Peridroplet mitochondria are associated with the severity of MASLD and the prevention of MASLD by diethyldithiocarbamate.

Mitochondria can contact lipid droplets (LDs) to form peridroplet mitochondria (PDM) which trap fatty acids in LDs by providing ATP for triglyceride synthesis and prevent lipotoxicity. However, the role of PDM in metabolic dysfunction associated steatotic liver disease (MASLD) is not clear. Here, the features of PDM in dietary MASLD models with different severity in mice were explored. Electron microscope photographs show that LDs and mitochondria rarely come into contact with each other in normal liver. In mice fed with high-fat diet, PDM can be observed in the liver as early as the beginning of steatosis in hepatocytes. For the first time, we show that PDM in mouse liver varies with the severity of MASLD. PDM and cytosolic mitochondria were isolated from the liver tissue of MASLD and analyzed by quantitative proteomics. Compared with cytosolic mitochondria, PDM have enhanced mitochondrial respiration and ATP synthesis. Diethyldithiocarbamate (DDC) alleviates choline-deficient, L-amino acid-defined diet-induced MASLD, while increases PDM in the liver. Similarly, DDC promotes the contact of mitochondria-LDs in steatotic C3A cells in vitro. Meanwhile, DDC promotes triglyceride synthesis and improves mitochondrial dysfunction in MASLD. In addition, DDC upregulates perilipin 5 both in vivo and in vitro, which is considered as a key regulator in PDM formation. Knockout of perilipin 5 inhibits the contact of mitochondria-LDs induced by DDC in C3A cells. These results demonstrate that PDM might be associated with the progression of MASLD and the prevention of MASLD by DDC.

Adverse cardiac remodeling augments adipose tissue ß-adrenergic signaling and lipolysis counteracting diet-induced obesity.

Cardiac triacylglycerol accumulation is a common characteristic of obesity and type 2 diabetes and strongly correlates with heart morbidity and mortality. We have previously shown that cardiomyocyte-specific perilipin 5 overexpression (Plin5-Tg) provokes significant cardiac steatosis via lowering cardiac lipolysis and fatty acid (FA) oxidation. In strong contrast to cardiac steatosis and lethal heart dysfunction in adipose triglyceride lipase deficiency, Plin5-Tg mice do not develop heart dysfunction and show a normal life span on chow diet. This finding prompted us to study heart function and energy metabolism in Plin5-Tg mice fed high-fat diet (HFD). Plin5-Tg mice showed adverse cardiac remodeling on HFD with heart function only being compromised in one-year-old mice, likely due to reduced cardiac FA uptake, thereby delaying deleterious cardiac lipotoxicity. Notably, Plin5-Tg mice were less obese and protected from glucose intolerance on HFD. Changes in cardiac energy catabolism in Plin5-Tg mice increased ß-adrenergic signaling, lipolytic, and thermogenic protein expression in adipose tissue ultimately counteracting HFD-induced obesity. Acute cold exposure further augmented ß-adrenergic signaling in Plin5-Tg mice, whereas housing at thermoneutrality did not protect Plin5-Tg mice from HFD-induced obesity albeit blood glucose and insulin levels remained low in transgenic mice. Overall, our data suggest that the limited capacity for myocardial FA oxidation on HFD increases cardiac stress in Plin5-Tg mice, thereby stimulating adipose tissue ß-adrenergic signaling, triacylglycerol catabolism, and thermogenesis. However, long-term HFD-mediated metabolic stress causes contractile dysfunction in Plin5-Tg mice, which emphasizes the importance of a carefully controlled dietary regime in patients with cardiac steatosis and hypertrophy.

Perilipin 5 protects the mitochondrial oxidative functions and improves the alcoholic liver injury in mice.

