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2016-03-01

Emerging evidence for beneficial functions in and obesity-induced insulin resistance

Timothy P. Fitzgibbons University of Massachusetts Medical School

Et al.

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Repository Citation Fitzgibbons TP, Czech MP. (2016). Emerging evidence for beneficial macrophage functions in atherosclerosis and obesity-induced insulin resistance. University of Massachusetts Medical School Faculty Publications. https://doi.org/10.1007/s00109-016-1385-4. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1067

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REVIEW

Emerging evidence for beneficial macrophage functions in atherosclerosis and obesity-induced insulin resistance

Timothy P. Fitzgibbons 1 & Michael P. Czech2

Received: 12 November 2015 /Revised: 14 January 2016 /Accepted: 19 January 2016 /Published online: 4 February 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract The discovery that obesity promotes macrophage demonstrating that abrogation of certain macrophage func- accumulation in visceral fat led to the emergence of a new tions, in particular autophagy, exacerbates both atherosclerosis field of inquiry termed Bimmunometabolism^.Thisbroad and obesity-induced insulin resistance. Insulin signaling field of study was founded on the premise that through mammalian target of rapamycin (mTOR) is a crucial and the corresponding increase in macrophage number and regulatory node that links nutrient availability to macrophage activity was a pathologic feature of metabolic diseases. autophagic flux. A more precise understanding of the meta- There is abundant data in both animal and human studies that bolic substrates and triggers for macrophage autophagy may supports this assertation. Established adverse effects of in- allow therapeutic manipulation of this pathway. These obser- flammation in visceral fat include decreased glucose and fatty vations underscore the complexity of the field acid uptake, inhibition of insulin signaling, and ectopic tri- Bimmunometabolism^, validate its importance, and raise glyceride accumulation. Likewise, in the atherosclerotic many fascinating and important questions for future study. plaque, macrophage accumulation and activation results in plaque expansion and destabilization. Despite these facts, Keywords Visceral adipose . Atherosclerosis . Insulin there is an accumulating body of evidence that resistance . Autophagy . Macrophage . Cholesterol . mTOR also have beneficial functions in both atherosclerosis and vis- ceral obesity. Potentially beneficial functions that are common to these different contexts include the regulation of Background efferocytosis, lipid buffering, and anti-inflammatory effects. Autophagy, the process by which cytoplasmic contents are The idea that inflammation, and specifically the macrophage delivered to the lysosome for degradation, is integral to many foam cell, is vital to the initiation and progression of athero- of these protective biologic functions. The macrophage uti- sclerosis dates back to initial observations by the pathologist lizes autophagy as a molecular tool to maintain tissue integrity Rudolph Virchow and studies in rabbits in 1958 [1]. A seminal and homeostasis at baseline (e.g., bone growth) and in the face paper by Hotamisligil and colleagues in 1993 demonstrated of ongoing metabolic insults (e.g., fasting, hypercholesterol- increased tumor necrosis factor alpha (TNF-α) messenger emia, obesity). Herein, we highlight recent evidence RNA (mRNA) expression in the visceral adipose of obese mice and neutralization of TNF-α led to improved insulin stimulated glucose uptake [2]. They postulated that there * Timothy P. Fitzgibbons [email protected] may be a link between obesity, insulin resistance (IR), and inflammation [2]. However, the cellular source of TNF-α was not clear until 2003, when two simultaneously published 1 Cardiovascular Division, Department of Medicine, University of papers demonstrated that obesity results in inflammation and Massachusetts Medical School, 55 Lake Avenue North, macrophage accumulation in epididymal adipose tissue [3, 4]. Worcester, MA 01655, USA Since that time, there have been hundreds of papers demon- 2 Program in Molecular Medicine, University of Massachusetts strating that abrogation of inflammation can improve or pre- Medical School, Worcester, MA 01655, USA vent atherosclerosis or obesity-associated insulin resistance 268 J Mol Med (2016) 94:267–275

