Many studies have demonstrated a detrimental effect of smoking on insulin action. Data from epidemiologic (7
) as well as cross-sectional studies report decreased insulin sensitivity in middle-aged smokers (4
). We are the first to report chronic cigarette smoking promotes insulin resistance in young, otherwise healthy college-aged men and women. Major findings from this study suggest saturation of intramuscular lipids, independent of concentration or turnover, may be related to insulin action. Further, smoking resulted in basal inhibition of insulin signaling. The induction of such metabolic defects at a young age will surely result in profound and premature untoward health effects.
In addition to lower insulin action, smokers have greater delivery of plasma FFA (16
) and lipoproteins (16
) to skeletal muscle. Therefore, we hypothesized smoking may promote IMTG and DAG accumulation. However, we found no differences in IMTG or DAG concentration between groups. These data add to other studies finding a dissociation between IMTG concentration and insulin action (31
) and support the notion that IMTG may only be a marker of insulin resistance rather than a cause. Similar DAG concentrations were surprising considering data linking DAG accumulation with insulin resistance (33
). Supporting a lack of DAG-induced insulin resistance were similar PCKζ and θ content between groups. However, these data are from whole-cell lysates, and it is possible that differences in cytosolic versus sarcolemmal localization went unnoticed. Together, our data suggest increased skeletal muscle IMTG and DAG concentrations are not related to insulin resistance in college-aged chronic smokers.
We hypothesized IMTG and DAG FSR would be positively related to insulin sensitivity (14
). Increased synthesis rates of IMTG may enhance FFA clearance and decrease ceramide and long-chain acyl-CoA synthesis, which may increase insulin action (34
). In support of this, recent evidence indicates increasing IMTG synthesis in rodents and humans protects against fat-induced insulin resistance (31
). In the current study, however, we found no differences in IMTG and DAG FSR between smokers and nonsmokers, suggesting rates of IMTG and DAG synthesis may not be related to insulin sensitivity in this population. Further, we found no change in whole-body glucose kinetics between groups similar to some (17
) but not all previous studies (36
). Smokers had increased plasma palmitate turnover as has been shown before in older smokers (17
). Our data on breath V13
C palmitate incorporation into IMTG suggests that the increased plasma palmitate disposal in smokers is oxidized and not stored as IMTG. These data suggest decreased mitochondrial FFA oxidation and mitochondrial dysfunction does not play a major role in smoking-induced insulin resistance.
Interestingly, IMTG and DAG were significantly more saturated in smokers compared with nonsmokers. Initially, we thought this could be explained by increased habitual consumption of saturated fat in smokers. However, there were no differences in overall phospholipid composition, which has been used as a surrogate measure of dietary lipid intake, between groups (37
). Less muscle lipid desaturation in smokers is unlikely, as the expression and protein content of SCD1, which converts saturated palmitoyl-CoA and stearoyl-CoA to monounsaturated palmitoleoyl-CoA and oleoyl-CoA, respectively, was not different between groups. Although indirect, a common surrogate for SCD1 activity are ratios of 16:1 to 16:0 and 18:1 to 18:0 (38
). None of these ratios were significantly different for IMTG and DAG pools between smokers and nonsmokers. Therefore, it is unlikely that changes in SCD1 content or activity explain alterations in muscle lipid saturation, but the exact mechanism by which smoking changes neutral lipid storage is not known. The importance of saturated lipids on tissue insulin action was highlighted by a study that developed a transgenic Elovl6 (elongation of long-chain fatty acids family member 6) knockout mouse (39
). They found no change in hepatic triglyceride content but a significant change in triglyceride composition and increased hepatic insulin action. We extend these data to humans and suggest IMTG and DAG composition may influence insulin sensitivity in skeletal muscle.
We analyzed many potential mediators of intracellular insulin resistance to explain why smokers were insulin resistant. There was no indication of plasma inflammation, lipid toxicity, lipid peroxidation, or endoplasmic reticulum stress. The only measure significantly different between groups was greater IRS-1 Ser636 phosphorylation in smokers compared with nonsmokers. This difference may explain decreased insulin action in smokers because of basal inhibition of insulin signaling.
