PI3K, PTEN and AktThomas F. Franke, Daniel C. Berwick

PI3K, PTEN and Akt

Thomas F. Franke, Daniel C. Berwick

20

20.1

Introduction

Research over the past decade has elucidated mo- lecular mechanisms involved in the regulation of Akt kinases by upstream signaling events, exam- ined their importance for the regulation of cellular signaling in response to extracellular stimuli and defined their roles in organismal function. Because of its ubiquitous and evolutionarily conserved func- tion as a key mediator of signals originating from the extracellular environment, Akt activation can result in diverse, even tissue-specific changes in animal and human physiology. While recent results point to novel and specific consequences of Akt activation in brain [194] and heart function [98], a mainstay of Akt function still relates to the regulation of me- tabolism, very often associated with insulin action and diabetes (for review see [197]). Another impor- tant cellular function of Akt concerns its effects on cell viability, which are relevant to the role of Akt in cell growth and survival (for review see [62]). Not surprisingly, because of its involvement in multiple cellular responses, numerous studies have impli- cated Akt in the pathobiology of human disease, including hereditary and sporadic forms of cancer (for review see [119]), metabolic disorders such as diabetes (for review see [172]), neurodegenerative diseases such as Alzheimer’s disease and spinoc- erebellar ataxia (for review see [211]), and cardiac dysfunction (for review see [123]). Here, we aim to provide our readership with an outline of the basic molecular mechanisms that govern the regulation of this signal transduction pathway, and seek to uti- lize this understanding to shed light on its specific implications in normal liver function and clinical liver disease.

20.2

Akt Kinases:

A Family of Homologous Kinases

The Akt gene is the cellular homolog of the v-akt oncogene transduced by AKT8, an acute transform- ing retrovirus in mice that was originally described in 1977 [174]. In 1991, three independent research groups cloned and characterized Akt kinases. The group of Philip Tsichlis identified v-akt as the gene transduced by rodent retrovirus AKT8 [14] and sub- sequently showed that its cellular homolog, then named c-akt, encoded the cytoplasmic serine-thre- onine protein kinase Akt in mice [15]. Also in 1991, two research groups in England and Switzerland identified Akt and similar kinases when searching for novel kinases related to protein kinases A and C [38, 87]. Thus, Akt has also been called RAC-PK (a protein kinase related to protein kinases A and C) and today, Akt is frequently referred to as protein kinase B (PKB; a kinase similar to protein kinases A and C).

All Akt serine/threonine kinases share a com-

mon structure that consists of an N-terminal pleck-

strin homology (PH) domain [56], a hinge region

connecting the PH domain to a kinase domain with

serine/threonine specificity [2], and a C-terminal

region required for the induction and maintenance

of its kinase activity [28]. In mammals, there are

three closely related Akt isoforms, Akt1 (PKB α),

Akt2 (PKB β) and Akt3 (PKBγ), which are encoded

by individual genes. Whereas Akt1 is almost ubiq-

uitously expressed at high levels [15, 38, 87], Akt2

is highly expressed in insulin-sensitive tissues in-

cluding the liver, skeletal muscle and adipose tis-

sue [86, 102]. In addition, the expression of Akt2 is

drastically increased during the differentiation of

adipose tissue and skeletal muscle [74, 191]. Akt3 is

expressed the highest in brain and testis, and exhib-

its lower expression levels in intestinal organs and

muscle tissue [137]. Since different Akt isoforms are

activated by comparable mechanisms and phospho-

rylate similar downstream substrates with almost

equal efficiency, it has been postulated that the dif- ferent Akt isoforms are functionally redundant [61].

Indeed, the fact that animals with deleted expres- sion of single Akt isoforms are viable and lack any severe developmental defects underscores the re- dundancy of Akt signaling and the potential of dif- ferent Akt isoforms to compensate for each other in cells. One explanation for the apparent phenotypic differences observed between Akt1

mice, which are growth retarded but normoglycemic [36], and

mice, which are of normal body weight but insulin resistant and hyperglycemic [35], may be that they are the consequences of altered total Akt expression in specific tissues. However, several re- cent findings point to more subtle functional differ- ences between various Akt isoforms. These include the observation that over-expression of Akt2, but not Akt1, is sufficient to restore insulin-mediated glucose uptake in Akt2

adipocytes [8]; that glu- cose uptake in wild-type adipocytes is potently in- hibited by RNAi knockdown of Akt2 but not Akt1 [84]; and the observation that only the expression of human Akt2 and Akt3 is frequently pathologically increased in human cancer, suggesting the involve- ment of specific Akt isoforms in the onset or propa- gation of cancer [34, 138].

20.3

Phosphatidylinositol 3-Kinases:

Signaling Networks Upstream of Akt

Critical to the understanding of Akt regulation in cells was the finding that Akt kinase activity is in- duced following activation of phosphatidylinositol 3-kinase (PI3K) in growth factor receptor-medi- ated signaling cascades [21, 57, 100]. At the plasma membrane, PI3K phosphorylates phosphoinositides on the D3 position of the inositol ring to generate the second messenger products phosphatidylinosi- tol-3,4-bisphosphate (PI-3,4-P

) and phosphatidyli- nositol-3,4,5-trisphosphate (PIP

), using phosphati- dyinositol-4-phosphate (PI-4-P) and phosphatidyli- nositol-4,5-bisphosphate (PI-4,5-P

) as substrates, respectively (for review see [157]). While it had al- ready been demonstrated that the levels of D3-phos- phorylated phosphatidylinositols change following growth factor stimulation of cells in culture [7], it was not until 1992 that a cDNA for a kinase capa- ble of phosphorylating phosphatidylinositol (PI) at the 3’-OH (D3) position was identified [73]. Subse- quently, PI3K was shown to consist of an 85-kDa regulatory and a 110-kDa catalytic subunit. To date, four catalytic subunits that can phosphorylate PI, PI-4-P and PI-4,5-P

in vitro have been cloned [26,

179]. Class Ia PI3Ks (p110 α, p110β and p110δ) asso- ciate with various regulatory subunits to form active heterodimers with distinct expression and activity.

These regulatory subunits include p85 α, p55α and p50 α, which are all encoded by the pik3r1 gene and have identical C-terminal regions, p85 β encoded by

γ encoded by pik3r3 (for review see [157]).

