Resveratrol-induced remodelling of myocellular lipid stores: A study in metabolically compromised humans.
ABSTRACT: 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'.
Project description:In contrast to insulin-resistant individuals, insulin-sensitive athletes possess high intramyocellular lipid content (IMCL), good mitochondrial function and high perilipin 5 (PLIN5) levels, suggesting a role for PLIN5 in benign IMCL storage. We hypothesised a role for PLIN5 in modulating fasting-mediated insulin resistance.Twelve men were fasted for 60 h, before and after which muscle biopsies were taken and stained for lipid droplets (LDs), PLIN5 and laminin. Confocal microscopy images were analysed for LD size, number, PLIN5 association and subcellular distribution.Fasting elevated IMCL content 2.8-fold and reduced insulin sensitivity (by 55%). Individuals with the most prominent increase in IMCL showed the least reduction in insulin sensitivity (r?=?0.657; p?=?0.028) and mitochondrial function (r?=?0.896; p?=?0.006). During fasting, PLIN5 gene expression or PLIN5 protein content in muscle homogenates was unaffected, microscopy analyses revealed that the fraction of PLIN5 associated with LDs (PLIN5+) increased significantly (+26%) upon fasting, suggesting PLIN5 redistribution. The significant increase in LD number (+23%) and size (+23%) upon fasting was entirely accounted for by PLIN5+ LDs, not by LDs devoid of PLIN5. Also the association between IMCL storage capacity and insulin resistance and mitochondrial dysfunction was only apparent for PLIN5+ LDs.Fasting results in subcellular redistribution of PLIN5 and promotes the capacity to store excess fat in larger and more numerous PLIN5-decorated LDs. This associates with blunting of fasting-induced insulin resistance and mitochondrial dysfunction, suggesting a role for PLIN5 in the modulation of fasting-mediated lipotoxicity.trialregister.nl NTR 2042.
Project description:<h4>Aims/hypothesis</h4>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.<h4>Methods</h4>Trained ([Formula: see text] 71.0?±?1.6 ml O<sub>2</sub> [kg lean body mass (LBM)]<sup>-1</sup> min<sup>-1</sup>), normoglycaemic (fasting glucose 5.1?±?0.1 mmol/l) individuals and untrained ([Formula: see text] 36.8?±?1.5 ml O<sub>2</sub> [kg LBM]<sup>-1</sup> min<sup>-1</sup>) 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 ?mol [kg LBM]<sup>-1</sup> min<sup>-1</sup> 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.<h4>Results</h4>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.<h4>Conclusions/interpretation</h4>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.
Project description:Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identified. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate ?-oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for ?-oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA flux to protect mitochondria against excessive exposure to FA during physiological stress.
Project description:Neutral lipids are packed into dedicated intracellular compartments termed lipid droplets (LDs). LDs are spherical structures delineated by an unusual lipid monolayer and they harbor a specific set of proteins, many of which function in lipid synthesis and lipid turnover. In mammals, LDs are covered by abundant scaffolding proteins, the perilipins (PLIN1-5). LDs in yeast are functionally similar to that of mammalian cells, but they lack the perilipins. We have previously shown that perilipins (PLIN1-3) are properly targeted to LDs when expressed in yeast and that they promote LD formation from the ER membrane enriched in neutral lipids. Here we address the question whether PLIN5 (OXPAT) has a similar function. Both human and murine PLIN5 were properly targeted to yeast LDs, but the protein localized to the cytosol and its steady-state level was reduced when expressed in yeast mutants lacking the capacity to synthesize storage lipids. When expressed in cells containing high levels of neutral lipids within the membrane of the endoplasmatic reticulum, PLIN5 promoted the formation of LDs. Interestingly, PLIN5 was properly targeted to LDs, irrespective of whether these LDs were filled with triacylglycerol or steryl esters, indicating that PLIN5 did not exhibit targeting specificity for a particular subtypes of LDs as was reported for mammalian cells.
Project description:This study investigated the effects of elevated fatty acid (FA) supply from adipose tissue on the ultrastructure of cardiac lipid droplets (LDs) and the expression and organization of LD scaffold proteins perilipin-2 (PLIN2) and perilipin-5 (PLIN5). Stimulation of adipocyte lipolysis by fasting (24 h) or β3-adrenergic receptor activation by CL316, 243 (CL) increased cardiac triacylglycerol (TAG) levels and LD size, whereas CL treatment also increased LD number. LDs were tightly associated with mitochondria, which was maintained during LD expansion. Electron tomography (ET) studies revealed continuity of LD and smooth endoplasmic reticulum (SER), suggesting interconnections among LDs. Under fed ad libitum conditions, the cristae of mitochondria that apposed LD were mostly organized perpendicularly to the tangent of the LD surface. Fasting significantly reduced, whereas CL treatment greatly increased, the perpendicular alignment of mitochondrial cristae. Fasting and CL treatment strongly upregulated PLIN5 protein and PLIN2 to a lesser extent. Immunofluorescence and immuno-electron microscopy demonstrated strong targeting of PLIN5 to the cardiac LD-mitochondrial interface, but not to the mitochondrial matrix. CL treatment augmented PLIN5 targeting to the LD-mitochondrial interface, whereas PLIN2 was not significantly affected. Together, our results support the concept that the interface between LD and cardiac mitochondria represents an organized and dynamic "metabolic synapse" that is highly responsive to FA trafficking.
