Insulin Resistance Is Associated With Higher Intramyocellular Triglycerides in Type I but Not Type II Myocytes Concomitant With Higher Ceramide Content

Subjects for this nested case-controlled study were selected from ongoing clinical investigations of skeletal muscle insulin resistance. Only female subjects were included to provide a sex-controlled analysis. Women aged 60–75 years were recruited through advertisements in the Pittsburgh, PA, area. A brief phone screening initially determined eligibility for study participation. Potential subjects who were determined to be sedentary (exercise ≤1 day/week), weight stable (±3 kg in the previous 6 months), with a BMI >30 kg/m², and nonsmokers were further evaluated at the Clinical Translational Research Center (CTRC) at the University of Pittsburgh. Exclusionary criteria included uncontrolled hypertension (>150/95 mmHg), anemia (hematocrit <34%), elevated liver enzymes (25% above normal), proteinuria, or hypothyroidism (sensitive thyroid-stimulating hormone [TSH] 8 mIU/l). Potential subjects were also excluded if they reported taking chronic medications known to affect glucose homeostasis. After the medical screening, potential subjects then completed a 2-h 75-g oral glucose tolerance test to determine glucose tolerance status. Subjects with type 2 diabetes determined by fasting glucose >126 mg/dl were excluded. All subjects gave written consent to the protocol, which was approved by the University of Pittsburgh's Institutional Review Board.

Insulin sensitivity was determined by a 4-h hyperinsulinemic (40 mU · m⁻² · min⁻¹) euglycemic clamp and [6,6-²H₂]glucose infusion as previously described (28). Briefly, subjects reported to the CTRC the evening before the clamp and after a 48-h period, in which they refrained from any vigorous physical activity. A continuous intravenous infusion of insulin (Humulin; Eli Lilly, Indianapolis, IN) was begun at ∼7:00 a.m., and euglycemia (target 90 mg/dl) was maintained using an adjustable infusion of 20% dextrose. To measure rate of insulin-stimulated glucose disposal and residual endogenous glucose production, a primed (0.22 μmol/kg) continuous (17.6 μmol · kg⁻¹ · min⁻¹) infusion of [6,6-²H₂]glucose was administered (29). [6,6-²H₂]glucose enrichment in plasma was determined by gas chromatography/mass spectrometry (6890 Network/5973 Series; Agilent, Santa Clara, CA) as previously described (30). Plasma glucose was measured by the glucose oxidase method (2300 Stat Plus; Yellow Springs Instruments, Yellow Springs, OH). Fasting plasma insulin concentration was determined by an enzyme-linked immunosorbent assay (Millipore, Billerica, MA). Rate of glucose disposal (R_d) was calculated using the tracer:tracee method with a modified version of the Steele et al. equation (31).

Percutaneous muscle biopsy samples were obtained in the fasted state before the glucose clamp, as described previously (32). Briefly, muscle biopsies were obtained from the vastus lateralis, 15 cm above the patella. A portion of the specimen (∼30 mg) was snap-frozen in liquid nitrogen and stored at −80°C for sphingolipid and diacylglycerol analysis. A second portion (∼20 mg) was snap-frozen and stored at −80°C for gene expression analysis. A third portion of the specimen for histochemistry was mounted on a small piece of cork with mounting medium (Shandon Cryochrome; Thermo Electron, Pittsburgh, PA), frozen in isopentane cooled with liquid nitrogen for 2–3 min (−160°C), and then placed into liquid nitrogen. All samples were stored at −80°C until analysis.

Briefly, liquid nitrogen–frozen samples (∼30 mg) were homogenized in ice-cold buffer (250 mmol/l sucrose, 25 mmol/l KCl, 50 mmol/l Tris, and 0.5 mmol/l EDTA, pH 7.4). Total content as well as the various molecular species of diacylglycerol and sphingolipid were measured by high-performance liquid chromatography–tandem mass spectrometry as previously described in detail (33).

Plasma free fatty acid concentrations were determined by gas chromatography with flame ionization detection (6890N; Agilent, Santa Clara, CA) using heptanoic acid as an internal standard. Free fatty acids were derivatized with dimethylamine using Deoxo-Fluor reagent (Sigma-Aldrich, St. Louis, MO).