For class Ia PI3Ks, a simple model for activa- tion by growth factors has been proposed. Upon tyrosine kinase receptor activation, PI3K complexes are recruited to the plasma membrane through the phosphorylation-specific interaction between SH2 domains in their regulatory subunit and the phos- pho-tyrosine residues on the receptor. As a result, the catalytic subunits are stabilized and brought into the presence of phosphoinositides in the plasma membrane, thus resulting in the subsequent genera- tion of D3-phosphorylated lipid products (for re- view see [24]). In the case of class Ib PI3K, only one regulatory subunit (p101) has been shown to inter- act with the p110 γ catalytic subunit. Class Ib PI3K is primarily regulated by small G proteins such as Ras and by the β/γ subunits of G protein-coupled recep- tors (GPCRs). The activation of the p110 γ subunits involves the direct binding of the catalytic subunit to the small G protein Ras [118], and its activity also requires the presence of the p101 subunit [180].

In addition to lipid kinase activity, class I PI3Ks also exert serine/threonine protein kinase activity against protein substrates. However, the physiologi- cal relevance of their protein kinase activity differs significantly between the different members of class I PI3Ks. More specifically, while only a few exam- ples of class Ia-dependent protein phosphorylation such as the phosphorylation of insulin receptor sub- strate (IRS)-1 are known [108], the protein kinase activity of class Ib has significant implications for the downstream activation of the mitogen-activated protein kinase cascade [118]. Other members of the

“extended” family of PI3Ks include class II-type ki- nases that only phosphorylate PI and PI-4-P, but not PI-4,5-P

[193], and class III PI3Ks such as mVps34 that are primarily involved in intracellular vesicle transport and sorting [165].

20.4

Activation of Akt Downstream of Phosphatidylinositol 3-Kinase

Phosphatidylinositol 3-kinases operate at the criti-

cal interface between the extracellular environment

and intracellular signaling by modulating the lo-

cal concentrations of D3-phosphorylated phosph-

oinositides at the plasma membrane. These rafts of concentrated second messenger signals define the starting point of several intracellular signaling cascades. Most prominent among these is the Akt pathway, which has been thoroughly studied. Akt activation is initiated by the specific binding of the Akt regulatory PH domain to PI-3,4-P

and PIP

[60, 63]. Between these, Akt shows a relatively higher af- finity for the binding of PI-3,4-P

when compared to other PH domain-containing proteins, which may be relevant for achieving a sustained signal of Akt activity in cells [60]. Supporting the importance of membrane localization for Akt function in cells, the oncogenic mutation found in v-akt induces growth factor-independent plasma membrane binding by N-terminal myristoylation, presumably by circum- venting the initial translocation step, and thus ren- dering v-akt constitutively active [57, 128].

While phosphoinositide binding downstream of activated PI3Ks initiates Akt activation by induc- ing a conformational change [60, 131], additional phosphorylation events are required to activate Akt fully. Phosphorylations of Akt at a threonine residue (threonine residue-308 in mouse Akt1) in a flexible peptide loop in the kinase domain (T-loop) close to the catalytic core, and at a second site within the hydrophobic motif (HM) in the C-terminal tail (serine residue-473 in mouse Akt1), potently acti- vate the enzyme [3]. The structural analysis of the Akt kinase domain has unraveled the precise nature of the phosphorylation-dependent mechanisms of Akt activation [201, 202]. These studies also pro- vide an excellent rationale for the requirement of HM phosphorylation to induce full Akt activity, since the phosphorylation event facilitates a disor- der-to-order transition within the structure of the Akt kinase domain and enables the proper align- ment of substrates within its active site. Additional phosphorylation sites in Akt exist, but their func- tional consequences are less defined (for review see [164]). These include activating phosphorylation of Akt by PKA [55], and phosphorylations on tyrosine residue-315 [83] and tyrosine residue-474 [39] (all positions numbered according to the amino acid se- quence of Akt1).

20.5

Akt Kinase Kinases:

PDK1 and PDK2

While other kinases including calmodulin-depend- ent kinase kinase (CaMKK) have been proposed as possible kinases to phosphorylate the critical T-loop residues in Akt kinases [203], clear biochemical and

genetic evidence has confirmed that in most bio- logical paradigms this activity is conferred through phosphoinositide-dependent kinase-1 (PDK1) [5, 178]. PDK1 is similar to Akt in that it contains a PH domain and a serine/threonine kinase domain.

However, the PH domain in PDK1 is located at its C-terminus and it binds to PIP3 at a higher affinity than does the PH domain of Akt when binding to PI-3,4-P

and PIP

. As a consequence, under physi- ological conditions, PDK1 is already present in the plasma membrane of non-stimulated cells, where it resides as a constitutively activated enzyme. In con- trast, Akt, its substrate, shuttles from the cytoplasm to the plasma membrane only after the intracellular levels of D3-phosphoinositides are increased follow- ing stimulation of PI3K [178]. Notably, many other members of the AGC “superfamily” of kinases, which consists of protein kinases from the protein kinase A, G and C families, are phosphorylated by PDK1 on corresponding T-loop motifs within their catalytic domain (although by using different mech- anisms for co-localization of PDK1 with its targets), thus defining PDK1 as a central molecular link be- tween D3-phosphoinositide levels and protein ki- nase phosphorylation (for review see [188]).

While the mechanisms regulating phosphoryla- tion of the T-loop residue in Akt are well understood, the molecular mechanisms governing the regulation of the HM phosphorylation in Akt are much less de- fined. Thus, the identity of the HM kinase (often re- ferred to as PDK2) has not yet been resolved. Exper- imental data support both mechanisms involving Akt autophosphorylation [187], or phosphorylation by alternative serine kinases including integrin- linked kinase (ILK)-1 [150] and PDK1 [9, 18]. How- ever, since PDK2 activities distinct from PDK1 and ILK have been isolated, further studies are required to determine the molecular identity of HM kinase activities in cells [75, 76].

The basic mechanisms underlying Akt activa- tion are summarized in Fig. 20.1.

20.6

Regulation of PI3K/Akt in Liver Cells

Like the regulation of Akt signaling in other tis-

sues, a PI3K-dependent activation of Akt has also

been observed in liver cells. In hepatocytes, similar

to many other cell systems, Akt activity is induced

downstream of activated receptor tyrosine kinases

through mechanisms that involve PI3K, as indicated

by specific inhibitor experiments. The various fac-

tors leading to Akt induction include insulin-like

growth factors (IGFs) and insulin in the context of

cell growth and glucose homeostasis (for review see [197]), and also hepatocyte growth factor (HGF), which is the ligand of the c-met receptor tyrosine kinase [199]. The pleiotropy of Akt-activating extra- cellular stimuli comes as little surprise when con- sidering that many receptor systems are very effi- cient inducers of PI3K activity. Furthermore, there is no conclusive evidence for the existence of signal- ing cascades that induce PI3K activity but not that

of Akt. As such, Akt has become a central player in processes that transform the elevated levels of PI3K products in cells into the specific phosphorylation and regulation of protein substrates. The finding of a concomitant activation of PI3K and Akt in many signaling paradigms raises an important question of how tissue specificity is achieved. One possibility for achieving tissue specificity is to utilize tissue- specific receptor systems, including neurotrophins [10] and cytokines [173]. However, as more often is the case, humoral factors such as insulin exert tis- sue-specific effects in different target tissues (e.g., liver, adipocytes, muscle, etc.), making it increas- ingly difficult to determine how shared upstream signals induce tissue-specific responses.