There are several known mechanisms that lead to phosphorylation of Ser636 on IRS-1. Increased mammalian target of rapamycin (mTOR) and/or p44/42 mitogen-activated protein kinase (MAPK, a.k.a. ERK1/2) activity promotes insulin resistance by phosphorylating this specific site on IRS-1. We measured the phosphorylation state of the downstream kinase for mTOR/p70s6k and found no differences between groups. Further, we measured ERK1/2 phosphorylation and found no differences between groups. Therefore, these data imply chronic signaling through mTOR or ERK1/2 cannot explain increased IRS-1 Ser636 phosphorylation in smokers compared with nonsmokers. However, our data are consistent with transitory nicotine-stimulated ERK1/2 activity that could result in basal IRS-1 Ser636 phosphorylation.
The effect of smoking to decrease insulin action is likely because of nicotine, which promotes insulin resistance following direct infusion in humans (40
). However, the mechanism behind smoking or nicotine-induced insulin resistance is not known. Recent evidence indicates nicotine increases ERK1/2 phosphorylation in a wide variety of cells (41
). Further, the increase in ERK1/2 phosphorylation is transient, starting after 2 min of nicotine exposure, peaking after ~10 min, and decreasing to basal values after 15–30 min of continued exposure (41
phosphorylation of IRS-1 induced by ERK1/2 activity has been reported in primary human muscle cell cultures from individuals with type 2 diabetes (44
) and following TNFα-induced insulin resistance in 3T3L-1 adipocytes (45
). Interestingly, ERK1/2 phosphorylation can decrease to basal levels before measuring increased serine phosphorylation of IRS-1 in 3T3L1 adipocytes (45
). The current data are consistent with nicotine exposure during tobacco smoking promoting insulin resistance via Ser636
phosphorylation of IRS-1, induced via transient ERK1/2 stimulation.
Skeletal muscle RT-PCR analysis supports other mechanisms may also be working to promote insulin resistance in smokers. Expression of MCP-1 was increased in skeletal muscle from smokers compared with nonsmokers. MCP-1 is a chemokine produced by adipocytes, vascular cells, and skeletal muscle, which promotes macrophage entry into tissues and itself may promote insulin resistance (46
). These data suggest smoking may promote macrophage infiltration in skeletal muscle, possibly promoted by smoking-induced endothelial damage (47
), which may result in local cytokine production and insulin resistance (48
). PPAR-γ expression was also decreased in smokers compared with nonsmokers. We found a trend for decreased RXR-γ expression, which dimerizes with PPAR-γ in the nucleus, and a nonsignificant increase in FOXO1 expression in smokers, which negatively regulates PPAR-γ/RXR promoter activity (49
). Muscle-specific knockout of PPAR-γ results in an insulin-resistant phenotype (50
). Therefore, these changes point to alterations in downstream targets of PPAR-γ as potential mechanisms in smoking-induced insulin resistance. Abbasi et al. (51
) treated insulin-resistant smokers for 12 weeks with pioglitazone and found increased insulin sensitivity and decreased plasma triglyceride concentrations. These data imply increased PPAR-γ stimulation may reverse part of the insulin resistance in smokers and suggest decreased muscle PPAR-γ may be important in insulin resistance in smokers.
There are several limitations to this study. The sample size studied was small, which may have limited our ability to measure small differences between groups. We chose subjects who were sedentary and engaged in planned physical activity less than 3 h/week. Even though succinate dehydrogenase content was similar between groups, it is possible that differences in physical activity may have influenced our results.
In conclusion, chronic cigarette smokers were less insulin sensitive compared with control subjects that did not smoke. There were no differences in IMTG or DAG content or synthesis rates, but smokers had increased saturation of skeletal muscle lipids. Insulin resistance in smokers may be because of increased basal inhibition of insulin signaling via Ser636 phosphorylation of IRS-1.