STATE-OF-THE-ART REVIEW IN ENDOCRINOLOGY
The GDF3-ALK7 signaling axis in adipose tissue: a possible therapeutic target for obesity and associated diabetes?
2023 Volume 70 Issue 8 Pages 761-770
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2023 Volume 70 Issue 8 Pages 761-770
ALK7, a type I receptor for the transforming growth factor-β superfamily, is known to be predominantly expressed in adipocytes in both mice and humans. The present review describes recent findings suggesting that ALK7 plays a major role in regulating lipid metabolism and fat mass. Furthermore, the ligands and upstream regulators that activate ALK7 signaling are discussed. The focus is on findings in mice and their derivative tissues and cells that harbor the mutations of ALK7 and related molecules. Particular attention is paid to the contradictory nature of the current literature about the loss-of-function phenotypes and the relationship with insulin secretion and sensitivity. Additional attention is paid to the ALK7 gene variants found in humans and their associated traits. The goal is to seek a parsimonious, and preferably singular and unified, description of the underlying mechanism. This review also introduces recent promising findings about ALK7 neutralizing treatment to obese mice.
ALK7 was cloned as the last member among seven mammalian type I transforming growth factor-beta (TGF-beta) receptors, as suggested by the nomenclature. Like ALK4 and ALK5, ALK7 signals through phosphorylation of Smad2 and Smad3, which in turn interact with Smad4 and are translocated to the nucleus to regulate gene expression (see review [1]). Because this Smad2/3 signaling pathway is commonly used by many other TGF-beta superfamily members, including TGF-betas, activins, and a subset of growth/differentiation factors (GDFs), it is important to investigate the unique biological functions of ALK7 in a physiological in vivo context. For example, receptor reconstitution experiments in Xenopus embryos indicate that ALK7 mediates signaling of a TGF-beta family member, Nodal, which plays crucial roles in mesoderm formation and left-right patterning during vertebrate development [2]. However, global ALK7-knockout mice are viable and fail to show the developmental abnormality found in Nodal-knockout mice [3]. Furthermore, they display normal histological organization of the cerebellum, cortex, and hippocampus, despite significant ALK7 expression in these brain regions. ALK7 transcripts are also detected in rodent islets and several β-cell lines [4-6] and in white adipose tissue (WAT), brown adipose tissue (BAT), and a preadipocyte cell line, 3T3-L1, during the late phase of adipocyte differentiation [7-9]. These expression patterns suggest that ALK7 may have endocrine and/or metabolic functions. In fact, recent findings suggest that ALK7 plays a major role in regulating lipid metabolism and fat mass. Although there is a consensus that the inactivation of ALK7 signaling reduces fat mass in obese mice, the underlying mechanism and the possible side effects are controversial. Particularly, its relationship with insulin secretion and sensitivity is contradictory. Furthermore, several ALK7 ligands and upstream regulators have been reported. This review tries to resolve these matters.
ALK7-knockout mice in a C57BL/6 genetic background display hyperinsulinemia and subsequently develop insulin resistance, liver steatosis, and impaired glucose tolerance [10]. In these mice, higher fasting levels of insulin in serum starts at 2 weeks of age and precedes the overt glucose intolerance and liver steatosis detected at 2 months of age and the increased β-cell mass detected at 5 months of age. Therefore, it was suggested that primary hyperinsulinemia causes late-onset, insulin resistant features in ALK7-deficient mice. However, pancreatic islets from the knockout mice show a relatively minor change in insulin secretion under the specific experimental condition: the mutant islets isolated at 2 months of age exhibit higher insulin secretion only under sustained glucose stimulation. This may reflect an in vivo stimulated state in response to the overt glucose intolerance already occurring at this age. Furthermore, the hypersecretion found after prolonged glucose stimulation in isolated islets does not explain the observed hyperinsulinemia after overnight fasting in mice. If insulin hypersecretion indeed occurs as a primary defect of β cells, lower fasting blood glucose levels should occur in young ALK7-knockout mice fed regular chow, which has not been reported. Moreover, insulin hypersecretion should increase fat mass by suppressing lipolysis in adipocytes. However, the same research group reported that ALK7-knockout mice in an Sv129OlaHsd background fed a high-fat diet (HFD) exhibited reduced fat accumulation, as well as hyperinsulinemia, insulin resistance, and liver steatosis [11], although the underlying mechanism or the responsible cells (β cells, adipocytes, or other cells) for the reduced fat accumulation were not explored. Thus, it is likely that the observed fasting hyperinsulinemia in these ALK7-knockout mice secondarily results from the decreased insulin sensitivity due to latent lipid accumulation in liver or other tissues that becomes overt with advanced age.