B “M1” “MMe” “M2” (IR) in mice. Searching PUBMED for the terms myeloid Metabolic ^ Antigen Presentation Fibrosis, Anti-inflammatory, specific knockout AND metabolism returns 2259 references, & T cell activation Response Angiogenesis half of which have been published in the last 5 years. There are many excellent reviews on the adverse effects of chronic in- ABCA1 flammation in these diseases and that will not be the focus of CD319 CD36 CD163 this paper [1, 5, 6].However,thefactthatclinicaltrialsofanti- CD274 PLIN2 CD206 inflammatory medications in diabetes have not been over- CD38 ACSL1 TFRC TGFβ1 whelmingly positive suggests that the pathophysiology is DHCR7 more complex than it may appear [7]. In normal conditions, both arteries and adipose tissue have a population of resident macrophages [8]. White adipose tis- TNFα, IL1, IL6 ? IL10, ARG1, RELMα sue (WAT) includes the subcutaneous or inguinal (iWAT) and Fig. 1 The spectrum of macrophage polarization. Although macrophage visceral adipose tissue (VAT), the latter being more prone to function has typically been dichotomized into classical (M1) or alterna- tive (M2), recent data highlights the limitations of this classification and inflammation. VAT includes the epididymal (eWAT), underscores the potential functional utility of Bmetabolically^ (MMe) parametrial (pWAT), and mesenteric (mWAT) depots, among activated subsets [13, 14, 30]. Such a subset is characterized by proteins others. Macrophages exert multiple and varied effects in these responsible for lipid uptake (CD36), storage (PLIN2), and export to high- tissues depending upon the physiologic conditions. Their density lipoproteins (ABCA1) number and activity is in constant flux, via recruitment of monocytes from the blood, proliferation of local tissue mac- IL10 and are dependent upon the transcription factors rophages, egress to lymphatics, and cell death. This makes STAT6, IRF-4, and PPARγ for their development. In mice, studies examining an endpoint at a single point in time (e.g., tissue resident M2 macrophages are generally characterized atherosclerosis or IR after 12 weeks of high fat diet (HFD)) by the cell surface markers F480+,CD11b+,andCD11c- very difficult to interpret. For example, silencing of TNFα [14]. In obesity, blood monocytes infiltrate eWAT, at least expression in the eWAT macrophages of ob/ob mice improves partially via CCR2 receptors in response CCL2 ligand, and glucose tolerance [9]. Yet, silencing of lipoprotein lipase differentiate into M1 macrophages (F480+,Cd11b+,and (LPL) in eWAT macrophages reduces foam cell formation Cd11c+ cells) [14]. Again, it should be emphasized that these and worsens glucose tolerance [10]. Furthermore, although distinctions are very complex, dynamic, and time dependent. short-term HFD (less than 1 week) results in obesity and For example, there are at least three populations of recruited eWAT inflammation, IR is not improved by depleting macro- interstitial macrophages after 8 weeks of HFD (Mgl1+/ phages or lymphocytes under these conditions [11]. In fact, it CD11c-,Mgl1Med/CD11c+, and Mgl1-/CD11c+)[15]. The lat- appears that the adipocyte plays a cell-autonomous role in the ter subset is typically M1 polarized and associated with development of IR that is dependent primarily upon cell size Bcrown-like structures^; but many CD11c + cells express both but also its metabolic substrates (saturated vs. unsaturated fat- M1 and M2 type mRNAs, and M2 polarization increases with ty acids) [12]. Hence, the role that macrophages play in IR and longer duration of HFD (12 weeks) [15]. It is still helpful to other pathologies is very complex, dependent upon time, con- think of M1 macrophages as those responding to a Th1 re- text, and other host-specific factors. sponse, with anti-microbial activity, and expressing inflamma- tory cytokines