Increase In Intramyocellular Lipids Research Articles

Myofiber “Lipotoxicity” Does Not Disrupt Cellular Biophysical Properties

Obesity is associated with pathological changes in skeletal muscle that contribute to functional loss and disability. While it is unclear which specific factors cause this impairment in muscle performance, intramyocellular lipid (IMCL) within the muscle fibers, termed “lipotoxicity”, is implicated. Some studies have suggested the mechanism for this association is mitochondrial dysfunction, whereas others propose the increase in IMCL leads to structural changes within the muscle fibers. The objective of this study was to determine whether IMCL, independent of obesity, impacted active force generation and passive viscoelasticity of the myofiber sarcomere lattice. This study utilized soleus muscles from wildtype (WT) and lipodystrophic (LD) mice and calf muscle biopsies from individuals with (DPN) and without (CTL) type 2 diabetes over a range of obesity status to isolate the effects of IMCL from the effects of obesity in general. IMCL was quantified for each fiber type by co-registration of Oil Red O (ORO) stained cryosections or fluorescent BODIPY stained section and myosin heavy chain isoform stained sections. BODIPY stained sections were used with confocal microscopy to quantify individual lipid droplet metrics (average area, total number). Individual muscle fibers were isolated from soleus muscles and calf muscle biopsies and chemically permeabilized to isolate the myofibrillar lattice and were tested in a specialized system (1400A; Aurora Scientific). Active and passive forces were recorded, fit with a 3-element model, and correlated with metrics of IMCL accumulation by fiber type. LD muscle had 2-3 fold higher IMCL accumulation (p<0.05) in type 2a and 2x fibers than WT quantified by ORO and BODIPY. This was due to an increase in both lipid droplet area and number. DPN muscle had ~2 fold higher IMCL accumulation (p<0.05) in type 2 fibers compared with CTL. Both type 1 and type 2 fibers had significantly increased area of lipid droplets, but only in type 2 fibers was there an increase in lipid droplet number and consequently in area fraction. Despite the large accumulation of IMCL, LD permeabilized muscle fibers had no deficit in active force generation and no change in any of the 5 measures of passive viscoelasticity compared with WT. Conversely, in humans, DPN type 2 fibers had significantly lower (-18%) active force generation and both type 1 and type 2 DPN fibers had elevated resting viscosity (+5%) compared with CTL. Across human samples, active force generation was significantly correlated with average lipid droplet area (r2=0.53, p=0.04), but no other correlations were significant. This study suggests that an increase in IMCL accumulation in and of itself does not physically disrupt sarcomere contraction. Dramatic accumulation of IMCL in LD mouse fibers did not impact active or passive forces, likely due to an absence of systemic complications (obesity, advanced diabetes). Human fibers of comparable IMCL accumulation have a deficit in active force generation correlated with IMCL parameters, but we conclude this relationship is more likely correlative than causative as type 1 fibers with similar lipid droplet areas have no association between lipid droplet area and active force generation. These data suggest that while accumulation of IMCL is associated with contractile disfunction across a number of conditions, the mechanism is likely either indirect or biochemical rather than biophysical. Shriners Hospitals for Children, P30 AR074992, AR075773, AG15768, AG46927, AR072999, AR073752. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Resveratrol-induced remodelling of myocellular lipid stores: A study in metabolically compromised humans.

In non‐athletes, insulin sensitivity correlates negatively with intramyocellular lipid (IMCL) content. In athletes, however, a pattern of benign IMCL storage exists, which is characterized by lipid storage in type I muscle fibres, in small and numerous lipid droplets (LDs) preferable coated with PLIN5, without affecting insulin sensitivity. Administration of resveratrol has been promoted for its beneficial effects on glucose homeostasis. We observed that 30 days of oral resveratrol administration (150 mg/day) in metabolically compromised individuals showed a 33% increase in IMCL (placebo vs. resveratrol; 0.86 ± 0.090 AU vs. 1.14 ± 0.11 AU, p = 0.003) without impeding insulin sensitivity. Thus, the aim of the present study was to examine if a resveratrol‐mediated increase in IMCL content, in metabolically compromised individuals, changes the LD phenotype towards the phenotype we previously observed in athletes. For this, we studied IMCL, LD number, LD size, subcellular distribution and PLIN5 coating in different fibre types using high‐resolution confocal microscopy. As proof of concept, we observed a 2.3‐fold increase (p = 0.038) in lipid accumulation after 48 h of resveratrol incubation in cultured human primary muscle cells. In vivo analysis showed that resveratrol‐induced increase in IMCL is predominantly in type I muscle fibres (placebo vs. resveratrol; 0.97 ± 0.16% vs. 1.26 ± 0.09%; p = 0.030) in both the subsarcolemmal (p = 0.016) and intermyofibrillar region (p = 0.026) and particularly in PLIN5‐coated LDs (p = 0.024). These data indicate that administration of resveratrol augments IMCL content in metabolically compromised individuals towards a LD phenotype that mimics an ‘athlete like phenotype’.

