Insulin Pathway
Miran Kim, Jack R. Wands
9
9.1
Introduction
Insulin is the principal hormone controlling blood glucose levels. Insulin stimulates the uptake of glu- cose, amino and fatty acids into cells and increases the expression and/or activity of enzymes that en- hance glycogen, lipid and protein synthesis, while inhibiting the activity or expression of those en- zymes that catalyze degradation of glycogen [47].
The increase in circulating insulin levels stimu- lates glucose transport into peripheral tissues and inhibits hepatic gluconeogenesis. Decreased secre- tion of insulin, coupled with tissue resistance re- sults in type 2 diabetes and also is associated with central obesity, hypertension, polycystic ovarian syndrome, dyslipidemia and atherosclerosis. In ad- dition, insulin has a role as a hepatotrophic factor and promotes hepatocyte proliferation, although the mechanisms by which it stimulates liver growth are not completely understood. At the cellular level, insulin action is characterized by diverse effects, including changes in vesicle trafficking, stimula- tion of protein kinases and phosphatases, promo- tion of cellular growth and differentiation, as well as activation or repression of gene transcription [4, 63]. The stimulation of the insulin/insulin receptor substrate-1 (IRS-1) system activates a number of in- tracellular signaling cascades that ultimately lead to important downstream biologic effects critical for cell function (Fig. 9.1). This complexity of cel- lular actions implies that insulin stimulation must involve multiple signaling pathways that diverge at or near the activation of receptor tyrosine kinase.
Indeed, it is likely that even individual effects of the hormone require the activities of multiple signal- ing cascades. Although understanding of the signal transduction pathways that underlie insulin’s major physiologic effects is still incomplete, remarkable advances have occurred in the last decade. It is now clear that activation of insulin receptor tyrosine ki- nase, acting through the insulin receptor substrate (IRS) proteins as multisite docking molecules, cre-
ates binding sites that enable the IRSs to recruit and activate multiple, independent intracellular signal generators [68]. In this chapter, we discuss some of the known structural and functional features of the insulin receptor and IRS proteins and focus on re- cent advances in understanding of the role of IRS proteins in insulin signaling effects. We will sum- marize the evidence regarding the potential role of IRS-1 in the pathogenesis of hepatocellular car- cinoma and explore insulin action on hepatocyte proliferation and liver development in the setting of chronic ethanol abuse.
9.2
Insulin Receptor
The insulin receptor, a tetrameric glycoprotein com- posed of two α- and β-subunits, is highly expressed in adipocytes and hepatocytes. The α-subunit com- prises the extracellular domain and contains the
Fig. 9.1. Insulin produces diverse biological effects on cells through the insulin receptor and downstream signal transduc- tion cascades
ligand binding site(s), whereas the intracellular portion of the β-subunit has tyrosine kinase activ- ity. The unoccupied α-subunit inhibits the tyrosine kinase activity of the β-subunit and removal of the α-subunits by deletional mutagenesis reverses this inhibition. Tyrosyl autophosphorylation after re- ceptor–ligand interaction is a key mechanism that activates insulin signaling pathways. These cascades transmit the insulin signal by promoting binding to Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of downstream signaling molecules such as IRS-1, IRS-2, and SH2 domain-containing (Shc) proteins, and growth factor receptor-bound protein 10 (Grb10) [16, 17, 36, 58]. Thus, it is clear that the intrinsic tyrosine kinase activity of the in- sulin receptor is essential for insulin action. Natu- ral occurring mutations of the insulin receptor in humans may cause partial inhibition of tyrosine kinase activity and are associated with severe insu- lin resistance. Without insulin receptors, mice die shortly after birth, while humans survive for a short time with severe growth retardation and diabetes [42].
9.3
IRS Proteins
9.3.1 Overview
The IRS proteins function as insulin receptor-spe- cific docking proteins that engage multiple down- stream signaling molecules. These proteins contain several common structural features: (1) an N-termi- nal pleckstrin homology (PH) and/or PTB domains that mediates protein–lipid or protein–protein in- teractions; (2) multiple C-terminal tyrosine residues that create SH2-protein binding sites; (3) proline- rich regions to engage SH3 or WW domains; and (4) serine/threonine-rich regions, which may regulate overall IRS function through other protein–protein interactions [52]. Such functional domains amplify receptor signals by directly recruiting SH2 proteins to their phosphorylation sites. These adaptor pro- teins also dissociate the intracellular signaling com- plex from endocytic pathways that are involved in the recycling of the insulin receptor. This property may be especially important for insulin-stimulated biological effects such as glucose uptake.
