Unraveling the roles of PLIN5: linking cell biology to physiology.

TEM-1007; No. of Pages 9

Review

Unraveling the roles of PLIN5: linking cell biology to physiology Rachael R. Mason and Matthew J. Watt Department of Physiology, Monash University, Clayton, Victoria 3800, Australia

The discovery of perilipin (PLIN) 1 provided a major conceptual shift in the understanding of adipose tissue lipolysis and generated intense interest in lipid droplet biology research. The subsequent discovery of other PLIN proteins revealed unique tissue distribution profiles, subcellular locations, and lipid-binding properties and divergent cellular functions. PLIN5 is highly expressed in oxidative tissues such as skeletal muscle, liver, and heart and is central to lipid homeostasis in these tissues. Studies in cell systems have ascribed several metabolic roles to PLIN5 and demonstrated interactions with other proteins that are requisite for these functions. We examine recent in vivo studies and ask whether the evidence from the cell biology approaches is consistent with the physiological roles of PLIN5. Introduction Lipolysis is a highly conserved process that involves the liberation of fatty acids (FAs) from stored triacylglycerol (TAG). Matching lipolysis to energetic demands is essential for systemic energy balance and also to prevent excessive free FA (FFA) delivery to tissues, which can result in metabolic dysfunctions including hepatosteatosis, cardiomyopathies, and type 2 diabetes (T2D) [1]. Lipolysis occurs at the surface of intracellular organelles known as lipid droplets (Box 1). Lipolysis is controlled by a complex interplay between neurohumoral regulators that invoke intracellular signaling networks and phosphorylation of lipolytic proteins by protein kinase A. This in turn regulates the hydrolase activity of key lipases, the translocation of lipolytic proteins to the surface of the lipid droplets, and protein–protein interactions that facilitate maximal lipolysis (Box 1). Recently, our understanding of the factors controlling adipose tissue lipolysis has undergone two major conceptual shifts: the first was the discovery of perilipin 1 (PLIN1) by the Londos laboratory [2,3] and the second was the concomitant discovery of adipose triglyceride lipase (ATGL) by three separate laboratories in 2004 [4–6]. The early work on PLIN1 (Box 2) led to the discovery of four other proteins that have now been classified as part of the PLIN superfamily (PLIN1–5; for nomenclature Corresponding author: Watt, M.J. ([email protected]). Keywords: lipid metabolism; triglyceride; lipid droplet; animal physiology; perilipin; mitochondrial biogenesis. 1043-2760/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2015.01.005

see Box 3). The grouping of these proteins was based on a 100-amino acid region of high sequence homology near their N termini [7]. Each PLIN protein has unique tissue distribution, subcellular location, and lipid-binding properties, indicative of divergent cellular functions [8,9]. Initial findings showed that PLIN5 is highly expression in oxidative tissues such as the heart, liver, and skeletal muscle [10–12], and suggested an apparent paradoxical role in regulating several components of FA metabolism [10,11]. PLIN5 was shown to interact with other critical modulators of lipid metabolism [13–15] and to modulate metabolic programs [16,17]. Here we describe the multiple metabolic roles of PLIN5, its involvement in adaptation to environmental stressors, and its potential role in health and disease. This review compares the information generated from cell biology approaches with recent in vivo studies conducted in animal models and humans and attempts to place the known PLIN5 functions in a physiological context. Using cell biology approaches to delineate the metabolic roles of PLIN5 Early studies in cells ectopically expressing PLIN5 indicated paradoxical roles of PLIN5 in lipid metabolism, with PLIN5 increasing FA uptake [10], decreasing lipolysis [11], and increasing FA oxidation [10] (Table 1). Supporting possible divergent roles was the observation of PLIN5 localization to the cytosol, lipid droplets, and mitochondria [10–12,18]. Later studies in immortalized and primary cell lines including hepatocytes, skeletal muscle, and cardiomyocytes led to the view that PLIN5 protects against ‘lipotoxic stress’ by regulating lipolysis. PLIN5 expression increases in response to FA exposure [19–21], which is likely to occur via the activation of peroxisome proliferatoractivated receptor (PPAR) a and/or d [19,22]. PLIN5 expression increases under lipid-loaded conditions, which many biologists use to mimic an ‘obesogenic environment’ [19,20,22–24], suggesting a protective role for PLIN5 against lipid-induced stress. This appears to be mediated by PLIN5 preventing uncontrolled lipolysis via interactions with the key lipolytic proteins ATGL [13,14], hormone-sensitive lipase (HSL) [15], and comparative gene identification-58 (CGI-58) [13]. Some studies have shown that PLIN5 independently interacts with ATGL or CGI-58 to promote interaction between ATGL and CGI-58, thereby driving lipolysis [14,25]. However, the general consensus is that the opposite is true: PLIN5 binding to ATGL [11,13] or PLIN5 binding to CGI-58 prevents the interaction between ATGL and CGI-58 and reduces lipolysis [26] (Figure 1). Trends in Endocrinology and Metabolism xx (2015) 1–9