Effects of regular-moderate exercise on high-fat diet-induced intramyocellular lipid accumulation in the soleus muscle of Sprague-Dawley rats.

Previously, we monitored the expression level of the pro-apoptotic proteins caspase-3 and cleaved poly-ADP-ribose polymerase in the skeletal muscle of high-fat diet-induced obese rats in order to assess muscle damage. In this study, we analyzed whether exercise or dietary adjustment was more effective at preventing high-fat diet-induced muscle damage. High-fat diet-induced obese rats were divided into three groups: the high-fat diet (HFD), the combined high-fat diet and exercise (HFD+EXE), and the dietary adjustment (DA) groups. For 6 weeks, the HFD+EXE group was subjected to exercise on an animal treadmill. Capsase-3 protein was quantified, and histopathology of the soleus muscle was performed. Both the HFD+EXE and DA interventions resulted in a reduction of lipid accumulation in the soleus muscle, and nucleus infiltration was significantly lower in the DA group. The inflammatory response, caspase-3 level, and relative muscle weight were significantly higher in the HFD+EXE group compared to the HFD group. An increase in intramyocellular lipids in the soleus muscle by obesity and exercise stimulated apoptosis. When the rats exercised, muscle growth was normal and unrelated to the effects of lipid accumulation. These data indicate that exercise was more effective than dietary adjustment in reducing lipid accumulation and increasing muscle metabolism.

Effect of acute physiological free fatty acid elevation in the context of hyperinsulinemia on fiber type-specific IMCL accumulation

It is well described that increasing free fatty acids (FFAs) to high physiological levels reduces insulin sensitivity. In sedentary humans, intramyocellular lipid (IMCL) is inversely related to insulin sensitivity. Since muscle fiber composition affects muscle metabolism, whether FFAs induce IMCL accumulation in a fiber type-specific manner remains unknown. We hypothesized that in the setting of acute FFA elevation by lipid infusion within the context of a hyperinsulinemic-euglycemic clamp, IMCL will preferentially accumulate in type 1 fibers. Normal-weight participants (n = 57, mean ± SE: age 24 ± 0.6 yr, BMI 22.2 ± 0.3 kg/m2) who were either endurance trained or sedentary by self-report were recruited from the University of Minnesota (n = 31, n = 15 trained) and University of Pittsburgh (n = 26, n = 14 trained). All participants underwent a hyperinsulinemic-euglycemic clamp in the context of a 6-h infusion of either lipid or glycerol control. A vastus lateralis muscle biopsy was obtained at baseline and end-infusion (6 h). The muscle biopsies were processed and analyzed at the University of Pittsburgh for fiber type-specific IMCL accumulation by Oil-Red-O staining. Regardless of training status, acute elevation of FFAs to high physiological levels (~400-600 meq/l) increased IMCL preferentially in type 1 fibers (+35 ± 11% compared with baseline, +29 ± 11% compared with glycerol control: P < 0.05). The increase in IMCL correlated with a decline in insulin sensitivity as measured by the hyperinsulinemic-euglycemic clamp (r = -0.32, P < 0.01) independent of training status. Regardless of training status, increase of FFAs to a physiological range within the context of hyperinsulinemia shows preferential IMCL accumulation in type 1 fibers.NEW & NOTEWORTHY This novel human study examined the effects of FFA elevation in the setting of hyperinsulinemia on accumulation of fat in specific types of muscle fibers. Within the context of the hyperinsulinemic-euglycemic clamp, we found that an increase of FFAs to a physiological range sufficient to reduce insulin sensitivity is associated with preferential IMCL accumulation in type 1 fibers.

