The tissue-specific metabolic effects of the PPARα agonist ciprofibrate in insulin-resistant male individuals: a double-blind, randomized, placebo-controlled crossover study.

OBJECTIVE: Insulin resistance is characterized by ectopic fat accumulation leading to cardiac diastolic dysfunction and nonalcoholic fatty liver disease. The objective of this study was to determine whether treatment with the peroxisome proliferator-activated receptor-α (PPARα) agonist ciprofibrate has direct effects on cardiac and hepatic metabolism and can improve insulin sensitivity and cardiac function in insulin-resistant volunteers. METHODS: Ten insulin-resistant male volunteers received 100 mg/d of ciprofibrate and placebo for 5 weeks in a randomized double-blind crossover study. Insulin-stimulated metabolic rate of glucose (MRgluc) was measured using dynamic 18 F-fluorodeoxyglucose-positron emission tomography (18 F-FDG-PET). Additionally, cardiac function, whole-body insulin sensitivity, intrahepatic lipid content, skeletal muscle gene expression, 24-hour blood pressure, and substrate metabolism were measured. RESULTS: Whole-body insulin sensitivity, energy metabolism, and body composition were unchanged after ciprofibrate treatment. Ciprofibrate treatment decreased insulin-stimulated hepatic MRgluc and increased hepatic lipid content. Myocardial net MRgluc tended to decrease after ciprofibrate treatment, but ciprofibrate treatment had no effect on cardiac function and cardiac energy status. In addition, no changes in PPAR-related gene expression in muscle were found. CONCLUSIONS: Ciprofibrate treatment increased hepatic lipid accumulation and lowered MRgluc, without affecting whole-body insulin sensitivity. Furthermore, parameters of cardiac function or cardiac energy status were not altered upon ciprofibrate treatment.

PCr/ATP ratios and mitochondrial function in the heart. A comparative study in humans.

Cardiac energy status, measured as phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio with 31P-Magnetic Resonance Spectroscopy (31P-MRS) in vivo, is a prognostic factor in heart failure and is lowered in cardiometabolic disease. It has been suggested that, as oxidative phosphorylation is the major contributor to ATP synthesis, PCr/ATP ratio might be a reflection of cardiac mitochondrial function. The objective of the study was to investigate whether PCr/ATP ratios can be used as in vivo marker for cardiac mitochondrial function. We enrolled thirty-eight patients scheduled for open-heart surgery in this study. Cardiac 31P-MRS was performed before surgery. Tissue from the right atrial appendage was obtained during surgery for high-resolution respirometry for the assessment of mitochondrial function. There was no correlation between the PCr/ATP ratio and ADP-stimulated respiration rates (octanoylcarnitine R2 < 0.005, p = 0.74; pyruvate R2 < 0.025, p = 0.41) nor with maximally uncoupled respiration (octanoylcarnitine R2 = 0.005, p = 0.71; pyruvate R2 = 0.040, p = 0.26). PCr/ATP ratio did correlate with indexed LV end systolic mass. As no direct correlation between cardiac energy status (PCr/ATP) and mitochondrial function in the heart was found, the study suggests that mitochondrial function might not the only determinant of cardiac energy status. Interpretation should be done in the right context in cardiac metabolic studies.

Twenty-four hour rhythmicity in mitochondrial network connectivity and mitochondrial respiration; a study in human skeletal muscle biopsies of young lean and older individuals with obesity.

OBJECTIVE: Mitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day-night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h METHODS: Muscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day RESULTS: Young healthy lean individuals displayed a day-night rhythmicity in mitochondrial network morphology, which aligned with the day-night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity CONCLUSIONS: Our data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.

Effect of ß2-agonist treatment on insulin-stimulated peripheral glucose disposal in healthy men in a randomised placebo-controlled trial.