In fact, we found no alternations in glucose-stimulated insulin secretion from isolated islets of genetically ALK7-deficient mice [12] or mice treated with ALK7 neutralizing antibody [13]. Furthermore, the protein and mRNA levels of ALK7 in islets were negligible compared with those in WAT, which is consistent with the previous findings that ALK7 transcripts are predominantly expressed in adipose tissues compared with other tissues in both mice and humans [14, 15]. Therefore, it is likely that ALK7-deficiency in β cells causes a minimal effect, if any, on insulin secretion and on metabolism generally.
In addition to findings from the above gene engineering approach, spontaneous ALK7 mutation was found through genetic analyses of obese mice. Our group performed a genome-wide screen for loci linked to glucose homeostasis and body weight using F1 and F2 progeny between BALB/cA mice and Tsumura, Suzuki, Obese Diabetes (TSOD) mice, which spontaneously develop obesity, hyperglycemia, and hyperinsulinemia, and identified a quantitative trait locus involved in body and fat weights on mouse chromosome 2 [16, 17]. The actual genetic alteration in this locus was found as a nonsense mutation of the Acvr1c gene encoding ALK7 [12]. The mutation is unexpectedly derived from the genome of control BALB/cA mice, which results in C-terminal deletion of the kinase domain. The congenic T.B-Nidd5/3 mice containing this loss-of-function mutation in the genetic background of TSOD mice show significantly reduced body and fat weights. Furthermore, they have smaller-sized adipocytes without a change in their number [17], suggesting that ALK7 is not directly involved in adipocyte differentiation or proliferation but may modulate lipid metabolism.
In fact, ALK7 dysfunction upregulates the adipose master regulators, C/EBPα and PPARγ, and promotes expression of a wide range of proteins involved in lipid metabolism [12] (Fig. 1). Conversely, expression of a constitutive active form of ALK7 or Smad3 markedly decreases the activities of the adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) promoters and the binding of these transcription factors. Although ALK7-deficient adipocytes display increases in both triglyceride (TG) synthesis and breakdown, they become smaller with a net decrease in lipid storage in an obese state. Because the rate of TG breakdown by lipolysis, but not that of TG synthesis, depends on total fat mass, as well as the activities of the involved enzymes, C/EBPα and PPARγ appear to promote a net TG breakdown in large mature adipocytes, whereas they induce a net TG synthesis in small preadipocytes during differentiation [18]. Smad3, which lies downstream of ALK7 signaling, can bind C/EBPα in vitro and repress its transcription function in cultured adipocytes [19]. Therefore, it seems that activated Smad3 by ALK7 titrates C/EBPα to inhibit its transcriptional activity and subsequently downregulates both C/EBPα and PPARγ by disrupting a positive-feedback loop for the expression between the two transcription factors [12]. Alternatively, the activation of Smad2/3 may subsequently inhibit Smad1/5/8 and suppress PPARγ [20], although it is unknown whether ALK7 indeed induces this pathway in adipocytes.