Abstract IA27: Exercise, energy balance, and cancer

Abstract IA27: Exercise, energy balance, and cancer

Effect of aging on muscle mitochondrial substrate utilization in humans

Previous studies have implicated age-associated reductions in mitochondrial oxidative phosphorylation activity in skeletal muscle as a predisposing factor for intramyocellular lipid (IMCL) accumulation and muscle insulin resistance (IR) in the elderly. To further investigate potential alterations in muscle mitochondrial function associated with aging, we assessed basal and insulin-stimulated rates of muscle pyruvate dehydrogenase (VPDH) flux relative to citrate synthase flux (VCS) in healthy lean, elderly subjects and healthy young body mass index- and activity-matched subjects. VPDH/VCS flux was assessed from the (13)C incorporation from of infused [1-13C] glucose into glutamate [4-13C] relative to alanine [3-13C] assessed by LC-tandem MS in muscle biopsies. Insulin-stimulated rates of muscle glucose uptake were reduced by 25% (P<0.01) in the elderly subjects and were associated with ∼70% (P<0.04) increase in IMCL, assessed by 1H magnetic resonance spectroscopy. Basal VPDH/VCS fluxes were similar between the groups (young: 0.20±0.03; elderly: 0.14±0.03) and increased approximately threefold in the young subjects following insulin stimulation. However, this increase was severely blunted in the elderly subjects (young: 0.55±0.04; elderly: 0.18±0.02, P=0.0002) and was associated with an ∼40% (P=0.004) reduction in insulin activation of Akt. These results provide new insights into acquired mitochondrial abnormalities associated with aging and demonstrate that age-associated reductions in muscle mitochondrial function and increased IMCL are associated with a marked inability of mitochondria to switch from lipid to glucose oxidation during insulin stimulation.

Mitochondrial dysfunction and insulin resistance from the outside in: extracellular matrix, the cytoskeleton, and mitochondria

Insulin resistance in skeletal muscle is a prominent feature of obesity and type 2 diabetes. The association between mitochondrial changes and insulin resistance is well known. More recently, there is growing evidence of a relationship between inflammation, extracellular remodeling, and insulin resistance. The intent of this review is to propose a potentially novel mechanism for the development of insulin resistance, focusing on the underappreciated connections among inflammation, extracellular remodeling, cytoskeletal interactions, mitochondrial function, and insulin resistance in human skeletal muscle. Several sources of inflammation, including expansion of adipose tissue resulting in increased lipolysis and alterations in pro- and anti-inflammatory cytokines, contribute to the insulin resistance observed in obesity and type 2 diabetes. In the experimental model of lipid oversupply, an inflammatory response in skeletal muscle leads to altered expression extracellular matrix-related genes as well as nuclear encoded mitochondrial genes. A similar pattern also is observed in "naturally" occurring insulin resistance in muscle of obese nondiabetic individuals and patients with type 2 diabetes mellitus. More recently, alterations in proteins (including α-actinin-2, desmin, proteasomes, and chaperones) involved in muscle structure and function have been observed in insulin-resistant muscle. Some of these cytoskeletal proteins are mechanosignal transducers that allow muscle fibers to sense contractile activity and respond appropriately. The ensuing alterations in expression of genes coding for mitochondrial proteins and cytoskeletal proteins may contribute to the mitochondrial changes observed in insulin-resistant muscle. These changes in turn may lead to a reduction in fat oxidation and an increase in intramyocellular lipid, which contributes to the defects in insulin signaling in insulin resistance.

Morning to evening changes of intramyocellular lipid content in dependence on nutrition and physical activity during one single day: a volume selective 1H-MRS study

Intramyocellular lipids (IMCL) were shown to be metabolically highly active. In order to get insight into short-term regulation of IMCL and to reveal related problems with standardization in metabolic studies using the common signal ratio IMCL/Cr3, relative concentration changes from morning to evening in the same day were examined under four different nutritional and exercise conditions. Twelve healthy male volunteers participated in an interventional program, comprising single days of fasting (F), low-caloric/low-fat diet (LC), or high-caloric/high-fat diet (HC), combined with low physical activity. A forth day course consisted of unchanged nutrition and extensive exercise (EX). (1)H-MRS of tibialis anterior (TA) and soleus muscle (SOL) was performed on a 3 T whole-body imager in the early morning and 12 h later after the intervention applying a single voxel STEAM technique. Interventions resulted in a clear reduction of IMCL/ Cr3 after F (IMCL/Cr3(TA): -28.1 ± 4.9%, IMCL/Cr3(SOL): -21.0 ± 3.7%) and EX (IMCL/Cr3(TA): -33.9 ± 4.9%, IMCL/Cr3(SOL): -18.3 ± 3.9%). LC led to slightly decreased IMCL/Cr3 ratio in the evening (IMCL/Cr3(TA): -8.7 ± 4.4%, IMCL/Cr3(SOL): -7.3 ± 2.7%), whereas negligible changes were detectable after HC (IMCL/Cr3(TA): + 0.6 ± 2.3%, IMCL/Cr3L(SOL): -0.2 ± 1.3%). Only high-caloric/high-fat diet combined with low physical activity led to nearly unchanged IMCL/Cr3 ratios in the evening when compared to corresponding measurements in the morning. In contrast, low-caloric/low-fat diet and especially fasting led to increasingly depleted IMCL stores in the evening. This depletion seems to be further emphasized by increased physical activity. An interesting aspect is the marked reduction of IMCL/Cr3 after 12 h of fasting, since a dramatic increase in IMCL has been reported after starvation over several days. Results of this study imply that highly standardized conditions regarding diet and physical activity are necessary for a proper assessment of IMCL data in metabolic studies.

