Endocrine Journal 2014, 61 (6), 529-538

Review Cbl-b and obesity-induced insulin resistance

Tomoki Abe1), Katsuya Hirasaka1), Shohei Kohno2), Arisa Ochi1), Naoko Yamagishi1), Ayako Ohno1), Shigetada Teshima-Kondo1) and Takeshi Nikawa1)

1)Department of Nutritional Physiology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan 2)Department of Pharmacology and Toxicology, College of Pharmacy, University of Texas, Texas 78712-0125, USA

Abstract. Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.

Key words: Cbl-b, Obesity, Insulin resistance, TLR4, Macrophage

Obesity is increasing worldwide and causes diseases 4]. Increasing evidence indicates that obesity-induced such as type II diabetes, fatty liver, atherosclerosis inflammation in adipose tissue plays critical roles in and cardiovascular disease [1]. Aging and nutritional aging-induced and high-fat diet (HFD)-induced insu- excess are major risk factors for obesity. Excessive lin resistance [5, 6]. The pro-inflammatory cytokines nutrients accumulate as triglycerides in adipose tissue, secreted by adipose tissue directly interfere with insu- resulting in adipocyte hypertrophy and hyperplasia. lin signaling in insulin-sensitive organs, such as liver We now know that adipose tissue acts as an endocrine and skeletal muscle, while pro-inflammatory cytokines organ as well as in energy storage. The expansion of cause insulin resistance through activation of nuclear adipose tissue causes insulin resistance in rodents and factor κB (NF-κB) and Jun N-terminal kinase (JNK) humans through dysregulation of adipokine secretion in these organs. Recently, the activation of the NF-κB [1, 2], and recent studies indicate that obesity induces and JNK pathways was reported in the adipose tissue chronic low-grade inflammation in adipose tissue [3, of obese rodents and humans [7-9].

Submitted Jan. 31, 2014; Accepted Feb. 4, 2014 as EJ14-0048 acid; GTT, glucose tolerance test; HFD, high-fat diet; JNK, Released online in J-STAGE as advance publication Mar. 9, 2014 Jun N-terminal kinase; KLF, Krüppel-like factor; LCFA, long chain fatty acid; LPS, lipopolysaccharide; MCP, monocyte Correspondence to: Takeshi Nikawa, M.D., Ph.D., Department chemoattractant ; ND, normal diet; PI3K, phosphoinositide of Nutritional Physiology, Institute of Health Biosciences, The 3-kinase; PKC, protein kinase C; PTK, phospho-tyrosine kinase; University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, RING, really interesting new ; SH, Src-homology; SOCS, Tokushima 770-8503, Japan. E-mail [email protected] suppressor of cytokine signaling; TCR, T-cell receptor; TKB, Abbreviations: AMPK, 5’-adenosine monophosphate-activated tyrosine kinase-binding; TLR, toll-like receptor; TRIF, Toll/IL-1 protein kinase; BMT, bone marrow transplantation; Cbl, Casitas receptor-domain-containing adapter-inducing interferon-β; UBA, B-lineage lymphoma; CCR, C-C chemokine receptor; FA, fatty ubiquitin-associated domain ©The Japan Endocrine Society 530 Abe et al.