Histochemical analysis was performed on serial sections using a modified version of methods previously used in our laboratory (32). Briefly, mounted biopsy samples were sectioned (10 μm) on a cryostat (Cryotome E; Thermo Shandon, Pittsburgh, PA) at −20°C and placed on a glass slide. Sections were then stained in a filtered solution of Oil Red O (300 mg/ml in 36% triethylphosphate) for 30 min at room temperature. Thereafter, sections were incubated with primary antibodies for anti-human myosin heavy chain (MYH)-7 (type I myocytes) and MYH2 (type IIa myocytes) overnight at room temperature and subsequently incubated with fluorescein (FITC) (type IIa myocytes) and rhodamine (type I myocytes) conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Type IIx fibers remained unstained. Images were visualized using a Leica microscope (Leica DM 4000B; Leica Microsystems, Bannockburn, IL), digitally captured (Retiga 2000R camera; Q Imaging, Surrey, Canada), and analyzed using specialized software (Northern Eclipse, v6.0; Empix Imaging, Cheektowaga, NY). Oil red O staining intensity and cross-sectional area were determined in type I and type II myocytes. Analysis is based on ∼200 fibers per section (range 130–350).

Gene expression analysis was carried out in a subset of subjects from each group, based on availability of vastus lateralis biopsy specimen (six IR and six IS). Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA). Isolated RNA was then further purified using a silicon-membrane spin column including an on-column DNase I treatment (Qiagen, Valencia, CA). Total RNA was reverse-transcribed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (SABiosciences, Frederick, MD). Gene expression was determined by quantitative real-time PCR (qRT-PCR) using SYBR green-based custom RT² Profiler PCR Arrays (SABiosciences, Frederick, MD). qRT-PCR was performed on an ABI prism 7900 (Applied Biosystems, Foster City, CA). Baseline, threshold cycle (C_t) and threshold were determined and set for all samples. The relative level of mRNA expression for each gene in each sample was normalized to the average expression of β2-microglobulin, hypoxanthine phosphoribosyltransferase (HPRT1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB) and expressed as 2^−ΔCt.

Total body fat and lean mass were assessed using dual-energy X-ray absorptiometry (GE Lunar Prodigy and Encore 2005 software v9.3). Subjects performed a VO₂ peak test on an electronically braked cycle ergometer to determine aerobic fitness (Sensor Medics, Yorba Linda, CA) as previously described (34).

Group comparisons were made by an independent two-tailed Student's t test. Correlations between variables were determined by Person's correlation coefficient. All data are means ± SE unless otherwise stated. Statistical significance was set at P < 0.05.

Ten insulin-sensitive (IS) and 12 insulin-resistant (IR) women were matched for age, body mass, BMI, and physical fitness (VO₂ peak) (Table 1). There was no significant group difference in fasting plasma glucose, insulin, or free fatty acid levels. However, by design, R_d during the hyperinsulinemic-euglycemic clamp was greater (P < 0.01) in the IS group when compared with the IR group (Table 1). The 2-h plasma glucose levels during the oral glucose tolerance test were also significantly higher in the IR group compared with the IS group (P = 0.016). The IR (n = 6) and IS (n = 6) subgroups for gene expression analysis had similar characteristics as their respective groups overall (data not shown).

Type I and II myocyte content and myocyte average cross-sectional area are presented in Table 2. The IS group had a higher proportion of type I myocytes (P = 0.002) and a lower proportion of type IIa (P = 0.014 and type IIx (P = 0.02) than the IR group. When data from both groups were combined, there was a higher proportion of type IIa compared with type IIx myocytes (P = 0.008). No differences were observed in myocyte average cross-sectional area. With both groups combined, there was a significant positive correlation between the proportion of type I myocytes and insulin sensitivity and a negative correlation between the proportion of type II myocytes (type IIa and IIx combined) and insulin sensitivity (Table 3).

