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Cell Metabolism

Article

Inactivation of Hepatic Foxo1 by InsulinSignaling Is Required for Adaptive NutrientHomeostasis and Endocrine Growth RegulationXiaocheng C. Dong,1 Kyle D. Copps,1 Shaodong Guo,1 Yedan Li,1 Ramya Kollipara,2 Ronald A. DePinho,2

and Morris F. White1,*1Howard Hughes Medical Institute, Division of Endocrinology, Children’s Hospital Boston, Karp Family Research Laboratories, Room 4210,

300 Longwood Avenue, Harvard Medical School, Boston, MA 02115, USA2Department of Medical Oncology, Center for Applied Cancer Science, Belfer Foundation Institute for Innovative Cancer Science,

Dana-Farber Cancer Institute, Department of Medicine, Department of Genetics, Harvard Medical School, Boston, MA 02115, USA

*Correspondence: [email protected]

DOI 10.1016/j.cmet.2008.06.006

SUMMARY

The forkhead transcription factor Foxo1 regulatesexpression of genes involved in stress resistanceand metabolism. To assess the contribution ofFoxo1 to metabolic dysregulation during hepatic in-sulin resistance, we disrupted Foxo1 expression inthe liver of mice lacking hepatic Irs1 and Irs2 (DKOmice). DKO mice were small and developed diabetes;analysis of the DKO-liver transcriptome identifiedperturbed expression of growth and metabolicgenes, including increased Ppargc1a and Igfbp1,and decreased glucokinase, Srebp1c, Ghr, andIgf1. Liver-specific deletion of Foxo1 in DKO mice re-sulted in significant normalization of the DKO-livertranscriptome and partial restoration of the responseto fasting and feeding, near normal blood glucoseand insulin concentrations, and normalization ofbody size. These results demonstrate that constitu-tively active Foxo1 significantly contributes to hyper-glycemia during severe hepatic insulin resistance,and that the Irs1/2 / PI3K / Akt / Foxo1 branchof insulin signaling is largely responsible for hepaticinsulin-regulated glucose homeostasis and somaticgrowth.

INTRODUCTION

Hyperglycemia and dyslipidemia owing to hepatic insulin resis-

tance are key pathologic features of type 2 diabetes (Brown

and Goldstein, 2008; Zimmet et al., 2001). In mice, near total he-

patic insulin resistance can be introduced via the systemic or

liver-specific knockout of key insulin signaling genes (Michael

et al., 2000; Cho et al., 2001; Okamoto et al., 2007; Mora et al.,

2005; Dong et al., 2006). Among these approaches, the com-

pound suppression or deletion of the insulin receptor substrates,

Irs1 and Irs2, is the least complicated by defective insulin clear-

ance or liver failure (Taniguchi et al., 2005; Dong et al., 2006). Irs1

and Irs2 link the insulin receptor tyrosine kinase to activation of

the PI3K / Akt cascade, which phosphorylates and inactivates

numerous proteins to facilitate adaptation of hepatocytes to the

fed state. Targets of Akt include inhibitors of macromolecular

synthesis such as GSK3-b (glycogen synthesis) and Tsc2 (pro-

tein synthesis); it also phosphorylates mediators of fasting

gene expression such as Foxo1 and Crtc2 by SIK2, resulting in

their degradation or exclusion from nuclei (Barthel et al., 2005;

Dann et al., 2007; Dentin et al., 2007; Jope and Johnson, 2004).

The program of gene expression directed by Foxo1 and its co-

factors ordinarily protects cells, as well as whole organisms,

from the life-threatening consequences of nutrient, oxidative,

and genotoxic stresses (van der Horst and Burgering, 2007).

For example, Daf16—the C. elegans ortholog of Foxo1—ex-

tends life span in nutrient-deprived worms in part by upregulat-

ing superoxide dismutase and catalase expression (Murphy

et al., 2003). Foxo1 and paralogous forkhead box O family mem-

bers counter DNA damage and growth-factor withdrawal by

suppressing cell-cycle progression via upregulation of p27kip

and increasing expression of GADD45 and DDB1 to facilitate

DNA repair (van der Horst and Burgering, 2007).

