Anne Gemmink

Chemical imaging of lipid droplets in muscle tissues using hyperspectral coherent Raman microscopy.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

Metabolic disturbances of non-alcoholic fatty liver resemble the alterations typical for type 2 diabetes

Non-alcoholic fatty liver (NAFL) is an independent risk factor for the development of type 2 diabetes (T2DM). We examined metabolic perturbations in patients with NAFL, patients with T2DM, and control (CON) subjects with normal intrahepatic lipid (IHL) content.A two-step (10 mU/m2 /min; 40 mU/m2/min) hyperinsulinemic-euglycemic clamp was performed in 11 NAFL, 13 T2DM, and 11 CON subjects, all matched for BMI, and aerobic fitness. IHL content was measured using proton magnetic resonance spectroscopy. Because of high IHL content variability in T2DM patients, this group was separated into a high IHL content group (IHL ≥ 5.0%, T2DM+NAFL) and a normal IHL content group (IHL < 5.0%, T2DM-non-NAFL) for further analysis.IHL content was increased in NAFL and T2DM+NAFL subjects (P<0.050 versus CON and T2DM-non-NAFL subjects). Adipose tissue insulin sensitivity index (Adipo-IRi) was higher in NAFL (P<0.050 versus CON and T2DM-non-NAFL subjects) and in T2DM+NAFL subjects (P=0.055 versus CON subjects, P<0.050 versus T2DM-non-NAFL subjects). Suppression of plasma-free fatty acids (P=0.046) was lower in NAFL compared with CON subjects, with intermediate values for T2DM-non-NAFL, and T2DM+NAFL subjects. Suppression of endogenous glucose production (EGP) and insulin-stimulated glucose disposal (ΔRd) was comparable between NAFL, T2DM-non-NAFL, and T2DM+NAFL subjects (all P>0.05), and was lower in comparison with CON subjects (all P<0.01). Metabolic flexibility was lower in T2DM-non-NAFL subjects (P=0.047) and NAFL subjects (P=0.059) compared with CON subjects. Adipo-IRi (r=0.652, P<0.001), hepatic insulin resistance index (HIRi) (r=0.576, P=0.001), and ΔRd (r=-0.653, P<0.001) correlated with IHL content.Individuals with NAFL suffer from metabolic perturbations to a similar degree as T2DM patients. NAFL is an important feature leading to severe insulin resistance and should be viewed as a serious health threat for the development of T2DM. ClinicalTrials.gov: NCT01317576.

Intramyocellular lipid droplets and insulin sensitivity, the human perspective

Skeletal muscle can store excess fat as subcellular lipid droplets (LDs). While originally viewed as uninteresting static balls of triacylglycerol, it is now clear that myocellular LDs play an active role in myocellular (patho)physiology. In this review we aim to discuss the role of LDs in muscle cell insulin sensitivity and identify parameters which appear to affect this relationship. Moreover, we discuss the application of novel tools permitting detailed examination of these parameters. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity.

Aims/hypothesisDissipating energy via mitochondrial uncoupling has been suggested to contribute to enhanced insulin sensitivity. We hypothesised that skeletal muscle mitochondria of endurance-trained athletes have increased sensitivity for fatty acid (FA)-induced uncoupling, which is driven by the mitochondrial protein adenine nucleotide translocase 1 (ANT1).MethodsCapacity for FA-induced uncoupling was measured in endurance-trained male athletes (T) and sedentary young men (UT) in an observational study and also in isolated skeletal muscle mitochondria from Zucker diabetic fatty (ZDF) rats and C2C12 myotubes following small interfering RNA (siRNA)-mediated gene silencing of ANT1. Thus, fuelled by glutamate/succinate (fibres) or pyruvate (mitochondria and myotubes) and in the presence of oligomycin to block ATP synthesis, increasing levels of oleate (fibres) or palmitate (mitochondria and myotubes) were automatically titrated while respiration was monitored. Insulin sensitivity was measured by hyperinsulinaemic–euglycaemic clamp in humans and via insulin-stimulated glucose uptake in myotubes.ResultsSkeletal muscle from the T group displayed increased sensitivity to FA-induced uncoupling (p = 0.011) compared with muscle from the UT group, and this was associated with elevated insulin sensitivity (p = 0.034). ANT1 expression was increased in T (p = 0.013). Mitochondria from ZDF rats displayed decreased sensitivity for FA-induced uncoupling (p = 0.008). This difference disappeared in the presence of the adenine nucleotide translocator inhibitor carboxyatractyloside. Partial knockdown of ANT1 in C2C12 myotubes decreased sensitivity to the FA-induced uncoupling (p = 0.008) and insulin-stimulated glucose uptake (p = 0.025) compared with controls.Conclusions/interpretationIncreased sensitivity to FA-induced uncoupling is associated with enhanced insulin sensitivity and is affected by ANT1 activity in skeletal muscle. FA-induced mitochondrial uncoupling may help to preserve insulin sensitivity in the face of a high supply of FAs.Trial registrationwww.trialregister.nl NTR2002

