerol comprised of predominantly unsaturated FFAs is a potent activator of certain intracellular substrates (e.g., protein kinase C), while that composed of saturated fatty acids is a relatively poor agonist.[80,81] An important future step will be to determine whether ameliorating ceramide accumulation, using either pharmacological or genetic manipulation strategies, quantitatively improves insulin sensitivity in intact organisms, such as insulin-resistant rodents.

TNF-α is a proinflammatory cytokine that inhibits insulin-stimulated glucose uptake and/or hepatic glucose production when administered to either cultured cells[82] or animals.[83] Obesity increases expression of TNF-α in white adipose tissue, prompting speculation that the cytokine induces insulin resistance in cases of increased adiposity. Although concentrations found in the circulation of obese patients are typically low, even in obese individuals, researchers have speculated that TNF-α functions in an autocrine or paracrine manner and is secreted from fat cells pervading muscle tissue in obese animals[84] or directly from muscle.[85] Neutralization of TNF-α activity, either by infusing fa/fa Zucker rats with a soluble TNF receptor lgG fusion protein or by crossing insulin-resistant mice with knockout mice lacking either TNF-α or TNF-α receptors, was shown previously to increase peripheral insulin sensitivity.[86,87] Although the systemic administration of a TNF-α-neutralizing antibody failed to improve insulin sensitivity in humans,[88] the effectiveness of this strategy at negating TNF-α activity could not be confirmed directly, and scientists speculate that increased TNF-α levels could exacerbate the insulin-resistant condition.

Under conditions whereby TNF-α inhibits insulin signaling, the cytokine promotes ceramide accumulation in brown adipocytes,[60] 3T3-L1 adipocytes, and C2C12 myotubes (A. Chavez, S.A.S., unpublished observation). Moreover, like ceramides, TNF-α has been shown to block insulin signaling at the level of IRS-1 and Akt/PKB, depending on the cell type being examined,[57,60,89,90] and to decrease the expression of IRS-1 and GLUT4.[91] Could ceramide mediate the inhibitory effects of TNF-α on insulin signaling? In myeloid 32D progenitor cells and 3T3-L1 adipocytes, the effects of TNF-α on IRS-1 were recapitulated by the addition of exogenous bacterial sphingomyelinase or ceramides.[57] In brown adipocytes, TNF-α was shown to promote the dephosphorylation of Akt/PKB by activating PP2A.[60] In this cell type, exogenous ceramides again recapitulated these TNF-α effects, and the PP2A inhibitor okadaic acid prevented the effects of both antagonists. The authors concluded that ceramide was the principle mediator of the signaling pathway linking TNF-α to the inhibition of insulin signaling.

When added to cultured cells or isolated tissues, glucocorticoids block glucose uptake[12,13,14,15,92,93,94,95] and glycogen and protein[96,97,98] synthesis, but the mechanisms underlying these inhibitory effects remain unclear. Studies both in vitro and in vivo have demonstrated that dexamethasone decreases expression and/or activation of insulin receptors, IRS-1, or PI3K,[99,100,101] but others have failed to see inhibition by dexamethasone at these early signaling steps.[102,103,104,105] The inconsistencies between studies done in vivo may be explained by differing degrees of hyperinsulinemia under the different treatment regimes. When the compensatory increase in insulin levels was prevented, the effects on insulin receptor levels or binding affinity were abolished.[106] Further downstream, corticosteroids have been shown to blunt the phosphorylation of Akt/PKB, 4E-BP1, p70S6K, and glycogen synthase[97,98,100,107,108,109] and to inhibit the translocation and expression of GLUT4.[14,15] Elucidating the mechanisms underlying these antagonistic actions of glucocorticoids is not only important for comprehending the pathogenesis of Cushing's syndrome, but is also extremely relevant for understanding the complications associated with exogenous glucocorticoid therapy.

As described above, glucorticoids activate synthetic pathways promoting sphingolipid formation in a wide variety of tissues, including insulin-responsive ones. In adipocytes, for example, low doses of dexamethasone, which antagonize insulin-stimulated glucose uptake,[110] selectively increase sphingolipid levels.[41] By contrast, adrenalectomy, which increases insulin sensitivity in adipocytes,[111] markedly decreases adipocyte sphingolipid levels.[44] An important aspect of these studies is the remarkable size and specificity of the glucocorticoid effect. In 3T3-L1 preadipocytes, for example, glucocorticoids induce a 50% increase in membrane sphingomyelin levels within 3 h after their addition, without affecting phospholipids or cholesterol.[112] Based on studies in cultured cells,[23,24] one would predict that increasing ceramide levels by this amount would be likely to block insulin action.

Though sphingolipids have been shown to be obligate intermediates in the pathways linking glucocorticoids to the regulation of various biological processes (e.g., thymocyte apoptosis [46]), researchers have yet to perform analogous studies determining whether ameliorating ceramide accumulation abates the glucocorticoid effect on insulin signaling or action. Nonetheless, two studies suggest that dexamethasone and ceramide inhibit insulin sig-naling and action using a common pathway. In L6 myoblasts, the effects of dexamethasone on distal constituents of the PI3K/Akt signaling pathway {i.e., ribosomal protein S6 kinase [p70(S6k)] and the cap-dependent translational repressor, eukaryotic initiation factor 4E (eIF4E)} were blocked by okadaic acid and calyculin A,[109] which, as described above, have been shown to reverse the effects of ceramide on insulin signaling.[24,60,66] Moreover, in 3T3-L1 cells, the inhibitory effects of sphingosine, sphinganine, or dexamethasone on glucose transport were not additive.[113] Collectively, these studies suggest the involvement of redundant intracellular mechanisms linking both sphingolipids and glucocortiocids to the regulation of insulin signaling or action.