Macrophages that infiltrate adipose tissue play indicates that Cbl family regulate the activation critical roles in obesity-caused inflammation [10]. of protein tyrosine kinases (PTKs) [12]. Three homo- The majority of pro-inflammatory cytokines in adi- logues of Cbl, namely c-Cbl, Cbl-b and Cbl-c, have been pose tissue are secreted by macrophages. Therefore, identified as ubiquitin ligases in mammals. Since Cbl a molecule that regulates macrophage activation is proteins contain multiple binding motifs, many different an attractive potential therapeutic target for treating proteins may be targets of Cbl proteins. All Cbl homo- obesity-induced insulin resistance. However, little is logues have highly conserved tyrosine kinase binding known about the mechanisms underlying aging-in- (TKB) and really interesting new gene (RING) finger duced and HFD-induced macrophage activation. We catalytic domains (Fig. 1). The TKB domain, which is found that Casitas B-lineage lymphoma (Cbl)-b reg- found only in Cbl proteins, interacts directly with phos- ulates aging-induced and HFD-induced macrophage phorylated tyrosine-containing proteins through Src- activation in the adipose tissue of mice. In this review, homology (SH) 2 domains [13]. The RING finger cat- we focus on the importance of Cbl-b in the pathogen- alytic domain is responsible for the E3 ubiquitin ligase esis of insulin resistance in aging-induced and HFD- activity of Cbl proteins and binds to E2 ubiquitin-con- induced obesity. jugating enzymes [14]. The C terminal domain is less conserved among Cbl protein family members than the 1. Cbl proteins N terminal domain, and the C terminal domains of Cbl proteins contain proline-rich domains that interact with The ubiquitin-proteasome system, which constitutes the SH3 domains of target proteins [15]. The ubiquit- an intracellular protein degradation pathway, is regu- in-associated domain (UBA) interacts with monoubiq- lated by three types of enzymes: a ubiquitin-activating uitinated or polyubiquitinated chains. Human Cbl-b is enzyme E1, a ubiquitin-conjugating enzyme E2 and a encoded by the CBLB gene and is located on chromo- ubiquitin ligase E3. First, E1 enzymes activate ubiq- some 3q13.11. The combined efforts of numerous labo- uitin proteins in an ATP-dependent manner. Second, a ratories have documented the negative regulatory roles ubiquitin-binding E1 enzyme transfers a ubiquitin pro- of Cbl-b using Cbl-b-deficient (Cbl-b-/-) mice [16-19]. tein to a cysteine residue on an E2 enzyme. Finally, Cbl-b is highly expressed in immune cells, and muta- the target protein is ubiquitinated, a process that is tions associated with the loss-of-function of CBLB have mediated by an interaction between E2 enzymes and been implicated in many autoimmune diseases [20, 21]. E3 ubiquitin ligases. Proteins tagged with ubiquitin These reports indicate that Cbl-b is critical for regulat- selectively dock to proteasomes and are degraded. ing immune cell activation. Therefore, E3 ubiquitin ligases are critical for deter- mining which specific target proteins will be degraded 2. the role of Cbl-b in immune cells via ubiquitination [11]. The Cbl protein family comprises a class of evolution- Cbl-b is the first E3 ligase to be identified as a sup- arily-conserved ubiquitin ligases. Increasing evidence pressor of T-cell activation in vivo [22, 23]. Both an