BACKGROUND AND AIMS: Alcohol consumption is a well-established risk factor for the onset and progression of hepatic steatosis. Perilipin 5 (Plin5), a lipid droplet protein, is an important protective factor against hepatic lipotoxicity induced by excessive lipolysis, but its role and molecular mechanism in alcoholic liver disease (ALD) are not fully elucidated. METHODS: The optimized National Institute on Alcohol Abuse and Alcoholism model was used to construct ALD model mice. Automatic biochemical analyser was used for Biochemical Parameters. The primary hepatocytes and Plin5-overexpressed HepG2 cells (including full-length Plin5 and Plin5 deleting 444-464 aa) were used for in vitro experiment. Haematoxylin and Eosin staining, Oil Red O staining, Bodipy 493/503 staining, Periodic Acid-Schiff staining, immunohistochemistry and JC-1 staining were used to evaluate cell morphology, lipids, glycogen, inflammation and membrane potential. Commercially kits are used to detect glycolipid metabolites, such as triglycerides, glycogen, glucose, reactive oxygen species, lactic acids, ketone bodies. Fluorescently labelled deoxyglucose, NBDG, was used for glucose intake. An XF96 extracellular flux analyser was used to determinate oxygen consumption rate in hepatocytes. The morphological and structural damage of mitochondria was evaluated by electron microscopy. Classical ultracentrifugation is used to separate the subcellular organelles of tissues and cells. Immunoblotting and qPCR were used to detect changes in mRNA and protein levels of related genes. RESULTS: Our results showed that the expression of Plin5 in mouse livers was enhanced by alcohol intake, and Plin5 deficiency aggravated the alcohol-induced liver injury. To clarify the mechanism, we found that Plin5 deficiency significantly elevated the hepatic NADH levels and ketone body production in the alcohol-treated mice. As NADH elevation could promote the reduction of pyruvate into lactate and then inhibit the gluconeogenesis, alcohol-treated Plin5-deficient mice exhibited more lactate production and severer hypoglycemia. These results implied that Plin5 deficiency impaired the mitochondrial oxidative functions in the presence of alcohol. In addition, we demonstrated that Plin5 could be recruited onto mitochondria by alcohol, while Plin5 without mitochondrial targeting sequences lost its mitochondrial protection functions. CONCLUSION: Collectively, this study demonstrated that the mitochondrial Plin5 could protect the alcohol-induced mitochondrial injury, which provides an important new insight on the roles of Plin5 in highly oxidative tissues.

Endocrine modulation of brain-skeleton axis driven by neural stem cell-derived perilipin 5 in the lipid metabolism homeostasis for bone regeneration.

Factors released from the nervous system always play crucial roles in modulating bone metabolism and regeneration. How the brain-driven endocrine axes maintain bone homeostasis, especially under metabolic disorders, remains obscure. Here, we found that neural stem cells (NSCs) residing in the subventricular zone participated in lipid metabolism homeostasis of regenerative bone through exosomal perilipin 5 (PLIN5). Fluorescence-labeled exosomes tracing and histological detection identified that NSC-derived exosomes (NSC-Exo) could travel from the lateral ventricle into bone injury sites. Homocysteine (Hcy) led to osteogenic and angiogenic impairment, whereas the NSC-Exo were confirmed to restore it. Mecobalamin, a clinically used neurotrophic drug, further enhanced the protective effects of NSC-Exo through increased PLIN5 expression. Mechanistically, NSC-derived PLIN5 reversed excessive Hcy-induced lipid metabolic imbalance and aberrant lipid droplet accumulation through lipophagy-dependent intracellular lipolysis. Intracerebroventricular administration of mecobalamin and/or AAV-shPlin5 confirmed the effects of PLIN5-driven endocrine modulations on new bone formation and vascular reconstruction in hyperhomocysteinemic and high-fat diet models. This study uncovered a novel brain-skeleton axis that NSCs in the mammalian brain modulated bone regeneration through PLIN5-driven lipid metabolism modulation, providing evidence for lipid- or bone-targeted medicine development.

Perilipin 5 ameliorates high-glucose-induced podocyte injury via Akt/GSK-3ß/Nrf2-mediated suppression of apoptosis, oxidative stress, and inflammation.