Lipid droplet dynamics and insulin sensitivity upon a 5-day high-fat diet in Caucasians and South Asians.

A 5-day High-Fat High-Calorie diet (HFHC-diet) reduces insulin-stimulated glucose disposal (Rd) in South Asian, but not Caucasian healthy lean males. We aimed to investigate if differences in myocellular lipid handling are underlying this differential response. A two-step hyperinsulinemic-euglycemic clamp and muscle biopsies were performed in 12 healthy lean Caucasian and South Asian males (BMI < 25 kg/m2, 19–25 years) before and after a 5-day HFHC-diet (regular diet + 375 mL cream/day; 1275 kcal/day; 94% fat). Triglyceride extractions and Western Blots for lipid droplet and mitochondrial proteins were performed. Intramyocellular lipid content and HFHC-diet response were similar between ethnicities (group effect: P = 0.094; diet effect: +~30%, P = 0.044). PLIN5 protein content increased upon the HFHC-diet (P = 0.031) and tended to be higher in South Asians (0.87 ± 0.42 AU vs. 1.35 ± 0.58 AU, P = 0.07). 4-HNE tended to increase in South Asians upon the HFHC-diet (interaction effect: P = 0.057). In Caucasians ΔPLIN5 content correlated with ΔRd (Caucasians: r = 0.756, P = 0.011; South Asians: r = −0.085, P = 0.816), while in South Asians Δ4-HNE associated with ΔPLIN5 content (Caucasians: r = 0.312, P = 0.380; South Asians: r = 0.771, P = 0.003). These data indicate that in Caucasians, PLIN5 may be protective against HFHC-diet induced insulin resistance, which for reasons not yet understood is not observed in South Asians, who possess increased lipid peroxidation levels.

Exercise training reduces intrahepatic lipid in people with and people without non-alcoholic fatty liver

Exercise training reduces intrahepatic lipid (IHL) content in people with elevated liver fat content. It is unclear, however, whether exercise training reduces IHL content in people with normal liver fat content. Here, we measured the effect of exercise training on IHL content in people with and people without nonalcohol fatty liver. We further measured changes in insulin sensitivity and hepatic energy metabolism. Eleven males with nonalcoholic fatty liver (NAFL) and 11 body mass index-matched individuals without nonalcoholic fatty liver (CON) completed a 12-wk supervised exercise training program. IHL content (proton magnetic resonance spectroscopy), maximal oxidative capacity (V̇o2max, spiroergometry), total muscle strength, body composition, insulin sensitivity (hyperinsulinemic-euglycemic clamp), hepatic ATP-to-total phosphorus ratio, and the hepatic phosphomonoester-to-phosphodiester (PME/PDE) ratio (phosphorus magnetic resonance spectroscopy) were determined. IHL content reduced with exercise training ( P = 0.014) in the whole study population. The relative reduction in IHL content was comparable in NAFL (-34.5 ± 54.0%) and CON (-28.3 ± 60.1%) individuals ( P = 0.800). V̇o2max ( P < 0.001), total muscle strength ( P < 0.001), and skeletal muscle insulin sensitivity ( P = 0.004) increased, whereas adipose tissue ( P = 0.246) and hepatic ( P = 0.086) insulin sensitivity did not increase significantly. Hepatic ATP-to-total phosphorus ratio ( P = 0.987) and PME/PDE ratio ( P = 0.792) did not change. Changes in IHL content correlated with changes in body weight ( r = 0.451, P = 0.035) and changes in hepatic PME/PDE ratio ( r = 0.569, P = 0.019). In conclusion, exercise training reduced intrahepatic lipid content in people with nonalcoholic fatty liver and in people with normal intrahepatic lipid content, and the percent reduction in intrahepatic lipid content was similar in both groups.