The data for muscle content of sphingolipid and diacylglycerol species is presented in Fig. 1. We observed higher total levels of ceramide (555 ± 53 vs. 293 ± 54 pmol/mg protein, P = 0.004), total unsaturated ceramide (198 ± 29 vs. 114 ± 21 pmol/mg protein, P = 0.034), and total saturated ceramide (361 ± 29 vs. 179 ± 34 pmol/mg protein, P = 0.001) in the IR group compared with the IS group. We also observed higher levels of specific ceramide species containing the following fatty acids: myristic (C14:0) (4.0 ± 0.8 vs. 1.9 ± 0.4 pmol/mg protein, P = 0.044), palmitic (C16:0) (58 ± 14 vs. 18 ± 5 pmol/mg protein, P = 0.023), and stearic (C18:0) (72 ± 10 vs. 40 ± 7 pmol/mg protein, P = 0.02) in IR compared with IS muscle. There also tended to be greater content of sphingolipids containing palmitoleic (C18:1) (43 ± 11 vs. 19 ± 3 pmol/mg protein, P = 0.077) and lignoceric (C24:0) (201 ± 25 vs. 123 ± 29 pmol/mg protein, P = 0.06) acids and sphingosine (3.4 ± 0.6 vs. 2.2 ± 0.3 pmol/mg protein, P = 0.08) in IR muscle. When data were combined from both groups, we observed a significant negative correlation between total unsaturated, total saturated, and total ceramide content and insulin sensitivity (Table 3). Total ceramide (R = 0.517, P < 0.05) and total saturated ceramide (R = 0.523, P < 0.05) were positively correlated with type II myocyte percentage. However, no significant differences in total or individual diacylglycerol species were observed between IS and IR subjects.

Type I and II myocyte–specific IMTG content are shown in Fig. 2. Total muscle IMTG was greater in the IR group compared with the IS group (P = 0.009). Type I myocyte–specific IMTG content was also greater in the IR group compared with the IS group (P = 0.005). There was no difference in type II myocyte–specific IMTG content between IS and IR groups, nor was IMTG content different in IR within subtypes of type IIa and type IIx myocytes. When both groups were combined, we observed a significant negative correlation between type I myocyte IMTG content and insulin sensitivity, whereas there was no relationship between insulin sensitivity and type II myocyte (type IIa and IIx combined) IMTG content (Fig. 2).

The gene expression data for IR and IS subgroups is presented in Fig. 3. Expression levels of key genes, including facilitated GLUT member 4 (GLUT4), insulin receptor substrate (IRS)-1, and IRS2 were lower in the IR group, indicating insulin resistance in this group at the tissue level in accord with their insulin resistance assessed in vivo. The expression of genes relating to oxidative phosphorylation transcription factors, fatty acid metabolism, ceramide metabolism, and lipid droplet associated proteins were examined. We found lower expression of the transcription factors peroxisome proliferator–activated receptor (PPAR)-α and PPAR-γ in the IR group compared with the IS group. Also, expression of PPAR-γ coactivator 1α (PGC-1α) and nuclear respiratory factor 1 (NRF1) tended to be lower in IR muscle. Furthermore, we observed a lower expression of carnitine palmitoyltransferase 1B (CPT1B) and malonyl-CoA decarboxylase (MLYCD), genes that are related to fatty acid metabolism. Gene expression of lipid droplet–associated proteins hormone-sensitive lipase (HSL), adipose differentiation–related protein (ADRP), adipose triglyceride lipase (ATGL), and synaptosomal-associated protein 23 (SNAP23) was lower in the IR group compared with the IS group. Interestingly, the expression of genes relating to ceramide metabolism was not different between groups. The expression of tumor necrosis factor (TNF)-α tended to be higher in the IR group compared with the IS group (P = 0.09).

Despite a preponderance of evidence from cell culture and animal models suggesting a role for both ceramide and diacylglycerol in skeletal muscle insulin resistance (7,8), there is actually no consensus as to whether this is relevant to human insulin resistance (12,13,15,17,19). This study was conducted for three purposes: 1) to determine whether diacylglycerol or sphingolipid species are elevated in IR human skeletal muscle, 2) to investigate whether the associations between intramyocellular lipids are dependent on muscle fiber type, and 3) to explore whether the skeletal muscle expression of several key genes relating to lipid metabolism are altered in IR muscle corresponding to intramyocellular lipid content.