In the liver, a closer examination of the Akt path- way has revealed several mechanisms that account for tissue specificity and may help to answer the question of how this is achieved. Thus, while Akt activity in many tissues is induced by the canoni- cal pathway comprised of PI3K, PTEN and PDK1 ac- tivities, additional molecules with distinct patterns of expression modulate Akt activation by forming specific protein–protein interactions (for review see [19]). These include the Tcl-1 protein that acts as an activator of Akt to facilitate Akt dimerization, primarily in lymphatic cells [107, 148]. They also include inhibitors that bind to Akt and impair its accessibility by upstream kinases. Examples of this latter category include the carboxy-terminal modu- lator protein (CTMP) [121] and, specifically in the liver, one of the mammalian homologs of Drosophila tribbles (TRB3) [50]. While CTMP and TRB3 are not directly related, both proteins still bind to the Akt C-terminus and inhibit phosphorylation of the HM motif, which effectively blocks Akt activation. Im- portantly, TRB3 is regulated on the transcriptional level and its expression is stimulated under fasting conditions [50]. Thus, a model has been proposed in which TRB3 binds and inhibits Akt under fasting conditions in order to promote glucose output from the liver, even in the presence of residual insulin sig- naling.

20.7

Inactivation of Akt Signaling by the PTEN Tumor Suppressor

Akt has been linked to numerous disease states, many of which are sustained by pathologically in- creased Akt activity (for review see [62]). One reason for Akt hyperactivation in tumors is pathologically increased PI3K activity. Thus, mutant p85 regula- tory subunits have been isolated from lymphoma

Fig. 20.1. PI3K, Akt and PTEN signaling. Tyrosine autophospho- rylation of receptor tyrosine kinases such as the insulin receptor induces the recruitment of the p85 regulatory subunit of class Ia PI3Ks, leading to PI3K activation. In contrast, the activation of class Ib PI3K is induced by interaction with small G proteins such as Ras. Once activated, the p110 catalytic subunits phosphor- ylate plasma membrane-bound phosphoinositides (PI-4-P and PI-4,5-P₂) on the D3 position of their inositol rings. The products of this PI3K-dependent reaction are PI-3,4-P₂ and PI-3,4,5-P₃ (also called PIP₃). The tumor suppressor activity of the PTEN lipid phosphatase opposes the activity of PI3K, in that PTEN dephos- phorylates specifically those phosphoinositides that are being generated by PI3K. PTEN, in turn, plays an important role in tox- in- or hepatitis-induced tumorigenesis of the liver, as discussed in Sect. 20.8. The phosphoinositide products of PI3K form high- affinity binding sites for the PH domains of intracellular signal- ing molecules to translate extracellular signals into intracellular responses. The serine/threonine-specific protein kinases PDK1 and Akt are two of the many targets of PI3K activity in cells. Fol- lowing localization to the plasma membrane, Akt is phospho- rylated by PDK1. A second kinase activity has tentatively been termed PDK2 (to indicate that it is also dependent on PI3K activ- ity) and phosphorylates Akt on the C-terminal HM motif, which is required for full Akt activation. At present, the molecular identity of PDK2 is not known. In the liver, specific mechanisms that can modify Akt signaling exist to achieve organ-specific activation.

One such mechanism involves TRB3 that inhibits Akt activity un- der fasting conditions by binding to the HM motif. Mechanisms with direct implications for the function of the PI3K/PTEN/Akt pathway in the liver are indicated in bold

cell lines [85, 88], and the p110 α catalytic subunit is amplified in ovarian tumor cells [167]. Moreover, frequently found mutations in upstream signaling molecules such as receptor tyrosine kinases (e.g., Her/ErbB2 [149]) and small G proteins (e.g., Ras [159]) result in increased PI3K activity as a common endpoint of oncogenic transformation, and thereby increase the PI3K-dependent generation of D3- phosphorylated phosphoinositides. As an example, the small GTPase Ras can activate PI3K via the di- rect interaction with the p110-kDa catalytic subunit [159]. These data implicate PI3K signaling in Ras- mediated transformation, and suggest that in many tumors PI3K activity is induced by Ras mutations, which have been found in ~30% of human tumors (for review see [51]).

Strong support for the involvement of the PI3K/

Akt signaling pathway in tumorigenesis is provided by studies on the gene encoding the phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/

mutated in multiple advanced cancers 1 (MMAC1) [111, 177]. Originally, the PTEN gene was identified as a tumor suppressor gene because it is frequently affected by germline and somatic mutations in hu- man cancer (for reviews see [23, 134]). However, while it was initially assumed that its intrinsic phos- photyrosine phosphatase activity was most relevant, in an influential study Maehama and Dixon [120]

showed that PTEN is also a phosphoinositide phos- phatase, which specifically dephosphorylates the D3 positions of PI3K products. Additional support for the importance of PTEN in regulating the PI3K/

Akt signaling cascade in normal and pathological cell function is obtained from transgenic animal studies [45, 175], and from genetic epistasis studies of a PTEN homolog in controlling Akt signaling in

20.8

Implications of PI3K/PTEN/Akt Signaling in Liver Tumors

Since PTEN dephosphorylates those phosphoi- nositide products of PI3K that otherwise will trig- ger the activation of Akt, inactivating mutations in PTEN or deletion of PTEN expression lead to in- creased levels of PI3K products. These, in turn, result in increased and prolonged Akt activity, which may also play a role in increased proliferation because of accelerated cell cycle progression, apoptosis re- sistance, metastasis and oncogenic transformation.

Thus, the inhibitory effects of PTEN on PI3K/Akt signaling explain why PTEN was originally identi- fied as a tumor suppressor gene: PTEN phosphatase

activity counteracts the pro-oncogenic effects of elevated PI3K kinase activity by decreasing the in- tracellular levels of Akt-activating phospholipids (for review see [23]). To date, countless papers have analyzed the expression and function of PTEN in human cancer cell lines and primary tumors from various tissue origins (for review see [126]). While hepatocellular carcinomas (HCC) are among the most common and aggressive malignancies in the world, specifically when including third-world countries where man-made environmental toxins such as arsenic lead to malignant liver tumors (for review see [31]), it is surprising that only a few pub- lications have investigated the role of genetic altera- tions of the PTEN/PI3K/Akt signaling cascade in its pathogenesis. In initial studies, deletion of PTEN was reported to correlate with the occurrence of liver cancer [47, 66, 92, 151, 206, 208], while intrahepatic metastasis and anchorage-independent growth was shown to be PI3K-dependent [200], thus supporting the critical involvement of this pathway in the tum- origenesis and progression of liver tumors.