During prolonged starvation, hepatic Foxo1 ensures the pro-

duction of sufficient glucose to prevent life-threatening hypogly-

cemia (Matsumoto et al., 2007). In healthy animals, the de-

creased insulin concentration during fasting promotes the

nuclear localization of Foxo1, where it interacts with Ppargc1a

and Creb/Crtc2 to increase the expression of the key gluconeo-

genic enzymes G6pc and Pck1 (Dentin et al., 2007; Koo et al.,

2005; Puigserver et al., 2003; Schilling et al., 2006; Barthel

et al., 2005; Mounier and Posner, 2006). Foxo1 also coordinates

decreased nutrient availability with reduced somatic growth by

increasing the hepatic expression of Igfbp1—a secreted factor

that limits the bioavailability of Igf1 (Barthel et al., 2005). Finally,

in conjunction with Creb/Crtc2, Foxo1 increases the expression

of Irs2 and reduces the expression of the Akt inhibitor Trib3,

which together can enhance fasting insulin sensitivity and aug-

ment the insulin response upon eventual feeding (Canettieri

et al., 2005; Matsumoto et al., 2006).

These salutary effects of Foxo1 may, however, be abrogated

by the presence of hepatic insulin resistance, in which case the

persistent nuclear activity of Foxo1 and its cofactors might block

adaptation of hepatocytes back to the fed state (Matsumoto

et al., 2007; Samuel et al., 2006; Zhang et al., 2006). To establish

genetically the degree to which hepatic Foxo1 alone contributes

Cell Metabolism 8, 65–76, July 2008 ª2008 Elsevier Inc. 65

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Cell Metabolism

Foxo1 Critically Regulates Nutrient Homeostasis

to hyperglycemia and diabetes during insulin resistance, we dis-

rupted the Irs1 and Irs2 genes in hepatocytes of mice and deter-

mined the extent of their recovery upon the coincident disruption

of Foxo1.

RESULTS

PI3K / Akt Cascade Requires Irs1 or Irs2for Activation in LiverWe used a Cre-loxP strategy to produce mice lacking hepatic

Irs1 (LKO1 mice), Irs2 (LKO2 mice), or both genes (DKO mice)

(see Experimental Procedures and Figure S1 in the Supplemen-

tal Data). At 6 weeks of age, Irs1 mRNA and protein were unde-

tectable in liver extracts from the LKO1 and DKO mice, and Irs2

mRNA and protein were undetectable in extracts from the LKO2

and DKO mice (Figures 1A–1C). Irs1 and Irs2 expression was not

affected in other tissues including adipocytes, skeletal muscle,

and brain in the knockout mice compared to the corresponding

control mice (data not shown).

To assess insulin signaling in the liver, we analyzed liver ex-

tracts from mice treated with insulin or saline. Insulin stimulated

the phosphorylation of Akt at the catalytic (T308) and hydropho-

bic (S473) sites in control liver extracts, and in liver extracts from

LKO1 or LKO2 mice (Figure 1D). Consistent with this, the insulin-

stimulated phosphorylation of Akt substrates GSK3-a (S21),

GSK3-b (S9) and Foxo1 (T24 and S256) was also observed in

control, LKO1, or LKO2 livers (Figure 1D). By contrast, insulin-

stimulated phosphorylation of Akt (T308), GSK3-a/b, or Foxo1

was undetected in DKO-liver, and the phosphorylation of Akt

(S473) was reduced at least 70% (Figure 1D). Insulin failed to

stimulate Erk1 phosphorylation (T202/Y204) in the DKO liver,

whereas Erk1 phosphorylation was stimulated 3-fold in the other

groups of mice; the moderate insulin-stimulated phosphoryla-

tion of Erk2 was also absent in the DKO liver (Figure 1D). Thus,

either Irs1 or Irs2 was needed for the phosphorylation of hepatic

Akt (T308) and Erk1/2 during acute insulin treatment; however,

some alternative mechanisms might also contribute to about

30% of insulin-stimulated Akt (S473) phosphorylation in the

absence of Irs1 and Irs2, including a possible insulin receptor-

mediated mechanism, as the tyrosine autophosphorylation of

IR was stimulated normally by insulin in the liver of DKO mice

(Figure S1C).