such as TNFα, IL1β, and IL6 (Fig. 1). M1 Defining macrophages and activation states activation of macrophages is dependent upon the transcription factors STAT1 and IRF-5. Deletion of CCR2 on circulating Traditionally, macrophage polarization has been dichoto- monocytes reduces eWAT macrophage accumulation in obese mized into classical (M1) or alternative (M2) activation. mice by 40 % [16]. Therefore, although this chemotactic path- These distinctions were developed based upon the in vitro way is important, there must be additional adipose-specific response of macrophages to INFγ or IL4 and IL13, respec- cues that stimulate inflammation [16]. The fact that many tively [13]. We now know that this is an oversimplification, CD11c+ cells surround dying adipocytes (epididymal and and that in vivo macrophages are very adaptable and exhibit a mesenteric) in Bcrown-like structures^ suggests that the broad range of activation states. Nonetheless, this is a useful necro-apoptotic adipocyte may be one such cue [17, 18]. In framework that we shall expand upon. M2 activation is a addition to infiltration, proliferation of tissue resident macro- characteristic feature of tissue resident macrophages; which phages also contributes to a local pro-inflammatory subset in account for approximately 10–15 % of stromal cells in lean eWAT and atherosclerotic plaques [19, 20]. The net result is a eWAT [13]. M2 macrophages remodel extracellular matrix dramatic increase in the total number of macrophages in obese (ECM), regulate angiogenesis, and have anti-inflammatory as compared to lean eWAT, numbering 45–60 % of the stromal effects, essential functions for normal growth of adipose tissue fraction [13]. Chronic inflammation in eWAT results in phys- (Fig. 1). They secrete anti-inflammatory cytokines such as iological dysfunction of adipocytes, excess free fatty acid J Mol Med (2016) 94:267–275 269 release, hepatic and systemic inflammation, and ectopic lipid The role of adipocyte autonomous inflammation deposition (liver, heart, skeletal muscle) [21]. Many studies have contributed to this notion; for example, ablation of Whereas decreasing monocyte recruitment to VAT is benefi- CD11c + macrophages improves HFD-induced IR [22]. cial in mouse models of DIO, Asterholm et al. recently pub- lished a provocative paper which demonstrates that reducing inflammatory chemokines elaborated by adipocytes them- M2 polarization and adaptive thermogenesis selves during short-term HFD impairs adipose tissue growth and expansion, and results in a worsening of IR [29]. The The importance of tissue resident M2 macrophages in adipose authors used three different mouse models to abrogate inflam- tissue biology has been highlighted by a growing body of mation specifically in adipose tissue. First, HFD-fed mice ex- literature that demonstrates their role in supporting brown pressing a dominant negative TNFα (dnTNFα) transgene adipose tissue (BAT) function [23–27]. In contrast to WAT, driven by the aP2 promoter were leaner but more IR than which stores triglyceride, BAT oxidizes fatty acids via control littermates [29]. This was thought to be due to de- uncoupled oxidative phosphorylation to generate heat via creased adipogenesis in the eWAT and iWAT of dnTNFα non-shivering thermogenesis. A third type of adipocyte, mice. In a second model, the authors expressed a novel termed the Bbeige^ or Bbrite^ adipocyte, is found in WAT adipose-specific transgene (RID) that broadly inhibits canon- depots, but can be recruited via catecholamines to induce a ical pro-inflammatory pathways. RID mice were also lean and thermogenic gene program (e.g. UCP1, PGC1α). The activa- had smaller iWAT, eWAT, and mWAT depots. Interestingly, tion of brown or Bbeige^ adipocytes hold great promise as a there was greater fibrosis in the RID adipose tissue, suggesting treatment for obesity and there is growing interest in finding that a function of acute low-grade inflammation in adipose methods to stimulate their growth/function. Interestingly, ex- tissue is to allow for degradation of extracellular matrix posure to the cold results in M2 activation of tissue resident (ECM) and adipogenesis [29]. Finally, they