Dissociation of intramyocellular lipid storage and insulin resistance in trained athletes and type 2 diabetes patients; involvement of perilipin 5?

Intramyocellular lipid storage is negatively associated with insulin sensitivity. However, endurance trained athletes and type 2 diabetes mellitus (T2DM) patients store similar amounts of lipids in their muscle; the so‐called athletes paradox. Compared to T2DM, trained athletes possess higher levels of perilipin 5 (PLIN5), a lipid droplet (LD) coating protein. We examined whether coating LD with PLIN5 affects the pattern of muscle lipid (LD size and number) in relation to the athletes paradox. Despite differences in PLIN5 protein content, we observed that coating the LD with PLIN5 could not explain the observed differences in LD size and number between athletes and T2DM. PLIN5‐coated LDs were positively associated with oxidative capacity but not with insulin sensitivity. We conclude that coating of LDs with PLIN5 cannot causally explain the athletes paradox.

Distinct lipid droplet characteristics and distribution unmask the apparent contradiction of the athlete's paradox

Objective Intramyocellular lipid (IMCL) storage negatively associates with insulin resistance, albeit not in endurance-trained athletes. We investigated the putative contribution of lipid droplet (LD) morphology and subcellular localization to the so-called athletes paradox. Methods We performed quantitative immunofluorescent confocal imaging of muscle biopsy sections from endurance Trained, Lean sedentary, Obese, and Type 2 diabetes (T2DM) participants (n = 8/group). T2DM patients and Trained individuals were matched for IMCL content. Furthermore we performed this analysis in biopsies of T2DM patients before and after a 12-week exercise program (n = 8). Results We found marked differences in lipid storage morphology between trained subjects and T2DM: the latter group mainly store lipid in larger LDs in the subsarcolemmal (SS) region of type II fibers, whereas Trained store lipid in a higher number of LDs in the intramyofibrillar (IMF) region of type I fibers. In addition, a twelve-week combined endurance and strength exercise program resulted in a LD phenotype shift in T2DM patients partly towards an ‘athlete-like’ phenotype, accompanied by improved insulin sensitivity. Proteins involved in LD turnover were also more abundant in Trained than in T2DM and partly changed in an ‘athlete-like’ fashion in T2DM patients upon exercise training. Conclusions Our findings provide a physiological explanation for the athletes paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.

Super-resolution microscopy localizes perilipin 5 at lipid droplet-mitochondria interaction sites and at lipid droplets juxtaposing to perilipin 2

OBJECTIVE Intramyocellular lipid droplets (LD) and their coat proteins PLIN2 and PLIN5 are involved in lipolysis, with a putative role for PLIN5 in mitochondrial tethering. Reportedly, these proteins co-localize and cover the surface of the LD. To provide the spatial basis for understanding how these proteins possess their distinct roles, we examined the precise location of PLIN2 and PLIN5 and explored PLIN5 presence at LD-mitochondria contact sites using Stimulated emission depletion (STED) microscopy and correlative light-electron microscopy (CLEM) in human skeletal muscle sections. METHODS LDs were stained by MDH together with combinations of mitochondrial proteins and PLINs. Subcellular distribution and co-localization of PLIN proteins and mitochondria was imaged by STED microscopy (Leica TCS SP8) and quantified using Pearsons correlation coefficients and intensity profile plots. CLEM was employed to examine the presence of PLIN5 on mitochondria-LD contact sites. RESULTS Both PLIN2 and PLIN5 localized to the LD in a dot-like, juxtaposed fashion rather than colocalizing and covering the entire LD. Both STED and CLEM revealed a high fraction of PLIN5 at the LD-mitochondria interface, but not at mitochondrial cristae, as suggested previously. CONCLUSION Using two super-resolution imaging approaches, this is the first study to show that in sections of human skeletal muscle PLIN2 and PLIN5 localize to the LD at distinct sites, with abundance of PLIN5 at LD-mitochondria tethering sites. This novel spatial information uncovers that PLIN proteins do not serve as lipolytic barriers but rather are docking sites for proteins facilitating selective lipase access under a variety of lipolytic conditions.

What are the benefits of being big

Like all eukaryotic cell types, skeletal muscle can store free fatty acids (FFA) as triacylglycerol (TG) in lipid droplets (LDs), which in three dimensions can be viewed as balls of lipid ranging in size. This article is protected by copyright. All rights reserved