ALK7 signaling and function in adipocytes
Because ALK7 deficiency reduces fat mass specifically in mice fed an HFD [11] and in genetically obesity-prone mice [12], the ALK7 signal is thought to be activated under nutrient-excess conditions, such as after food intake and in an obese state. ALK7 activates Smad2/3 directly [12] and/or might subsequently inhibit Samd1/5/8 indirectly [20], which suppresses the expression and transcriptional activities of C/EBPα and PPARγ in adipocytes and leads to downregulation of a broad range of genes encoding proteins involved in lipid metabolism, such as FA transport (aP2/FABP4, FATP), FA esterification (GPAT3, APGAT2, Lipin1, DGAT1, DGAT2), lipolysis (ATGL, HSL) [12], and β-ARs [23]. The net effect on fat mass varies in different sizes of adipocytes, because the rate of lipolysis, but not that of TG synthesis, depends on the existing fat mass [18]. Thus, ALK7 activation in large mature adipocytes results in net fat accumulation due to a stronger inhibitory effect on TG breakdown. Conversely, ALK7 expression is not found in an early stage of adipocyte differentiation in 3T3-L1 preadipocyte and stromal-vascular fraction cells [9, 12], possibly because it would prevent fat accumulation by a strong inhibitory effect on TG synthesis in small differentiating adipocytes.
ALK7-deficinet adipocytes express fewer inflammatory cytokines, such as MCP-1 and TNFα, and more insulin-sensitizing adiponectin [18], which may result from the reduced cell size [21]. At the whole-body level, T.B-Nidd5/3 mice display improved insulin sensitivity and glucose tolerance compared with parental TSOD mice, which is accompanied by increased O2 consumption and decreased respiratory quotients as measured in a metabolic cage. These phenotypes contrast with those of ALK7 knockout mice in either a C57BL/6 or an Sv129OlaHsd background when fed regular chow, and they display ectopic lipid accumulation in liver and reduced insulin sensitivity despite having a normal body weight [10, 11]. The phenotypic discrepancy is not due to differences in the genetic mutations, because C57BL/6 mice harboring the same nonsense mutation found in BALB/cA mice, and thus in T.B-Nidd5/3 mice, also display liver steatosis at least when fed an HFD (our unpublished observation). Therefore, differences in the genetic background of mouse strains likely account for the discrepancy. In fact, C57BL/6 mice are known to be susceptible to HFD-induced obesity, insulin resistance, and non-alcoholic fatty liver disease [22]. In addition, because the body weight of outbred ddY mice, from which inbred TSOD and T.B-Nidd5/3 strains are derived, is roughly twice that of C57BL/6 mice, it may be that larger mice tolerate increased lipid efflux by enhanced lipolysis in WAT.
Subsequently, Guo et al. generated fat-specific ALK7-knockout mice and found enhanced β-adrenoreceptor (β-AR) expression in WAT of both global and fat-specific ALK7-knockout mice fed an HFD [23]. Because catecholamine (CA) stimulates lipolysis and lipid oxidation in adipose tissue, the authors suggested that ALK7 signaling contributes to diet-induced CA resistance in adipose tissue by downregulating β-ARs. Consistently, C/EBPα, which, as described above, is upregulated in ALK7-deficient adipocytes, has been shown to play a critical role in the transcription of β3-AR gene [24].
However, given the simultaneous upregulation of a broad range of adipose proteins involved in fatty acid (FA) uptake and esterification as well as lipolysis in ALK7-deficient adipocytes [12] (Fig. 1), the enhanced expression of β-ARs likely reflects some contribution from the conditions induced by the activation of C/EBPα and PPARγ. In fact, we observed proportional increases in lipolysis between basal and isoproterenol-stimulated states in ALK7-deficient adipocytes compared with ALK7-intact adipocytes [12, 25]. Guo et al. also showed increased basal lipolysis in ALK7-deficient mice fed an HFD, elevated total protein levels of HSL in ALK7-deficient adipocytes, and proportionally decreased basal and CA-stimulated lipolysis in wild-type (WT) adipocytes treated by activin B for ALK7 activation [23], all of which suggest that ALK7 does not specifically inhibit CA-responsive lipolysis, but suppresses global lipolysis by downregulating adipose lipases.