Hyperglycemia-induced podocyte damage contributes to the onset of diabetic nephropathy, a severe complication of diabetes. Perilipin 5 (Plin5) exerts a vital role in numerous pathological conditions via affecting cell apoptosis, oxidative stress, and inflammation. However, whether Plin5 plays a role in regulating podocyte damage of diabetic nephropathy has not been fully determined. This work aimed to explore the role of Plin5 in mediating high glucose (HG)-induced injury of podocytes in vitro. Our results demonstrated that Plin5 expression was markedly decreased in mouse podocytes challenged with HG. Plin5 overexpression markedly suppressed HG-induced apoptosis, reactive oxygen species (ROS) production, and the pro-inflammatory response in podocytes. On the contrary, Plin5 silencing produced the opposite effects. Further mechanistic analysis demonstrated that Plin5 upregulation remarkably increased the levels of phospho-Akt and phospho-glycogen synthase kinase-3ß (GSK-3ß) in HG-exposed podocytes. Moreover, Plin5 overexpression increased the levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and enhanced the activation of Nrf2 signaling. Akt inhibition markedly blocked Plin5-mediated activation of Nrf2, while GSK-3ß inhibition reversed Plin5-silencing-induced suppressive effects on Nrf2 activation. Notably, Nrf2 suppression significantly blocked Plin5-mediated protective effects against HG-induced podocyte injury. In summary, our work indicates a vital role for Plin5 in protecting against HG-induced apoptosis, oxidative stress, and inflammation in podocytes via modulation of Akt/GSK-3ß/Nrf2 signaling. This study suggests that Plin5 may participate in modulating podocyte damage in diabetic nephropathy.

Understanding the Role of Perilipin 5 in Non-Alcoholic Fatty Liver Disease and Its Role in Hepatocellular Carcinoma: A Review of Novel Insights.

Consumption of high-calorie foods, such as diets rich in fats, is an important factor leading to the development of steatohepatitis. Several studies have suggested how lipid accumulation creates a lipotoxic microenvironment for cells, leading cells to deregulate their transcriptional and translational activity. This deregulation induces the development of liver diseases such as non-alcoholic fatty liver disease (NAFLD) and subsequently also the appearance of hepatocellular carcinoma (HCC) which is one of the deadliest types of cancers worldwide. Understanding its pathology and studying new biomarkers with better specificity in predicting disease prognosis can help in the personalized treatment of the disease. In this setting, understanding the link between NAFLD and HCC progression, the differentiation of each stage in between as well as the mechanisms underlying this process, are vital for development of new treatments and in exploring new therapeutic targets. Perilipins are a family of five closely related proteins expressed on the surface of lipid droplets (LD) in several tissues acting in several pathways involved in lipid metabolism. Recent studies have shown that Plin5 depletion acts protectively in the pathogenesis of liver injury underpinning the importance of pathways associated with PLIN5. PLIN5 expression is involved in pro-inflammatory cytokine regulation and mitochondrial damage, as well as endoplasmic reticulum (ER) stress, making it critical target of the NAFLD-HCC studies. The aim of this review is to dissect the recent findings and functions of PLIN5 in lipid metabolism, metabolic disorders, and NAFLD as well as the progression of NAFLD to HCC.

Leptin Reduces Plin5 m6A Methylation through FTO to Regulate Lipolysis in Piglets.

Perilipin5 (Plin5) is a scaffold protein that plays an important role in lipid droplets (LD) formation, but the regulatory effect of leptin on it is unclear. Our study aimed to explore the underlying mechanisms by which leptin reduces the N6-methyladenosine (m6A) methylation of Plin5 through fat mass and obesity associated genes (FTO) and regulates the lipolysis. To this end, 24 Landrace male piglets (7.73 ± 0.38 kg) were randomly sorted into two groups, either a control group (Control, n = 12) or a 1 mg/kg leptin recombinant protein treatment group (Leptin, n = 12). After 4 weeks of treatment, the results showed that leptin treatment group had lower body weight, body fat percentage and blood lipid levels, but the levels of Plin5 mRNA and protein increased significantly in adipose tissue (p < 0.05). Leptin promotes the up-regulation of FTO expression level in vitro, which in turn leads to the decrease of Plin5 M6A methylation (p < 0.05). In in vitro porcine adipocytes, overexpression of FTO aggravated the decrease of M6A methylation and increased the expression of Plin5 protein, while the interference fragment of FTO reversed the decrease of m6A methylation (p < 0.05). Finally, the overexpression in vitro of Plin5 significantly reduces the size of LD, promotes the metabolism of triglycerides and the operation of the mitochondrial respiratory chain, and increases thermogenesis. This study clarified that leptin can regulate Plin5 M6A methylation by promoting FTO to affect the lipid metabolism and energy consumption, providing a theoretical basis for treating diseases related to obesity.