A primary finding of our study was that total as well as saturated and unsaturated ceramide content was approximately twofold higher in IR skeletal muscle in human subjects independent of obesity, physical fitness, and plasma free fatty acid concentration. These results extend the findings of Adams et al. (13), who observed elevated ceramide content in skeletal muscle of obese subjects. Studies by Straczkowski et al. (12,14) also found that ceramide accumulation in skeletal muscle was inversely related to insulin sensitivity. They also suggested that higher ceramide content in lean offspring of patients with type 2 diabetes was due to decreased ceramide degradation, whereas IR of obesity was caused by an increase in de novo ceramide synthesis (12,14). Not all studies, however, have observed an association between muscle ceramide content and insulin resistance (18,19). These discrepant results could be attributed to differences in the subject population studied, e.g., age, sex, physical activity, and methodological differences, in assessing ceramide content and insulin sensitivity (11). Thus, our study provides important new information about the relationship between elevated ceramide content and muscle insulin resistance. This is the first to a comprehensive profile of the content of the various molecular species of ceramide in human IR muscle using tandem mass spectrometry while controlling for obesity, physical fitness, and age. This is the first study to demonstrate elevated levels of both saturated and unsaturated ceramides, as well as specific ceramides containing C14:0, C16:0, and C18:0 fatty acids in IR human muscle.

None of the prior studies examining ceramide in human muscle have accounted for heterogeneity in muscle type that could account for differences in insulin sensitivity. Our novel finding that ceramide content was associated with a higher percentage of type II myocytes suggests that ceramide accumulation may be fiber-type dependent. Further investigation is needed to confirm this. In addition, the complementary observations that both the IMTG content in type I myocytes and the lower proportion of type I myocytes are related to insulin resistance provide insight into muscle heterogeneity in lipid metabolism in insulin resistance. These data suggest a possibility that type I myofibers may have a greater capacity to buffer increases in free fatty acids by partitioning to lipid droplet IMTG, whereas type II fibers have a reduced capacity to incorporate excess fatty acids into IMTG and are more inclined to partition to lipotoxic mediators such as ceramide species. An impairment of skeletal muscle oxidative capacity has been suggested to initiate or potentiate insulin resistance (35). Our data indicate that the proportion of type I and type II myocytes is associated with higher ceramide content and insulin resistance and that the expression levels of several genes related to mitochondrial biogenesis and fatty acid metabolism are lower in insulin resistance. Therefore, our complementary results support the concept that a low oxidative capacity of muscle may be associated with ceramide accumulation and insulin resistance. This is consistent with previous reports that the increase in whole muscle IMTG content in obesity or type 2 diabetes is primarily due to increased IMTG in type I myocytes (25) and that differences in fiber type proportion also play a role in insulin resistance of obesity and type 2 diabetes (36,37). These observations are supported by a number of reports suggesting that type I myocytes are more insulin responsive than type II myocytes (26,27,38). Yet, differences in fiber type proportion between obese subjects either with or without type 2 diabetes have been reported in some (36,37), but not all previous studies (22,24).

Despite evidence in IR animal models supporting a role for DAG in insulin resistance (8,9), we did not observe an increased level of DAG in insulin-resistant muscle. Straczkowski et al. (12) have also reported elevated levels of ceramide, but not DAG, in obese impaired glucose tolerant versus obese normal glucose tolerant (NGT) subjects. A possible reason for lipid-induced insulin resistance by ceramide and not DAG, and visa versa, may be that higher levels of palmitate result in greater ceramide accumulation, while unsaturated fatty acids partition toward DAG (39). Thus, both ceramides and DAG can potentially induce insulin resistance depending on the degree of saturation of the lipid overload. Our finding that C16:0 ceramide was more than threefold higher in IR muscle is consistent with this. Thus, it is plausible that basal levels of ceramide and DAG in muscle are influenced by the degree of saturation and palmitate content in the diet of obese individuals. Further studies are needed to address this attractive hypothesis.

We also examined the differential expression of several other genes relating to lipid metabolism, which could underlie accumulation of IMTG and other bioactive species, i.e., DAG and ceramide. We found that many genes involved in ceramide synthesis and degradation were not differentially expressed in IR muscle with increased ceramide content. A recent report suggests that over expression of acid ceramidase prevents the inhibitory effect of saturated fatty acids on insulin signaling (40). Also, inhibition of serine palmitoyltranseferase in Zucker diabetic fatty (ZDF) rats, by treatment with the amino acid antibiotic myriocin, prevented the onset of type 2 diabetes and inhibited an increase in ceramide content in muscle, when compared with nontreated ZDF rats (41). Our results suggest that factors other than increased enzyme content, possibly increased enzyme activity or fatty acid flux, contribute to ceramide accumulation in IR muscle. In support of this, Straczkowski et al. (12) demonstrated that the activity of neutral sphingomyelinase, the enzyme that converts sphingomyelin to ceramide, is increased in muscle of obese impaired glucose tolerant subjects compared with obese normal glucose tolerant subjects, concomitant with higher ceramide content. Finally, the observation that gene expression of TNF-α tended to be elevated in IR muscle, albeit statistically nonsignificant, concomitant with elevated ceramide content is consistent with data demonstrating that TNF-α can stimulate de novo ceramide biosynthesis (42).