ALK7 is significantly expressed in BAT and its deficiency reduces the cell size of brown adipocytes in T.B-Nidd5/3 mice [12, 17]. The specific function in BAT was investigated in mice lacking ALK7 in brown adipocytes expressing Cre recombinase under the regulatory sequence of the Ucp1 gene [26]. The knockout mice show no apparent phenotypes under a normal feeding condition, but display reduced BAT mass and cold exposure-induced hypothermia after 14 h of fasting. They did not show the increased level of β-ARs or HSL phosphorylated by protein kinase A, the mediator of CA effects, in BAT, in contrast to the findings in WAT of ALK7-knockout mice fed regular chow or HFD [23]. The underlying mechanism was suggested to be fasting-induced upregulation of KLF15 and its targeting genes regulating amino acid catabolism in BAT. However, this scenario postulates the opposite regulation and function of KLF15 between WAT and BAT, because KLF15 expression is decreased by fasting in WT white adipocytes, and because mice lacking KLF15 in both white and brown adipocytes display decreased WAT mass with an increased level of a phosphorylated form of HSL and accelerated lipolysis [27]. Instead, the common mechanism might explain the reduced BAT mass by ALK7 deficiency, because ALK7-knockout mice exhibit elevated basal lipolysis with increased C/EBPα and ATGL expression in BAT [26], as found in ALK7-deficient WAT [12]. In any case, it should be kept in mind that this BAT-specific ALK7-knockout mouse may display phenotypes indirectly or partly reflecting ALK7 deficiency in WAT, because UCP1 is expressed in ‘beige’ or ‘brite’ adipocytes in WAT [28], especially after fasting or cold exposure. In fact, we found that UCP1 is induced in WAT of mice treated with ALK7 neutralizing antibody [13].
Activin AB and activin B, but not activin A, activate ALK7 signaling in heterologous cells [5]. Furthermore, exogenously added activin B decreases expression of β-ARs, HSL, and PPARγ in adipocytes derived from WT mouse embryonic fibroblasts, but not in those from ALK7-deficient fibroblasts [23]. However, there is no direct in vivo evidence that activin AB or B functions as an ALK7 ligand. When fed regular chow, activin B-knockout mice in a mixed 129/Sv-C57BL/6 background exhibit less body weight and fat mass with age [29], whereas ALK7-knockout mice exhibit normal body weight and fat mass [10, 11]. Other activin family members may also function as ALK7 ligands. Recently, activin C was shown to signal through ALK7 by cell-based reporter assays [30]. It also stimulated phosphorylation of Smad2/3 and downregulation of ATGL in mature adipocytes, although it was not confirmed whether these effects are lost in ALK7-deficient adipocytes. It remains unknown from which cells activin B or C is derived to act on ALK7 in adipocytes. On the other hand, mutations of genes encoding hepatokine, activin E, and ALK7 are commonly associated with fat distribution and diabetes risk in humans [31, 32], although there is no direct biochemical evidence for their ligand-receptor relationship.
GDF3 activates ALK7 signaling in heterologous cells in the presence of the coreceptor Cripto [11, 25]. Furthermore, recombinant GDF3 protein phosphorylates and activates Smad3 and suppresses lipolysis only in ALK7-intact adipocytes, but not in ALK7-deficient adipocytes [25]. Although a high dose (400 ng/mL) of commercially available recombinant GDF3 protein was used in this ex vivo experiment, it was also shown that ~100 pmol/L of endogenous GDF3 released into the supernatant of cultured ATMs is enough to activate Smad3 and to inhibit lipolysis in ALK7-intact adipocytes, but not in ALK7-deficient adipocytes. The recombinant GDF3 may be less active than the endogenous GDF3, because it is not well understood how GDF3 is processed to the active form [33, 34]. In fact, a native GDF3 construct was poorly processed in HEK293T cells, and the prodomain of GDF3 was replaced with that of bone morphogenic protein (BMP) 2 to produce mature GDF3 protein more efficiently [35].
