I Glycogen Synthesis
I Glucose Oxidation
Fig. 2. Free fatty acids induce skeletal muscle insulin resistance in humans by inhibition of insulin signaling.
induce multiple defects in the insulin signaling cascade, at the level of glycogen synthesis, insulin-induced glucose transport and phosphorylation of the insulin receptor, insulin receptor substrate IRS-1, IRS-1-associated phosphatidylinositol (PI) 3-kinase activity, and of Akt (142, 151, 166, 171-175), but this did not happen when lipid was infused to already insulin-resistant obese subjects (175), suggesting that lipotoxicity may be already established in such individuals.
The role of FFA to cause insulin resistance and impair insulin signaling comes from a number of studies that show that an increase in fatty acid supply increases intramyocellular lipids (IMCL) and promotes the formation of a variety of fat-derived toxic metabolites, including fatty acyl-CoAs, cera-mides, and diacylglycerol (DAG) with activation of the NF-k^ pathway. Boden et al. (176) and Bachmann et al. (177) quantified IMCL by 1H-MRS (magnetic resonance imaging and spectroscopy) in soleus and tibialis anterior muscles during lipid infusion studies and measured insulin sensitivity by means of the gold-standard euglycemic insulin clamp technique. Both groups showed that IMCL levels increased significantly within ~2 h of an intravenous lipid infusion, and continued to increase during the 4-6-h lipid infusion in parallel with a progressive reduction in insulin sensitivity throughout the lipid infusion period. Insulin sensitivity had a strong inverse correlation in these studies with the increase in IMCL, in both soleus and tibialis anterior muscles, suggesting an important role for triglyceride accumulation in the development of insulin resistance. These studies were consistent with the observation of increased IMCL in insulin-resistant nondiabetic subjects (140, 178-182) and in T2DM subjects (182-184) . It is now well established that lipid-derived metabolites [i.e, ceramide, DAG (185), and long-chain acyl-CoA] activate cellular serine kinases and inhibit key insulin signaling molecules within muscle. Inflammatory pathways including both protein kinase C (PKC) activation of PII and 5 isoforms in human skeletal muscle and Ik^/NFk^ pathways have been clearly implicated in the FFA-induced impairment of IRS-1 tyrosine phosphorylation, and other downstream intracellular key signaling steps (171, 186-188), being reversible by antiinflammatory agents, such as salicylates (189). DAG is a powerful allosteric activator of PKC, a serine/threonine kinase with several isoforms, and increased serine phosphorylation of IRS-1 has been shown to inhibit IRS signaling (129, 133, 134). Adams et al. (187) have showed that insulin sensitivity and stimulation of Akt phosphorylation by insulin is significantly impaired in obese nondiabetic subjects, being associated with a~twofold increase in muscle ceramide concentration. Moreover, ceramide content was significantly correlated with the plasma FFA concentration (r = 0.51, p < 0.05), providing another indication of the role of lipotoxicity in impairing insulin action in skeletal muscle.
More recently, there has been an increasing interest in toll-like receptor 4 (TLR4), the best characterized of the family of TLRs, as playing an essential role inflammation and insulin resistance in the setting of obesity, lipotoxicity, and T2DM. TLRs are important to the immune system by activating proinflammatory signaling pathways in response to microbial pathogens (66, 190) . TLR4 are activated by lipoplysaccharide (LPS) of bacterial walls and saturated fatty acids and play an important role in ligand recognition. IKK/Ik^/NFk^ and JNK, which belong to the family of stress-activated protein kinases, are inflammatory pathways downstream of the TLR receptor and susceptible to activation by FFA. FFA activate TLR-driven signaling activating both inflammatory pathways and it is subject to intense research, as TLRs are believed to play an important role in the pathogenesis of FFA-induced insulin resistance. Recently Shi et al. (191) reported that in adipose tissue from insulin resistant high-fat-fed ob/ob and db/db mice, TLR gene expression was markedly increased compared to control normal animals and that TLR4 was required for FFA to generate the IKE/IK^/NF^-mediated inflammatory response. Furthermore, the investigators were able to demonstrate that the ability of FFA to activate the IKK/Ik^/NFkP pathway and induce an inflammatory response could be blocked in TLR4 null mice. Activation of these same pathways and protection from a high-fat diet-induced insulin resistance at the level of muscle, liver, and adipose tissue has also been reported in mice with a loss-of-function mutation in TLR4 (192) . Preliminary data from within our group indicates that TLR4 gene expression in vastus lateralis muscle from obese nondiabetic and T2DM subjects is increased, suggesting that TLR4 signaling is required for FFA-induced inflammation, and possibly insulin resistance (N. Musi, personal communication). Taken together, while much remains to be understood, it is now apparent that a decrease FFA cause insulin resistance largely by the accumulation of triglyceride and other lipid-derived metabolites with subsequent activation of intramyocellular inflammatory pathways that impair insulin signaling at different levels, rather than by substrate competition, as initially believed.