ß2-agonist treatment improves skeletal muscle glucose uptake and whole-body glucose homeostasis in rodents, likely via mTORC2-mediated signalling. However, human data on this topic is virtually absent. We here investigate the effects of two-weeks treatment with the ß2-agonist clenbuterol (40 µg/day) on glucose control as well as energy- and substrate metabolism in healthy young men (age: 18-30 years, BMI: 20-25 kg/m2) in a randomised, placebo-controlled, double-blinded, cross-over study (ClinicalTrials.gov-identifier: NCT03800290). Randomisation occurred by controlled randomisation and the final allocation sequence was seven (period 1: clenbuterol, period 2: placebo) to four (period 1: placebo, period 2: clenbuterol). The primary and secondary outcome were peripheral insulin-stimulated glucose disposal and skeletal muscle GLUT4 translocation, respectively. Primary analyses were performed on eleven participants. No serious adverse events were reported. The study was performed at Maastricht University, Maastricht, The Netherlands, between August 2019 and April 2021. Clenbuterol treatment improved peripheral insulin-stimulated glucose disposal by 13% (46.6 ± 3.5 versus 41.2 ± 2.7 µmol/kg/min, p = 0.032), whereas skeletal muscle GLUT4 translocation assessed in overnight fasted muscle biopsies remained unaffected. These results highlight the potential of ß2-agonist treatment in improving skeletal muscle glucose uptake and underscore the therapeutic value of this pathway for the treatment of type 2 diabetes. However, given the well-known (cardiovascular) side-effects of systemic ß2-agonist treatment, further exploration on the underlying mechanisms is needed to identify viable therapeutic targets.

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism.

OBJECTIVE: SGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. METHODS: Twenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. RESULTS: Mitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 µm2 (0.01-0.06), p < 0.05) and number (0.003 µm-2 (-0.001-0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4-OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue-1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (-40.0774 (-64.4766, -15.6782) pmoles∗mg tissue-1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. CONCLUSION: Dapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux. The trial is registered with ClinicalTrials.gov, number NCT03338855.

Metabolic responses to mild cold acclimation in type 2 diabetes patients.

Mild cold acclimation for 10 days has been previously shown to markedly improve insulin sensitivity in patients with type 2 diabetes. Here we show in a single-arm intervention study (Trialregister.nl ID: NL4469/NTR5711) in nine patients with type 2 diabetes that ten days of mild cold acclimation (16-17 °C) in which observable, overt shivering was prevented, does not result in improved insulin sensitivity, postprandial glucose and lipid metabolism or intrahepatic lipid content and only results in mild effects on overnight fasted fat oxidation, postprandial energy expenditure and aortic augmentation index. The lack of marked metabolic effects in this study is associated with a lack of self-reported shivering and a lack of upregulation of gene expression of muscle activation or muscle contraction pathways in skeletal muscle and suggests that some form of muscle contraction is needed for beneficial effects of mild cold acclimation.

Hypoxia-inducible lipid droplet-associated induces DGAT1 and promotes lipid storage in hepatocytes.

OBJECTIVE: Storage of triglycerides in lipid droplets is governed by a set of lipid droplet-associated proteins. One of these lipid droplet-associated proteins, hypoxia-inducible lipid droplet-associated (HILPDA), was found to impair lipid droplet breakdown in macrophages and cancer cells by inhibiting adipose triglyceride lipase. Here, we aimed to better characterize the role and mechanism of action of HILPDA in hepatocytes. METHODS: We performed studies in HILPDA-deficient and HILPDA-overexpressing liver cells, liver slices, and mice. The functional role and physical interactions of HILPDA were investigated using a variety of biochemical and microscopic techniques, including real-time fluorescence live-cell imaging and Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM). RESULTS: Levels of HILPDA were markedly induced by fatty acids in several hepatoma cell lines. Hepatocyte-specific deficiency of HILPDA in mice modestly but significantly reduced hepatic triglycerides in mice with non-alcoholic steatohepatitis. Similarly, deficiency of HILPDA in mouse liver slices and primary hepatocytes reduced lipid storage and accumulation of fluorescently-labeled fatty acids in lipid droplets, respectively, which was independent of adipose triglyceride lipase. Fluorescence microscopy showed that HILPDA partly colocalizes with lipid droplets and with the endoplasmic reticulum, is especially abundant in perinuclear areas, and mainly associates with newly added fatty acids. Real-time fluorescence live-cell imaging further revealed that HILPDA preferentially localizes to lipid droplets that are being remodeled. Overexpression of HILPDA in liver cells increased the activity of diacylglycerol acyltransferases (DGAT) and DGAT1 protein levels, concurrent with increased lipid storage. Confocal microscopy coupled to FRET-FLIM analysis demonstrated that HILPDA physically interacts with DGAT1 in living liver cells. The stimulatory effect of HILPDA on lipid storage via DGAT1 was corroborated in adipocytes. CONCLUSIONS: Our data indicate that HILPDA physically interacts with DGAT1 and increases DGAT activity. Our findings suggest a novel regulatory mechanism by which fatty acids promote triglyceride synthesis and storage.