In addition to correlative studies investigating PTEN function, strong evidence for the specific role of the PI3K/Akt pathway in the pathogenesis of HCC can be derived from its involvement in mediating the biological effects of hepatitis B and C virus on cell survival [37, 109, 156, 182]. The hepatitis B and C viruses are not the only pathogenic viruses with the ability to manipulate the PI3K pathway toward enhanced survival of infected cells. Other examples include the human immunodeficiency virus-associ- ated tat protein [130], cytomegalovirus (CMV) ma- jor immediate-early proteins and simian virus-40 (SV40) large T-antigen [210]. The hepatitis B virus X (HBx) protein toxin has been shown to activate the PI3K/Akt pathway, either directly by activating PI3K [109], or indirectly by inhibiting the p53-dependent expression of PTEN [37]. Similar findings have been obtained for the hepatitis C virus, in which the NS5A protein activates PI3K-dependent survival [182].

Together with correlative studies that have found a negative correlation between PTEN expression and disease outcome of hepatitis C virus-positive HCC [156], these data suggest that the dysregulation of the PI3K/Akt signaling plays a crucial part in hepa- titis-induced liver cancer.

In summary, these findings suggest PI3K and

Akt to be propitious targets for novel drugs in HCC,

a tumor type that is often resistant to therapy.

20.9

Mechanics of Akt-Dependent Substrate Phosphorylation

The identification of Akt as a substrate downstream of PI3K has been a powerful starting point for deter- mining the basic cellular machinery that translates extracellular signals through the PI3K/Akt signal- ing cascade into specific biological responses. A logical second step has been to identify the protein substrates downstream of Akt that represent the di- rect molecular links between increased Akt activ- ity and altered downstream responses. As outlined in Fig. 20.2, the many consequences of Akt activa- tion include glucose transport, glycolysis, glycogen synthesis, suppression of gluconeogenesis, protein synthesis, lipogenesis, increased cell size, cell-cycle progression and cellular viability.

The first insights into the molecular conse- quences of increased Akt activation were derived from seminal studies that ultimately identified the

“orphan” proto-oncogene as an obligate interme- diate downstream of PI3K in the insulin-depend- ent metabolic control of glycogen synthesis. When searching for kinases that could regulate glycogen synthase kinase-3 (GSK3), the groups of Brian Hem- mings and Phil Cohen realized that Akt inhibited GSK3 activity by direct phosphorylation of an N- terminal regulatory serine residue in an insulin- stimulated and PI3K-dependent manner [40]. While other kinases besides Akt have also been implicated in GSK3 regulation (for an example see [166]), the identification of GSK3 as an Akt substrate helped to define a typical Akt phosphorylation motif. By sys- tematically permutating the amino acid sequence surrounding the Akt phosphorylation site in GSK3, Alessi et al. [4] derived an optimal peptide sequence for Akt phosphorylation (RXRXXS/T; where R is an arginine residue, S is serine, T is threonine and X is any possible amino acid).

The Akt consensus motif is a common feature of known substrates of Akt and the presence of this motif predicts reasonably well whether a given pro- tein may be phosphorylated by Akt enzyme in vitro (for review see [42]). Experiments that have em- ployed randomized permutations within the motif to optimize substrate peptides have defined further the requirement for optimal phosphorylation by Akt [142]. The preferred phosphoacceptor for Akt- dependent phosphorylation is a serine residue, but a synthetic substrate peptide with a threonine residue as phosphoacceptor instead (RPRAATF; P=proline, A=alanine, F=phenylalanine) is also easily phos- phorylated. The phosphoacceptor is best followed by a hydrophobic residue with a large side-chain

in the p+1 position, and preceded by a serine or threonine at the p-2 position. Other kinases such as serum and glucocorticoid-activated kinase (SGK1) and cytokine-independent survival kinase (CISK), which belong to the SGK protein kinase family, ex- hibit similar substrate specificities [99, 116].

Indeed, most of the validated Akt substrates con- tain a phosphorylation site similar to that found in GSK3. To date, many additional Akt substrates have been identified and even more have been predicted and await experimental verification (for review see [42]). However, the power of predicting possible downstream substrates solely based on a phospho- rylation motif may remain limited, since factors other than the primary peptide sequence, such as intracellular protein localization or access to the phosphorylation site, may better determine the ef- fectiveness of kinase–substrate interactions and substrate phosphorylation (for review see [164]).

Fig. 20.2. Biological consequences of Akt activation. Activated Akt modulates a variety of physiological responses in animal cells. Akt has been implicated in the regulation of cell growth, survival and proliferation. Moreover, Akt has also been implicat- ed in metabolic control, specifically as it is related to the insulin- dependent maintenance of glucose homeostasis. The various implications of Akt signaling are shown in a simplified schemat- ic representation, in which activating effects are indicated by pointed arrows, while inhibitory events are shown as flat-headed arrows. Some of these effects are mediated by the direct phos- phorylation of Akt substrate proteins (e.g., the inhibition of GSK3 by direct phosphorylation leads to increased glycogen synthe- sis), while other responses are indirect by the Akt-dependent regulation of transcription (e.g., glucose uptake is facilitated by the Akt-dependent increase of GLUT1 transporter transcription).

For details on the specific consequences of Akt signaling in the liver, please refer to the related sections

20.10

Metabolic Regulation Downstream of Activated Akt

The molecular implications of Akt in the regula- tion of glucose levels in mice [35, 69] point to the relevance of this pathway for metabolic liver func- tions. Not surprisingly, experiments directed at the specific disruption of the PI3K/PTEN/Akt signaling pathway in the liver have resulted in metabolic phe- notypes that are in agreement with the significant participation of hepatic insulin signaling in glucose homeostasis (for review see [141]). More explicitly, the liver-specific inhibition [22] or genetic ablation of the Akt antagonist PTEN [181] has resulted in in- creased insulin sensitivity in the liver and improved glucose tolerance. Accordingly, the liver-specific disruption of the insulin receptor-encoding gene [129], or gene targeting of PI3K function in the liver using the adenoviral delivery of a dominant-nega- tive signaling mutant of PI3K [132], have led to the expected opposite findings of insulin resistance and glucose intolerance.

