Adiponectin is inversely associated with intramyocellular and intrahepatic lipids in obese premenopausal women

Adiponectin, an adipokine secreted by adipocytes, exerts beneficial effects on glucose and lipid metabolism and has been found to improve insulin resistance by decreasing triglyceride content in muscle and liver in obese mice. Adiponectin is found in several isoforms and the high-molecular weight (HMW) form has been linked most strongly to the insulin-sensitizing effects. Fat content in skeletal muscle (intramyocellular lipids, IMCL) and liver (intrahepatic lipids, IHL) can be quantified noninvasively using proton magnetic resonance spectroscopy ((1)H-MRS). The purpose of our study was to assess the relationship between HMW adiponectin and measures of glucose homeostasis, IMCL and IHL, and to determine predictors of adiponectin levels. We studied 66 premenopausal women (mean BMI 31.0 ± 6.6 kg/m(2)) who underwent (1)H-MRS of calf muscles and liver for IMCL and IHL, computed tomography (CT) of the abdomen for abdominal fat depots, dual-energy X-ray absorptiometry (DXA) for fat and lean mass assessments, HMW and total adiponectin, fasting lipid profile and an oral glucose tolerance test (homeostasis model assessment of insulin resistance (HOMA(IR)), glucose and insulin area under the curve). There were strong inverse associations between HMW adiponectin and measures of insulin resistance, IMCL and IHL, independent of visceral adipose tissue (VAT) and total body fat. IHL was the strongest predictor of adiponectin and adiponectin was a predictor of HOMA(IR). Our study showed that in premenopausal obese women HMW adiponectin is inversely associated with IMCL and IHL content. This suggests that adiponectin exerts positive effects on insulin sensitivity in obesity by decreasing intracellular triglyceride content in skeletal muscle and liver; it is also possible that our results reflect effects of insulin on adiponectin.     

Proton magnetic resonance spectroscopy shows lower intramyocellular lipid accumulation in middle-aged subjects predisposed to familial longevity

Families predisposed to longevity show enhanced glucose tolerance and skeletal muscle insulin sensitivity compared with controls, independent of body composition and physical activity. Intramyocellular lipid (IMCL) accumulation in skeletal muscle has been associated with insulin resistance. Here, we assessed whether subjects enriched for familial longevity have lower IMCL levels. We determined IMCL levels in 48 subjects from the Leiden Longevity Study, comprising 24 offspring of nonagenarian siblings and 24 partners thereof as control subjects. IMCL levels were assessed noninvasively using short echo time proton magnetic resonance spectroscopy ((1)H-MRS) of the tibialis anterior muscle with a 7 Tesla human MR scanner. IMCL levels were calculated relative to the total creatine (tCr) CH3 signal. Physical activity was assessed using the International Physical Activity Questionnaire (IPAQ). After correction for age, sex, BMI, and physical activity, offspring of long-lived nonagenarian siblings tended to show lower IMCL levels compared with controls (IMCL/tCr: 3.1 ± 0.5 vs. 4.5 ± 0.5, respectively, P = 0.051). In a pairwise comparison, this difference reached statistical significance (P = 0.038). We conclude that offspring of nonagenarian siblings predisposed to longevity show lower IMCL levels compared with environmentally matched control subjects. Future research should focus on assessing what mechanisms may explain the lower IMCL levels in familial longevity.     

Metabolic Characterization of All-Trans-Retinoic Acid (ATRA)–Induced Craniofacial Development of Murine Embryos Using In Vivo Proton Magnetic Resonance Spectroscopy

Aim

To characterize the abnormal metabolic profile of all-trans-retinoic acid (ATRA)–induced craniofacial development in mouse embryos using proton magnetic resonance spectroscopy (¹H-MRS).

Methods

Timed-pregnant mice were treated by oral gavage on the morning of embryonic gestation day 11 (E11) with all-trans-retinoic acid (ATRA). Dosing solutions were adjusted by maternal body weight to provide 30, 70, or 100 mg/kg RA. The control group was given an equivalent volume of the carrier alone. Using an Agilent 7.0 T MR system and a combination of surface coil coils, a 3 mm×3 mm×3 mm ¹H-MRS voxel was selected along the embryonic craniofacial tissue. ¹H-MRS was performed with a single-voxel method using PRESS sequence and analyzed using LCModel software. Hematoxylin and eosin was used to detect and confirm cleft palate.