GDF3 is specifically upregulated in WAT of WT mice fed an HFD [36], and GDF3-knockout mice show partial resistance to HFD-induced obesity [11, 36]. The finding that GDF3-knockout mice show similar, but slightly milder, phenotypes compared with ALK7-knockout mice might suggest the presence of other ligands, such as activins, in vivo. However, Bu et al. showed that GDF3 is primarily expressed in CD11c+ adipose tissue macrophages (ATMs), and that depletion of macrophages including GDF3-producing cells by clodronate treatment upregulates PPARγ, C/EBPα, ATGL, and HSL and reduces fat mass specifically in ALK7-intact TSOD mice, but not in ALK7-deficient T.B-Nidd5/3 mice [25]. Because nonspecific macrophage depletion highlights the ALK7-dependent differences, the GDF3-ALK7 signaling pathway could represent a major link between macrophages and adipocytes in the regulation of whole-body lipid metabolism and fat accumulation. GDF3-producing ATMs are markedly decreased in WAT of both ALK7-deficient mice [25] and ALK7-intact mice treated with ALK7 neutralizing antibody [13]. Mechanistically, ALK7 signaling upregulates release of S100A8/A9 protein from adipocytes, which subsequently activates NLRP3 inflammasome and the downstream gene product, interleukin-1β (IL-1β), and upregulates GDF3 in ATMs [13] (Fig. 2). The presence of such a reciprocal pathway from ALK7 to GDF3 also suggests a functional link between them. These findings support the view that GDF3 is a principal candidate for an ALK7 ligand, at least in WAT.
ALK7 ligands and external and internal factors regulating their expression
Activin AB, B, and C can activate ALK7 signaling both in heterologous cells and in adipocytes [5, 23, 30], although it is unknown from which cells these activins are derived to act on ALK7 in vivo. Variants in activin E produced from liver and ALK7 are associated with a lower waist-to-hip ratio in humans [31, 32], although there is no direct evidence for the ligand-receptor relationship between them. GDF3 can also activate ALK7 signaling both in heterologous cells and in adipocytes [11, 25]. The effects of GDF3 on lipid metabolism and fat mass are absent in ALK7-deficient adipocytes [25]. GDF3 is produced from CD11c+ ATMs [25] and is upregulated under nutrient-excess conditions [36]. PPARγ is necessary for the expression of GDF3 in macrophages [49]. Insulin upregulates GDF3 specifically in ATMs, but not in macrophages from other tissues [25]. NLRP3 inflammasome and the downstream IL-1β are also involved in GDF3 expression [13, 37]. S100A8/A9 protein released from adipocytes by ALK7-dependent signals also upregulates GDF3 via the receptor for advanced glycation end products (RAGE) in ATMs, which forms the reciprocal positive feedback pathway from ALK7 to GDF3 [13].
However, there are reports suggesting that GDF3 may act through other receptors in WAT. Camell et al. showed that fasted aged mice display a reduction in adipocyte lipolysis, which is accompanied with NLRP3 inflammasome activation and GDF3 upregulation in ATMs [37]. Furthermore, deletion of GDF3 in inflammasome-activated macrophages improves lipolysis by decreasing levels of monoamine oxidase A (MAOA), which degrades CA synthesized by the neighboring sympathetic nerve. Therefore, GDF3-induced MAOA in ATMs appears to suppress CA-stimulated lipolysis in adipocytes by increasing CA degradation, rather than by inducing CA-signaling resistance. This cell-autonomous action of GDF3 is unlikely to be mediated via ALK7, because ATMs do not express ALK7 [12, 23]. However, it is notable that the fasted aged mice in this study showed decreases in ATGL and HSL expression, as well as the level of phosphorylated HSL, in WAT, which implies the presence of ALK7 activation in adipocytes. Because ALK7-dependent signals from adipocytes is critical for GDF3 production in ATMs as described above (Fig. 2), the observed blunt lipolysis due to upregulation of GDF3 and MAOA in ATMs might also be indirectly induced by the ALK7 activation in neighboring adipocytes.