Various Akt-dependent mechanisms achieve the regulation of insulin-dependent glucose homeosta- sis via the PI3K/Akt pathway. In the liver, these may include the insulin-mediated increase in glycogen synthesis and suppression of glycogenolysis via the Akt-dependent phosphorylation and inactivation of glycogen synthase kinase 3 (GSK3), similar to the regulation of glycogen synthesis by Akt in muscle and fat. Unphosphorylated active GSK3 decreases the activity of glycogen synthase, which is the rate- limiting enzyme in glycogen synthesis, through a phosphorylation-dependent mechanism, while Akt-dependent phosphorylation blocks GSK3 activ- ity [40]. Also in the liver, the suppression of hepatic glucose production and glycogenolysis may be fur- ther enhanced by the insulin-mimicking peripheral effects of leptin, which induces Akt activity in vitro and in vivo in pathways parallel to those utilized in insulin signaling [97, 183], and antagonizes the increase of cAMP levels by glucagon, presumably by inducing the Akt-dependent phosphorylation of phosphodiesterase 3B (PDE3B) [214].

Other metabolic effects of Akt activation include the phosphorylation and activation of 6-phosphof- ructo-2-kinase (PFK2), which has been described in cardiac myocytes and results in an increased rate of glycolysis [44]. In addition, Akt activation has also been shown to cause the translocation of hexoki- nase to the mitochondria for the purpose of eliciting the capture of intracellular glucose by phosphor- ylation [70]. Furthermore, Akt has been implicated in increased lipogenesis through the phosphoryla-

tion of ATP-citrate lyase [17]. Finally, in addition to modifying glucose metabolism, Akt activity also increases the transcription of the glucose transport- ers GLUT1 [11] and the translocation of GLUT4 to the plasma membrane in muscle and fat cells, thus increasing the uptake of glucose into cells [101].

20.11

Implications for Akt Signaling in Metabolic Liver Function

A specific involvement of PI3K/Akt in hepatic me- tabolism is achieved by the regulation of hepatic gluconeogenesis, which is primarily achieved by blocking the level of transcription of genes encoding the enzymes that govern the rate-limiting steps in this process: phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). The idea that Akt activity suppresses their expression and, in turn, hepatic gluconeogenesis, was inspired by the finding of an insulin response element (IRE) in the promoter regions of both genes [12, 72, 135, 207]. IREs have been shown to be under the control of the FoxO family of transcription factors (Foxo1, Foxo3a, Foxo4 and Foxo6), which are established Akt substrates that are suppressed by Akt-depend- ent phosphorylation. Phosphorylation of FoxO proteins by Akt results in their transcriptional in- activation through cytoplasmic retention following their Akt-induced binding to 14-3-3 proteins [190].

While conflicting conclusions concerning the role of Akt in the regulation of PEPCK and G6Pase gene transcription were originally derived [1, 114], more recent studies have identified peroxisome prolifera- tor-activated receptor γ co-activator (PGC)-1α as one of the missing pieces in the puzzle [155]. Since this co-activator has to be present in a Foxo1–PGC- 1 α complex to achieve an optimal transcriptional activation of the gluconeogenic target genes, differ- ences in its tissue distribution may explain differ- ences in the Akt-dependent regulation of gene tran- scription downstream of FoxOs. Taken together, these findings build a simple model for the regula- tion of PEPCK and G6Pase gene transcription by in- sulin, through the inhibition of FoxO transcription factors by Akt phosphorylation and the disruption of Foxo1–PGC-1 α complexes [155].

The possibility of a liver-specific involvement of Akt in the regulation of these genes has been under- scored by recent findings that the Foxo-related tran- scription factor Foxa2 is also an Akt substrate [198].

Foxa2 has been known previously as hepatocyte nu-

clear factor (HNF)-3 β and regulates metabolic gene

expression as well as liver development [89]. Foxa2

belongs to the forkhead family of transcription fac- tors that consists of Foxo1, Foxo3a, Foxo4 and Foxo6, as well as Foxa1, Foxa2 and Foxa3 [90]. However, while all FoxOs contain three Akt phosphorylation sites, Foxa2 (but not Foxa1 or Foxa3) contains only a single threonine site that is phosphorylated by Akt and regulates nuclear/cytoplasmic shuttling [155].

Whether this regulation involves binding to 14-3-3 proteins, similar to the regulation of nuclear locali- zation of FoxOs, is presently unknown, even though it is probably unlikely when considering how much the amino acid sequence of the Akt phosphoryla- tion site in Foxa2 differs from the Akt consensus phosphorylation motif and 14-3-3 binding sites in other proteins. While the exact biochemical details of Foxa2 regulation by Akt require further clarifica- tion, the possibility of an Akt-dependent regulation of Foxa2 in concert with Foxo1 may explain the pro- found defects that are observed in the liver of Akt2 mutant animals when examining the insulin-de- pendent suppression of glucose output [35].

The principles governing the activation of mem- bers of the forkhead family of transcription factors in the liver resemble the evolutionarily conserved mechanisms of their regulation in lower eukaryotes [147]. Other pathways downstream of activated PI3K within the context of liver function are extremely specialized. Examples include molecules involved in the transport of conjugated bile acids starting from sinusoidal uptake and leading to canalicular secretion [91]. Through pharmacological inhibitor experiments, PI3K has been directly implicated in the translocation of the Na

-taurocholate-co-trans- porting peptide (NTCP) that is the major Na

-TC co- transporter at the sinusoidal membrane [195, 196].

However, while other functions downstream of acti- vated PI3K in the liver are primarily attributable to Akt activation, the primary target of PI3K-activated PDK1 in this biological context is PKC ζ [127], which is another member of the AGC family of kinases reg- ulated by PDK1-dependent T-loop phosphorylation (also see Sect. 20.5). Still, Akt is ultimately activated through its intracellular crosstalk with PKC ζ [127]

and may mediate the anti-apoptotic effects of tau- rine-conjugated bile acids on hepatocytes [163].

20.12

Pathological PI3K/Akt Signaling in Hepatic Insulin Resistance

Additional mechanisms of regulating PI3K/Akt ac- tivation specifically in the liver are obtained from studies of insulin resistance in animal models such as ob/ob mice [94], which lack a functional leptin re-

ceptor, and in lipodystrophic mice [168]. The find- ing of decreased IRS-2 but not IRS-1 expression in these animal models points to the prominent role of IRS-2 that leads to increased glycogen synthesis [162]. The hypothesis of a primary role for IRS-2 is further supported by gene-targeting experiments, in which it has been demonstrated that the in- creased hepatic insulin resistance and the suppres- sion of hepatic glucose output by inhibiting PEPCK and G6Pase is primarily regulated by IRS-2, rather than by IRS-1 [95]. Taken together, IRS-2 is the most likely candidate for translating signals from the ac- tivated insulin receptor into the intracellular activa- tion of PI3K/Akt. IRS-2 expression is also increased due to transcriptional upregulation by forkhead-re- lated transcription factors (FoxOs), which in turn are inhibited by Akt-dependent phosphorylation [213]. A negative feedback mechanism can thus be proposed, in which IRS-2 expression is decreased by increased Akt activity in an effort to blunt the signaling strength of the InsR/IRS-2/PI3K cascade leading to Akt activation.