Result

¹H-MRS revealed elevated choline levels in embryonic craniofacial tissue in the RA70 and RA100 groups compared to controls (P<0.05). Increased choline levels were also found in the RA70 and RA100 groups compared with the RA30 group (P<0.01). High intra-myocellular lipids at 1.30 ppm (IMCL13) in the RA100 group compared to the RA30 group were found (P<0.01). There were no significant changes in taurine, intra-myocellular lipids at 2.10 ppm (IMCL21), and extra-myocellular lipids at 2.30 ppm (EMCL23). Cleft palate formation was observed in all fetuses carried by mice administered 70 and 100 mg/kg RA.

Conclusions

This novel study suggests that the elevated choline and lipid levels found by ¹H-MRS may represent early biomarkers of craniofacial defects. Further studies will determine performance of this test and pathogenetic mechanisms of craniofacial malformation.     

Evaluation of intramyocellular lipid breakdown during exercise by biochemical assay, NMR spectroscopy, and Oil Red O staining

The study compared the net decline of intramyocellular lipids (IMCL) during exercise (n = 18) measured by biochemical assay (BIO) and Oil Red O (ORO) staining on biopsy samples from vastus lateralis muscle and by (1)H-MR spectroscopy (MRS) sampled in an 11 x 11 x 18-mm(3) voxel in the same muscle. IMCL was measured before and after a 2-h cycling bout ( approximately 75% V(.)(O(2) peak)). ORO and MRS measurements showed substantial IMCL use during exercise of 31 +/- 12 and 47 +/- 6% of preexercise IMCL content. In contrast, use of BIO for IMCL determination did not reveal an exercise-induced breakdown of IMCL (2 +/- 9%, P = 0.29) in young healthy males. Correlations between different measures of exercise-induced IMCL degradation were low. Coefficients were 0.48 for MRS vs. ORO (P = 0.07) and were even lower for BIO vs. MRS (r = 0.38, P = 0.13) or ORO (r = 0.08, P = 0.78). This study demonstrates that different methods to measure IMCL in human muscles can result in different conclusions with regard to exercise-induced IMCL changes. MRS has the advantage that it is noninvasive, however, not fiber type specific and hampered by an at least 30-min delay in measurements after exercise completion and may overestimate IMCL use. BIO is the only quantitative method but is subject to variation when biopsies have different fiber type composition. However, BIO yields lower IMCL breakdown compared with ORO and MRS. ORO has the major advantage that it is fiber type specific, and it therefore provides information that is not available with the other methods.     

Utilisation of intramyocellular lipids (IMCLs) during exercise as assessed by proton magnetic resonance spectroscopy (1H-MRS).

Recently, a 1H-MRS method became available to quantify intramyocellular lipids (IMCL) non-invasively. Currently, little is known about the regulation of this lipid pool. During prolonged exercise of moderate intensity, non-plasma-derived fatty acids play an important role as an energy source; lipids located within the skeletal muscle are considered to be a major source for these fatty acids. To see whether IMCL are reduced by exercise, 12 male runners were studied before and after exercising at different workloads and duration. Six subjects participated in a non-competitive run (NCR), three runners in a competitive half marathon (HM, 21 km) and another three in a competitive marathon (M, 42 km). Intra- and extramyocellular lipids were quantified by 1H-MR spectroscopy in the tibialis anterior (TA) and soleus (SOL) muscles prior to and after the exercise bout. Moderate intensity (MI; 60-70% VO2max in NCR) with a mean exercise time (MET) ranging between 105-110 min decreased IMCL by 10 - 36% in both muscles. Prolonged MI exercise (MET 210-240 min; 68-70% VO2max in M) reduced IMCL by 42-57% in TA and 27 - 56% in SOL. In contrast, high intensity exercise (HI; MET 80-120 min; 83-85% VO2max in HM) did not alter IMCL in either muscle. Extramyocellular lipids (EMCL) did not show any significant change in any group. The data show that one bout of moderate-intensity (60-70% VO2max) aerobic exercise markedly reduces the IMCL in TA and SOL muscles in a time-dependent fashion as assessed by 1H-MRS. However, exercise of similar duration but higher workload (> 80% VO2max) does not reduce IMCL. These data suggest that both exercise duration and workload are important factors in determining the reduction of IMCL.     