Table 9.1. Summary of IRS function as determined by knockout mouse models
Gene Phenotype Reference
IRS-1 Significant growth inhibition [1, 41, 58, 71]
Mild insulin resistance, glucose tolerance does not develop due to compensatory hyperinsulinemia
IRS-2 Insulin resistance in muscle and liver coupled with abnormal β-cell development lead to type 2 diabetes
[25, 26, 68]
Males develop dehydration, hyperosmolar coma leading to death
IRS-3 Normal growth, normal glucose tolerance [30]
IRS-4 Mild defects in growth in male mice [13]
Mild defects in reproduction, slight impairment in glucose homeostasis
IRS-1/IRS-3 Lipoatrophic diabetes [28]
9.3.2
Members of the IRS Protein Family
Six IRS proteins have been identified that differ with respect to tissue distribution, subcellular localiza- tion, developmental expression, binding to the in- sulin receptor, and interaction with SH2 domain- containing proteins. Although the IRS proteins are highly homologous, they serve complementary roles in insulin signaling rather than represent redun- dant molecules, as shown by studies in knockout mice (Table 9.1). For example, IRS-1 and IRS-2 are the best characterized members of this family and are widely expressed in muscle, liver, fat, and pan- creatic islet cells [57, 58]. In this regard, IRS-1 null (–/–) mice are stunted in growth but do not develop diabetes because an alternate substrate such as IRS- 2 (pp190) compensates for the lack of IRS-1 in the liver [1, 59, 65]. In contrast, IRS-2 –/– mice develop insulin resistance in the liver and skeletal muscle and lose their ability to regulate glucose homeosta- sis [69]. In contrast, IRS-3 expression is restricted to adipose tissue and β-cells in rodents. This gene has not yet been identified in the human genome [29]. The IRS-4 gene is expressed predominantly in brain, thymus, and kidney, where it may bind to and transmit signals via phosphoinositide 3-kinase (PI3K) and growth factor receptor-bound protein 2 (Grb2)-mediated cascades. The IRS-4 –/– mice ap- pear normal with the exception of reduced fertility [14]. Finally, IRS-5 and IRS-6 are most abundantly expressed in kidney, liver and skeletal muscle, re- spectively [6].
9.3.3 IRS-1
Human (h) IRS-1 was cloned from an overexpress- ing hepatocellular carcinoma (HCC) cell line and serves as the prototype docking protein for the in- sulin receptor. It was initially detected in insulin- stimulated Fao hepatoma cells by immunoprecipi- tation with anti-phosphotyrosine antibody [7]. The IRS-1 protein has a calculated molecular mass of 132 kDa, but due to extensive phosphorylation it mi- grates at 185 kDa on sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) [38].
More important, IRS-1 is tyrosyl phosphorylated by insulin receptor tyrosine kinase activity [60].
Tyrosyl-phosphorylated IRS-1 transduces various growth and metabolic signals through interaction with downstream SH2-containing molecules that bind to specific IRS-1 motifs, namely the p85 subu- nit of PI3K [3, 35], Grb2 [54], SH2 domain-contain-
ing protein tyrosine phosphatase-2 (SHP2 or Syp) [56], and phospholipase C γ (PLCγ) [67].
9.3.4
The IRS-1 Gene
and Hepatocellular Carcinoma
There is evidence to suggest that hIRS-1 may have transforming properties as well as play a prominent role in normal hepatic growth. The hIRS-1 protein found in hepatocytes is highly overexpressed in multiple HCC cell lines and clinical tumor samples.
This observation suggests that hIRS-1 may function as a signal transduction molecule during the molec- ular pathogenesis of HCC [31, 33, 38, 62, 63]. Thus, highly expressed and phosphorylated hIRS-1 may enhance intracellular growth signals and contrib- ute to the multistep process of hepatic oncogenesis.