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Trends in Endocrinology and Metabolism xxx xxxx, Vol. xxx, No. x

Box 1. Lipids droplets and lipolysis Lipid droplets Lipid droplets are essential storage organelles found in nearly all cells of the body. Lipid droplets comprise an outer phospholipid monolayer that encases a neutral lipid core comprising mainly sterols, cholesterol esters, and TAG. The phospholipid monolayer is studded with proteins that regulate lipid metabolism, signaling, cytoskeletal proteins, and chaperone proteins [67]. Lipid droplets serve to protect the cell by sequestering lipids into a protective environment and preventing excessive accumulation of lipid intermediates such as ceramide, diglyceride, and sphingolipids, which cause ‘lipotoxic’ outcomes such as endoplasmic reticulum (ER) stress, activation of proinflammatory signaling, insulin resistance, and apoptotic cell death. Cellular control of lipolysis Lipolysis occurs at the surface of cytoplasmic lipid droplets and involves the hydrolysis of triglycerides by specific lipases, resulting in the liberation of three FAs and one glycerol. The FAs enter the circulation where they are taken up by other tissues and used to fuel energy production. The three major lipases controlling lipolysis are ATGL, HSL, and monoacylglycerol lipase (MAGL). The lipases have specific substrate specificities and work in sequence to fully degrade triglycerides. Lipolysis is increased following the activation of badrenoceptors by catecholamines, which activates PKA, which in turn phosphorylates the lipases ATGL and HSL, and the lipiddroplet-associated protein PLIN1. These phosphorylation events promote lipolysis by increasing lipase recruitment to the lipid droplet and the interaction of lipases with coactivator proteins [68].

Importantly, many cell-based studies examining PLIN5 function were undertaken in cell systems with no or very little endogenous PLIN5 mRNA or protein, such as CHO [11,18], Sol8 [19], C2C12 [19], 3T3-L1 [10,12,27], L6 myoblast [12], and HEK [28] cells. In addition, some cell systems lack other lipid metabolism proteins (for example, CHO cells lack HSL [15]) that may be necessary

to faithfully replicate the protein–protein interactions required for in vivo lipolysis and other metabolic functions. PLIN5 and mitochondrial function Emerging evidence from cell-based studies suggests an intimate association between PLIN5, the regulation of intracellular lipid fluxes, and mitochondrial function/biogenesis. Evidently, these observations are relevant for the understanding of cellular adaptations to stress such as exercise training and for potential therapeutic agents, because mitochondrial dysfunction in skeletal muscle is proposed to contribute to reduced FA oxidation, lipid deposition, and the development of insulin resistance in obese individuals [29]. Pioneering work by Sztalryd and colleagues indicates that PLIN5 provides physical and metabolic linkage of lipid droplets and mitochondria [30]. Using reconstituted cell culture systems, the authors made two important observations to support this model: first, they provided evidence that PLIN5 recruits mitochondria to the lipid droplet surface; and second, PLIN5 inhibits TAG hydrolysis and channels FAs into lipid droplets under basal conditions, but increases TAG hydrolysis and directs FAs to mitochondrial oxidation under protein kinase A (PKA)-stimulated conditions (Figure 1). Justifiably, the conclusions drawn from this work are that PLIN5 matches FA demand to cellular needs, protecting mitochondria from excessive FA flux in states of low energetic demand and promoting FA mobilization and oxidation in states of high energy demand [30] (reviewed in [9]). A secondary hypothesis suggests that PLIN5, by regulating lipolysis, may enhance FA oxidative capacity by enhancing mitochondrial biogenesis, most likely via PPARa/d-mediated transcription [9,31–33].

Table 1. Characteristics and functions of PLIN5 Chromosome (human) Protein size (kDa) Cellular location Tissue distribution Known functions Binding partners

Transgenic mice

Phenotype of transgenic mice

Loss/gain of function intervention

2

19 54 Cytosol, ER, mitochondria, lipid droplets Ubiquitous: primarily oxidative tissues, skeletal muscle, cardiac muscle, and liver Increases FA uptake, decreases lipolysis, increases FA oxidation ATGL HSL CGI-58 Plin5/: gene disruption exon 2–9 Plin/: gene disruption exon 2, 3, 4 Plin5/: gene disruption exon 5, 6, 7 (insertion of an expression cassette in the targeted locus) Cardiac-specific overexpression: Plin5 cDNA under the control of the cardiomyocytespecific a-myosin heavy chain promoter Plin5/: decreased lipid droplets in the heart of fasted mice, increased cardiac boxidation, increased reactive oxygen species (ROS) production, cardiac dysfunction Plin5/: improved glucose tolerance, hepatic insulin sensitivity yet skeletal muscle insulin resistance, ceramide and sphingolipid accumulation in muscle Plin5/: hepatic injury, inflammation Cardiac-specific overexpression: cardiac steatosis, cardiac hypertrophy, and cardiac dysfunction Overexpression by electroporation of Plin5 cDNA: increased IMTG content, increased Plin5 localization on mitochondria Overexpression with adeno-associated virus harboring Plin5 cDNA in islets: stores FAs in TAG during fasting, releases FAs during feeding to augment glucose-stimulated insulin secretion