Insulin resistance,intramyocellular lipid content,and plasma adiponectin in patients with type 1 diabetes

Insulin resistance is a key pathogenic factor of type 2 diabetes (T2DM); in contrast, in type 1 diabetes (T1DM) it is considered a secondary alteration. Increased intramyocellular lipid (IMCL) content accumulation and reduced plasma adiponectin were suggested to be pathogenic events of insulin resistance in T2DM. This study was designed to assess whether IMCL content and plasma adiponectin were also associated with the severity of insulin resistance in T1DM. We studied 18 patients with T1DM, 7 older and overweight/obese patients with T2DM, and 15 nondiabetic, insulin-resistant offspring of T2DM parents (OFF) and 15 healthy individuals (NOR) as appropriate control groups matched for anthropometric features with T1DM patients by means of the euglycemic hyperinsulinemic clamp combined with the infusion of [6,6-2H2]glucose and 1H magnetic resonance spectroscopy of the calf muscles. T1DM and T2DM patients showed reduced insulin-stimulated glucose metabolic clearance rate (MCR: 5.1 +/- 0.6 and 3.2 +/- 0.8 ml x kg(-1) min(-1)) similar to OFF (5.3 +/- 0.4 ml x kg(-1) x min(-1)) compared with NOR (8.5 +/- 0.5 ml x kg(-1) min(-1), P < 0.001). Soleus IMCL content was increased in T1DM (112 +/- 15 AU), T2DM (108 +/- 10 AU) and OFF (82 +/- 13 AU) compared with NOR (52 +/- 7 AU, P < 0.05) and the result was inversely proportional to the MCR (R2 = 0.27, P < 0.001); an association between IMCL content and Hb A1c was found only in T1DM (R2 = 0.57, P < 0.001). Fasting plasma adiponectin was reduced in T2DM (7 +/- 1 microg/ml, P = 0.01) and OFF (11 +/- 1 microg/ml, P = 0.03) but not in T1DM (25 +/- 6 microg/ml), whose plasma level was increased with respect to both OFF (P = 0.03) and NOR (16 +/- 2 microg/ml, P = 0.05). In conclusion, in T1DM, T2DM, and OFF, IMCL content was associated with insulin resistance, demonstrating that IMCL accretion is a marker of insulin resistance common to both primary genetically determined and secondary metabolic (chronic hyperglycemia) alterations. The increased adiponectin levels in insulin-resistant patients with T1DM, in contrast to the reduced levels found in patients with T2DM and in OFF, demonstrated that the relationship of adiponectin to insulin resistance in humans is still unclear.     

Interethnic differences in muscle, liver and abdominal fat partitioning in obese adolescents

The prevalence of insulin resistance and type 2 diabetes (T2D) in obese youth is rapidly increasing, especially in Hispanics and African Americans compared to Caucasians. Insulin resistance is known to be associated with increases in intramyocellular (IMCL) and hepatic fat content. We determined if there are ethnic differences in IMCL and hepatic fat content in a multiethnic cohort of 55 obese adolescents. We used (1)H magnetic resonance spectroscopy (MRS) to quantify IMCL levels in the soleus muscle, oral glucose tolerance testing to estimate insulin sensitivity, magnetic resonance imaging (MRI) to measure abdominal fat distribution. Liver fat content was measured by fast-MRI. Despite similar age and % total body fat among the groups, IMCL was significantly higher in the Hispanics (1.71% [1.43%, 2.0%]) than in the African-Americans (1.04% [0.75%, 1.34%], p = 0.013) and the Caucasians (1.2% [0.94%, 1.5%], p = 0.04). Liver fat content was undetectable in the African Americans whereas it was two fold higher than normal in both Caucasians and Hispanics. Visceral fat was significantly lower in African Americans (41.5 cm(2) [34.6, 49.6]) and was similar in Caucasians (65.2 cm(2) [55.9, 76.0]) and Hispanics (70.5 cm(2) [59.9, 83.1]). In a multiple regression analysis, we found that ethnicity independent of age, gender and % body fat accounts for 10% of the difference in IMCL. Our study indicates that obese Hispanic adolescents have a greater IMCL lipid content than both Caucasians and African Americans, of comparable weight, age and gender. Excessive accumulation of fat in the liver was found in both Caucasian and Hispanic groups as opposed to virtually undetectable levels in the African Americans. Thus, irrespective of obesity, there seem to be some clear ethnic differences in the amount of lipid accumulated in skeletal muscle, liver and abdominal cavity.     