Impairment of Muscle Insulin Signaling and Insulin Sensitivity by FFA is Dose-Dependent in Humans: Clinical Implications
A limitation of most, but not all (155, 160, 161, 164, 193), studies of humans examining the role of an increase in plasma FFA was the use of high-dose lipid infusion rates, which elevated plasma FFA usually >1,500 ^mol/L. These levels are considerably higher than the usual FFA levels observed in obese and T2DM subjects. Fasting plasma FFA levels in healthy subjects range between ~300 and 400 ^mol/L and increase to between ~800 and 1,100 ^mol/L only under certain extreme conditions such as fasting for 2-3 days (194, 195). As discussed earlier, in obese nondiabetic individuals (145, 196, 197) and in patients with T2DM (198, 199), fasting and day-long plasma FFA levels are usually elevated (~600-700 ^mol/L) because of resistance of adipose tissue to the antilipolytic effect of insulin, but plasma FFA usually rarely exceed ~1,000 ^mol/L even in the presence of severe hypertriglyceri-demia (with or without concomitant diabetes) (86) or poorly controlled diabetes (200-202). Because few studies had used these lower lipid infusion doses (155, 160, 161, 164, 193) . one could argue against the clinical day-to-day relevance of FFA in human disease. These early studies did suggest that a small increase of plasma FFA levels in healthy subjects-induced insulin resistance, impaired glucose oxidation, and glycogen synthase activity (160, 161) . but no information was available on earlier steps of insulin signaling at plasma FFA elevations within the physiological range observed in T2DM. To better understand how FFA interacted with early molecular steps responsible for insulin action in muscle, we performed acute dose-response studies in healthy subjects at FFA spanning from plasma levels typically seen in obesity and T2DM (600-700 ^mol/L), through the upper range of the physiological spectrum (~ 1,000-1,200 ^mol/L) and on into the pharmacological range (~ 1,700 ^mol/L) (151). Of note, heparin was not coinfused to resemble as closely as possible physiological conditions, as it is uncertain whether a plasma FFA elevation achieved by dislodging lipoprotein lipase with heparin really resembles the physiologic delivery of FFA to tissues under normal living conditions. As observed in Fig. 3, FFA-induced insulin resistance in a dose-dependent manner, with most of the insulin resistance developing already with the low, physiological increase in plasma FFA concentrations (i.e., 695 ^mol/L) observed in obesity and T2DM. Moreover, as observed in Fig. 4, there was a clear gradient of inhibition of all proximal insulin signaling steps ranging from the physiological to the pharmacological range.
This was the first demonstration in humans that plasma FFA inhibits insulin signal transduction in a dose-dependent manner. An important observation was that 70% of the maximal inhibition of insulin signaling was observed within just a few hours and at plasma FFA concentrations that were well within the physiological range (Fig. 3). Moreover, there was a close correlation between the plasma FFA concentration fasting and during the euglycemic insulin clamp and insulin sensitivity (Fig. 5). The clinical relevance of such a finding has far-reaching implications because it tells us that it takes only this small increase in plasma FFA to cause a broad inhibition of the signaling cascade, from the insulin receptor through Akt phosphorylation. This data also helps to understand how FFA can cause a significant impairment in skeletal muscle insulin action even in the presence of a mild expansion in
Insulin-stimulated glucose uptake (mg/m2/min)
~70% of maximal inhibition achieved at plasma FFA levels seen in obese and T2DM patients
Fig. 3. Free fatty acid (FF4)-induced insulin resistance in a dose-dependent manner, with most of the insulin resistance developing already with the low, physiological increase in plasma FFA concentrations (i.e., 695 |mol/L) observed in obesity and type 2 diabetes mellitus (T2DM).
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