utilized inducible macrophages in iWAT and BAT [25]. In fact, these resident expression of IκBα to inhibit NFκB signaling specifically in cells may be required for expression of a thermogenic gene adipocytes. In comparison to wild type mice, transgenic mice program in BAT and lipolysis of triglyceride in iWAT, which had decreased eWATweight, increased liver weight, and were is the fuel for BAT thermogenesis [25]. Whereas it has always more glucose intolerant after 8 weeks of HFD [29]. The au- been assumed that sympathetic innervation of fat depots pro- thors concluded that adipocyte initiated inflammation is nec- vided catecholamines to stimulate lipolysis and thermogene- essary for adipogenesis, because macrophages are needed to sis, M2 ATMs induce tyrosine hydrolase (TH) upon cold remodel ECM and promote angiogenesis, allowing for fat pad exposure and may produce, surprisingly, a significant amount growth [29]. There may be depot-specific differences in the of adipose tissue catecholamines [25]. Depletion of BAT tis- role of adipocyte inflammation; for example, inflammation in sue resident macrophages via clodronate, or abrogation of M2 mWAT stimulates angiogenesis and growth of this depot, polarization in IL4 receptor knockout macrophages allowing for proper intestinal barrier function in obese condi- (IL4RαLoxP/LoxP LysMCre mice), reduced induction of thermo- tions [29]. genic genes in BAT and prevented maintenance of core body temperature upon exposure to 4 °C [25]. More recent work BMetabolic^ activation of macrophages has unraveled a network of immune cells that may orchestrate this process [26, 27]. M2 polarization of ATMs is promoted In addition to this beneficial role of inflammation in adipose by paracrine secretion of IL4 by local eosinophils. tissue biology, recent evidence suggests that our definition of Eosinophils, in turn, may be stimulated by endocrine circu- Binflammation^ in metabolic contexts may be incomplete lating factors released from muscle or fat, during exercise or [30]. Using a proteomics approach to study M1 macrophage cold exposure, respectively [26, 27]. Reducing environmental cell surface proteins in vitro, Kratz et al. found a panel of three temperature from 28 to 4 ° C dramatically alters the BAT markers for M1 activation that are common to human and transcriptome [28]. Among the many regulated gene net- mouse macrophages; CD274, CD38, and CD319 [30] works, upregulation of AMPK and downregulation of (Fig. 1). These three markers were validated on macrophages mTOR signaling pathways was noted [28]. Thus, cold stress from the airway of patients with , which are is analogous to fasting conditions and could potentially stim- chronically exposed to bacterial pathogens. However, none ulate autophagic pathways in macrophages or other cells (see of these markers were increased in the stromal fraction of below). To summarize, just as prevention of M1 macrophage VAT from obese humans (omental) or mice (eWAT), despite polarization or infiltration may be beneficial to adipose tissue increased mRNA levels of TNFα and IL1β from these same metabolism, stimulation of M2 polarization in ATMs may be samples [30]. The authors hypothesized that obesity does not beneficial in iWAT and BAT to promote adaptive thermogen- induce M1 macrophage polarity per se, but perhaps a distinct esis and energy expenditure. macrophage phenotype [30]. By exposing macrophages to 270 J Mol Med (2016) 94:267–275 high levels of palmitate, insulin, and glucose, they found a macrophage utilizes autophagy to metabolize lipid in condi- panel of markers (Abca1, Cd36, Plin2)thatwereuniquefor tions of fatty acid excess [14, 30, 37]. How and why might this Bmetabolic activation (MMe)^, and did not overlap with occur? known M1 or M2 markers. Other groups have also found that In fasting conditions, when adipocyte lipolysis is maximal- markers of pro-inflammatory macrophages (M1) are not as ly activated, there is a sudden and abundant increase in eWAT abundant in obese VATas originally thought, especially when lipid-laden ATMs [38, 39] (Fig. 2). It is thought that these total macrophage number is accounted for [3, 4, 14]. macrophages are recruited