To further characterize intramyocellular lipid content and metabolism in IR skeletal muscle, we determined the expression of selected genes coding for lipid droplet proteins and proteins involved in lipid droplet hydrolysis. ATGL and HSL expression/activity have been demonstrated to contribute to adipose tissue and skeletal muscle triacylglycerol lipolysis, elevated plasma fatty acid levels, and accumulation of ectopic lipid deposits associated with type 2 diabetes (43). Our observation that ATGL and HSL mRNA are lower in IR muscle is in line with reports of lower expression of these lipases in obesity and/or insulin resistance (44,45). We also found lower expression of ADRP in IR muscle. Consistent with this, Phillips et al. showed that skeletal muscle protein expression of ADRP was upregulated in response to improvements in insulin action induced by weight loss and insulin sensitizer pharmacotherapy (46). Furthermore, ATGL and ADRP have been reported to be more highly expressed in type I muscle fibers (47,48). The fiber type–specific expression of ATGL and ADRP, and our observation of reduced type I fibers in obese IR subjects, potentially explains the lower level of ATGL and ADRP observed in whole muscle in the present study. It is tempting to speculate the same may be true for other lipases and that fiber type composition plays a greater role in insulin resistance than previously thought. Furthermore, the trend toward higher TNF-α gene expression in IR muscle is in line with reports that TNF-α represses ATGL expression (49). We also observed for the first time in human muscle that the expression of SNAP23, a member of the SNARE family of proteins whose function is to mediate intracellular vesicle fusion events including GLUT4 transport to the plasma cell membrane and lipid droplet fusion (50), is lower in IR muscle. This novel finding potentially linking intramyocellular lipid droplets and glucose transport is supported by others describing an association between SNAP23 and fatty acid–induced insulin resistance (50).

Future studies should be conducted to examine whether or not interventions such as exercise or weight loss affect oxidative capacity, DAG, ceramide, and insulin sensitivity to better understand these associations in humans. Although we substantiated our insulin resistance phenotype with expression of key genes involved in insulin signaling and glucose transport, additional studies are needed to link intramyocellular lipids, fiber type, oxidative capacity, and activation of specific insulin-signaling proteins. Taken together, however, these results suggest a role for muscle fiber type proportion and/or the oxidative capacity of muscle in insulin resistance, possibly through partitioning muscle lipids into either triglycerides or ceramides.

In summary, ceramide but not diacylglycerol is elevated in IR human skeletal muscle independent of obesity, physical activity, or aerobic fitness. Moreover, muscle fiber type as well as fiber type–specific lipid content is associated with insulin resistance in muscle. Importantly, key genes associated with fatty acid metabolism and lipid droplet regulation are decreased in IR muscle. Together, these data suggest that mechanisms underlying the associations between intramyocellular lipids and insulin resistance likely involve ceramide, which in turn may depend on the muscle fiber type or its oxidative capacity. Future mechanistic studies are needed to further elucidate these important findings.

This study was supported by the University of Pittsburgh CTRC (M01RR00056) and Obesity and Nutrition Research Center (1P30DK46204), the National Institute on Aging (R01-AG-20128), and an American Diabetes Association Clinical Research Award (to B.H.G.).

No potential conflicts of interest relevant to this article were reported.

The results of this study were presented in abstract form at the 69th annual Scientific Sessions of the American Diabetes Association, New Orleans, Louisiana, 5–9 June 2009 (Abstract 253-OR).

The authors thank all subjects for participating in the study; acknowledge the excellent technical assistance of Krista Clark, Steve Anthony, and the nursing staff at the University of Pittsburgh CTRC; and appreciate the assistance of Jim DeLany and Kelly McCoy with plasma free fatty acid and plasma glucose enrichment measures.