Refs [11] [10–12] [10–12] [10–12] [10–12,35] [13,14] [15] [13] [26] [34] [35] [37,38] [34] [35] [26] [37,38] [17,28,44]


Review


Box 2. PLIN1, lipid droplets, and lipolysis Early studies identified PLIN1 as a major regulator of adipocyte lipolysis. As PLIN1 is constitutively bound to lipid droplets, it was proposed to act as a scaffolding protein facilitating lipase access to the lipid droplet. Originally identified as a major PKA substrate in adipocytes, PLIN1 is now known to have multiple PKA consensus sites – five identified in humans and six in rodents [69] – and phosphorylation plays a major role in its regulation [70]. In the absence of PLIN1 phosphorylation by PKA, lipolysis is maintained at low rates. This is mediated by PLIN1 associating with CGI-58, which prevents the capacity for CGI-58 to bind to ATGL and facilitate ATGLmediated triglyceride hydrolase activity. PKA-mediated phosphorylation of PLIN1 triggers several events that are essential for complete lipolysis: PLIN1 disassociates from CGI-58 to allow CGI58 to bind and activate ATGL and facilitates HSL translocation to the lipid droplet [71]. The generation of Plin1 null mice confirmed the importance of PLIN1 for adipocyte lipolysis and whole-body energy homeostasis. Plin1 null mice have a dramatic reduction (70%) in adipose tissue mass that was attributed to increased basal adipocyte lipolysis [72,73]. When they were challenged with physiological and pharmacological procedures that stimulate lipolysis, such as prolonged fasting, b-adrenoceptor agonist administration, and cold exposure, they had a blunted lipolytic response [73]. Plin1 null mice had higher resting energy expenditure due to an increase in thermogenesis associated with high basal lipolysis [72,73] and were resistant to diet-induced obesity [73]. However, the mice become glucose intolerant and insulin resistant [72], most likely due to an increase in hepatic glucose production (i.e., through gluconeogenesis) and a decrease in glucose disposal rate due to insulin resistance in adipose tissue [74]. Hence, PLIN1 is required to translate energy demand signals to the lipid droplet to regulate FA supply and metabolic homeostasis [36].

β-adrenergic smulaon

Basal FA

TAG FA

ATGL PLIN5 PLIN5 P P

PLIN5 CGI-58

PLIN5 P

CGI-58 HSL

ATGL P CGI-58

FA

PLIN5 FA

TAG PPAR

OxPhos genes

PLIN5

β-oxidaon

Key: FA uptake FA oxidaon P

Phosphate

Elucidating the role of PLIN5 in vivo using transgenic models The generation and phenotypic characterization of wholebody Plin5 null (Plin5/) mice by three independent laboratories has shed light on PLIN5 function in heart [34], skeletal muscle, [35] and liver [26] (Table 1). The mouse lines were generated by standard recombination techniques resulting in complete PLIN5 mRNA and protein ablation. A potential limitation of the Plin5 deletion model is disruption to Plin3 and Plin4, which are located in close proximity to Plin5 in the genome [36]. However, PLIN3 and PLIN4 protein content were not affected by PLIN5 deletion [26,35]. Further studies in mice harboring cardiac-specific overexpression of PLIN5 (CM-PLIN5) using the a-myosin heavy chain promoter [37,38] has shed light on PLIN5’s function in the heart. PLIN5 and whole-body metabolism Whole-body Plin5/ mice have normal growth rates, organ weights, and lean and fat masses compared with their wild type littermates when fed either standard chow or high-fat diets (HFDs) [26,34,35]. PLIN5 ablation did not impact food intake [34,35] and had no effect on [35] or modestly increased energy expenditure that did not impact systemic energy balance [34]. Whole-body carbohydrate oxidation was either unchanged [34] or increased in Plin5/ mice fed a standard chow diet, but this effect was not observed in HFD-fed mice [35]. The increased carbohydrate oxidation and decreased FA oxidation is at odds with the consensus that PLIN5 restricts lipolysis and increases FA oxidation in cells [10,26,34]; hence, in Plin5/ mice the expectation would be for increased FA availability and thereby increased FA oxidation. Metabolic substrates including blood glucose, FAs, TAG, and ketone bodies are similar between Plin5/ mice and their wild type littermates [26,34,35], indicating no major metabolic perturbations. PLIN5 is most important during prolonged nutrient deprivation. Overnight fasting of whole-body Plin5/ mice results in complete depletion of lipid droplets in cardiac muscle [34] and a reduction in the size and number of lipid droplets in skeletal muscle [35], indicating marked intramyocellular lipolysis. Hence, PLIN5 may protect myocytes from depletion of intramyocellular fuels and/or control intracellular FA flux during fasting to prevent lipotoxic stress [34,35]. This fasting-mediated depletion of lipid stores does not occur in the liver [34,35], although later studies indicate that PLIN5 ablation may cause lipotoxic liver injury, especially in mice fed a HFD [26] (see below).