Direct evidence for this concept was provided by the construction of a dominant negative mutant that in- terfered with endogenous hIRS-1 tyrosyl phosphor- ylation. The C-terminal truncated hIRS-1 molecule (dominant-negative mutant) inhibited tyrosyl phos- phorylation of endogenous hIRS-1 and Shc proteins.
Subsequently the activity of downstream signal- ing molecules such as PI3K and mitogen-activated protein kinase (MAPK) were inhibited. More im- portant, stable transfection of this dominant-nega- tive mutant into HCC cells reversed the malignant phenotype as characterized by inhibition of trans- formed foci formation, loss of anchorage-independ- ent growth in soft agar, inability to form tumors in nude mice, and strikingly reduced cell proliferate activity [64].
The Grb2 and SHP2 proteins also contributed to the cellular transforming activity of hIRS-1. Stable transfection and overexpression of the hIRS-1 gene in NIH 3T3 cells leads to increased hIRS-1 tyrosyl phosphorylation, enhanced binding of Grb2 and SHP2 but not PI3K, and persistent or constitutive activation of the downstream MAPK cascade. Such transfected 3T3 cells develop a phenotype charac- terized by transformed foci formation, induction of anchorage-independent cell growth and increased cell proliferation. When such cells were injected into nude mice, they were highly tumorigenic [20].
Further studies have revealed that hIRS-1-mediated
mitogenic signals are directly regulated by interac-
tion with Grb2 and Syp, and these interactions were
followed by the activation of MAPK cascade [32, 35,
39, 70, 72]. Mutant (Y897F and Y1180F) hIRS-1 con-
structs reduced the intracellular interaction of IRS-
1 with Grb2 and Syp proteins, respectively. Single
IRS-1 mutant molecules did not completely reduce
the insulin-dependent transforming activity. How-
ever, a double mutant (Y897F/Y1180F) construct strikingly attenuated the transforming activity of hIRS-1. Therefore, hIRS-1-induced cellular trans- formation required an interaction with both Grb2 and Syp signal transduction molecules [60].
It is of interest that approximately 40% of human HCC tumors had enhanced hIRS-1 gene expression compared with adjacent non-involved liver tissue.
Moreover, there was a significant relationship be- tween the level of hIRS-1 overexpression and the tumor size. The overexpressed hIRS-1 protein was found to be tyrosyl phosphorylated and interacted with PI3K, Grb2 and SHP2 proteins and there was constitutive activation of both the PI3K and MAPK signal transduction cascades. Moreover, overex- pression of hIRS-1 in the liver of a transgenic mouse model led to increased hepatocyte DNA synthesis.
These investigations support a possible role for hIRS-1 in tumor growth [61].
9.3.5
IRS and Hepatocyte Proliferation
Insulin is one of the regulators of normal hepatocyte proliferation and subsequent liver growth [23, 67].
The cellular pathways responsible for transmitting the insulin-mediated signal from the cell surface to the nucleus in the context of fetal liver growth are under active investigation. Most recently, the role of IRS-mediated growth cascades has been studied in rapid-growing fetal rat liver; expression and tyrosyl phosphorylation of IRS-1 was reduced compared with the adult liver. These developmental changes resulted in a lack of sensitivity to insulin stimula- tion and subsequent downstream activation of the PI3K and MAPK cascades until they become func- tional in the postneonatal period. In contrast, a high level of IRS-2 expression and tyrosyl phosphoryla- tion was present as early as embryonic day 15 with robust PI3K binding and activation, which may en- hance hepatocyte survival during the rapid growth phase of the liver. In addition, IRS-2 was found to propagate the insulin signal via PI3K in the late-ges- tation fetal liver. Therefore, IRS-2 is the dominant substrate for insulin receptor kinase activity with respect to tyrosyl phosphorylation and downstream PI3K pathway activation during fetal life and may enhance hepatocyte survival signals. These investi- gations lead us to believe that IRS-1 may have a ma- jor role in the adult liver with respect to mediating hepatic growth via the MAPK pathway [24]. How- ever, the IRS-1 signal transduction pathway does not play a major role in fetal liver growth because IRS-2 functions as the major insulin-responsive molecule.
