Search

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., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

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., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 μm 2 vs. 0.32 ± 0.02 μm 2 , 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., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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/m 2 , 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 ΔR d (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., Competing Interests: The authors declare no competing financial interests.

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.