TRENDS in Endocrinology & Metabolism

Figure 1. Metabolic functions of perilipin 5 (PLIN5): evidence from cell-based studies. This schematic represents the key cellular functions of PLIN5 and is based on studies conducted in various cell types. Under basal conditions (left) PLIN5 appears to be integral to fatty acid (FA) metabolism in two ways. First, PLIN5 independently binds to adipose triglyceride lipase (ATGL) and its coactivator comparative gene identification-58 (CGI-58) to prevent their interaction and thereby reduce lipolysis. Second, PLIN5 facilitates sequestering of FA into lipid droplets to prevent excessive FA oxidation. Under b-adrenoceptor-stimulated conditions (right) and/or when energy demand is increased, PLIN5 facilitates increased lipase interaction on the lipid droplet surface while also directing FAs to the mitochondria for b-oxidation. The released FAs can also activate a peroxisome proliferator-activated receptor (PPAR) a or d transcription program (OxPhos genes) that increases the oxidative capacity of cells. In addition, PLIN5 may be required for maintaining close lipid droplet–mitochondrial contact (denoted by blue arrow).

PLIN5 and cardiac metabolism and function Kuramoto et al. [34] showed that whole-body Plin5 deletion increases ATGL lipase activity and cardiomyocyte lipolysis, which was associated with increased FA oxidation and oxidative stress. Heart function declined in aging Plin5/ mice and alleviating oxidative stress via N-acetylcysteine administration prevented these functional declines. Thus, PLIN5 is important for heart function by preventing excessive lipolysis, FA oxidation, and oxidative stress (Figure 2). In support of the major conclusions from the Plin5/ mice, CM-PLIN5 caused a reduction in 3


Review


Box 3. PLIN nomenclature The designation of protein names for PLIN1–5 has created a concise standard for the field [75]. Before the new nomenclature, many names were given to the same protein: PLIN1 was previously perilipin, PERI, or PLIN; PLIN2 was previously adipocyte differentiation-related protein (ADRP), ADFP, or adipophilin [76,77]; PLIN3 was called tail-interacting protein 47 (TIP47), placental protein 17b (PP17), or mannose 6-phosphate receptor-binding protein 1 (M6PRBP1) [78,79]; PLIN4 was previously S3-12 [80]; and PLIN5 was known as OXPAT, myocardial lipid droplet protein (MLDP), or lipid storage droplet protein 5 (LSPD5) [10–12].

ATGL- and HSL-mediated lipolysis that was coupled to decreased FA oxidation [37]. CM-PLIN5 overexpressor mice have cardiac steatosis, increased heart weight, left ventricular hypertrophy, and mild cardiac dysfunction [37,38]. These metabolic effects in CM-PLIN5 mice are reminiscent of the ATGL null mouse [39], albeit not as dramatic, and highlight the importance of regulating lipid flux from lipid droplets in preventing cardiomyopathies. Taken together, the PLIN5 null and overexpression models demonstrate that PLIN5 is required for normal cardiac metabolism and function, but too much PLIN5 may contribute to cardiomyopathy. Human studies are needed to address this issue. PLIN5 and skeletal muscle metabolism The role of PLIN5 in skeletal muscle was recently investigated. It was shown that, while Plin5 ablation depleted muscle TAG (most prominently in red oxidative muscle), the increased lipolytic flux was not precisely matched by increased oxidation of TAG-derived FAs, resulting in the accumulation of ceramide and sphingomyelin [35]. The

inability to oxidize all TAG-derived FAs suggests that PLIN5 might be important in directing FAs to the mitochondria for oxidation in skeletal muscle, which is not the case in the heart where the increased lipolysis is accompanied by increased FA oxidation and oxidative stress in Plin5/ mice [34]. Nevertheless, the increase in myocellular sphingolipids was associated with the development of skeletal muscle insulin resistance. Surprisingly, the livers of Plin5/ mice remained insulin sensitive [35], and clearance of a glucose load (glucose tolerance) by Plin5/ mice was more efficient than in wild type littermates when fed either low-fat diets or HFDs. Collectively, these studies showed that PLIN5 is required to match the lipolysis of intramyocellular TAG to metabolic demands, which helps to maintain insulin sensitivity in skeletal muscle (Figure 2). While ablating PLIN5 increases intramyocellular lipolysis [35], the importance of PLIN5 in regulating substrate availability during contraction/exercise is unclear. Studies using coimmunoprecipitation techniques have reported PLIN5 associations with ATGL and CGI-58 in resting skeletal muscle that are unchanged following acute electrically induced contractions in rats [40] or after moderateintensity exercise in humans [41]. One caveat to these studies is that the interaction of lipolytic proteins occurs on lipid droplets, is transient, and may not survive immunoprecipitation or preparation for immunohistochemical analysis. Thus, while PLIN5 is unequivocally important in limiting lipolysis by binding to ATGL and CGI-58 [34,35], the lack of change in protein–protein associations during increased metabolic demand questions PLIN5’s importance in a normal physiological context.