During liver regeneration induced by partial hepatectomy, there was tyrosyl phosphorylation of the insulin receptor β-subunit and IRS-1 followed by an association with PI3K; these events occurred prior to the onset of DNA synthesis in the late G
0phase of the cell cycle [48]. In another setting, IRS- 1 protein was significantly increased in cirrhosis compared to normal liver, which may favor en- hanced hepatic growth [55]. In the early stage of rat liver regeneration, IRS-1 expression was increased, a finding consistent with a stimulatory role in the regenerative process, whereas it returned to baseline levels 7 days later when the hepatic growth process was complete. The reduced IRS-1 level occurred in the setting of increased IRS-2 and IRS-4 expres- sion. Given that 1 and 7 days after partial hepate- ctomy, isolated hepatocytes responded similarly to insulin in terms of cell proliferation, a compensa- tory role was proposed for the induction of IRS-2/4.
Since IRS-4 is activated by insulin stimulation of rat hepatocytes, it seems likely that expression and tyrosyl phosphorylation of IRS-4 was a compensa- tory mechanism to augment liver regeneration. In support of this argument was the association of IRS- 4 with PI3K, SHP2, and protein kinase C ζ (PKCζ), subsequently to transmit the insulin signal [13].
9.3.6
Insulin Signaling Pathways Through IRS Proteins
While more detailed information regarding the in- sulin signaling cascade is provided elsewhere (see Chaps. 19 and 20), we will present a synopsis of the key steps that result from insulin action (Fig. 9.2).
PI3K, one of the SH2 domain-containing molecules,
interacts with tyrosyl phosphorylated IRS proteins,
thereby activating this enzyme [53, 66] to generate
phosphatidylinositol-3,4,5-triphosphate (PIP3). In-
creasing PIP3 concentrations bring protein kinase
B (PKB)/Akt into proximity with another PH-do-
main-containing protein kinase, namely phosph-
oinositide-dependent kinase 1 (PDK1), resulting in
Akt phosphorylation at residues The 308 and Ser
473 [49, 69]. The identification of Akt substrates
has been of great interest to understand the mecha-
nisms by which this kinase impacts cell growth and
programmed cell death pathways. The glycogen
synthase kinase 3 β (GSK3β) protein is a ubiquitous-
ly expressed serine/threonine protein kinase and is
one of the principal Akt substrates [2, 21]. The Akt-
induced phosphorylation of GSK3 β results in GSK3β
inactivation and leads to decreased phosphoryla-
tion and increased glycogen synthase activity [15,
49]. In addition, GSK3 β overexpression elicits ap-
optosis that can be blocked by Akt-mediated GSK3 β phosphorylation [41]. Akt has also been implicated in the regulation of Ras protein-specific guanine nucleotide-releasing factor 2 (Raf) and provides possible crosstalk between PI3K and MAPK signal transduction cascades. Other recently identified targets of Akt include Bcl2-antagonist of cell death (BAD), forkhead box protein 01A (FKHR), forkhead box protein 03A (FKHRL1), forkhead box protein 04 (AFX), endothelial nitric oxide synthase (eNOS) and mammalian target of rapamycin (mTOR) [49].
Activation of the MAPK/endothelial signal-reg- ulated kinase (ERK) pathway is another major effec- tor mechanism for insulin action [66]. This pathway involves the tyrosine phosphorylation of IRS pro- teins and Shc [16], which interact with Grb2 thereby recruiting Son-of-Sevenless (SOS) exchange protein to the plasma membrane for activation of Ras. The activation of Ras also requires the stimulation of SHP2 through its interaction with Grb2-associated binding protein 2 (Gab-1) or IRS1/2. Once activated, Ras operates as a molecular switch stimulating a ser- ine kinase cascade through the stepwise activation of Raf, MAPK/ERK kinase 1 (MEK) and ERK. Ac- tivated ERK can translocate into the nucleus where it catalyzes the phosphorylation of transcription
factors such as p62
TCFimportant for initiating gene expression required for cellular proliferation. Block of this pathway with dominant-negative mutants or pharmacological inhibitors prevents cell growth in- duced by insulin signaling but has no effect on the metabolic actions of this hormone [47].