Effects of a short-term overfeeding with fructose or glucose in healthy young males

Consumption of simple carbohydrates has markedly increased over the past decades, and may be involved in the increased prevalence in metabolic diseases. Whether an increased intake of fructose is specifically related to a dysregulation of glucose and lipid metabolism remains controversial. We therefore compared the effects of hypercaloric diets enriched with fructose (HFrD) or glucose (HGlcD) in healthy men. Eleven subjects were studied in a randomised order after 7 d of the following diets: (1) weight maintenance, control diet; (2) HFrD (3.5 g fructose/kg fat-free mass (ffm) per d, +35 % energy intake); (3) HGlcD (3.5 g glucose/kg ffm per d, +35 % energy intake). Fasting hepatic glucose output (HGO) was measured with 6,6-2H2-glucose. Intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) were measured by 1H magnetic resonance spectroscopy. Both fructose and glucose increased fasting VLDL-TAG (HFrD: +59 %, P < 0.05; HGlcD: +31 %, P = 0.11) and IHCL (HFrD: +52 %, P < 0.05; HGlcD: +58 %, P = 0.06). HGO increased after both diets (HFrD: +5 %, P < 0.05; HGlcD: +5 %, P = 0.05). No change was observed in fasting glycaemia, insulin and alanine aminotransferase concentrations. IMCL increased significantly only after the HGlcD (HFrD: +24 %, NS; HGlcD: +59 %, P < 0.05). IHCL and VLDL-TAG were not different between hypercaloric HFrD and HGlcD, but were increased compared to values observed with a weight maintenance diet. However, glucose led to a higher increase in IMCL than fructose.

The Increase in Intramyocellular Lipid in Leg Skeletal Muscle is Associated With Lowered Aerobic Exercise Capacity in Heart Failure Patients

The Increase in Intramyocellular Lipid in Leg Skeletal Muscle is Associated With Lowered Aerobic Exercise Capacity in Heart Failure Patients

Murine intramyocellular lipids quantified by NMR act as metabolic biomarkers in burn trauma

It has been suggested that intramyocellular lipids (IMCLs) may serve as biomarkers of insulin resistance and mitochondrial dysfunction. Using a hind-limb mouse model of burn trauma, we tested the hypothesis that severe localized burn trauma involving 5% of the total body surface area causes a local increase in IMCLs in the leg skeletal muscle. We quantified IMCLs from ex vivo intact tissue specimens using High-Resolution Magic Angle Spinning (HRMAS) 1H NMR and characterized the accompanying gene expression patterns in burned versus control skeletal muscle specimens. We also quantified plasma-free fatty acids (FFAs) in burn versus control mice. Our results from HRMAS 1H NMR measurements indicated that IMCL levels were significantly increased in mice exposed to burn trauma. Furthermore, plasma FFA levels were also significantly increased, and gene expression of Glut4, insulin receptor substrate 1 (IRS1), glycolytic genes, and PGC-1beta was downregulated in these mice. Backward stepwise multiple linear regression analysis demonstrated that IMCL levels correlated significantly with FFA levels, which were a significant predictor of IRS1 and PGC-1beta gene expression. We conclude from these findings that IMCLs can serve as metabolic biomarkers in burn trauma and that FFAs and IMCLs may signal altered metabolic gene expression. This signaling may result in the observed burn-induced insulin resistance and skeletal muscle mitochondrial dysfunction. We believe that IMCLs may therefore be useful biomarkers in predicting the therapeutic effectiveness of hypolipidemic agents for patients with severe burns.

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.

Muscle type dependent increase in intramyocellular lipids during prolonged fasting of human subjects: a proton MRS study.