Effects of Simvastatin on Lipid Metabolism in Wild-Type Mice and Mice with Muscle PGC-1α Overexpression.

Previous studies suggest that statins may disturb skeletal muscle lipid metabolism potentially causing lipotoxicity with insulin resistance. We investigated this possibility in wild-type mice (WT) and mice with skeletal muscle PGC-1α overexpression (PGC-1α OE mice). In WT mice, simvastatin had only minor effects on skeletal muscle lipid metabolism but reduced glucose uptake, indicating impaired insulin sensitivity. Muscle PGC-1α overexpression caused lipid droplet accumulation in skeletal muscle with increased expression of the fatty acid transporter CD36, fatty acid binding protein 4, perilipin 5 and CPT1b but without significant impairment of muscle glucose uptake. Simvastatin further increased the lipid droplet accumulation in PGC-1α OE mice and stimulated muscle glucose uptake. In conclusion, the impaired muscle glucose uptake in WT mice treated with simvastatin cannot be explained by lipotoxicity. PGC-1α OE mice are protected from lipotoxicity of fatty acids and triglycerides by increased the expression of FABP4, formation of lipid droplets and increased expression of CPT1b.

Hepatic PLIN5 signals via SIRT1 to promote autophagy and prevent inflammation during fasting.

Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization.

Skeletal muscle lipid droplets are resynthesized before being coated with perilipin proteins following prolonged exercise in elite male triathletes.

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg-1·min-1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (-53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers (P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.

Disruption of Plin5 degradation by CMA causes lipid homeostasis imbalance in NAFLD.

BACKGROUND & AIMS: The pathological hallmark of nonalcoholic fatty liver disease (NAFLD) is an imbalance in hepatic lipid homeostasis, in which lipophagy has been found to play a vital role. However, the underlying molecular mechanisms remain unclear. We investigated the role of chaperone-mediated autophagy (CMA) in the pathogenesis of NAFLD. METHODS: CMA activity was evaluated in liver tissues from NAFLD patients and high-fat diet (HFD)-fed mice. Liver-specific LAMP2A-knockout mice and HepG2 cells lacking LAMP2A [L2A(-) cells] were used to investigate the influence of CMA on lipolysis in hepatocytes. The expression of Plin5, a lipid droplet (LD)-related protein, was also evaluated in human and mouse liver tissues and in [L2A(-)] cells. RESULTS: Here, we found disrupted CMA function in the livers of NAFLD patients and animal models, displaying obvious reduction of LAMP2A and concurrent with decreased levels of CMA-positive regulators. More LDs and higher serum triglycerides accumulated in liver-specific LAMP2A-knockout mice and L2A(-) cells under high-fat challenge. Meanwhile, deleting LAMP2A hindered LD breakdown but not increased LD formation. In addition, the LD-associated protein Plin5 is a CMA substrate, and its degradation through CMA is required for LD breakdown. CONCLUSIONS: We propose that the disruption of CMA-induced Plin5 degradation obstacles LD breakdown, explaining the lipid homeostasis imbalance in NAFLD.

Perilipin 5 alleviates HCV NS5A-induced lipotoxic injuries in liver.