Protein kinase C ε (PKCε) and protein kinase C δ (PKCδ) are involved in the downregulation of insulin signaling through IRS-1. In HepG2 human HCC cells, treatment with high glucose concentra- tions resulted in phosphorylation of serine residues on IRS-1. The high glucose treatment attenuated the insulin-induced association of IRS-1 with PI3K and downstream phosphorylation of Akt. This phenom- enon was associated with the translocation of PKC ε and PKC δ from the cytosol to the plasma membrane in association with IRS-1. In contrast, insulin-in- duced association of Shc and Grb2 to the insulin re- ceptor was not inhibited. Therefore, PKC ε and PKCδ may function as inhibitors of the insulin signaling pathway via regulating the phosphorylation of IRS- 1 [37].
Fig. 9.2. Cartoon of the insulin signaling pathways through the IRS proteins. Note the importance of signaling through the PI3K and MAPK/ERK cascades
9.4
Inhibition of Insulin Signaling
Insulin signaling can be inhibited by several mech- anisms including phosphotyrosine dephospho- rylation, serine/threonine phosphorylation, and degradation of IRS proteins. The protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. For exam- ple, PTP1B overexpression in L6 myocytes and Fao HCC cells blocked tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and PI3K. Thus, there was inhibition of downstream Akt and MAPK phosphorylation as well [12]. Reduction of PTP1B protein expression by specific antisense oligodeoxynucleotides in Fao cells also increased insulin-stimulated phosphorylation of Akt and GSK3 β without any noticeable change in protein expression levels. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor [8]. In insulin-resistant rats, the increase of PTP1B expression and interaction with the insulin recep- tor contributed to impaired insulin signaling in the liver [18]. In addition, serine/threonine phosphor- ylation of IRS proteins decreases tyrosyl phosphor- ylation and thereby attenuates insulin signaling [19, 22, 26]. Liver from insulin-resistant rodents showed enhanced serine kinase activity for IRS-1 as demon- strated by a specific Ser789-phosphorylation of IRS- 1 [43, 44]. However, the identity of the serine kinase responsible for this specific phosphorylation event is still not known. Interleukin-6 (IL-6) is one of the several proinflammatory cytokines that have been associated with insulin resistance and type 2 diabe- tes. IL-6 exposure reduces tyrosine phosphorylation of IRS-1 and the association with PI3K in both pri- mary mouse hepatocytes and the HepG2 cells [50].
Suppressors of cytokine signaling (SOCS) proteins are induced by inflammation. Among them, SOCS1 or SOCS3 targets IRS-1 and IRS-2 for ubiquitin- mediated degradation and therefore blocks insulin signaling. Indeed, SOCS1 and SOCS3 were found to bind both recombinant and endogenous IRS-1 and IRS-2 protein and promote their ubiquitination and subsequent degradation in multiple cell types [46].
Interestingly, the IL-6-dependent induction of in- sulin resistance is mediated by SOCS proteins. In mice exposed to IL-6 hepatic SOCS3, expression was increased and it was associated with inhibition of insulin-dependent insulin receptor autophosphor- ylation and IRS-1 tyrosyl phosphorylation as well.
Induction of SOCS3 in liver may be an important mechanism to explain IL-6-mediated insulin resist-
ance [51]. Moreover, ubiquitin/proteasome-medi- ated degradation of IRS-2 but not IRS-1 in L1 and Fao HCC cells occurs via a PI3K/Akt-dependent pathway and is closely associated with inhibition of insulin signaling [45]. Ethanol also affects insulin signaling through inhibition of IRS-1 tyrosyl phos- phorylation. Therefore, ethanol reduces the interac- tion between Syp and tyrosyl phosphorylated IRS-1 [5, 34, 71]. High ethanol intake is considered a ma- jor factor for impaired insulin sensitivity. Acute and chronic ethanol-exposed rats resulted in reduced tyrosyl phosphorylation of insulin receptors, IRS- 1 and IRS-2 proteins. In addition, chronic ethanol exposure impairs survival mechanisms in the liver because of its inhibitory effect on insulin signaling through PI3K/Akt. Finally, chronic ethanol con- sumption increases the hepatocyte levels of phos- phatase and tensin homolog deleted on chromo- some 10 (PTEN), a major negative regulator of the PI3K/Akt signal transduction cascade [40, 73].