to buffer the acute local increase Expression of the consensus MMe markers was validated in in FFA concentrations because depletion of macrophages after VAT from obese humans and mice, and their induction was a 24 h fast results in a dramatic rise in plasma FFA [38]. shown to be independent of type 1 interferon or toll-like re- Whether or not macrophage autophagy is activated in these ceptor signaling [30]. Instead, PPARγ and sequestosome-1 circumstances is not known. (p62) regulated expression of the MMe markers CD36, On the other hand, autophagy may be stimulated in eWAT ABCA1, and PLIN2 [30]. p62-/- macrophages, and macro- ATMs during obesity. This possibility was recently raised by phages treated with the PPARγ antagonist T0070907, failed Xu et al., who performed microarrays of eWAT in lean and to induce MMe markers after palmitate treatment and obese mice and correlated the results with three phenotypes: overproduced TNFα and IL1β. Therefore, there may be a link body mass, fasting insulin, or macrophage-specific gene ex- between metabolic activation and anti-inflammatory effects. pression [14]. Different transcriptional networks were regulat- The role of PPARγ was not surprising, given its known effects ed for each condition, but only one was upregulated in all as a regulator of metabolic and anti-inflammatory pathways. three; lysosomal biogenesis [14]. Proteins, important for au- However, the mechanism by which p62 suppresses expression tophagy and lysosomal biogenesis, were enriched in macro- of TNFα and IL1β was less clear. p62 is normally degraded in phages from the stromal fraction of eWAT from obese mice. In the autophagolysosome and accumulates when autophagy is comparison to lean mice, ATMs from obese mice doubled blocked [30]. Upregulation of p62 suggested that autophagy their lysosome content, as measured by Lysotracker accumu- was activated but flux was reduced in MMe macrophages; this lation (26 vs. 57 %). Although lysosomes accumulated in both upregulation was correlated with palmitate uptake. Indeed, F480+/Cd11b+ and F480+/Cd11b+/Cd11c+ cells, they were increasing palmitate concentrations increased the levels of greater in F480+/Cd11b+/Cd11c+ macrophages. Microscopic p62, in a CD36-independent manner [30]. To summarize, analysis of these lipid-laden cells demonstrated two classes of there appears to be an additional subset of macrophages acti- lipid droplets: larger bodipy positive droplets with and without vated by metabolic factors (MMe) that is distinct from tradi- surrounding lysosomes and smaller more numerous droplets tional M1 or M2 classifications (Fig. 1). The accumulation of that co-localized with lysosomal markers [14]. Exposure of palmitate in macrophage autophagolysosomes may contribute bone marrow-derived macrophages to eWAT in vitro induced to the anti-inflammatory properties of this novel subset. The expression of genes involved in lipid uptake (Msr1, Plin2)and precise mechanism of palmitate uptake, and the biological lysosomal biogenesis (Atp6v0d2, Lipa, Ctsk), but not classic significance of MMe ATMs, has yet to be elucidated. M1 activation (TNFα, Tlr2, Tlr4)[14]. Inhibition of lysosom- al function using either chloroquine or Bafilomycin A1 result- Macrophage autophagy and lipid metabolism ed in a marked accumulation of macrophage lipid content. These results were consistent with the subsequent studies by There are three main types of autophagy [31–33]. Kratz et al., suggesting that obesity results in a unique activa- Macroautophagy refers to the engulfment of cytosolic con- tion profile of ATMs, characterized by induction of lipid up- tents into double vesicles called autophagosomes. take and lysosomal pathway encoding genes [30]. This process can be selective or non-selective [31]. A second These data suggest that macrophages in eWAT take up and type of autophagy is microautophagy, which is the direct store lipid released from dysfunctional adipocytes, leading to of cytosolic material by the lysosomal mem- the formation of macrophage Bfoam cells^. Stored lipid is brane. Finally, a third form of autophagy is termed likely metabolized predominantly by lysosomal degradation; chaperone-mediated