Decoration of myocellular lipid droplets with perilipins as a marker for in vivo lipid droplet dynamics: A super-resolution microscopy study in trained athletes and insulin resistant individuals.

In many different cell types neutral lipids can be stored in lipid droplets (LDs). Nowadays, LDs are viewed as dynamic organelles, which store and release fatty acids depending on energy demand (LD dynamics). Proteins like perilipin 2 (PLIN2) and PLIN5 decorate the LD membrane and are determinants of LD lipolysis and fat oxidation, thus affecting LD dynamics. Trained athletes and type 2 diabetes (T2D) patients both have high levels of intramyocellular lipid (IMCL). While IMCL content scales negatively with insulin resistance, athletes are highly insulin sensitive in contrast to T2D patients, the so-called athlete's paradox. Differences in LD dynamics may be an underlying factor explaining the athlete's paradox. We aimed to quantify PLIN2 and PLIN5 content at individual LDs as a reflection of the ability to switch between fatty acid release and storage depending on energy demand. Thus, we developed a novel fluorescent super-resolution microscopy approach and found that PLIN2 protein abundance at the LD surface was higher in T2D patients than in athletes. Localization of adipocyte triglyceride lipase (ATGL) to the LD surface was lower in LDs abundantly decorated with PLIN2. While PLIN5 abundance at the LD surface was similar in athletes and T2D patients, we have observed previously that the number of PLIN5 decorated LDs was higher in athletes, indicating more LDs in close association with mitochondria. Thus, in athletes interaction of LDs with mitochondria was more pronounced and LDs have the protein machinery to be more dynamic, while in T2D patients the LD pool is more inert. This observation contributes to our understanding of the athlete's paradox.

Label-free CARS microscopy reveals similar triacylglycerol acyl chain length and saturation in myocellular lipid droplets of athletes and individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Intramyocellular lipid (IMCL) content associates with development of insulin resistance, albeit not in insulin-sensitive endurance-trained athletes (trained). Qualitative and spatial differences in muscle lipid composition may underlie this so-called athlete's paradox. Here we studied triacylglycerol (TAG) composition of individual myocellular lipid droplets (LDs) in trained individuals and individuals with type 2 diabetes mellitus. METHODS: Trained ([Formula: see text] 71.0 ± 1.6 ml O2 [kg lean body mass (LBM)]-1 min-1), normoglycaemic (fasting glucose 5.1 ± 0.1 mmol/l) individuals and untrained ([Formula: see text] 36.8 ± 1.5 ml O2 [kg LBM]-1 min-1) individuals with type 2 diabetes (fasting glucose 7.4 ± 0.5 mmol/l), with similar IMCL content (3.5 ± 0.7% vs 2.5 ± 0.3%, p = 0.241), but at opposite ends of the insulin sensitivity spectrum (glucose infusion rate 93.8 ± 6.6 vs 25.7 ± 5.3 µmol [kg LBM]-1 min-1 for trained individuals and those with type 2 diabetes, respectively) were included from our database in the present study. We applied in situ label-free broadband coherent anti-Stokes Raman scattering (CARS) microscopy to sections from skeletal muscle biopsies to measure TAG acyl chain length and saturation of myocellular LDs. This approach uniquely permits examination of individual LDs in their native environment, in a fibre-type-specific manner, taking into account LD size and subcellular location. RESULTS: Despite a significant difference in insulin sensitivity, we observed remarkably similar acyl chain length and saturation in trained and type 2 diabetic individuals (chain length: 18.12 ± 0.61 vs 18.36 ± 0.43 number of carbons; saturation: 0.37 ± 0.05 vs 0.38 ± 0.06 number of C=C bonds). Longer acyl chains or higher saturation (lower C=C number) could be detected in subpopulations of LDs, i.e. large LDs (chain length: 18.11 ± 0.48 vs 18.63 ± 0.57 carbon number) and subsarcolemmal LDs (saturation: 0.34 ± 0.02 vs 0.36 ± 0.04 C=C number), which are more abundant in individuals with type 2 diabetes. CONCLUSIONS/INTERPRETATION: In contrast to reports of profound differences in the lipid composition of lipids extracted from skeletal muscle from trained and type 2 diabetic individuals, our in situ, LD-specific approach detected only modest differences in TAG composition in LD subpopulations, which were dependent on LD size and subcellular location. If, and to what extent, these modest differences can impact insulin sensitivity remains to be elucidated. Graphical abstract.