GDF3 is also proposed to functions as an inhibitor of BMPs possibly by its extracellular interaction with BMPs, as originally proposed during vertebrate development [38]. Hall et al. showed that HEK293T cells expressing GDF3 do not activate Smad2/3 phosphorylation, but instead reduce Smad1/5/8 phosphorylation, whereas these cells expressing activin B activate Smad2/3 phosphorylation, but do not affect Samd1/5/8 phosphorylation [39]. Furthermore, HEK293T and C2C12 myoblasts expressing GDF3 inhibit the activation of the Smad1/5/8 pathway monitored by luciferase reporter containing the BMP-responsive element. However, these cells may not express ALK7 and/or Cripto at significant levels. At least in HEK293T cells, exogenous expression of these receptors is required to detect GDF3-dependent activation of a luciferase reporter containing the Smad2/3-responsive element [25]. Furthermore, HEK293T cells do not process GDF3 properly and efficiently, as described before [35]. It remains unknown whether GDF3 inhibits Smad1/5/8 in adipocytes, but if this was the case, it is also unknown just what kinds of BMPs in the medium or BMP receptors on adipocytes the GDF3 traps or binds. A recent work using inhibitors of Smad signaling suggests that activation of Smad2/3 may lead to inhibition of Smad1/5/8, which in turn suppresses PPARγ expression and 3T3-L1 adipogenesis [20]. If this is the case, the inhibition of Smad1/5/8 by GDF3 does not need to postulate other pre-existing BMP signals.
As described above, GDF3 is expressed in ATMs. Camell et al. showed that GDF3 is strongly downregulated in NLRP3-deficient ATMs [37]. As shown in Fig. 2, we also found that activation of NLRP3 inflammasome and the downstream IL-1β in ATMs are involved in the reciprocal positive feedback signals between GDF3 and ALK7. NLRP3-induced GDF3 may play a critical role in forming an inflammatory and insulin resistant state under nutrient-excess conditions.
Bu et al. showed that ATMs specifically and significantly express insulin receptor (IR), in contrast to macrophages derived from other tissues, and that a physiological concentration of 61 pmol/L of insulin administered ex vivo induces GDF3 in ATMs [25]. These findings raise the possibility that insulin inhibits lipolysis and accumulates fat indirectly by upregulation of GDF3 in ATMs. Consistently, the in vivo effects of insulin to suppress lipolysis and to accumulate fat were eliminated in ALK7-deficient mice and WT mice receiving transplantation of GDF3-deficient bone marrow, which provides direct evidence that both ALK7 and GDF are required for this insulin action in vivo. Although insulin is generally believed to regulate lipolysis by directly acting on adipocytes, Bu et al. found that a much higher concentration (25 nmol/L) is required for insulin to inhibit lipolysis in adipocytes ex vivo. In fact, high concentrations (1–100 nmol/L) of insulin have been administered to adipocytes to detect suppression of the cAMP-mediated signaling pathway [40-43] or the adipose lipase expression [44-46]. Given that a physiological concentration of serum insulin is less than 1 nmol/L, the direct action of insulin in adipocytes is considered to occur only at a sufficient concentration of insulin. These findings reveal a previously unrecognized mechanism of insulin to inhibit lipolysis via the GDF3-ALK7 signaling in WAT, and indicate that ATMs not only modulate inflammation or insulin sensitivity in adiposity but directly regulate fat metabolism and mass. The indirect action may also function in humans, because insulin infused into human subjects has been reported to suppress lipolysis at markedly low concentrations (half-maximal suppression at 101 pmol/L) [47]. It is possible that this highly insulin-sensitive pathway in ATMs may induce insulin resistance in other tissues by promoting fat accumulation and inflammatory reactions.
Hall et al. found that GDF3 expression in WAT is markedly reduced in knockin mice, in which serine 273 of PPARγ is replaced with alanine when fed an HFD [39]. The phosphorylation of PPARγ at this site induced by HFD in WT mice is thought to be independent of PPARγ receptor agonism [48]. However, it should be noted that PPARγ itself is necessary for the expression of GDF3 in macrophages [49]. Furthermore, the epigenetic regulator, BRD4, bound to the promoter and enhancers of GDF3 facilitates PPARγ-dependent GDF3 expression in bone marrow derived macrophages [50]. Moreover, like ALK7-deficient mice, myeloid lineage-specific BRD4-knockout mice under HFD display decreased GDF3 expression in ATMs and less fat accumulation with elevated lipolysis in WAT. Therefore, the straightforward interpretation of these data is that PPARγ upregulates GDF3 via its receptor agonism, and that the serine 273 phosphorylation selectively increases the transcription of GDF3 in ATMs possibly via interaction with other co-regulators. This view may explain why ex vivo administration of the PPARγ ligand rosiglitazone, which inhibits this phosphorylation [48], partially reduced HFD-induced upregulation of GDF3 in WAT of WT mice [39]. It should be noted that, although GDF3 can downregulate PPARγ via ALK7 in adipocytes, it cannot do so in ATMs that do not express ALK7.