However, in an elegant study, Ide et al. [79] have

recently shown that IRS-2 expression is also regu-

lated by sterol regulatory element-binding proteins

(specifically SREBP-1a and -1c) that bind to the IRS-

transcription to FoxO-dependent transcriptional

activation: SREBPs decrease IRS-2 expression levels,

while FoxOs increase its expression [79]. In the liver,

SREBPs may therefore serve as molecular switches

that respond to the fed or fasting states. They are

absent in the fasting state where IRS-2 levels are in-

duced to increase basal Akt activity and promote

hepatic insulin sensitivity. Re-feeding with a carbo-

hydrate-rich diet induces SREBP expression, which

in turn facilitates the expression of cholesterol syn-

thesis genes and lipogenic enzymes. However, this

induction of SREBP also promotes conditions of in-

creased hepatic insulin resistance, since IRS-2 levels

are lowered by the concomitant SREBP-dependent

competition for FoxO-binding sites on the IRS-2

promoter [79]. Under pathological conditions, in-

cluding after a high-carbohydrate-containing diet,

which is a known exacerbating factor leading to

insulin resistance, the transient regulation of IRS-

2 expression may be sustained and lead to chronic

insulin resistance and hyperglycemia (as discussed

in [79]). The decrease in IRS-2 expression following

the SREBP-dependent transcriptional suppression

of the IRS-2 promoter will lead to reduced Akt activ-

ity, which in turn will enhance the nuclear localiza-

tion of FoxOs (as outlined above). As a result of their

increased residual nuclear localization, FoxOs will

elicit transcription of the PEPCK and G6Pase gene

promoters and thus increase the hepatic glucose

output, which will further promote SREBP activity [124]. The endpoints of this self-promoting regula- tory loop are pathologically increased hepatic glu- cose output and hyperglycemia. They are accompa- nied by accelerated lipogenesis (“fatty liver”), due to the inherent lipogenic properties of SREBPs [146], and insulin resistance, because of the suppression

of IRS-2 expression [79]. Fatty livers and insulin re- sistance are phenotypes in the frequently observed metabolic syndrome, as indicated in Fig. 20.3.

Fig. 20.3. Implications of the PI3K/Akt pathway in liver-specific biological responses. Signals downstream of activated PI3K are mediated by multiple downstream effectors. In the liver, the known mediators of PI3K signaling include Akt and PKCζ, which does not, however, preclude the fact that other PI3K targets in the liver may be numerous and diverse. PKCζ is another AGC fam- ily member and by genetic experiments in cell lines, it has been implicated in the regulation of bile acid transport, as discussed in Sect. 20.11. PKCζ is phosphorylated by PDK1 downstream of activated PI3K, which may explain the finding of concomitant regulation of PKCζ and Akt activities in the liver. Once activated, Akt exerts biological responses through its downstream targets.

These functions of Akt in cells include the regulation of glycogen synthesis, gluconeogenesis, proliferation, cell size and cell sur- vival. The finding of a wide phenotypic diversity of Akt functions can be explained by the existence of multiple protein substrates.

In enzymatic substrates of Akt, phosphorylation can result in ac- tivation (e.g., PDE2) or inhibition (e.g., GSK3). Non-enzymatic targets of Akt comprise apoptotic proteins, transcription factors and other regulatory proteins. In some instances, Akt phosphor- ylation of its targets generates high-affinity binding sites for the phospho-serine/threonine-dependent binding of 14-3-3 mol- ecules that bind to Akt-phosphorylated proteins in the cytosol

(e.g., BAD, Foxo1). Additional consequences of Akt-dependent phosphorylation include changes in the nuclear localization and activity of transcription factors (e.g., Foxo2, CREB). Furthermore, Akt phosphorylation can impair the formation and stability of macromolecular aggregates (e.g., TSC2). Direct mechanisms of regulation are depicted by open arrows. Closed arrows depict mechanisms that may require several intermediate steps. Direct phosphorylation by Akt is indicated by (P). Even when substrates are directly inhibited by Akt, this event may still lead to a factual increase of signaling downstream of Akt if the substrate has been an inhibitor itself. For example, BAD, which reduces cell survival, in turn is inhibited by Akt. As a result, the negative effects of BAD on cell survival are negotiated and cell survival results. Similar dualistic mechanisms regulate mTOR activity, where inhibition of TSC2 results in secondary activation of mTOR by removing an inhibitory input, here indicated by a dashed line. Of note is the existence of a negative feedback loop that is designed to im- prove PI3K/Akt signaling under low Akt activity conditions: IRS-2 expression is induced through activation of FoxOs when Akt ac- tivity is reduced. This feedback mechanism has been discussed in Sect. 20.12 and has significant implications for the pathogen- esis of the metabolic syndrome

20.13

PI3K/Akt-Dependent Regulation of Liver Cell Viability

Multiple lines of research have supported the cen- tral role of PI3K in the suppression of apoptosis [46, 103, 204, 205]. Thus, the initial discovery of Akt as a proto-oncogene has made it a logical candidate for mediating PI3K-dependent survival responses. As a result, Akt has been examined as a key anti-apoptot- ic enzyme in many different cell death paradigms, including withdrawal of extracellular signaling fac- tors, oxidative and osmotic stress, irradiation and treatment of cells with chemotherapeutic drugs, and ischemic shock (for review see [59]). Many studies have connected Akt to apoptosis suppression, either by demonstrating its downregulation following pro- apoptotic insults, or by using gene transfer experi- ments that have either transduced activated mutants to promote survival, or used inactive mutants of Akt to interfere with the function of its endogenous sig- naling to cause cell death even when survival sig- nals are present.