Fig. 1 The primary structure and domain organization of Cbl proteins. Cbl proteins contain highly conserved tyrosine kinase-binding (TKB), linker (L), RING finger (RF) and proline-rich domains. 4H, four-helix bundle; SH2, Src-homology 2; UBA, ubiquitin- associated domain. Cbl-b regulates insulin sensitivity 531 antigen-activated T-cell receptor (TCR) signal and 3. Cbl-b in aging-induced macrophage a CD28-induced co-stimulatory signal are required activation to induce T-cell activation [24]. Cbl-b acts to induce anergy and thus to prevent autoimmune responses in Aging causes the accumulation of visceral fat in the absence of CD28 ligation. TCR activation induces rodents and humans by decreasing energy consump- Cbl-b expression, and this induction is mediated by tion and endocrine function [2]. Aging induces insulin calcium ions [25]. Phosphorylation of Vav1 is required resistance by increasing pancreatic β-cell dysfunction, for T-cell activation. Cbl-b suppresses the TCR- and oxidative stress and chronic inflammation [31-34]. The CD28-induced phosphorylation of Vav1, which is accumulation of excess visceral fat increases the secre- independent of ubiquitination and Vav1 degradation tion of pro-inflammatory cytokines by adipocytes and [22, 23]. Cbl-b binds and ubiquitinates p85, a subunit macrophages. Although various immune cells infil- of phosphoinositide 3-kinase (PI3K), which causes trate and are activated in adipose tissue, macrophages Vav1 phosphorylation [15]. Protein kinase C θ (PKCθ) are the largest population of immune cells in visceral plays a critical role in inhibiting Cbl-b in the presence fat. Recent studies demonstrated that elevated levels of CD28 ligation [26]. Specifically, after TCR activa- of free fatty acids and chemokines are associated with tion induces an interaction between Cbl-b and PKC θ, macrophage infiltration into adipose tissue [35]. The CD28 ligation causes PKC θ -dependent Cbl-b ubiq- expansion of adipocytes thus induces the infiltration uitination and degradation [26]. Thus, the mechanisms of macrophages into adipose tissue, a process which that are responsible for the negative control of T-cell is at least partially mediated by monocyte chemotac- activation by Cbl-b are well established. However, lit- tic protein (MCP)-1 [36]. MCP-1 is a CC chemokine, tle is known about the roles of Cbl-b in macrophage and most CC chemokines act on monocytes, T-cells, activation. eosinophils and neutrophils [37]. Circulating pro- Keane et al. [27] first reported that differentia- inflammatory (Ly-6Chigh) monocytes that express high tion of the HL60 and U937 cell lines towards a mac- levels of CC chemokine receptor type 2 (CCR2), an rophage/monocyte lineage up-regulates Cbl-b expres- MCP-1 receptor, infiltrate adipose tissue and differen- sion. Further, Cbl-b directly associates with the SH3 tiate into pro-inflammatory macrophages [38]. MCP-1 domains of several proteins. These results indicate that also promotes the release of pro-inflammatory mono- Cbl-b negatively regulates signal transduction in mac- cytes from bone marrow. The increased infiltration of rophages. Choi et al. [28] later found that thioglycol- macrophages into adipose tissue is a major contributor late-induced peritonitis is exacerbated by an increase to insulin resistance. in macrophage recruitment into the peritoneal cavity Hirasaka et al. [39] identified Cbl-b as a critical reg- of Cbl-b-/- mice. Cbl-b deficiency promotes the LFA- ulator of aging-associated macrophage infiltration and 1-mediated adhesion of bone marrow-derived mac- activation in the adipose tissue of mice. Compared with rophages to endothelial cells in vitro [28]. Recently, wild-type (Cbl-b+/+) mice, 30-week-old Cbl-b-/- mice Han et al. [29] reported that Cbl-b suppresses the show adiposity, hepatic steatosis, hyperinsulinemia, TLR4-induced activation of macrophages in a CD11b- glucose intolerance and insulin resistance [39]. These dependent manner. These reports indicate that Cbl-b results indicate that Cbl-b deficiency is associated with is involved in integrin-associated inside-out signaling type II diabetes in mice. Yokoi et al. [20, 40] reported activation in macrophages. Cbl-b is also an important that loss of function due to mutated CBLB is associated regulator of osteoclast differentiation and activation. with type I diabetes in rodents and humans. CBLB muta- Deletion of Cbl-b promotes RANK-RANKL signal- tion causes autoimmune disease by impairing anergy ing and bone resorption by osteoclasts, and osteopenia induction in T-cells. Hirasaka et al. [39] also observed is observed in Cbl-b-/- mice [30]. These reports indi- the infiltration of various immune cells into B cells in cate that Cbl-b negatively regulates macrophage acti- Cbl-b-/- mice aged >20 weeks. Hyperinsulinemia might vation. However, the targets of Cbl-b in macrophage be caused in part by the infiltration of CD4+, CD8+ and activation remain unclear. Therefore, we investigated CD68+ immune cells into the islets of Langerhans of the importance of Cbl-b in aging-induced and HFD- Cbl-b-/- mice aged >20 weeks. induced macrophage activation in mice. Consistent with an increase in adipose tissue weight, leptin and adiponectin expression is impaired in the adi- 532 Abe et al.