autophagy (CMA). Specific motif- however, it is possible that stored lipid is also metabolized by containing proteins are recognized by chaperones such as heat selective macro-autophagy or Blipophagy^. This process was shock cognate protein of 70 kDa (hsc70) and then delivered to originally described by Singh et al. in 2009 [40]. Hepatocytes the surface of the lysosome [31]. CMA has recently been deficient in autophagy-related protein 5 (Atg5), when chal- shown to contribute to lipid droplet metabolism, although lenged with oleic acid, have markedly enlarged lipid droplets. the degradation of lipids by selective macroautophagy, or Animals with hepatocyte-specific autophagy-related protein 7 Blipophagy^, is thought to be most operative in metabolic (Atg7) deletion developed marked hepatosteatosis due to disease, and that is the process that we refer to here [34–36]. blocked autophagy and resultant reduced lipolysis [40]. The findings by Kratz et al. and others suggest that the Therefore, autophagy appears to serve a protective effect in J Mol Med (2016) 94:267–275 271

ATP MACROPHAGE

2 FA Lipases Glucose 1 TG Phagophore CholE 4 AMPK ↑↑AMP

mTORC1 ↑↑ATP Autophagosome

Palmitate, oxLDL, etc. 3 p62 A 5 NPC1/2 B CholE Chol C HDL LAL Chol A 1 Lysosome Autolysosome

Fig. 2 Regulation of lipid metabolism in foam cells and tissue resident acid lipase (LAL) hydrolyzes cholesterol ester (CholE) to cholesterol macrophages. (step 1, blue) In basal conditions, macrophages take up (Chol). Whether or FA generated from the hydrolysis of CholE or TG glucose to generate energy via oxidative metabolism, storing excess are available for beta-oxidation is not yet known (not shown). However, glucose in lipid droplets after de novo lipogenesis. (step 2, green)In the export of cholesterol is dependent on this process. The export of fasting conditions, neutral lipases at the lipid droplet surface hydrolyze cholesterol is facilitated by the proteins of Niemann-Pick disease, type stored triglyceride (TG) to fatty acids (FA), which undergo beta-oxidation C1,2 (NPC1/2), and member 1 of human transporter subfamily ABCA in adjacent mitochondria to generate adenosine triphosphate (ATP). (Step (ABCA1). Depending upon the substrates within the autolysosome au- 3, red) In obesity or hypercholesterolemia, macrophages in the plaque or tophagy may be blocked at this step. This will result in the accumulation visceral adipose tissue may endocytose lipoproteins/lipid via scavenger of sequestosome-1 (p62) and lipid in the autolysosome and prevent cho- receptors (CD36, SRA) or via micro . Engulfed lipoproteins lesterol efflux to high density lipoprotein (HDL). Decreased autophagic are metabolized via lysosomal degradation. (step 4, black)Somestored flux will also cause leakage of autolysosome contents into the cytoplasm lipid may also be metabolized via autophagy, which is initiated by for- (cathespins, reactive oxygen species) and cause activation of the mation of a phagophore. AMP activated protein kinase (AMPK) and inflammasome or unfolded protein response. These latter effects contrib- mammalian target of rapamycin (mTORC1), respectively, activate and ute to worsening of local inflammation or programmed cell death inhibit this process. The autophagosome fuses with the lysosome to gen- erate the autolysosome. (step 5, orange) In the autolysosome lysosomal the liver by preventing hepatosteatosis in the setting of HFD. treatment with the mTOR inhibitors everolimus and sirolimus, Furthermore, this protection is at least partially mediated by reduce atherosclerosis in mice [37]. lysosomal acid lipase (LAL) and not only neutral cytoplasmic Razani et al. tested the hypothesis that autophagy defective lipases (HSL, Nceh1, CEH) [41, 42]. Autophagy in mice on the apoliprotein E null (Apoe-/-) background might preadipocytes regulates adipogenesis, and its inhibition pre- develop worse atherosclerosis [37]. Beclin-1/Atg6 heterozy- vents adipocyte differentiation in vitro and in vivo [43]. gous deficient mice, which are only haploinsufficient for au- Whole body Atg5-/- and Atg7-/- mice die at birth, but tophagy, did not develop worse atherosclerosis than Apoe-/- adipocyte-specific