ALK7-deficient mice display no apparent fatal or serious abnormalities when fed a regular chow, and show a significant reduction in fat mass when fed an HFD. Therefore, anti-ALK7 therapy may be a beneficial treatment for obesity and associated diabetes. Furthermore, it activates PPARγ selectively in adipocytes, which may avoid adverse events induced by systemic activation of PPARγ, as in the case of thiazolidinedione administration in humans, including weight gain and fat accumulation [51]. To explore the promise of anti-ALK7 therapy, a neutralizing monoclonal antibody toward the extracellular domain of ALK7 (ALK7 mAb) that blocks binding of the endogenous ligands was administrated to obese mouse models (Fig. 3). ALK7 mAb did not alter food intake, but potently and specifically reduced adiposity in ALK7-intact TSOD mice to levels equivalent to those found in ALK7-deficient T.B-Nidd5/3 mice [13]. Furthermore, ALK7 mAb-treated TSOD mice showed a markedly decreased number of GDF3-producing ATMs, as found in T.B-Nidd5/3 mice without antibody treatment, by the previously described mechanism (Fig. 2). Therefore, this therapy not only blocks ALK7 signaling but also suppresses production of its ligand, GDF3, which may further strengthen its efficiency to reduce adiposity. Similar significant effects of this antibody treatment were observed in outbred ddY mice fed an HFD. Lipids released from adipocytes by elevated lipolysis could be redistributed to other tissues, as liver steatosis is observed in aged or HFD-fed ALK7-knockout mice [10, 11]. In fact, the antibody treatment for 6 weeks increased hepatic TG content. However, the longer treatment for 15 weeks largely eliminated the difference in liver. Histological examination revealed no signs of steatosis, fibrosis, or inflammation. Furthermore, the antibody-treated mice exhibited increased O2 consumption and preferential lipid usage, as found in ALK7-deficient mice [12]. Skeletal muscle showed elevated FA oxidation with upregulation of genes involved in this pathway, including PPARα and PPARδ. Because ATGL-dependent WAT lipolysis has been shown to control PPARα activity in liver [52], a similar mechanism may function in muscle of ALK7 mAb-treated mice. The obese mice treated for 18 weeks displayed significant improvement in glucose tolerance or insulin sensitivity, although those treated for 6 weeks showed no such changes. Elevated lipolysis and the resultant increase in circulating FA levels are often thought to reduce insulin sensitivity in peripheral tissues and insulin secretion in pancreatic β cells, which has been termed ‘lipotoxicity.’ However, ALK7 mAb treatment did not increase circulating FA levels, possibly because lipid release from adipocytes is efficiently used in peripheral tissues and declines as fat mass decreases. Overall, although ALK7 mAb treatment may temporarily lead to ectopic fat accumulation in non-adipose tissues, it eventually reduces fat mass and improves insulin sensitivity and glucose tolerance.
ALK7 as a target of obesity therapy
Treatment with ALK7 neutralizing antibody potently reduced adiposity and significantly enhanced insulin sensitivity and glucose tolerance in both genetic and dietary obese mouse models [13]. FA released from adipocytes by elevated lipolysis was efficiently broken down by FAO in muscle. Anti-ALK7 therapy is expected to make mature large adipocytes smaller by elevated lipolysis, which leads to increased lipid turnover, changes in cytokine repertoires in adipocytes and ATMs, and suppression of chronic inflammation [18].