BACKGROUND: The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. METHODS: HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. RESULTS: One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P < 0.05) observed in Plin5-null mice. Moreover, Plin5 deficiency in the liver and hepatocytes aggravated the elevation of MDA and 4-HNE levels induced by NS5A expression (P < 0.01). The triglyceride (TG) content was increased approximately 25% by NS5A expression in the wild-type liver and hepatocytes but was unchanged in the Plin5-null liver and hepatocytes. More importantly, Plin5 deficiency in the liver and hepatocytes exacerbated the elevation of non-esterified fatty acids (NEFAs) stimulated by NS5A expression (P < 0.05 and 0.01 respectively). Using triacsin C to block acyl-CoA biosynthesis, we found that Plin5 deficiency aggravated the NS5A-induced lipolysis of TG. In contrast, Plin5 overexpression in HepG2 cells ameliorated the NS5A-induced lipolysis and lipotoxic injuries. Immunofluorescent staining demonstrated that NS5A expression stimulated the targeting of Plin5 to the surface of the LDs in hepatocytes without altering the protein levels of Plin5. By co-IP, we found that the N-terminal domain (aa 32-128) of Plin5 was pivotal for its binding with NS5A. CONCLUSIONS: Our data highlight a protective role of Plin5 against hepatic lipotoxic injuries induced by HCV NS5A, which is helpful for understanding the steatosis and injuries in liver during HCV infection.

Perilipin 5 promotes hepatic steatosis in dairy cows through increasing lipid synthesis and decreasing very low density lipoprotein assembly.

Fatty liver is a common metabolic disorder in dairy cows during the transition period. Perilipin 5 (PLIN5), a lipid droplet coat protein, plays important roles in the development of hepatic steatosis in mice and humans. Whether PLIN5 plays a role in the development of fatty liver in dairy cows is unknown. An in vivo study consisting of 10 healthy and 10 cows with fatty liver was performed to harvest liver tissue and blood samples. In addition, hepatocytes isolated from calves were infected with PLIN5 overexpression adenovirus for 48 h; treated with 0, 0.6, 1.2, or 2.4 mM nonesterified fatty acids (NEFA) for 24 h; or infected with PLIN5 silencing adenovirus for 48 h and then treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and ß-hydroxybutyrate were greater in cows with fatty liver. Milk production and plasma glucose concentrations were lower in cows with fatty liver. The results revealed that PLIN5 is highly expressed in steatotic liver and localized to lipid droplets. The abundance of fatty acid and triacylglycerol (TAG) synthesis-related proteins including sterol regulatory element binding protein-1c, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 was greater in the liver of cows with fatty liver. In contrast, the abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100, and apolipoprotein E was lower in the liver of cows with fatty liver. Consequently, cows with fatty liver exhibited severe hepatic TAG accumulation and lower blood concentration of very low density lipoprotein apolipoprotein B (VLDL-ApoB). Overexpression of PLIN5 and exogenous NEFA in cultured hepatocytes increased the abundance of sterol regulatory element binding protein-1, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 but decreased the abundance of microsomal triglyceride transfer protein, apolipoprotein B100, and apolipoprotein E, which promoted TAG synthesis and inhibited VLDL-ApoB assembly, inducing lipid accumulation. Importantly, knockdown of PLIN5 attenuated the upregulation of TAG synthesis and downregulation of VLDL-ApoB assembly induced by NEFA. Overall, these data suggest that NEFA activate PLIN5, leading to TAG accumulation and inhibition of VLDL assembly. As such, these mechanisms explain in part the development of hepatic steatosis in dairy cows.

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.

Widespread expression of perilipin 5 in normal human tissues and in diseases is restricted to distinct lipid droplet subpopulations.