Plin5 –/–

TAG lipolysis Fay acid ATGL acvity Ceramide Fay acid Fay acid oxidaon Insulin resistance Oxidave stress Key:

TAG

Fay acid

Cardiac dysfuncon

Lipid droplet Mitochondria Glucose Insulin

ER stress

Liver damage Inﬂammatory cells

ALT

Inhibitory acon TRENDS in Endocrinology & Metabolism

Figure 2. Tissue-specific roles of perilipin 5 (PLIN5) in vivo. This schematic is a simplified representation of the key effects of PLIN5 deletion in metabolic tissues. From left to right, deletion of PLIN5 in muscle increases triacylglycerol (TAG) lipolysis, which in turn increases the amount of fatty acids (FAs) in the tissue. The accumulation of FAs results in accumulation of sphingolipids, especially ceramide, and causes insulin resistance. In the liver, the increased lipolysis results in free FA (FFA) accumulation, which is associated with increased endoplasmic reticulum stress, increased inflammatory cell numbers, and liver damage. In the heart, increased adipose triglyceride lipase (ATGL) activity increases lipolysis and mitochondrial FA oxidation. This causes oxidative stress and results in cardiac dysfunction.

4


Review PLIN5 and liver metabolism Wang et al. focused their analysis of Plin5/ mice on lipid metabolism in the liver [26]. In their colony, Plin5/ mice stored less TAG and more FFAs in the liver compared with wild type littermates, although similar findings were not reported in other Plin5/ mice of similar age and sex [34,35]. Nevertheless, the Plin5/ mice of Wang et al. [26] displayed morphological and biochemical changes consistent with liver damage, including abnormal lobular architecture, increased binucleate cells, intrahepatic cholestasis, and increased alanine aminotransferase. The livers were also characterized by increased inflammatory cell numbers, endoplasmic reticulum stress, and lipid peroxidation [26] (Figure 2). While these data support the conclusion that PLIN5 is required to prevent FFA accumulation, lipotoxicity, and liver damage, similar work in wholebody Plin5/ mice generated by others showed no evidence of steatosis or liver damage [35], necessitating further work to conclusively delineate the roles of PLIN5 in the liver in vivo. A direct role of PLIN5 in modulating very-low-density lipoprotein (VLDL) assembly and secretion is also unclear. Preformed triglyceride pools are used for VLDL assembly via a process involving lipolysis/re-esterification and is likely to involve TAG hydrolase [42] rather than the PLIN5interacting lipase ATGL or HSL [43]. Hence, while VLDL secretion is decreased in cultured hepatocytes derived from Plin5/ mice [26], the circulating VLDL-TAG does not differ between Plin5/ and wild type mice [26,34,35], suggesting no role for PLIN5 in VLDL-TAG secretion and/or enhanced TAG clearance in Plin5/ mice in vivo. PLIN5 and pancreatic b cell function PLIN5 was recently shown to regulate postprandial insulin secretion [44]. Using adeno- and adeno-associated virus to overexpress PLIN5 in cultured b cells and islets of mice, respectively, the authors showed that PLIN5 overexpression promotes TAG storage in fasting mice and increases lipolysis when cAMP levels are increased during refeeding. Mobilizing FA under these conditions augments glucosestimulated insulin secretion (GSIS), suggesting that PLIN5 traps FAs in b cells during fasting, which provides FA substrate to drive GSIS during feeding. Furthermore, the enhanced intracellular lipolytic signaling generated by PLIN5 amplifies G protein-coupled receptor 40 (GPR40) signaling, which contributes up to 50% of the FA-mediated augmentation of GSIS [45]. The mechanisms underpinning this complex regulation are unresolved but, unexpectedly, may involve transport of FA from b cells to activate cellsurface GPR40. These studies again highlight how PLIN5 function varies with nutritional state and future studies should examine whether impaired PLIN5 function contributes to islet dysfunction in T2D. PLIN5 and brown adipose tissue (BAT) Early studies reported marked Plin5 expression in BAT of mice [10], BAT being a highly oxidative tissue essential for uncoupled respiration. Given that FAs derived from intracellular TAG are essential for efficient uncoupled respiration and thermogenesis, studies examining PLIN5 in BAT function are warranted. However, arguing against an important role of PLIN5 in BAT thermogenesis is the