It has been reported in obese human subjects that adipose tissue expression of ALK7 is downregulated, whereas that of inhibin-βB, which forms activin B, is upregulated [14], although it remains unknown whether these changes contribute to development of obesity or occur secondarily. Direct genetic evidence in humans has come from a study reporting that three heterozygous ACVR1C variants with missense mutations in the intracellular domain of ALK7 (N150H, I195T, and I482V), as well as one noncoding variant, rs72927479, are associated with reduced waist-to-hip ratio adjusted for body mass index (WHRadjBMI) and a lower risk of types 2 diabetes [53], although the functional consequences of these variants are unknown. The same study also showed that the missense variants in PNPLA2 and ABHD15 are associated with higher WHRadjBMI. Because PNPLA2 and ABHD15 encode ATGL and a possible lipase with the alpha beta hydrolase domain [54], respectively, their dysfunction is expected to result in decreased lipolysis. These findings are consistent with the notion that fat mass and distributions are regulated by lipolysis.
Association of protein-coding variants in the ACVR1C gene with WHRadjBMI was confirmed by multiple studies [31, 32, 55]. Furthermore, loss-of-function variants in the INHBE gene that encodes activin E, which is highly and specifically expressed in hepatocytes, are not only associated with lower WHRadjBMI but also with protection from diabetes and liver damage traits [31, 32], although it is unknown whether activin E functions as an ALK7 ligand. These findings suggest that ALK7 and its potential ligands play a conserved regulatory role in lipid metabolism and fat mass between rodents and humans, and could be a target for therapy of obesity and/or diabetes in humans. ALK7 inhibition in humans influences body fat distribution, but does not appear to have a major effect on body weight itself, as found in ALK7 mAb-treated obese mice. This reflects the fact that a blockade of ALK7 signals does not directly alter energy intake or expenditure, but does induce preferential utilization of lipids among nutrients. It should also be noted that ALK7 deficiency does not constitutively activate lipolysis, but differentially promotes TG synthesis and breakdown via upregulation of C/EBPα and PPARγ in a signal- and context-dependent manner [12, 18]. Although anti-ALK7 therapy may not have a major effect on body weight reduction, the most important purpose of obesity treatment is prevention of obesity-associated metabolic diseases, except in cases with extreme obesity. From this point of view, anti-ALK7 therapy should produce beneficial effects, because higher WHRadjBMI is strongly associated with increased risk of both type 2 diabetes and coronary heart disease [56].
TGF-beta superfamily members play fundamental and versatile roles in development. A total of 33 mammalian ligands are considered to bind seven type I and five type II receptors. Thus, deficiency in any TGF-beta receptor family member could induce phenotypes reflecting dysfunction of its multiple ligands. Indeed, knockout of these receptors in mice is often lethal or results in serious developmental abnormalities [57]. By contrast, ALK7-deficient mice display no major abnormalities, but exhibit protection from obesity and associated diabetes only under nutrient-excess conditions. Furthermore, blockage of ALK7 signals differentially control lipid usage and storage dependent upon the existing fat mass, which should not cause unidirectional changes in fat mass, such as lipodystrophy or extreme adiposity. This unique feature makes anti-ALK7 therapy a safe and promising treatment for obesity and diabetes. Of course, careful consideration is required prior to application in humans, because potentially undesirable side effects of ALK7 inhibition have been reported in ALK7-knockout C57BL/6 mice even when fed regular chow, which included conditions such as liver steatosis [10], female reproductive changes [58], and cardiac abnormalities [59, 60]. It is currently unknown whether these adverse effects are specific to C57BL/6 mice. Apart from these clinical considerations, it should be emphasized that this signaling axis, involving insulin from pancreatic β cells, GDF3 from ATMs, and ALK7 on adipocytes, plays important physiological roles in storing excess nutrient as fat. Further investigation is required to more fully elucidate the complete regulatory mechanisms in vivo.
T.I. wrote this review and takes responsibility for the integrity and the accuracy of the content.
I would like to appreciate help from Sachiko Shigoka for preparing a manuscript and the contribution of scientific findings generated by previous and current members of my laboratory. The author received research support from Eli Lilly Japan K.K., and Teijin Pharma Donation.
The author declares no conflict of interest.
Data sharing not applicable to this review article, because no datasets were generated or analyzed during the current study.
Not applicable.