Diseases associated with the accumulation of lipid droplets are increasing in western countries. Lipid droplet biogenesis, structure and degradation are regulated by proteins of the perilipin family. Perilipin 5 has been shown to regulate basal lipolysis in oxidative tissues. We examine perilipin 5 in normal human tissues and in diseases using protein biochemical and microscopic techniques. Perilipin 5 was constitutively located at small lipid droplets in skeletal myocytes, cardiomyocytes and brown adipocytes. In addition, perilipin 5 was detected in the epithelia of the gastrointestinal and urogenital tract, especially in hepatocytes, the mitochondria-rich parietal cells of the stomach, tubular kidney cells and ductal cells of the salivary gland and pancreas. Granular cytoplasmic expression, without a lipid droplet-bound localization was detected elsewhere. In cardiomyopathies, in skeletal muscle diseases and during hepatocyte steatogenesis, perilipin 5 was upregulated and localized to larger and more numerous lipid droplets. In steatotic human hepatocytes, perilipin 5 was moderately increased and colocalized with perilipins 1 and 2 but not with perilipin 3 at lipid droplets. In liver diseases implicated in alterations of mitochondria, such as mitochondriopathies, alcoholic liver disease, Wilson's disease and acute liver injury, perilipin 5 was frequently localized to small lipid droplets and less in the cytoplasm. In tumorigenesis, perilipin 5 was especially upregulated in lipo-, leio- and rhabdomyosarcoma and hepatocellular and renal cell carcinoma. In summary, our study provides evidence that perilipin 5 is not restricted to certain cell types but localizes to distinct lipid droplet subpopulations reflecting a possible function in oxidative energy supply in normal tissues and in diseases.

Atorvastatin reduces lipid accumulation in the liver by activating protein kinase A-mediated phosphorylation of perilipin 5.

Statins have been proven to be effective in treating non-alcoholic fatty liver disease (NAFLD). Recently, it was reported that statins decreased the hepatic expression of perilipin 5 (Plin5), a lipid droplet (LD)-associated protein, which plays critical roles in regulating lipid accumulation and lipolysis in liver. However, the function and regulation mechanism of Plin5 have not yet been well-established in NAFLD treatment with statins. In this study, we observed that atorvastatin moderately reduced the expression of Plin5 in livers without changing the protein level of Plin5 in the hepatic LD fraction of mice fed with high-fat diet (HFD). Intriguingly, atorvastatin stimulated the PKA-mediated phosphorylation of Plin5 and reduced the triglyceride (TG) accumulation in hepatocytes with overexpression of wide type (Plin5-WT) compared to serine-155 mutant Plin5 (Plin5-S155A). Moreover, PKA-stimulated FA release of purified LDs carrying Plin5-WT but not Plin5-S155A. Glucagon, a PKA activator, stimulated the phosphorylation of Plin5-WT and inhibited its interaction with CGI-58. The results indicated that atorvastatin promoted lipolysis and reduced TG accumulation in the liver by increasing PKA-mediated phosphorylation of Plin5. This new mechanism of lipid-lowering effects of atorvastatin might provide a new strategy for NAFLD treatment.

Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

During human heart failure, the balance of cardiac energy use switches from predominantly fatty acids (FAs) to glucose. We hypothesized that this substrate shift was the result of mitochondrial degeneration; therefore, we examined mitochondrial oxidation and ultrastructure in the failing human heart by using respirometry, transmission electron microscopy, and gene expression studies of demographically matched donor and failing human heart left ventricular (LV) tissues. Surprisingly, respiratory capacities for failing LV isolated mitochondria (n = 9) were not significantly diminished compared with donor LV isolated mitochondria (n = 7) for glycolysis (pyruvate + malate)- or FA (palmitoylcarnitine)-derived substrates, and mitochondrial densities, assessed via citrate synthase activity, were consistent between groups. Transmission electron microscopy images also showed no ultrastructural remodeling for failing vs. donor mitochondria; however, the fraction of lipid droplets (LDs) in direct contact with a mitochondrion was reduced, and the average distance between an LD and its nearest neighboring mitochondrion was increased. Analysis of FA processing gene expression between donor and failing LVs revealed 0.64-fold reduced transcript levels for the mitochondrial-LD tether, perilipin 5, in the failing myocardium (P = 0.003). Thus, reduced FA use in heart failure may result from improper delivery, potentially via decreased perilipin 5 expression and mitochondrial-LD tethering, and not from intrinsic mitochondrial dysfunction.-Holzem, K. M., Vinnakota, K. C., Ravikumar, V. K., Madden, E. J., Ewald, G. A., Dikranian, K., Beard, D. A., Efimov, I. R. Mitochondrial structure and function are not different between nonfailing donor and end-stage failing human hearts.