finding that Plin5/ and wild type littermates have similar tolerance to the cold [34]. More sensitive approaches using direct examination of BAT thermogenesis with PLIN5 modulation are required. PLIN5 and mitochondrial function in vivo Prominent PLIN5 localization is shown at the mitochondria in skeletal muscle of humans [41] and rats [28,46] and mitochondrial PLIN5 localization is increased after electrically induced contraction in rat skeletal muscle, concomitant with reduced intramyocellular TAG content [47]. This observation favors the interpretation that PLIN5 facilitates TAG-derived FA transfer from lipid droplets to mitochondria during muscle contraction. Notably, PLIN5 localization to the mitochondria was not increased after 5 or 60 min of moderate-intensity exercise in humans [41], suggesting that the intensity of exercise and thereby degree of metabolic stress may dictate PLIN5 cellular localization. Understanding the intracellular signals mediating PLIN5 mitochondrial localization will be important in understanding the coordination of PLIN5 function during contraction/exercise. There is some support for the premise that PLIN5 regulates mitochondrial function chronically. PLIN5 protein content correlates with mitochondrial function in Zucker diabetic fatty (ZDF) rats [28] and the protein content of the electron transport chain in healthy humans [28]. Koves et al. [16] provided further support by demonstrating close associations between PLIN5 expression, increased TAG-FA flux, and mitochondrial biogenesis in cell models, mice that underwent acute exercise, and endurance trained compared with untrained humans. Despite the morphological evidence that PLIN5 localizes to the mitochondria, and the in vitro evidence that PLIN5 promotes the close proximity of lipid droplets with the mitochondria [30], there is not yet a defined role for PLIN5 in the mitochondria. The few studies in murine models of PLIN5 ablation or overexpression do not provide conclusive support for PLIN5 in facilitating lipid droplet–mitochondrial interactions or modulating mitochondrial function. For example, recent studies show no differences in resting or stimulated mitochondrial oxygen consumption, expression of genes related to FA oxidation and oxidative phosphorylation, cellular mitochondrial content, maximal activities of mitochondrial enzymes, or mitochondrial-to-lipid-droplet contact in the skeletal muscle of Plin5/ and wild type littermates [35]. Moreover, the maximal running capacity of Plin5/ mice is comparable to that of wild type mice, indicating no functional decline in mitochondrial oxygen uptake or utilization. Hence, PLIN5 is not required to sustain normal mitochondrial function in muscle in vivo, where other contraction-responsive molecular signals are probably more important for mitochondrial adaptation [48]. By contrast, mitochondrial mass was increased in the livers of Plin5/ mice [26], suggesting potential tissue-specific roles of PLIN5 based on differences in the magnitude and patterning of energy stress. Even less clear is whether increasing PLIN5 impacts mitochondrial function. Mild PLIN5 overexpression by in vivo electroporation of cDNA into glycolytic rat muscle increased oxidative gene expression but did not increase mitochondrial respiratory chain protein content or, critically, alter FA oxidation measured in either isolated 5


Review


Table 2. Summary of studies examining PLIN5 responses to physiological and pathological stressors in rodents and humans (A) Studies of PLIN5 responses in rodents: role of high-fat feeding and genetic obesity Intervention 45% fat HFD 35.5% fat 42% kcal fat 38% energy from fat 61% energy from fat Transgenic mice

Species Mouse Mouse Mouse Human Mouse ob/ob Fatty liver dystrophic ob/ob ZDF rats (fa/fa)

Major finding " Protein (86%) " mRNA gene (290–450%) and " protein (% unreported) " mRNA (400%) and " protein (% unreported) = mRNA " mRNA (300%) and " protein (322%) = Versus wild type mice = mRNA and " protein (% unreported) " mRNA (400%) and " protein (% unreported) " Protein in fa/fa versus fa/+ (200%)

Refs [59] [19] [60] [55] [51] [60] [21] [62]

(B) PLIN5 responses to exercise and metabolic diseases in rodents and humans Intervention/cohort Ex vivo Exercise Acute Training

Athletes

Obese

Diabetic

Electrical stimulation 80 min (5 m/min to 15 m/min) 60 min 60% VO2max Strength: 11 weeks, 3/7 days; endurance: 10 weeks, 3/7 days Strength: 6 weeks, 3/7 days 8 weeks, 5/7 days; BMIa = 32.3 0.7 kg/m 2 12 weeks, 3/7 days; lean: BMI < 24.9 kg/m2, obese: BMI > 30 kg/m 2 Interval training: 6 weeks, 3/7 days; endurance: 6 weeks, 5/7 days 19 weeks; running wheel in cage Athlete: VO2peak = 53.8 2.0 ml/kg/min Sedentary: VO2peak = 41.5 2.8 ml/kg/min Trained: VO2max > 55 ml/kg/min Untrained: VO2max < 45 ml/kg/min + sedentary Lean: BMI = 22.5 0.5 kg/m2; obese: BMI = 30.9 0.9 kg/m 2 Lean: BMI = 24.5 0.9 kg/m2; obese: BMI = 23.9 0.5 kg/m 2 Lean: BMI < 24.9 kg/m2; obese I: BMI 30–34.9 kg/ m2; obese II: BMI > 35 kg/m 2 Lean: BMI < 24.9 kg/m2; obese: BMI > 30 kg/m 2 Lean: fasting glucose = 5.7 0.2 mmol/l; T2D: fasting glucose = 8.9 0.6 mmol/l Controls: fasting glucose = 5.9 0.5 mmol/l; T2D: fasting glucose = 8.5 2.1 mmol/l