IMCL area density, but not IMCL utilization, is higher in women during moderate-intensity endurance exercise, compared with men

Women use more fat during endurance exercise as evidenced by a lower respiratory exchange ratio (RER). The contribution of intramyocellular lipid (IMCL) to lipid oxidation during endurance exercise is controversial, and studies investigating sex differences in IMCL utilization have found conflicting results. We determined the effect of sex on net IMCL use during an endurance exercise bout using an ultrastructural evaluation. Men (n = 17) and women (n = 19) completed 90-min cycling at 63% Vo(2peak). Biopsies were taken before and after exercise and fixed for electron microscopy to determine IMCL size, # IMCL/area, IMCL area density, and the % IMCL touching mitochondria. Women had a lower RER and carbohydrate oxidation rate and a higher lipid oxidation rate during exercise (P < 0.05), compared with men. Women had a higher # IMCL/area and IMCL area density (P < 0.05), compared with men. Women, but not men, had a higher % IMCL touching mitochondria postexercise (P = 0.03). Exercise decreased IMCL area density (P = 0.01), due to a decrease in the # IMCL/area (P = 0.02). There was no sex difference in IMCL size or net use. In conclusion, women have higher IMCL area density compared with men, due to an increased # IMCL and not an increased IMCL size, as well as an increased % IMCL touching mitochondria postexercise. Endurance exercise resulted in a net decrease in IMCL density due to decreased number of IMCL, not decreased IMCL size, in both sexes.     

Relationship between visceral adiposity and intramyocellular lipid content in two rat models of insulin resistance

High visceral adiposity and intramyocellular lipid levels (IMCL) are both associated with the development of type 2 diabetes. The relationship between visceral adiposity and IMCL levels was explored in diet- and glucocorticoid-induced models of insulin resistance. In the diet-induced model, lean and fa/fa Zucker rats were fed either normal or high-fat (HF) chow over 4 wk. Fat distribution, IMCL content in the tibialis anterior (TA) muscle (IMCL(TA)), and whole body insulin resistance were measured before and after the 4-wk period. The HF diet-induced increase in IMCL(TA) was strongly correlated with visceral fat accumulation and greater glucose intolerance in both groups. The increase in IMCL(TA) to visceral fat accumulation was threefold greater for fa/fa rats. In the glucocorticoid-induced model, insulin sensitivity was impaired with dexamethasone. In vivo adiposity and IMCL(TA) content measurements were combined with ex vivo analysis of plasma and muscle tissue. Dexamethasone treatment had minimal effects on visceral fat accumulation while increasing IMCL(TA) levels approximately 30% (P < 0.05) compared with controls. Dexamethasone increased plasma glucose by twofold and increased the saturated fatty acid content of plasma lipids [fatty acid (CH2)n/omegaCH3 ratio +15%, P < 0.05]. The lipid composition of the TA muscle was unchanged by dexamethasone treatment, indicating that the relative increase in IMCL(TA) observed in vivo resulted from a decrease in lipid oxidation. Visceral adiposity may influence IMCL accumulation in the context of dietary manipulations; however, a "causal" relationship still remains to be determined. Dexamethasone-induced insulin resistance likely operates under a different mechanism, i.e., independently of visceral adiposity.     

Relationship between serum adiponectin concentration and intramyocellular lipid stores in humans.