The amount of intramyocellular lipids in skeletal muscle was assessed by proton magnetic resonance spectroscopy during a voluntary fasting period of 120 h in four healthy lean volunteers. The aim of the study was to determine whether muscular lipid uptake in the presence of high plasma lipid levels, or lipid oxidation due to lacking glycogen as a source of energy in musculature, are the dominant effects on intramyocellular lipid levels under fasting conditions in various muscle types. Intramyocellular lipids were quantified in the tibialis anterior (mixed type I and type II fibers, predominantly type II) and the soleus muscle (predominantly type I fibers) before and after 24 h, 72 h, and 120 h of fasting. An extreme increase in intramyocellular lipids to levels of 369 % (median) was found in the tibialis anterior muscle compared to baseline value (intramyocellular lipid level prior to fasting, set to 100 %; p = 0.02). The soleus muscle with clearly higher baseline content of intramyocellular lipids (2 - 4-fold compared to tibialis anterior) revealed slightly delayed and less pronounced uptake of intramyocellular lipids during fasting to 152 % (median) after 120 h (p = 0.02). The absolute increment in intramyocellular lipids (in terms of ratios between lipid and creatine signals) was also higher in tibialis anterior than in soleus (not statistically significant). These findings indicate augmentation of the intramyocellular lipid pool during long-term elevation of plasma FFA in the presence of low plasma insulin concentrations in both muscles investigated. The rate of muscular lipid oxidation during fasting is clearly lower than the increased uptake of FFA by myocytes.

Cellular mechanism of insulin resistance: potential links with inflammation.

Insulin resistance is a pivotal feature in the pathogenesis of type 2 diabetes, and it may be detected 10-20 y before the clinical onset of hyperglycemia. Insulin resistance is due to the reduced ability of peripheral target tissues to respond properly to insulin stimulation. In particular, impaired insulin-stimulated muscle glycogen synthesis plays a significant role in insulin resistance. Glucose transport (GLUT4), phosphorylation (hexokinase) and storage (glycogen synthase) are the three potential rate-controlling steps regulating insulin-stimulated muscle glucose metabolism, and all three have been implicated as being the major defects responsible for causing insulin resistance in patients with type 2 diabetes. Using (13)C/(31)P magnetic resonance spectroscopy (MRS), we demonstrate that a defect in insulin-stimulated muscle glucose transport activity is the rate-controlling defect. Using a similar (13)C/(31)P MRS approach, we have also demonstrated that fatty acids cause insulin resistance in humans due to a decrease in insulin-stimulated muscle glucose transport activity, which could be attributed to reduced insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, a required step in insulin-stimulated glucose transport into muscle. Furthermore, we have recently proposed that this defect in insulin-stimulated muscle glucose transport activity may be due to the activation of a serine kinase cascade involving protein kinase C theta and IKK-beta, which are key downstream mediators of tissue inflammation. Finally, we propose that any perturbation that leads to an increase in intramyocellular lipid (fatty acid metabolites) content such as acquired or inherited defects in mitochondrial fatty acid oxidation, defects in adipocyte fat metabolism or simply increased fat delivery to muscle/liver due to increased energy intake will lead to insulin resistance through this final common pathway. Understanding these key cellular mechanisms of insulin resistance should help elucidate new targets for treating type 2 diabetes.

Nicotinic acid-induced insulin resistance is related to increased circulating fatty acids and fat oxidation but not muscle lipid content

Insulin resistance is associated with increased circulating lipids and skeletal muscle lipid content. Chronic nicotinic acid (NA) treatment reduces insulin sensitivity and provides a model of insulin resistance. We hypothesized that the reduction in insulin sensitivity occurs via elevation of circulating nonesterified fatty acids (NEFAs) and an increase in intramyocellular lipid (IMCL). A total of 15 nondiabetic males (mean age 27.4 ± 1.6 years) were treated with NA (500 mg daily for 1 week, 1 g daily for 1 week). Insulin sensitivity (glucose infusion rate [GIR]) was determined pre- and post-NA by euglycemic-hyperinsulinemic clamp. Substrate oxidation was determined by indirect calorimetry. Skeletal muscle lipid was assessed by estimation of long-chain acyl-CoA (LCACoA) and triglyceride (TG) content and by 1H-magnetic resonance spectroscopy quantification of IMCL (n = 11). NA reduced GIR (P = .03) and nonoxidative glucose disposal (P < .01) and increased fasting NEFAs (P = .01). The decrease in GIR related significantly to the increase in fasting NEFAs (r2 = .30, P = .03). The intrasubject increase in basal and clamp fat oxidation correlated with the decrease in GIR (r2 = .45, P < .01 and r2 = .63, P < .01). There were no significant changes in muscle LCACoA, TG, or IMCL content. Therefore, induction of insulin resistance by NA occurs with increased availability of circulating fatty acids to muscle rather than with increased muscle lipid content.