Exercising your fat (metabolism) into shape: a muscle-centred view.

Fatty acids are an important energy source during exercise. Training status and substrate availability are determinants of the relative and absolute contribution of fatty acids and glucose to total energy expenditure. Endurance-trained athletes have a high oxidative capacity, while, in insulin-resistant individuals, fat oxidation is compromised. Fatty acids that are oxidised during exercise originate from the circulation (white adipose tissue lipolysis), as well as from lipolysis of intramyocellular lipid droplets. Moreover, hepatic fat may contribute to fat oxidation during exercise. Nowadays, it is clear that myocellular lipid droplets are dynamic organelles and that number, size, subcellular distribution, lipid droplet coat proteins and mitochondrial tethering of lipid droplets are determinants of fat oxidation during exercise. This review summarises recent insights into exercise-mediated changes in lipid metabolism and insulin sensitivity in relation to lipid droplet characteristics in human liver and muscle. Graphical abstract.

One-leg inactivity induces a reduction in mitochondrial oxidative capacity, intramyocellular lipid accumulation and reduced insulin signalling upon lipid infusion: a human study with unilateral limb suspension.

AIMS/HYPOTHESIS: Physical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown. METHODS: Here we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m2) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control. RESULTS: In vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to 14CO2 was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [14C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg. CONCLUSIONS/INTERPRETATION: Together, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans. TRIAL REGISTRATION: ClinicalTrial.gov NCT01576250. FUNDING: PS was supported by a 'VICI' Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).

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 Pearson's 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.

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 athlete's 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 athlete's paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.

Exercise training reduces intrahepatic lipid content in people with and people without nonalcoholic 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?

KEY POINTS: 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 athlete's 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 athlete's 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 athlete's paradox. ABSTRACT: Intramyocellular lipid (IMCL) hampers insulin sensitivity, albeit not in endurance-trained athletes (Trained). Compared to type 2 diabetes mellitus (T2DM) patients, Trained subjects have high levels of perilipin 5 (PLIN5). In the present study, we tested whether the fraction of PLIN5-coated lipid droplets (LDs) is a determinant of skeletal muscle insulin sensitivity and contributes to the athlete's paradox. Muscle biopsies were taken from eight Trained, Lean sedentary, Obese and T2DM subjects. Trained, Obese and T2DM subjects were matched for total IMCL content. Confocal images were analysed for lipid area fraction, LD size and number and PLIN5+ and PLIN5- LDs were measured. A stepwise linear regression was performed to identify factors explaining observed variance in glucose infusion rate (GIR). Trained and T2DM subjects stored IMCL differently; Trained subjects had a higher number of LDs compared to T2DM subjects (0.037 ± 0.004 µm-2 vs. 0.023 ± 0.003 µm-2 , P = 0.024) that were non-significantly smaller (0.27 ± 0.01 µm2 vs. 0.32 ± 0.02 µm2 , P = 0.197, Trained vs. T2DM). Even though total PLIN5 protein content was almost double in Trained vs. T2DM subjects (1.65 ± 0.21 AU vs. 0.89 ± 0.09 AU, P = 0.004), PLIN5 coating did not affect LD number or size significantly. Of the observed variance in GIR, the largest fraction by far (70.2%) was explained by maximal oxygen uptake. Adding PLIN5 protein content or PLIN5+ LDs increased the explained variance in GIR (74.7% and 80.7% for PLIN5 protein content and PLIN5+ LDs, respectively). Thus, the putative relationship between PLIN5 and insulin sensitivity is at best indirect and is apparent only in conjunction with maximal oxygen uptake. Hence, PLIN5 abundance cannot be causally linked to the athlete's paradox.

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.

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.

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.

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

AIMS/HYPOTHESIS: Dissipating 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). METHODS: Capacity 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. RESULTS: Skeletal 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/INTERPRETATION: Increased 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 REGISTRATION: www.trialregister.nl NTR2002.