Similar studies have been performed in cell cul- tures of hepatic cells and in animal livers [29, 48, 77, 110, 122]. Not surprisingly, especially when consid- ering the implications of PI3K/PTEN/Akt signaling in hepatic tumorigenesis, as outlined in Sect. 20.8, the pro-survival function of the PI3K/Akt pathway is found to be conserved in the liver. Thus, increased Akt signaling prevents apoptosis induction that is otherwise observed after treating hepatic cells with death factor receptor-stimulating ligands [33, 52, 53, 80, 158] or after exposure of hepatic cells to cytotox- ic agents [106, 110, 115, 209]. Notably, even liver-spe- cific mechanisms of PI3K/Akt-dependent survival signaling have been observed. For example, it has been observed that specific types of conjugated bile acids can promote liver cell viability by stimulating the PI3K/Akt cascade, thus moderating the negative effects of these hepatic products on cellular viability [122, 163].

20.14

Targets of Akt with Implications in Apoptosis Suppression

One of the first targets of Akt identified with direct implications for regulating cell survival is the pro- apoptotic Bcl-2-family member BAD (for review see [58]). When BAD is not phosphorylated, it will inhibit Bcl-x

and other anti-apoptotic Bcl-2 fam- ily members by direct binding of its Bcl-2 homol-

ogy (BH3) domain to their hydrophobic grooves [67]. Once phosphorylated, these phospho-serine residues of BAD form high-affinity binding sites for 14-3-3 molecules. As a result, phosphorylated BAD is retained in the cytosol where its pro-apoptotic activity is effectively neutralized [212]. The impor- tance of BAD as an integration point of survival signaling is underscored by it being a substrate for multiple independent kinase pathways in cells, not all of which phosphorylate BAD at the same site(s) as Akt [43]. The mechanisms of 14-3-3-depend- ent regulation of BAD function resemble the Akt- dependent inhibition of those FoxO transcription factors that regulate the transcription of specific pro-apoptotic genes [20]. One implication of FoxO transcriptional regulation is found in insulin-de- pendent metabolic responses, as discussed in Sect.

20.11. However, studies in primary human tumors and cell lines have also suggested that their nuclear exclusion correlates with apoptosis resistance and oncogenic transformation [71, 78, 82, 136].

Other Akt-dependent mechanisms similar to the inhibition of BAD, which alter the threshold of mitochondrial damage responses, include increased transcription of the anti-apoptotic Bcl-2 family member Mcl-1 in hepatic cell lines [104]. However, Akt functions not only at the level of maintaining mitochondrial integrity to keep cytochrome-c and other apoptogenic factors in the mitochondria [93].

Akt activity also mitigates the response of cells to the release of cytochrome-c into the cytoplasm.

While caspase-9 is an Akt substrate in human cells, where it may explain cytochrome-c resistance [25], it cannot be the only target since Akt-dependent cy- tochrome-c resistance is also observed in cell mod- els in which caspase-9 lacks an Akt phosphorylation site [65, 217]. Thus, other molecules of the post-mi- tochondrial machinery such as the X-linked inhibi- tor of apoptotic proteins (XIAP) have recently been suggested as potential Akt substrates [41].

Another important class of Akt substrates are proteins that are involved in the stress-activated/

mitogen-activated protein kinase (SAPK/MAPK) cascades. Increased Akt activity has been shown to suppress the JNK and p38 pathways in a number of cell systems [16, 27, 145]. Recently, it has been shown that ASK1 is regulated by Akt phosphorylation [96].

Thus, ASK1 is one of many points of convergence between PI3K/Akt signaling and stress-activated kinase cascades. Other examples include the Akt- dependent phosphorylation and regulation of other activators of the JNK pathway, including MLK3 [13]

and the small G protein Rac1 [105].

20.15

Mechanisms of Akt-Dependent Cell Cycle Regulation

A further consequence of Akt activation is the in- creased entry of cells into the cell cycle. Akt is known to be involved in the modulation of two key cell cycle regulator proteins: p21

and p27

. p21

WAF1

was shown to be stabilized after activation of the Akt pathway by direct phosphorylation and Akt- dependent inhibition of GSK3 [112, 160, 161]. Fur- thermore, Li and coworkers [215] showed that the phosphorylation of p21

by Akt inhibited its interaction with PCNA and promoted the assembly of the D1-CDK4 complex. Akt also phosphorylates p27

, which in turn is a target gene of FoxO tran- scription factors. Phosphorylation of p27

induc- es cytoplasmic localization and inhibition, similar to the regulation of p21

.

Additional evidence for the involvement of Akt in the regulation of cell proliferation is found at the interface between p53 tumor suppressor signaling and Akt activity. For example, Akt phosphorylates the ubiquitin ligase Mdm2, which is induced by p53 and blocks p53 function. Two alternative mecha- nisms of Akt-dependent regulation of Mdm2 in- clude an Akt-dependent increase in nuclear import [125] and Mdm2’s intrinsic ubiquitin ligase activity [6, 143, 216]. There is also crosstalk between PTEN and p53. PTEN not only regulates p53 protein levels via Akt and Mdm2, but also directly interacts with p53 and enhances its transcriptional activity [64].

Conversely, based on the finding of a p53 binding site within the PTEN promoter, Stambolic and col- leagues have presented evidence for regulation of PTEN transcription by p53 [176].

20.16

PI3K and Akt:

Increasing Cell Size Matters

Cell growth and proliferation are thought to be dis- tinct cellular processes, a view that has been sup- ported by seminal studies from the Raff laboratory [68]. However, the boundaries of this distinction are fluid when examining the PI3K/Akt pathway. Thus, while Akt promotes cell proliferation by regulating specific targets related to the cell cycle machinery and to DNA damage-dependent cell responses, a conserved function of PI3K/Akt signal transduction is also found in regulating cell volume (for review see [62]). For example, the expression of constitutively active mutants of PI3K or Akt, or dominant-negative

mutants of PI3K or Akt specifically in the cardiac tissue of transgenic mice results in enlarged or re- duced heart size, respectively [169, 170], while the cell growth is decreased in Akt1 knockout mice [36].

Such observations in mammals recapitulate find- ings in Drosophila, in which mutants of Akt [192] or its downstream target p76 S6 kinase (p70S6k) [133]

alter cell size, thus indicating a conserved signaling pathway that originates at the level of growth factor receptors and leads to increased cell volume through PI3K/Akt activation.