Project description:Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as at times when cellular energy needs and energy production from mitochondria fatty acid (FA) ?-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific plin5 over-expression (MHC-plin5), a member of the perilipin protein family. Hearts from MHC-plin5 mice expressed at least 4-fold higher levels of plin5 and exhibit a 3.5- fold increase in triglyceride content versus non-transgenic littermate. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of PPAR? target genes, decreased mitochondria function and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative induced genes via NF-E2-related factor 2 anti-oxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and promotes cardiac steatosis without major heart function impairment. Hearts from Four MCH-Plin5 mice and four control mice at the age of 12 weeks were harvested
Project description:Presence of ectopic lipid droplets (LDs) in cardiac muscle is associated to lipotoxicity and tissue dysfunction. However, presence of LDs in heart is also observed in physiological conditions, such as when cellular energy needs and energy production from mitochondria fatty acid ?-oxidation are high (fasting). This suggests that development of tissue lipotoxicity and dysfunction is not simply due to the presence of LDs in cardiac muscle but due at least in part to alterations in LD function. To examine the function of cardiac LDs, we obtained transgenic mice with heart-specific perilipin 5 (Plin5) overexpression (MHC-Plin5), a member of the perilipin protein family. Hearts from MHC-Plin5 mice expressed at least 4-fold higher levels of plin5 and exhibited a 3.5-fold increase in triglyceride content versus nontransgenic littermates. Chronic cardiac excess of LDs was found to result in mild heart dysfunction with decreased expression of peroxisome proliferator-activated receptor (PPAR)? target genes, decreased mitochondria function, and left ventricular concentric hypertrophia. Lack of more severe heart function complications may have been prevented by a strong increased expression of oxidative-induced genes via NF-E2-related factor 2 antioxidative pathway. Perilipin 5 regulates the formation and stabilization of cardiac LDs, and it promotes cardiac steatosis without major heart function impairment.
Project description:The surface of lipid droplets (LDs) in various cell types is coated with perilipin proteins encoded by the Plin genes. Perilipins regulate LD metabolism by selectively recruiting lipases and other proteins to LDs. We have studied the expression of perilipins in mouse muscle. The glycolytic fiber-enriched gastrocnemius muscle expresses predominantly Plin2-4. The oxidative fiber-enriched soleus muscle expresses Plin2-5. Expression of Plin2 and Plin4-5 is elevated in gastrocnemius and soleus muscles from mice fed a high-fat diet. This effect is preserved in peroxisome proliferator-activated receptor (PPAR)?-deficient mice. Mouse muscle derived C2C12 cells differentiated into glycolytic fibers increase transcription of these Plins when exposed to various long chain fatty acids (FAs). To understand how FAs regulate Plin genes, we used specific activators and antagonists against PPARs, Plin promoter reporter assays, chromatin immunoprecipitation, siRNA, and animal models. Our analyses demonstrate that FAs require PPAR? to induce transcription of Plin4 and Plin5. We further identify a functional PPAR binding site in the Plin5 gene and establish Plin5 as a novel direct PPAR? target in muscle. Our study reveals that muscle cells respond to elevated FAs by increasing transcription of several perilipin LD-coating proteins. This induction renders the muscle better equipped to sequester incoming FAs into cytosolic LDs.
Project description:Cytosolic lipid droplets (LDs) are present in most cell types, and consist of a core comprising neutral lipids, mainly triglycerides and sterol esters, surrounded by a monolayer of phospholipids. LDs are heterogeneous in their structure, chemical composition, and tissue distribution. LDs are coated by several proteins, including perilipins and other structural proteins, lipogenic enzymes, lipases and membrane-trafficking proteins. Five proteins of the perilipin (PLIN) family (PLIN1 (perilipin), PLIN2 (adipose differentiation-related protein), PLIN3 (tail-interacting protein of 47kDa), PLIN4 (S3-12), and PLIN5 (myocardial lipid droplet protein)), are associated with LD formation. More recently, the CIDE family of proteins, hypoxia-inducible protein 2 (HIG2), and patanin-like phospholipase domain-containing 3 (PNPLA3) have also gained attention in hepatic LD biology. Evidence suggests that LD proteins are involved in the pathophysiology of fatty liver diseases characterized by excessive lipid accumulation in hepatocytes. This review article will focus on how hepatic LDs and their associated proteins are involved in the pathogenesis of three chronic liver conditions: hepatitis C virus infection, non-alcoholic fatty liver disease, and alcoholic liver disease.
Project description:Aims/hypothesis: While lipid deposition in skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like skeletal muscle. Here we investigated the effects of PLIN5 overexpression M-bM-^@M-^S in comparison with effects of PLIN2 M-bM-^@M-^S on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in skeletal muscle promoted expression of a cluster of genes under control of PPARM-NM-1 and PGC1M-NM-1 involved in FA catabolism and mitochondrial oxidation. Rats received a high fat diet for 3 weeks; 2 weeks after start of the diet intervention Plin5 (OXPAT) or Plin2 (ADRP) were overexpressed in either the right or left tibialis anterior muscle. One week later pooled tibialis anterior muscle samples were analysed on microarrays.