Hepatic Irs1 or Irs2 Mediate Glucose HomeostasisDKO mice developed hyperglycemia by 5 weeks of age when

Irs1 and Irs2 deletion was nearly completed (Figure S1D). Con-

sistent with overlapping roles of Irs1 and Irs2 to mediate hepatic

insulin signaling, both LKO1 and LKO2 mice displayed normal

fasting glucose and normal postprandial glucose and insulin

concentrations at 8 weeks of age (Figures S2A–S2C). Compared

with the control mice, fasting and postprandial glucose and insu-

lin blood concentrations were significantly elevated in 8-week-

old DKO mice (Figures S2A–S2C). A glucose tolerance test con-

firmed that the DKO mice were diabetic at 8 weeks of age, as

fasting and 2 hr blood glucose concentrations were significantly

elevated (Figure 1E). By comparison, the LKO1 mice displayed

mild glucose intolerance, whereas glucose tolerance of the

LKO2 mice was indistinguishable from the control mice

(Figure 1E). Consistent with these results, an intraperitoneal

66 Cell Metabolism 8, 65–76, July 2008 ª2008 Elsevier Inc.

injection of insulin failed to reduce blood glucose in DKO mice,

whereas blood glucose decreased normally in the LKO2 mice

(Figure 1F). Compared to the control and LKO2 mice, the

LKO1 mice displayed a weaker—albeit not significantly

weaker—response to the injected insulin (Figure 1F).

To further confirm that diabetes in DKO mice resulted from

loss of hepatic Irs1 and Irs2, we infected the DKO mice by tail

vein injection with adenoviruses containing either control green

fluorescent protein (Ad-GFP) or Irs1 or Irs2 (Ad-Irs1, Ad-Irs2)

coding sequences. Ad-Irs1 or Ad-Irs2 restored the expression

of hepatic Irs1 or Irs2, respectively, as well as the phosphoryla-

tion of Akt (S473) and Foxo1 (S256) until the experiment was

terminated (Figure 2A). Moreover, Ad-Irs1 or Ad-Irs2—but not

Ad-GFP—restored the fasting blood glucose of DKO mice to

a normal concentration (Figure 2C). We conclude that the DKO

liver was not permanently damaged by 12 weeks of age, as glu-

cose homeostasis was normalized by acute restoration of the

Irs1 or Irs2 branch of the insulin signaling cascade.

Acute Inactivation of Foxo1 in DKO MiceSince Foxo1 was never inactivated in the DKO liver by insulin-

stimulated phosphorylation, we suppressed its expression with

an adenovirus encoding an siRNA against Foxo1 (Ad-siFoxo1)

to determine whether glucose homeostasis could be restored.

Two days after the tail-vein injection, Ad-siFoxo1, but not Ad-

siGFP, reduced Foxo1 expression by at least 75% in control

and DKO liver (Figure 2B and data not shown). Moreover, Ad-

siFoxo1 injection reduced the elevated fasting blood glucose

concentration in the DKO mice to the normal range (Figure 2C).

Thus, constitutive Foxo1 activity contributed to the dysregulated

glucose homeostasis in the DKO mice.

Gene Expression in the Liver of DKO and TKO MiceWe used the Cre-loxP strategy to produce ‘‘triple-knockout’’

(TKO) mice lacking Irs1, Irs2, and Foxo1 in hepatocytes. Irs1, Irs2,

and Foxo1 were barely detected in liver extracts of 8-week-old

TKO mice, confirming that the deletion of all three genes was ac-

complished (Figure 2D). As expected, insulin signaling assessed

by the phosphorylation of Akt (T308), GSK3-b (S9), or Erk1/2

(T202, Y204) was not detected in liver extracts from TKO mice

(Figure 2E). Whereas insulin-stimulated Akt (S473) phosphoryla-

tion was impaired in DKO liver, it was almost completely abol-

ished in the TKO liver. These results suggest that Foxo1 might

play a positive role in Akt (S473) phosphorylation independently

of Irs1 or Irs2 (Figures 1D and 2E).

Affymetrix GeneChips were used to establish the effect of

Foxo1 upon gene expression in the DKO liver. Analysis of RNA

samples from the liver of LKO1, LKO2, DKO, and TKO mice

and the corresponding control mice revealed 9824 significantly

changed probe sets (false discovery rate, FDR < 0.05) that cor-

responded to 5756 annotated genes of which 420 displayed

a maximal change of at least 1.5-fold (Table S1). Three principal

components (PC)—each with a positively and negatively corre-

lated gene cluster—accounted for 86% of the total expression

variance (Figures 3A–3C) (Sharov et al., 2005). Most of the signif-

icantly changed genes (86.7%) were associated with PC1, in-

cluding 3531 displaying increased and 593 displaying decreased

expression in the DKO liver (Figure 3A). The dysregulated ex-

pression of these genes in the DKO liver was largely restored

Cell Metabolism


Figure 1. Hepatic Deficiency of Irs1 and Irs2 Results in Insulin Resistance and Diabetes

(A and B) Irs1 or Irs2 mRNA levels in the liver of 6-week-old control (Irs1L/L, Irs2 L/L, 12 L/L for floxed Irs1, Irs2, or both, respectively) and liver-specific knockout mice

for Irs1 (LKO1), Irs2 (LKO2), and both (DKO) were analyzed by real-time PCR (n = 3). Error bars represent SEM.