Fig. 2 Suppression of aging-induced macrophage infiltration and activation by Cbl-b. Aging induces adipose tissue hypertrophy and induces macrophage infiltration and activation. The macrophages secrete pro-inflammatory cytokines and chemokines, such as MCP-1. Circulating MCP-1 is recognized by CCR2-expressing monocytes, which promote macrophage infiltration into adipose tissue. Phosphorylation of Vav1 is associated with macrophage migration, and Cbl-b negatively regulates macrophage infiltration into adipose tissue by regulating Vav1 phosphorylation. pose tissue of 20-week old Cbl-b-/- mice. In addition, 4. Cbl-b in HFD-induced macrophage MCP-1 expression is up-regulated in both adipocytes activation and macrophages in the adipose tissue of Cbl-b-/- mice. Interestingly, we also found an increase in macrophage Like aging, chronic nutritional excess induces obe- infiltration and in the expression of pro-inflammatory sity by adipocyte hypertrophy and hyperplasia. Diet- cytokines in the adipose tissue of 20-week-old Cbl-b-/- induced obesity induces the infiltration of immune mice. These results suggest that elevated expression of cells, such as CD8+ T cells, B cells, neutrophils and MCP-1 induces macrophage infiltration into the hyper- pro-inflammatory (M1) macrophages [6]. In contrast, trophied adipose tissue of Cbl-b-/- mice. In addition, CD4+ regulatory T cells, basophils and anti-inflam- we tested whether Cbl-b regulates chemotaxis-related matory (M2) macrophages are decreased in hypertro- signaling in macrophages. Previous reports demon- phied adipose tissue [6]. M2 macrophages are char- strated that Vav1 regulates macrophage migration [41]. acterized as alternatively activated macrophages and Phosphorylation of Vav1 at Tyr267 regulates Vav1 act to maintain homeostasis in lean adipose tissue. activity in neutrophils [42]. We found that in Vav1, However, obesity causes phenotypic switching of mac- tyrosine phosphorylation is enhanced in peritoneal rophages from M2 polarization to M1 polarization macrophages from Cbl-b-/- mice. We concluded that [43]. Importantly, Xu et al. [44] and Weisberg et al. Cbl-b suppresses MCP-1-induced macrophage migra- [45] reported that macrophages infiltrate into the adi- tion, which is mediated at least in part by the inhibition pose tissue of obese mice and humans. Infiltrating of Vav1 phosphorylation (Fig. 2). Finally, to confirm macrophages can increase the cells in the adipose tis- the importance of MCP-1-mediated macrophage infil- sue of mice by up to 40% [45]. These macrophages tration in insulin resistance, we administered an anti- play a critical role in inducing chronic inflammation. MCP-1 antibody to Cbl-b-/- mice. We found that this Most of these infiltrated macrophages are M1 macro- reduced the aging-induced infiltration of macrophages phages that express CD11c [46]. M1 macrophages in into adipose tissue and also reduced systemic insulin adipose tissue induce systemic insulin resistance by resistance in 20-week-old Cbl-b-/- mice [39]. These secreting pro-inflammatory cytokines such as TNF-α, results indicate that Cbl-b regulates insulin sensitivity IL-1β and IL-6 [47]. Using mice that expressed the by inhibiting aging-associated macrophage infiltration CD11c-diphtheria toxin receptor, Patsouris et al. [46] and activation in mice. reported that ablation of CD11c+ cells improved HFD- Cbl-b regulates insulin sensitivity 533 induced inflammation and insulin resistance. Another showed impaired insulin sensitivity compared to Cbl- recent study demonstrated that Krüppel-like factor 4 b+/+ mice fed the same diet. Previously, pro-inflamma- (KLF4) also controls macrophage polarization in mice tory cytokines were found to interfere with insulin sig- [48]. Depletion of KLF4 in myeloid cells induces a naling by inducing the expression of the suppressor of pro-inflammatory phenotype in macrophages, exacer- cytokine signaling-3 (SOCS-3) protein [57]. SOCS-3 bating HFD-induced obesity, inflammation and insulin inhibits insulin signaling by targeting insulin recep- resistance [48]. These results indicate that increased tor substrate-1/2. We found that SOCS-3 expression levels of pro-inflammatory macrophages in adipose was increased in the liver and skeletal muscle of Cbl- tissue