9.5
Insulin and Growth Hormone
The insulin signaling pathway is also linked to growth hormone (GH). Excess GH is associated with secondary hyperinsulinemia through alterations of the early steps of insulin action in the liver. Insulin receptors were reduced in a transgenic mouse model that overexpressed GH whereas insulin receptor and IRS-1 phosphorylation, the IRS-1/PI3K interaction and PI3K activity appeared to be maximally acti- vated. Under these conditions, it was not possible to further stimulate this signal transduction cascade in vivo due to a complete insensitivity to insulin action [9]. On the other hand, GH deficiency was associated with increased tissue sensitivity to insu- lin. In the liver of growth hormone receptor (GHR)-
"knockout" mice, the lack of GH effects was associ- ated with increased insulin receptor abundance and enhanced autophosphorylation following insulin binding. These alterations may represent an adap- tation to the low insulin concentrations leading to or contributing to increased insulin sensitivity [10].
The antagonistic action of GH on insulin signaling is not a consequence of a direct interaction with the insulin receptor. Instead, long-term exposure to GH leads to a reduction of insulin receptor levels and an impairment of tyrosine kinase activity. The signals induced by GH and insulin may converge on down- stream post-receptor proteins. Activation of PI3K appears to be an important site of convergence be- tween the signals generated by these two hormones.
Rodent models of chronic GH excess have been
useful to investigate the mechanisms by which GH induces insulin resistance. Decreased insulin re- ceptor, IRS-1, and IRS-2 tyrosyl phosphorylation in response to insulin stimulation was found in skel- etal muscle, whereas a chronic activation of the IRS/
PI3K pathway was found in liver. The induction of proteins that inhibit insulin receptor signaling such as SOCS-1 and -6 may also be involved in GH-medi- ated effects. The mechanisms of insulin resistance induced by GH involve uncoupling between PI3K and its downstream signaling mediators. Finally, GH may modulate the lifespan of cells by altering insulin sensitivity [11].
9.6
Perspectives
The molecular mechanisms of insulin action on cells have been under intense investigation. In this context, efforts to understand insulin effects on tis- sues have led to the discovery of the insulin receptor, its primary role as a tyrosine kinase, and more im- portantly how this tyrosine kinase phosphorylates IRS proteins, especially IRS-1. It is now appreciated that tyrosyl phosphorylated IRS-1 acts as a docking protein. It binds to and activates several cytosolic signaling molecules important in mediating down- stream growth and metabolic effects. These major accomplishments aid our understanding of the mo- lecular mechanisms involved in the insulin-signal- ing network, as exemplified in Fig. 9.2. Future efforts will need to focus on determining how the various IRS-1-associated proteins mediate growth signals related to the multistep process of hepatocarcino- genesis. Other significant areas of research include defining the role of chronic ethanol consumption on phosphorylation of IRS proteins in an attempt to understand better the inhibitory effect of ethanol on liver growth. Therefore, understanding insulin ac- tion may have direct relevance to the pathogenesis of acute and chronic liver diseases as well as the de- velopment of hepatocellular carcinoma.
Acknowledgments
This work was supported in part by NIH grants CA- 35711 (JWR) and COBRE RR-P20RR017695 (MK).
Selected Reading
White MF. The insulin signaling system and the IRS proteins.
Diabetologia 1997;40:S2–S17. (This paper reviews the insu- lin signaling network and IRS proteins, including functional motifs of IRS-1 and IRS-2.)
Sesti G, Federici M, Hribal ML et al. Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J 2001;15:2099–2111. (This review focuses on the structure and function of IRS proteins, their knockout mouse models.
In addition it reviews physiological roles of IRS-1 and IRS-2 from animal and human studies.)
Saltiel AR, Kahn CR. Insulin signaling and the regulation of glu- cose and lipid metabolism. Nature 2001;414:799–806. (This review article describes the regulation of glucose transport and lipid metabolism by insulin.)
Khamzina L, Gruppuso PA, Wands JR. Insulin signaling through insulin receptor substrate 1 and 2 in normal liver develop- ment. Gastroenterology 2003;125:572–585. (This recent pa- per shows the different role of IRS-1 and IRS-2 in normal liver development.)
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