The recently identified adipocytokine adiponectin has been shown to improve insulin action and decrease triglyceride content in skeletal muscle (by stimulating lipid oxidation) in mice. In the present study, we tested the hypothesis that high serum concentrations of adiponectin are associated with lower intramyocellular (IMCL) fat content by promoting lipid oxidation in humans. IMCL-content in predominantly non-oxidative tibialis anterior muscle and oxidative soleus was determined by proton magnetic resonance spectroscopy in a cross- sectional study involving 63 healthy volunteers. In a second set of experiments, changes in IMCL in both muscles were measured after a three days dietary lipid challenge (n = 18) and after intravenous lipid challenge (n = 12) with suppressed lipid oxidation under hyperinsulinemia. Adiponectin serum concentrations were found to be negatively correlated with IMCL in the oxidative soleus muscle (IMCL [sol]) (r = - 0.46, p < 0.001) independent of measures of obesity, but not with IMCL in the non-oxidative tibialis anterior muscle (IMCL [tib]) (p = 0.40). Adiponectin serum concentrations were negatively correlated with the observed increase in IMCL load after dietary lipid challenge in the tibialis (r = 0.53, p = 0.03) but not in the soleus muscle. During suppression of lipid oxidation by hyperinsulinemia, no effect of adiponectin on IMCL was observed in either soleus or tibialis muscle. Overall, the presented findings are consistent with the hypothesis that adiponectin promotes lipid oxidation in humans resulting in lower intracellular lipid content in human muscle. These results are consistent with animal data, where adiponectin could be shown to enhance lipid oxidation and reduce muscle triglycerides.     

Intramyocellular Lipid Content and Molecular Adaptations in Response to a 1‐Week High‐Fat Diet

Objective: To investigate molecular adaptations that accompany the elevation of intramyocellular lipid (IMCL) content on a high‐fat (HF) diet for 1 week. Research Methods and Procedures: Ten subjects consumed a normal‐fat (NF) diet for 1 week, followed by an HF diet for another week. After both dietary periods, we determined the IMCL content by proton magnetic resonance spectroscopy in the vastus lateralis muscle and quantified changes in gene expression, protein content, and activity in biopsy samples. We investigated genes involved in carbohydrate and fatty acid handling [lipoprotein lipase, acetyl‐coenzyme A carboxylase (ACC) 2, hormone‐sensitive lipase, hexokinase II, and glucose transporter 4] and measured protein levels of CD36 and phosphorylated and unphosphorylated ACC2 and the activity of adenosine monophosphate‐activated kinase. Results: IMCL content was increased by 54% after the HF period. Lipoprotein lipase mRNA concentration was increased by 33%, whereas ACC2 mRNA concentration tended to be increased after the HF diet. Hexokinase II, glucose transporter 4, and hormone‐sensitive lipase mRNA were unchanged after the HF diet. ACC2 and CD36 protein levels, phosphorylation status of ACC2, and adenosine monophosphate‐activated kinase activity did not change in response to the HF diet. Discussion: We found that IMCL content in skeletal muscle increased after 1 week of HF feeding, accompanied by molecular adaptations that favor fat storage in muscle rather than oxidation.     

Diet-induced modulation of mitochondrial activity in rat muscle

Growing evidence supports the theory that mitochondrial dysfunction is an underlying cause of intramyocellular lipid (IMCL) accumulation and insulin resistance. Here, we hypothesized that high dietary fat (HF) intake could trigger changes in mitochondrial activity such that fatty acid oxidation is impaired in muscle and contributes to an elevation in intramyocellular lipid (IMCL) levels. Muscle mitochondrial activity was determined in vivo through measurement of the F(1)F(0) ATP synthase flux, the terminal step in the oxidative phosphorylation process. An initial study comparing rats on normal chow diet with rats on an HF diet revealed strong correlations between muscle ATP synthesis rates, IMCL levels and whole body glucose tolerance. Results obtained from two latter studies showed multiphasic responses to dietary intervention. Initially, the ATP synthesis rates decreased as much as 50% within 24 h of raising the fat content in the diet to 60% of the caloric intake. These rates eventually returned to normal values after 2-3 wk on the HF regimen, seemingly to prevent further IMCL accumulation. Only beyond 1 mo on the HF diet did results consistently show ATP synthesis rates to diminish by 30-50% accompanied by steadily augmenting IMCL levels. Interestingly, switching back to a chow diet after 3 wk of HF feeding reversed the initial diet-induced changes. Although the muscle mitochondrial system may initially offer enough compliance to counteract lipid surplus, these in vivo data suggest a vicious long-term cycle among mitochondrial dysfunction, IMCL accumulation, and glucose intolerance in the rat.     