ablation of Atg7-/- results is reduced in littermates. However, macrophage-specific Atg5 knockout leaner, more insulin-sensitive mice, with more brown adipose mice on the Apoe-/- background (Atg5-mϕ Apoe-/-) developed tissue [43, 44]. Whether or not macrophage specific loss of dramatically worse atherosclerosis. Reduced autophagic flux autophagy exacerbates IR in obese mouse models is not yet was demonstrated by increased p62 protein levels in the aortas known. However, there is abundant recent evidence demon- of Atg5-mϕ Apoe-/- mice. In addition to the increased athero- strating that macrophage autophagy is protective against ath- sclerotic area, Atg5-mϕ Apoe-/- mice hyper-secreted IL1β in erosclerosis [37, 41, 45]. response to LPS and had markedly reduced cholesterol efflux [37]. Macrophage autophagy and atherosclerosis It is notable that cholesterol crystals in advanced plaques not only activate the inflammasome but also block autophagic The idea that macrophage autophagy might be beneficial in processing. This is reminiscent of the effect of palmitate to limiting atherosclerosis stemmed from the observation of inhibit autophagy and subsequently repress the anti- autophagosomes in atherosclerotic plaque on electron micros- inflammatory effects of autophagy [30]. Hence it appears that copy [46]. Additionally, as defective efferocytosis and the the metabolic substrates of autophagy, depending upon their accumulation of cholesterol crystals are hallmarks of the ath- suitability for lysosomal degradation, may determine autoph- erosclerotic plaque, it is intuitive that autophagy may be pro- agic flux. Poorly digestible substrates (oxidized LDL, tective, by potentially clearing this cellular debris. Finally, lipofuscin, ceroid) will result in stalled autophagy and stimu- interventions that activate autophagy, such as fasting and lation of pro-inflammatory pathways [30, 37, 47]. For 272 J Mol Med (2016) 94:267–275 example, the accumulation of undigested cholesterol crystals autophagy or Bself-digestion^ is not necessary for the provi- stimulates the inflammasome, whereas palmitate and other sion of metabolites for cellular growth and ATP synthesis [46]. saturated fatty acids activate the unfolded protein response In contrast, under fasting conditions, when there are lower [34, 48]. In this way, atherosclerosis, obesity, and amounts of extracellular nutrients and growth factors, hepatosteatosis may all represent acquired lysosomal storage mTOR1 is not activated, and Atg1 kinase activity is disorders [49]. Compounds that promote the mobilization of derepressed, therefore stimulating autophagy for the provision cholesterol crystals from the lysosome and into the cytoplasm of cellular nutrients. The importance of this pathway to cellu- (e.g.,β-cyclodextrins) have been promoted as potential thera- lar metabolism is reflected in its inter-species conservation, peutic compounds for these disorders [50–52]. This is in con- most dramatically seen in C.elegans, where fasting and stim- trast to oleate and other unsaturated fatty acids that stimulate, ulation of autophagy promote longevity and delay cellular but do not stall, flow through the autophagy pathway [34]. senescence [57]. These insights have led to the understanding Stalled autophagy not only triggers inflammation and lipid that reduced insulin signaling promotes longevity and might droplet accumulation, but it prevents the hydrolysis of choles- have other beneficial effects. terol ester and subsequent efflux of cholesterol to apo-A1 [41]. Interestingly, deletion of the insulin receptor specifically It was previously thought that cytoplasmic neutral cholesterol from macrophages, as opposed to whole body deletion, im- esterases (HSL, ATGL) were responsible for hydrolysis and proves atherosclerosis on the Apoe-/- background [58]. In con- mobilization of cholesterol from foam cells. However, trast, hepatic insulin receptor deletion promotes atherosclero- Ouitmet et al. showed that in lipid-loaded macrophages, cho- sis even in wild type mice, due to the development of lesterol efflux to apoA-1 is dependent on lysosomal acid li- Bselective^ insulin resistance [59]. Hepatic insulin