lead to HFD-induced inflammation and insulin b-/- mice fed a HFD for 5 weeks. These results indi- resistance. However, little is known about the mecha- cate that Cbl-b deficiency exacerbated obesity-induced nisms involved in macrophage activation in the hyper- insulin resistance by inducing chronic inflammation in trophied adipose tissue of HFD-induced obese rodents adipose tissue. and humans. Next we investigated the roles of Cbl-b in mac- Elevation in the levels of circulating fatty acids is one rophages in vitro. In adipose tissue, saturated fatty feature of obesity and is associated with insulin resis- acids cause macrophage activation that is mediated by tance. Recent studies revealed that fatty acid-induced TLR4 signaling. We found that in murine peritoneal activation of toll-like receptor 4 (TLR4) causes insu- macrophages, depletion of Cbl-b promotes saturated lin resistance via the expression and secretion of pro- fatty acid-induced IL-6 expression as well as the phos- inflammatory cytokines from macrophages that have phorylation of inhibitor of NF-κB (IκB) α and JNK. In infiltrated into the adipose tissue [49, 50]. In the innate contrast, Cbl-b overexpression inhibits saturated fatty immune system, TLR4 recognizes lipopolysaccharide acid-activated TLR4 signaling in RAW264.7 mac- (LPS), which is a component of the outer membrane of rophages. We also demonstrated that saturated fatty gram-negative bacteria [51]. CD14 and MD-2 are nec- acids regulate cell surface TLR4 protein levels via Cbl- essary for the activation of TLR4 by LPS [52]. After b-mediated TLR4 ubiquitination and degradation in LPS interacts with CD14, this complex is delivered to HEK293 cells and macrophages. These results indi- the MD-2-TLR4 complex. Although the mechanism cate that Cbl-b negatively regulates TLR4 signaling of LPS-induced TLR4 activation is known, fatty acid- in macrophages and that this regulation depends on induced activation of TLR4 seems to occur in a differ- proteasome activity (Fig. 3). Cbl-b can recognize and ent manner. In particular, some groups report that fatty bind to phosphorylated tyrosine residues in substrates. acids do not directly bind to the MD-2-TLR4 complex In fact, phosphorylation of Tyr674 and Tyr680 of human [53, 54]. A recent study elegantly revealed how fatty TLR4 is required for LPS-induced signal transduction acids activate TLR4 signaling in macrophages [55]. Pal [58]. It is unclear whether saturated fatty acids also et al. [55] demonstrated that fetuin-A, a glycoprotein induce the phosphorylation of TLR4 tyrosine residues secreted by the liver, acts as an endogenous ligand of in macrophages. Bachmaier et al. [59] reported that TLR4 in fatty acid-mediated insulin resistance. Taken in neutrophils, Cbl-b negatively regulates LPS-induced together, these findings suggest that TLR4 is a potent TLR4 activation by inhibiting the interaction between therapeutic target for obesity-induced insulin resis- TLR4 and MyD88 rather than by inhibiting ubiquit- tance. However, the intracellular regulation of fatty ination and degradation. In contrast, Han et al. [29] acid-activated TLR4 signaling remains unclear. reported that LPS causes Cbl-b-mediated ubiquitina- Recently, we reported that Cbl-b suppresses TLR4 tion and the degradation of phosphorylated MyD88 and signaling that is activated by saturated fatty acids in Toll/IL-1 receptor-domain-containing adapter-induc- macrophages [56]. Cbl-b-/- mice fed a HFD for 5 ing interferon-β (TRIF) in macrophages. Surprisingly, weeks showed increased body weight, adiposity and we found that saturated fatty acids did not induce the hyperlipidemia compared with Cbl-b+/+ mice fed the degradation of MyD88 and TRIF even in the presence same HFD. Feeding a HFD for only 5 weeks caused of Cbl-b [56]. These differences between LPS and sat- macrophage infiltration into adipose tissue of Cbl-b-/- urated fatty acids are not fully understood. The lower mice. Along with increased macrophage infiltration, affinity of saturated fatty acids versus LPS for TLR4 pro-inflammatory cytokine expression was up-regu- may be involved in these differences. Further studies lated in adipose tissue. Cbl-b-/- mice fed a HFD also are needed to address these questions. 534 Abe et al.