Intramyocellular lipid content in human skeletal muscle

Fat can be stored not only in adipose tissue but also in other tissues such as skeletal muscle. Fat droplets accumulated in skeletal muscle [intramyocellular lipids (IMCLs)] can be quantified by different methods, all with advantages and drawbacks. Here, we briefly review IMCL quantification methods that use biopsy specimens (biochemical quantification, electron microscopy, and histochemistry) and non-invasive alternatives (magnetic resonance spectroscopy, magnetic resonance imaging, and computed tomography). Regarding the physiological role, it has been suggested that IMCL serves as an intracellular source of energy during exercise. Indeed, IMCL content decreases during prolonged submaximal exercise, and analogously to glycogen, IMCL content is increased in the trained state. In addition, IMCL content is highest in oxidative, type 1 muscle fibers. Together, this, indeed, suggests that the IMCL content is increased in the trained state to optimally match fat oxidative capacity and that it serves as readily available fuel. However, elevation of plasma fatty acid levels or dietary fat content also increases IMCL content, suggesting that skeletal muscle also stores fat simply if the availability of fatty acids is high. Under these conditions, the uptake into skeletal muscle may have negative consequences on insulin sensitivity. Besides the evaluation of the various methods to quantify IMCLs, this perspective describes IMCLs as valuable energy stores during prolonged exercise, which, however, in the absence of regular physical activity and with overconsumption of fat, can have detrimental effects on muscular insulin sensitivity.     

Effect of weight loss on muscle lipid content in morbidly obese subjects

The purpose of this study was to test the hypothesis that weight loss results in a reduction in intramuscular lipid (IMCL) content that is concomitant with enhanced insulin action. Muscle biopsies were obtained from morbidly obese individuals [body mass index (BMI) 52.2 +/- 2.5 kg/m(2); n = 6] before and after gastric bypass surgery, an intervention that improves insulin action. With intervention, there was a 47% reduction (P < 0.01) in BMI and a 93% decrease in homeostasis model assessment, or HOMA (7.0 +/- 1.9 vs. 0.5 +/- 0.1). Histochemically determined IMCL content decreased (P < 0.05) by approximately 30%. In relation to fiber type, IMCL was significantly higher in type I vs. type II fibers. In both fiber types, there were reductions in IMCL and trends for muscle atrophy. Despite these two negating factors, the IMCL-to-fiber area ratio still decreased by approximately 44% with weight loss. In conclusion, despite differing initial levels and possible atrophy, weight loss appears to decrease IMCL deposition to a similar relative extent in type I and II muscle fibers. This reduction in intramuscular triglyceride may contribute to enhanced insulin action seen with weight loss.     

Fatty acid-induced defects in insulin signalling, in myotubes derived from children, are related to ceramide production from palmitate rather than the accumulation of intramyocellular lipid

The elevation of free fatty acids (FFAs), observed in childhood obesity results in intramyocellular lipid (IMCL) accumulation with consequent insulin resistance. Using in vitro differentiated myotubes from normal weight pre-pubertal children (n = 8), we examined the effects of saturated (palmitate) and unsaturated (oleate) FFAs on insulin-stimulated AKT phosphorylation (pAKT) and IMCL accumulation. Palmitate decreased pAKT (Mean [SEM] % change pAKT with palmitate 750 microM vs. control; pThr308 site -50.5% [28.7] and pSer473 site -38.7% [11.7]; P < 0.001) with no effect on IMCL formation. Equimolar bromopalmitate did not effect pAKT and blocking ceramide production abolished the palmitate-induced reduction in signalling, suggesting that ceramide synthesis is critical for palmitate's actions. Oleate did not effect pAKT (1,000 microM oleate; pSer473 site -3.4% [11.4]; P = NS) but increased IMCL accumulation (+32.3% [7.1%]; P < 0.001). Co-administration of oleate diminished the reduction in pAKT seen with palmitate (+36.4% [23.6] vs. -13.3% [13.6]; P = 0.28), with similar IMCL levels to oleate alone. Co-administration also caused a significant reduction in 14C-ceramide synthesis from 14C-palmitate (101.6 [21.6] vs. 371.5 [122.4] DPM/mg protein; P < 0.001). In summary, palmitate appears to cause insulin resistance in children's myotubes via its metabolism to ceramide, and this process appears unrelated to IMCL formation and is ameliorated by oleate.     