receptor pase (LAL) [41]. An inhibitor of LAL (lalistat) prevented knockout stimulates VLDL synthesis resulting in high blood cholesterol efflux to apo-A1. RCT in vivo was reduced in triglyceride-rich lipoprotein levels and atherosclerosis. dele- mice given lipid-loaded macrophages from Atg5-/- donors. tion of the insulin receptor in macrophages results in de- Therefore, mobilization of cholesterol for RCT from foam creased atherosclerosis and inflammatory cytokine production cells is dependent upon autophagy. Foam cells accumulate by macrophages, despite identical blood lipoprotein levels and lipid droplets in an unregulated fashion via non-selective up- metabolic features [58]. take of triglyceride rich lipoprotein particles (TRLP) by scav- Myeloid-specific insulin receptor knockout also provides pro- enger receptors (SR-A1, SR-B1, CD36), whereas non-lipid tection against DIO [60]. The mechanism was thought to be due laden macrophages take up TRLP in a regulated fashion, via to increased macrophage in the absence of insulin and/ lipoprotein receptors (LDLR). How do lipid-laden macro- or decreased macrophage infiltration into adipose tissue [60]. We phages sense the metabolic milieu and regulate autophagic speculate that an additional protective effect of myeloid specific pathways? insulin receptor knockout may be due to reduced mTOR1 acti- vation and resultant stimulation of autophagy. Numerous other MTOR links environmental cues to autophagy studies in mice and rabbits have demonstrated that inhibition of and nutritional status mTOR1 signaling by pharmacologic (rapamycin analogs) or genetic approaches (mTOR1 siRNA) leads to stimulation of Mammalian target of rapamycin (mTOR1) provides a link autophagy, a reduction in atherosclerotic lesion size, and stabili- between the presence of extracellular nutrients/growth factors zation of the fibrous plaque [46, 54]. and the stimulation of cell growth and protein synthesis [53]. The protective functions of macrophage autophagy extend Stimulation of the insulin receptor leads to activation of beyond Blipophagy^ in the atherosclerotic plaque or VAT. phosphatidylinositiol-3 kinase (PI3K), phosphorylation of Autophagy also helps to prevent apoptosis, promote Akt, and downstream activation of mTOR1. It is important efferocytosis of necrotic cells, and digest cellular components to note that the effect of PI3K on autophagy is class depen- (e.g., mitochondria) damaged by oxidative stress [61]. For dent; class 1 PI3 kinases inhibit autophagy (by activating example, 7-ketocholesterol has been shown to induce autoph- mTOR1), whereas class 3 PI3 kinases activate autophagy by agy and prevent induction of smooth muscle cell apoptosis by inducing beclin 1 [54]. Activation of mTOR1 inhibits autoph- fluvastatin, through yet to be identified mechanisms [62]. The agy by several mechanisms. mTOR1 phosphorylates ATG13 cardioprotective effects of adiponectin (Acrp30) may be par- and ULK1/2, which are components of the autophagy initiat- tially due to the stimulation of autophagy in foam cells of the ing UNC-5 like autophagy activating (ULK) complex [55]. atherosclerotic plaque [63]. Transplant of Acrp30-deficient mTOR1 also phosphorylates ATG14L, causing inhibition of perivascular fat to the carotid artery of Apoe-/- mice results in the associated class 3 PI3 kinase VPS34. Finally, mTOR1 worse atherosclerosis and decreased macrophage autophagy; phosphorylates autophagy/beclin 1 regulator 1 (AMBRA1) Acrp30 also stimulates autophagy in macrophages in vitro [46, 55, 56]. In this way, mTOR activation signals cells that [63]. This effect of Acrp30 may be mediated by AMP- environmental conditions favor cellular growth, and activated protein kinase (AMPK), a known antagonist of J Mol Med (2016) 94:267–275 273 mTOR1 signaling whose activity is increased by fasting [53]. 2. Hotamisligil GS, Shargill NS, Spiegelman BM (1993) Adipose AMPK mediates these effects by activating tuberous sclerosis expression of tumor necrosis factor-alpha: direct role in obesity- linked insulin resistance. Science 259:87–91 complex 2 (TSC2) and also by direct serine phosphorylation 3. 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