Fig. 3 Cbl-b mediates the inhibition of TLR4 activation by saturated fatty acids. Nutritional excess leads to obesity, which increases circulating fatty acid levels. Saturated fatty acids activate TLR4 in macrophages, which then induce the expression of pro- inflammatory cytokines via activation of JNK and NF-κB. Cbl-b suppresses TLR4 activation by saturated fatty acids. After stimulation by saturated fatty acids, Cbl-b induces the ubiquitination of TLR4 in macrophages, after which ubiquitinated TLR4 is degraded in a proteasome-dependent pathway.

We investigated the role of Cbl-b in macrophages deletion of Cbl-b promotes HFD-induced insulin resis- in vivo by bone marrow transplantation (BMT) of Cbl- tance without adiposity. Although adiposity causes the b+/+ or Cbl-b-/- hematopoietic cells into irradiated Cbl- development of insulin resistance and inflammation, it b+/+ mice [56]. Interestingly, hematopoietic cell-spe- remains unclear whether adiposity and hyperlipidemia cific Cbl-b deficiency did not affect body weight or the are necessary for insulin resistance. In fact, lipodystro- metabolic parameters of mice fed a HFD. Consistent phy causes severe inflammation in adipose tissue with- with our results, hematopoietic cell-specific deletion of out adiposity [61, 62]. TLR4 also had no effect on body weight gain in mice Finally, we investigated the importance of TLR4 in fed a HFD [60]. As in Cbl-b global knockout mice, the development of insulin resistance in Cbl-b-/- mice insulin sensitivity was impaired in hematopoietic cell- fed a HFD for 5 weeks by using Eritoran, a TLR4 antag- specific Cbl-b deficient (Cbl-b-/- BMT) mice fed a HFD onist [63]. Eritoran treatment improved fasting blood for 24 weeks compared with control (Cbl-b+/+ BMT) glucose and insulin levels in Cbl-b-/- mice fed a HFD. mice fed the same diet. We also found that hematopoi- HFD-induced macrophage infiltration and inflamma- etic cell-specific depletion of Cbl-b enhanced macro- tion in the adipose tissue of Cbl-b-/- mice fed a HFD phage infiltration and inflammation in the adipose tis- for 5 weeks were also decreased by Eritoran treatment. sue of Cbl-b-/- BMT fed a HFD for 24 weeks. These These results demonstrate that TLR4 is a potent thera- results indicate that Cbl-b plays a critical role in hema- peutic target for insulin resistance due to obesity. topoietic-derived cells including macrophages, T cells and B cells. We could not exclude the possibility that 5. Differences between c-Cbl and Cbl-b Cbl-b knockout affected T cells and B cells. In con- trast to the results from Cbl-b global knockout mice, We reported that Cbl-b deficiency exacerbates we found that hematopoietic cell-specific Cbl-b defi- aging-induced and HFD-induced insulin resistance ciency did not affect body weight gain due to a HFD. by enhancing macrophage activation in adipose tis- These results indicate that hematopoietic cell-specific sue. Although c-Cbl is a Cbl protein that has a struc- Cbl-b regulates insulin sensitivity 535