Species Rat Mouse Human Human

Major change in PLIN5 = Versus not contracted " mRNA (200%) versus pre-exercise = Protein versus pre-exercise = mRNA with training

Refs [46] [16] [41] [55]

Human Human Human

= Protein versus pre-training = mRNA, " protein (26%) versus pre-training = mRNA, " protein (20–85%) versus pretraining " Protein (200%) versus pre-training

[56] [52] [54]

" mRNA (50%), = protein versus pretraining " Protein in athletes versus sedentary (700%)

[51]

[16]

Human

" mRNA in trained compared with untrained (200–300%) = mRNA lean versus obese

Human

= Normal weight versus obese

[57]

Human

" Protein in obese with BMI = 30–34 kg/m2 versus lean (50%) = Protein lean versus obese = mRNA T2D versus lean

[63]

= Protein T2D versus controls # Protein in T2D with rosiglitazone (30%)

[62]

Human Mouse Human Human

Human Human Human

[53]

[57]

[61]

[54] [61]

a

Body mass index.

mitochondria or muscle homogenates [17,28]. Examination of FA metabolism in intact skeletal muscle overexpressing PLIN5 is required to definitively address this important issue. In contrast to the aforementioned studies in skeletal muscle, overexpression of PLIN5 in cardiac muscle reduces intracellular lipolysis and is associated with reduced mRNA content of oxidative phosphorylation proteins and mildly impaired mitochondrial respiration [37,38,49]. Both CM-PLIN5 mice strains exhibited tight clustering of mitochondria around lipid droplets [37,38], with increased mitochondrial size, increased abnormalities in the mitochondria, and intramitochondrial vacuoles in the heart [38]. Thus, it is clear that PLIN5 is localized to the mitochondria and appears to move to the mitochondria during energetic stress, such as during intense muscle contraction. While the in vitro data showing that PLIN5 promotes lipid droplet–mitochondrial interactions is teleologically appealing [30], the evidence that PLIN5 is required to modulate lipid droplet–mitochondrial FA shuttling or that PLIN5 6

promotes mitochondrial biogenesis in vivo is inconclusive. Deciphering the role of PLIN5 in mitochondria remains a major question in the field. Regulation of PLIN5 by phosphorylation There is accumulating evidence that PLIN5 is a PKA substrate, predicting involvement in processes associated with b-adrenergic activation. Recombinant PLIN5 protein is phosphorylated by the catalytic subunit of PKA in vitro [35] and [32P]orthophosphate labeling of PLIN5 is increased in cells on PKA activation [13]. Recent work by MacPherson and colleagues used a pan-serine antibody to demonstrate serine phosphorylation of PLIN5 in resting isolated skeletal muscle; surprisingly, no change in PLIN5 serine phosphorylation occurred after muscle contractions that decreased intramyocellular TAG content [50]. While PLIN5 phosphorylation was demonstrated using this approach, conclusions regarding PLIN5 phosphorylation/ function relationships are limited because neither the specific phosphorylation sites nor the upstream kinases