Insulin resistance in non-obese subjects is associated with activation of the JNK pathway and impaired insulin signaling in skeletal muscle

Background

The pathogenesis of insulin resistance in the absence of obesity is unknown. In obesity, multiple stress kinases have been identified that impair the insulin signaling pathway via serine phosphorylation of key second messenger proteins. These stress kinases are activated through various mechanisms related to lipid oversupply locally in insulin target tissues and in various adipose depots.

Methodology/Principal Findings

To explore whether specific stress kinases that have been implicated in the insulin resistance of obesity are potentially contributing to insulin resistance in non-obese individuals, twenty healthy, non-obese, normoglycemic subjects identified as insulin sensitive or resistant were studied. Vastus lateralis muscle biopsies obtained during euglycemic, hyperinsulinemic clamp were evaluated for insulin signaling and for activation of stress kinase pathways. Total and regional adipose stores and intramyocellular lipids (IMCL) were assessed by DXA, MRI and ¹H-MRS. In muscle of resistant subjects, phosphorylation of JNK was increased (1.36±0.23 vs. 0.78±0.10 OD units, P<0.05), while there was no evidence for activation of p38 MAPK or IKKβ. IRS-1 serine phosphorylation was increased (1.30±0.09 vs. 0.22±0.03 OD units, P<0.005) while insulin-stimulated tyrosine phosphorylation decreased (10.97±0.95 vs. 0.89±0.50 OD units, P<0.005). IMCL levels were twice as high in insulin resistant subjects (3.26±0.48 vs. 1.58±0.35% H₂O peak, P<0.05), who also displayed increased total fat and abdominal fat when compared to insulin sensitive controls.

Conclusions

This is the first report demonstrating that insulin resistance in non-obese, normoglycemic subjects is associated with activation of the JNK pathway related to increased IMCL and higher total body and abdominal adipose stores. While JNK activation is consistent with a primary impact of muscle lipid accumulation on metabolic stress, further work is necessary to determine the relative contributions of the various mediators of impaired insulin signaling in this population.     

DGAT1 overexpression in muscle by in vivo DNA electroporation increases intramyocellular lipid content

In adipose tissue, the microsomal enzyme 1,2-acyl CoA:diacylglyceroltransferase-1 (DGAT1) plays an important role in triglyceride storage. Because DGAT1 is expressed in skeletal muscle as well, we aimed to directly test the effect of DGAT1 on muscular triglyceride storage by overexpressing DGAT1 using in vivo DNA electroporation. A pcDNA3.1-DGAT1 construct in saline was injected in the left tibialis anterior muscle of rats, followed by the application of eight transcutaneous pulses, using the contralateral leg as sham-electroporated control. Electroporation of the DGAT1 construct led to significant overexpression of the DGAT1 protein. The functionality of DGAT1 overexpression is underscored by the pronounced diet-responsive increase in intramyocellular lipid (IMCL) storage. In chow-fed rats, DGAT1-positive myocytes showed significantly higher IMCL content compared with the control leg, which was almost devoid of IMCL (1.99 +/- 1.13% vs. 0.017 +/- 0.014% of total area fraction; P <0.05). High-fat feeding increased IMCL levels in both DGAT1-positive and control myocytes, resulting in very high IMCL levels in DGAT1-overexpressing myocytes (4.96 +/- 1.47% vs. 0.80 +/- 0.14%; P <0.05). Our findings indicate that DGAT1 contributes to the storage of IMCL and that in vivo DNA electroporation is a promising tool to examine the functional consequences of altered gene expression in mature skeletal muscle.     