table 1 Targets of c-Cbl or/and Cbl-b 6. Conclusions Target c-Cbl Cbl-b Ubiquitination Reference APS + - - [67] Both aging and a HFD induce insulin resistance via macrophage infiltration into adipose tissue. Therefore, CrkL + + + [68, 69] understanding the mechanisms underlying macrophage Grb2 + + - [16] activation in adipose tissue is important for prevent- IRS-1 - + + [70] ing insulin resistance. We found that the E3 ubiquitin PDGFR + - + [71] ligase Cbl-b plays a critical role in regulating macro- PI3K p85 + + + [15, 72] phage activation. Mechanistically, Cbl-b controls mac- rophage infiltration into adipose tissue by suppress- Sprouty2 + - - [73] ing Vav1 phosphorylation. Thus, Cbl-b deficiency Src + + - [74] enhances aging-induced macrophage accumulation in Syk + + + [29] adipose tissue, resulting in decreased insulin sensitivity. TLR4 - + + [56, 59] We also found that Cbl-b acts as a negative regulator of TRIF - + + [29] TLR4 signaling in macrophages. HFD-induced obesity leads to the expansion of adipose tissue and to hyperlip- Vav2 + - + [75] idemia. Elevated levels of circulating fatty acids, espe- Zap-70 + + + [76, 77] cially saturated fatty acids, cause macrophage activation that is mediated by TLR4 signaling. In the presence of saturated fatty acids, Cbl-b stimulates the ubiquitination ture similar to that of Cbl-b (Fig. 1), some of the targets and degradation of TLR4 in macrophages. These find- of c-Cbl and Cbl-b are distinct (Table 1). Surprisingly, ings may be useful for developing novel therapeutics depletion of c-Cbl improves aging-induced and HFD- for treating insulin resistance. induced insulin resistance in mice [64-66]. Molero et al. [64] reported that c-Cbl knockout (c-Cbl-/-) mice Conflicts of Interests show less adiposity and higher insulin sensitivity even though they are hyperphagic. Depletion of c-Cbl up- None of the authors have any potential conflicts of regulates energy expenditure by increasing mitochon- interest associated with this research. drial size and by increasing phosphorylated 5’-adenos- ine monophosphate-activated protein kinase (AMPK) Acknowledgments in the soleus muscle. The loss of the ubiquitin ligase activity of c-Cbl is associated with elevated phosphory- This study was part of the Ground Research lation of AMPK and with increased energy expenditure Announcement for Space Utilization which was pro- [66]. c-Cbl deficiency also prevents adiposity and the moted by the Japan Aerospace Exploration Agency and impairment of glucose metabolism due to HFD-induced the Japan Space Forum and which provided funding to -/- obesity [65]. c-Cbl mice fed a HFD show greater O2 T.N. Funding was provided to T.N. by the following: consumption and fatty acid oxidation. Taken together, the Ministry of Education, Culture, Sports, Science, these results indicate that c-Cbl plays a critical role in and Technology of Japan, who provided a grant-in-aid whole-body energy expenditure. However, the targets for scientific research; by the Seed Production Program of c-Cbl have not yet been identified. at the Japan Science and Technology Agency; and by There are many differences in the metabolic pheno- the Promotion of Basic Research-Oriented Technology types of c-Cbl-/- and Cbl-b-/- mice, and the role of Cbl-b Research Advancement Institution of Japan. This in energy homeostasis remains unclear. However, we study was also supported in part by a high-technology found that a HFD causes adiposity and hyperlipidemia research center grant from the Ministry of Education, in Cbl-b-/- mice [56]. These findings suggest that Cbl-b Culture, Sports, Science, and Technology of Japan to may be related either directly or indirectly to the reg- the Juntendo University Research Institute for Disease ulation of energy expenditure and lipid metabolism. of Old Ages, Tokyo, Japan. Further study is essential to elucidate the role of Cbl-b in energy homeostasis. 536 Abe et al.

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