Decoration of intramyocellular lipid droplets with PLIN5 modulates fasting-induced insulin resistance and lipotoxicity in humans.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS/INTERPRETATION: 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. TRIAL REGISTRATION: trialregister.nl NTR 2042.

The interplay of protein kinase A and perilipin 5 regulates cardiac lipolysis.

Defective lipolysis in mice lacking adipose triglyceride lipase provokes severe cardiac steatosis and heart dysfunction, markedly shortening life span. Similarly, cardiac muscle (CM)-specific Plin5 overexpression (CM-Plin5) leads to severe triglyceride (TG) accumulation in cardiomyocytes via impairing TG breakdown. Interestingly, cardiac steatosis due to overexpression of Plin5 is compatible with normal heart function and life span indicating a more moderate impact of Plin5 overexpression on cardiac lipolysis and energy metabolism. We hypothesized that cardiac Plin5 overexpression does not constantly impair cardiac lipolysis. In line with this assumption, TG levels decreased in CM of fasted compared with nonfasted CM-Plin5 mice indicating that fasting may lead to a diminished barrier function of Plin5. Recent studies demonstrated that Plin5 is phosphorylated, and activation of adenylyl cyclase leads to phosphorylation of Plin5, suggesting that Plin5 is a substrate for PKA. Furthermore, any significance of Plin5 phosphorylation by PKA in the regulation of TG mobilization from lipid droplets (LDs) is unknown. Here, we show that the lipolytic barrier of Plin5-enriched LDs, either prepared from cardiac tissue of CM-Plin5 mice or Plin5-transfected cells, is abrogated by incubation with PKA. Notably, PKA-induced lipolysis of LDs enriched with Plin5 carrying a single mutation at serine 155 (PlinS155A) of the putative PKA phosphorylation site was substantially impaired revealing a critical role for PKA in Plin5-regulated lipolysis. The strong increase in protein levels of phosphorylated PKA in CM of Plin5 transgenic mice may partially restore fatty acid release from Plin5-enriched LDs, rendering these hearts compatible with normal heart function despite massive steatosis.

Perilipin 5 is dispensable for normal substrate metabolism and in the adaptation of skeletal muscle to exercise training.

Cytoplasmic lipid droplets provide a reservoir for triglyceride storage and are a central hub for fatty acid trafficking in cells. The protein perilipin 5 (PLIN5) is highly expressed in oxidative tissues such as skeletal muscle and regulates lipid metabolism by coordinating the trafficking and the reversible interactions of effector proteins at the lipid droplet. PLIN5 may also regulate mitochondrial function, although this remains unsubstantiated. Hence, the aims of this study were to examine the role of PLIN5 in the regulation of skeletal muscle substrate metabolism during acute exercise and to determine whether PLIN5 is required for the metabolic adaptations and enhancement in exercise tolerance following endurance exercise training. Using muscle-specific Plin5 knockout mice (Plin5(MKO)), we show that PLIN5 is dispensable for normal substrate metabolism during exercise, as reflected by levels of blood metabolites and rates of glycogen and triglyceride depletion that were indistinguishable from control (lox/lox) mice. Plin5(MKO) mice exhibited a functional impairment in their response to endurance exercise training, as reflected by reduced maximal running capacity (20%) and reduced time to fatigue during prolonged submaximal exercise (15%). The reduction in exercise performance was not accompanied by alterations in carbohydrate and fatty acid metabolism during submaximal exercise. Similarly, mitochondrial capacity (mtDNA, respiratory complex proteins, citrate synthase activity) and mitochondrial function (oxygen consumption rate in muscle fiber bundles) were not different between lox/lox and Plin5(MKO) mice. Thus, PLIN5 is dispensable for normal substrate metabolism during exercise and is not required to promote mitochondrial biogenesis or enhance the cellular adaptations to endurance exercise training.