The most likely effector of Akt in this pathway is tuberin. Reduced tuberin (also called TSC2) func- tion alternates with loss of hamartin (also known as TSC1) in tuberous sclerosis (TSC), an autosomal dominant tumor disease of benign tumors affecting 1 in 6,000 individuals. TSC is characterized by an in- crease in cell size, although it rarely leads to highly malignant tumors as are observed after PTEN loss- of-function (for review see [30]). The exact conse- quences of the Akt-dependent phosphorylation of tuberin remain controversial, but some data point to a decrease in the stability of the TSC1/TSC2 com- plex, perhaps due to proteasomal degradation [81, 154], or the binding of 14-3-3 proteins [113, 117, 139, 171]. It is clear, however, that TSC2 phosphorylation by Akt induces the kinase activity of the mammalian target of rapamycin (mTOR), presumably by reliev- ing Rheb GTPase from the inhibition of the TSC1/

TSC2 complex [185]. As a result, ribosomal protein p70S6k and (eIF)4E-binding protein-1 (4E-BP1)/

PHAS-I are phosphorylated by mTOR [81, 184], thus leading to increased protein synthesis. The pivotal role of Akt in cell growth is also observed in hepato- cytes, as suggested by data that were obtained from stably transfected hepatoma cells in which expres- sion of constitutively activated Akt resulted in in- creased cell size by increasing protein translation and decreasing protein degradation [54].

Interestingly, the Akt-dependent differences in protein turnover may in turn result in functional changes that extend beyond increases in cell mass.

As an example, studies in hepatocytes and whole livers have indicated that Akt-dependent increases in mTOR activity may enhance the expression lev- els of cyclin D1 [140]. These data provide a direct link between the regulation of cell mass, cell cycling and proliferation. They also explain how on a mo- lecular level multiple cellular phenotypes such as cell growth and proliferation may be regulated by a single pathway.

Similarly, the metabolic functions of Akt are di-

rectly related to its function in cell survival. Thus,

contrary to the pro-survival proteins of the Bcl-2

family, survival downstream of Akt also relies on

glucose metabolism, particularly at the first step of

committing cells to glycolysis where it does not re- quire de-novo protein synthesis [70, 152]. Overall, these findings suggest that the distinctions between different cellular processes such as cell growth, sur- vival, metabolism and apoptosis regulation may be- come very diffuse when examining Akt (for review see [49]). The difficulties in attributing several of these functions to Akt are further compounded by findings that Akt not only modifies cell survival responses by phosphorylating specific substrates, which are easily measured, but also has subtle ef- fects on cellular homeostasis that are more difficult to detect [70, 152]. While these additional functions of Akt are of fundamental relevance for the mainte- nance of the cellular machinery, and for the produc- tion and availability of nutrients and other building blocks necessary for the execution of basic cellular functions, it is only very recently that studies have begun to examine more closely the relevance of Akt at the margins of cell survival, growth and metabo- lism (for review see [153]).

20.17 Conclusions

The data that we have reviewed here with special emphasis on the implications of PI3K/Akt signaling in liver function demonstrate the many and varied consequences of PI3K signaling, specifically at the level of Akt. Implicit in this effort is the dysregula- tion of this signaling cascade in numerous disease states, most notably in cancer and diabetes. From a review of the existing literature, it appears that the consequences of Akt dysregulation for hepatic glucose metabolism have been thoroughly stud- ied, while the few studies of its role in liver cancer have yet to be expanded. Specifically, the role of PI3K/PTEN/Akt in mediating oncogenic transfor- mation after persistent infections with hepatitis viruses defines the pathway as a pivotal target for the development of novel therapies targeting HCCs.

However, the pleiotropy of Akt-dependent cellular responses makes it also a risky target for therapeu- tic intervention. As a result, current work is focused on the identification of Akt substrates – molecules that regulate specific effects of Akt activation – and on determining which substrates are the most im- portant in mediating Akt function in physiological models of health and disease. It is likely that these molecules will hold the key to Akt function in vivo, and will ultimately unveil excellent drug targets, as long as they are thoroughly validated in tissue- and substrate-specific genetic models of Akt signaling in vivo.

Acknowledgments

T.F.F. is supported by Career Development Award DAMD17-0-1-0214 and a scholarship sponsored by the Mary Kay Ash Charitable Foundation. We espe- cially thank Ms Lisa Segev for her valuable help with the preparation of the manuscript.

Selected Reading

Du K, Herzig S, Kulkarni RN et al. TRB3: a tribbles homolog that inhibits Akt/PKB activation by insulin in liver. Science 2003;300:1574–1577. (The authors have characterized TRB3 as an Akt-interacting molecule in the liver that is expressed under fasting conditions and inhibits Akt activity. The iden- tification of a liver-specific modulation of Akt activity, as defined by TRB3, is an excellent example for the existence of tissue-specific regulators of Akt activity. Thus, the find- ings shown here give an insight into the mechanisms that may be involved in generating organ-specific sensitivity and consequences of insulin signaling in the liver [see also Sect. 20.6]).

Rahman MA, Kyriazanos ID, Ono T et al. Impact of PTEN expres- sion on the outcome of hepatitis C virus-positive cirrhotic hepatocellular carcinoma patients: possible relationship with COX II and inducible nitric oxide synthase. Int J Cancer 2002;100:152–157. (This paper investigates the importance of the PI3K/PTEN/Akt pathway in the pathogenesis of hepa- tocellular carcinoma, specifically with its implication in tu- morigenesis following persistent hepatitis virus infections.

Because of the grave implication of liver cancer for human health and in view of novel therapeutic strategies, addi- tional studies are desirable for further investigation of PI3K/

PTEN/Akt-dependent mechanisms in the pathogenesis of liver tumors, as outlined in Sect. 20.8.)

Stiles B, Wang Y, Stahl A et al. Live-specific deletion of negative regulator Pten results in fatty liver and insulin hypersensitiv- ity. Proc Natl Acad Sci USA 2004;101:2082–2087. (In this study, the authors have used the liver-specific genetic deletion of PTEN to characterize PTEN in murine liver function. The met- abolic phenotype resulting from this deletion supports the importance of PI3K/PTEN/Akt signaling for insulin-depend- ent regulation of glucose metabolism in the context of ani- mal metabolism. Thus, these studies help to clarify previous contradictory findings that have arisen from the somewhat puzzling phenotypes observed in PI3K mutant mice [32, 186, 189]. Further details are discussed in Sect. 20.11.)

Ide T, Shimano H, Yahagi N et al. SREBPs suppress IRS-2-mediated insulin signalling in the liver. Nat Cell Biol 2004;6:351–357.

(This study investigates the implication of SREBPs for the PI3K/Akt-dependent transcriptional regulation of gluconeo- genic genes in the liver. A mechanism by which SREBPs sup- press IRS-2 expression levels following carbohydrate-rich

diets is proposed. Based on these findings, a positive feed- back mechanism leading from high glucose to increased SREBP expression and reduced IRS-2 levels in liver cells is proposed, which may further increase hepatic glucose out- put. This feedback mechanism can explain metabolic phe- notypes observed as part of the metabolic syndrome, as outlined in Sect. 20.12. If validated, this self-sustaining loop has significant implications for human metabolic diseases, including diabetes.)

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