The Effect of Aerobic Exercise on Intrahepatocellular and Intramyocellular Lipids in Healthy Subjects

Background

Intrahepatocellular (IHCL) and intramyocellular (IMCL) lipids are ectopic lipid stores. Aerobic exercise results in IMCL utilization in subjects over a broad range of exercise capacity. IMCL and IHCL have been related to impaired insulin action at the skeletal muscle and hepatic level, respectively. The acute effect of aerobic exercise on IHCL is unknown. Possible regulatory factors include exercise capacity, insulin sensitivity and fat availability subcutaneous and visceral fat mass).

Aim

To concomitantly investigate the effect of aerobic exercise on IHCL and IMCL in healthy subjects, using Magnetic Resonance spectroscopy.

Methods

Normal weight, healthy subjects were included. Visit 1 consisted of a determination of VO_2max on a treadmill. Visit 2 comprised the assessment of hepatic and peripheral insulin sensitivity by a two-step hyperinsulinaemic euglycaemic clamp. At Visit 3, subcutaneous and visceral fat mass were assessed by whole body MRI, IHCL and IMCL before and after a 2-hours aerobic exercise (50% of VO_2max) using ¹H-MR-spectroscopy.

Results

Eighteen volunteers (12M, 6F) were enrolled in the study (age, 37.6±3.2 years, mean±SEM; VO_2max, 53.4±2.9 mL/kg/min). Two hours aerobic exercise resulted in a significant decrease in IMCL (−22.6±3.3, % from baseline) and increase in IHCL (+34.9±7.6, % from baseline). There was no significant correlation between the exercise-induced changes in IMCL and IHCL and exercise capacity, subcutaneous and visceral fat mass and hepatic or peripheral insulin sensitivity.

Conclusions

IMCL and IHCL are flexible ectopic lipid stores that are acutely influenced by physical exercise, albeit in different directions.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT00491582","term_id":"NCT00491582"}}NCT00491582     

Regional differences in intramyocellular lipids in humans observed by in vivo 1H-MR spectroscopic imaging.

Regional differences in the content of intramyocellular lipids (IMCL), extramyocellular lipids, and total creatine (TCr) were quantified in soleus (S), tibialis posterior (TP), and tibialis anterior (TA) muscles in humans using in vivo 1H proton spectroscopic imaging at 4 T. Improved spatial resolution (0.25-ml nominal voxel resolution) made it feasible to measure IMCL in S, TP, and TA simultaneously in vivo. The most significant regional difference was found in the content of IMCL compared with extramyocellular lipids or TCr. The concentrations of TCr were found to be 29-32 mmol/kg, with little regional variation. IMCL content was measured to be 4.8 +/- 1.6 mmol/kg tissue wt in S, 2.8 +/- 1.3 mmol/kg tissue wt in TP, and 1.6 +/- 0.9 mmol/kg tissue wt in TA in the order of S > TP > TA (P < 0.05). It is likely that these IMCL values are consistent with the known fiber types of these muscles, with S having the greatest fraction of type I (slow-twitch, oxidative) fibers and TA having a large fraction of type IIb (fast-twitch, glycolytic) fibers.     

Intramyocellular triacylglycerol in prolonged cycling with high- and low-carbohydrate availability.

Vastus lateralis intramyocellular lipid (IMCL) content was assessed by (1)H-magnetic resonance spectroscopy before and after prolonged time trial cycling bouts of approximately 3-h duration. Six highly trained male cyclists completed a double-blind, randomized, crossover design of two experimental trials after a strenuous exercise bout and 48 h of high (HC) (9.32 +/- 0.08 g. kg(-1). day(-1)) and low (LC) (0.59 +/- 0.21 g. kg(-1). day(-1)) dietary carbohydrate. Resting IMCL content was significantly higher after LC vs. HC (P < 0.01) and was reduced during exercise by 64 and 57%, respectively. IMCL was not different between conditions after exercise (P > 0.05). The approximately twofold increase in IMCL degradation in LC compared with HC suggests that higher rates of whole body lipid metabolism in LC were in part attributable to a greater IMCL utilization. Four subjects experienced reductions of IMCL in excess of 70% during exercise. To our knowledge, this is the first study to report near depletion of IMCL during prolonged cycling, indicating that IMCL, presumably the triacylglycerol component, may be exhausted by prolonged strenuous exercise.