Review were identified. In this regard, a functional role for the putative PKA site PLIN5 Ser155 was demonstrated in an in vitro system [49]. Lipid droplets prepared from COS-7 cells overexpressing PLIN5 S155A (rendering S155 phosphorylation defective) exhibited impaired PKA-stimulated lipolysis compared with lipid droplets expressing native PLIN5. The authors suggested that PKA regulation facilitates the release of CGI-58 from PLIN5, making it available to coactivate ATGL and activate lipolysis. Such regulation would be akin to PKA regulation of PLIN1mediated lipolysis and is consistent with PLIN5’s ability to increase lipolysis with PKA stimulation [49]. Thus, while PLIN5 can be phosphorylated by PKA and PLIN5 Ser155 appears to be a functionally relevant site for lipolysis regulation, the direct demonstration of specific PKA phosphorylation sites and the in vivo biological relevance of these sites remains unresolved and is an important line of future inquiry. Function of PLIN5 in health and disease The studies examining PLIN5 in health and diseases are summarized in Table 2. PLIN5 expression is rapidly increased after a single exercise bout in mice and remains elevated for at least 24 h [16]. Prolonged voluntary wheel running increased skeletal muscle PLIN5 mRNA and protein content by approximately threefold compared with sedentary controls [51]. Chronic exercise training in humans increases skeletal muscle PLIN5 protein content [52–54] but, interestingly, has no effect on Plin5 gene expression [52,55,56]. This apparent mismatch is consistent with the observation that post-translational stability by FAs and/or micro-lipid droplet formation is important in maintaining cellular PLIN5 protein [18]. Cross-sectional studies have reported increased PLIN5 content in the muscles of endurance-trained athletes compared with sedentary controls [16,57], supporting the likelihood that PLIN5 is an important regulator of muscle TAG levels. Clearly, PLIN5 is exercise responsive and given that PLIN5 expression increases coordinately with other molecular effectors of TAG metabolism, it may be a component Box 4. Outstanding questions Demonstrating changes in PLIN5 interactions with other lipolytic regulators during PKA stimulation and metabolic challenges in vivo and determining whether these associations are cell autonomous. Verification that PLIN5 can promote mitochondria–lipid droplet associations in vitro and the demonstration of this function in vivo. Determining whether, and how, PLIN5 promotes mitochondrial biogenesis. Understanding the intracellular signals mediating PLIN5 mitochondrial localization to decipher the coordination of PLIN5 function during contraction/exercise and other situations of increased energy demand. Determining whether PLIN5 regulates VLDL-TAG secretion from the liver. Delineating the role of PLIN5 in brown adipose tissue and its possible role in adaptive thermogenesis. Identification of PLIN5 phosphorylation sites and regulatory protein kinases (besides PKA) and understanding their relevance for PLIN5 function. Confirming PLIN5’s involvement (or not) in pathophysiological states in humans (e.g., its role in obesity, T2D, and hepatosteatosis).


of an exercise-induced transcription program that contributes to the so-called ‘athletes’ paradox’ (i.e., the maintenance of insulin sensitivity despite high cellular lipid levels) [58]. The role of PLIN5 in metabolic disease remains perplexing, owing to the lack of concordance between studies using similar experimental designs and interspecies differences. Plin5 gene expression and protein content is increased in the muscle and liver of mice fed a HFD [19,26,51,59,60]. By contrast, there is no change in skeletal muscle Plin5 mRNA expression in humans fed a HFD for 12 weeks [55], and most studies report normal PLIN5 protein content in obese humans [54,61–63]. Consistent with these observations, skeletal muscle PLIN5 expression/content does not differ between T2D patients and lean normoglycemic individuals [61,62] (Table 2). Thus, on the balance of evidence, PLIN5 expression is increased in the skeletal muscle of rodents fed a HFD but does not associate with metabolic disease in humans. Currently there is no evidence of SNPs in the PLIN5 gene conferring disease in the human population, unlike the incidence of PLIN1 SNPs [64–66]. We are only beginning to delineate PLIN5 responses to environmental/ physiological situations and further studies will provide a clearer picture of PLIN5’s functions in physiological and pathophysiological states in vivo. Concluding remarks and future perspectives Studies using cellular and molecular approaches show that PLIN5 interacts with ATGL and CGI-58 to suppress lipolysis and that PLIN5 regulates intracellular FA fluxes and may drive the reprogramming of cells to a more ‘oxidative phenotype’ capable of dealing with high lipid loads. The recent generation of several transgenic mice has enabled researchers to examine the relevance of these findings in the whole animal. PLIN5 is clearly required to apply a brake to intramyocellular lipolysis, which limits the production of lipid intermediates and cellular stress signals and prevents disruption to tissue morphology and functions including cardiac contraction, muscle insulin action, and liver integrity. While PLIN5 expression is ‘exercise responsive’ and part of an adaptive program in muscle that may facilitate more efficient lipid handling, it is not required for mitochondrial biogenesis, as previously hypothesized [9], and its importance may depend on the energy challenges unique to each tissue and the associated molecular signals regulating mitochondrial function. In addition, PLIN5’s role in regulating metabolic substrate use during (patho)physiological stress requires further clarification, again with a focus on tissue-specific responses. Finally, cell studies suggest that PLIN5 may prevent FA-induced ‘lipotoxicity’ and as such may be a therapeutic target for treating metabolic diseases such as T2D. While PLIN5 ablation causes muscle insulin resistance, hepatic inflammation, and mild cardiac dysfunction in mice, there is currently no conclusive evidence of PLIN5 dysfunction in regulating the components of metabolic disease in humans. Future studies (Box 4) integrating the molecular, cellular, and whole-body approaches are required to tease out the post-translational regulation of PLIN5 and whether this can be manipulated to modulate PLIN5 functions in vivo. 7


Review Acknowledgments The authors thank Dr Renea Taylor (Monash University) for critical reading of the manuscript. M.J.W. is supported by the National Health and Medical Research Council of Australia (1077703 and 1047138) and R.R.M. by a Paul McNamee Postgraduate award co-funded by the Faculty of Medicine, Nursing, and Health Sciences of Monash University and Monash Sport.

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