(C) Immunoblot analysis of Irs1 and Irs2 proteins in the liver of 6-week-old control and knockout mice.

(D–F) CNTR indicates the combined floxed gene control samples. (D) shows quantitative immunoblot analysis of insulin signaling in the liver extracts of 8-week-

old CNTR and liver-specific knockout mice (n = 3) injected via the vena cava with saline (�) or 5 units of insulin (+) for 4 min. Glucose (E) and insulin tolerance tests

(F) of 8-week-old male mice (n = 8–12) are shown. Individual areas under curves were analyzed by ANOVA; groups that share a vertical bar at the final time point

did not significantly differ. All other between-group comparisons in (E) and (F) were significant with p < 0.05. Error bars represent SEM.

in the TKO liver to the normal range displayed by the control,

LKO1, and LKO2 liver (Figure 3A).

PC2 accounted for the expression variance in 440 genes (9.1%

of total variance), which responded positively or negatively to

feeding regardless of the presence of Irs1, Irs2, or Foxo1 (Fig-

ure 3B). Many PC2 genes in the DKO liver displayed either

elevated expression under fasting conditions or an increased

response to feeding. Thus, PC2 genes are strongly regulated by

fasting and feeding, but also modulated by insulin/FOXO signal-

ing. PC3 accounted for the expression variance of 4.2% of the


Cell Metabolism


Figure 2. Hyperglycemia in DKO Mice Can Be Rescued by Irs1 or Irs2 Re-expression or Knockdown or Knockout of Foxo1

(A) Immunoblot analysis of liver extracts from 12-week-old floxed control (CNTR) and DKO mice treated with adenovirus-expressing control green fluorescent

protein (Ad-GFP) or Irs1 (Ad-Irs1) or Irs2 (Ad-Irs2).

(B) Immunoblot analysis of Foxo1 knockdown in the liver extracts of 12-week-old CNTR and DKO mice by adenovirus-mediated siRNA against Foxo1

(Ad-siFoxo1) or GFP (Ad-siGFP).

(C) Fasting (6 hr) blood glucose levels in 12-week-old mice treated with adenoviruses expressing GFP, Irs1, or Irs2, or siRNA against Foxo1 or control GFP (n = 4).

Error bars represent SEM.

(D) Immunoblot analysis of Irs1, Irs2, and Foxo1 in liver extracts of 8-week-old floxed control (CNTR) and liver-specific Irs1:Irs2:Foxo1 triple knockout (TKO) mice.

Actin is used as a loading control.

(E) Analysis of insulin signaling in 8-week-old floxed control (CNTR), TKO, and DKO liver extracts using phosphospecific and total protein antibodies.

significantly changed genes, which were largely dysregulated in

the liver of TKO mice (Figure 3C). Thus, expression of PC3 genes

was largely independent of Irs1 or Irs2 signaling, but sensitive to

the expression of Foxo1. Foxo1 itself—assessed by 30-directed

Affymetrix probe sets targeted against the deleted exon 2 in

Foxo1 gene in the TKO liver—was found in PC3 (Table S1).

Gene Set Enrichment Analysis (GSEA) revealed at least 50

transcription factor recognition sites, which were significantly

represented in the set of 5756 significantly changed genes (Table

S2). A FOXO recognition site (TTGTTT, p < 10�45) was relatively

abundant as it occurred in at least 560 genes—69 of the genes

that change by at least 1.5-fold (Table S1). Genes regulated by

cAMP and glucocorticoids play an important role in the response

to fasting; however, the consensus binding sites by CREB or GR

were not among the top 50 recognition sites (Table S2). Regard-


less, two genes contained both FOXO and CREB sites (Ppargc1a

and Maf), and two genes contained both FOXO and GR sites

(Txnip and Pik3r1). However, 83% of the genes that changed by

at least 1.5-fold did not contain a consensus FOXO recognition

site. It is possible that these 83% significantly changed genes

could still be regulated by Foxo1 through either a nonconsensus

recognition site or protein-protein interaction without Foxo1 di-

rectly binding to the gene promoters. Other transcription factors

or cofactors such as PGC-1a might indirectly contribute to the

effect of insulin and Foxo1 upon gene expression.

The Effect of Foxo1 upon Genes RegulatingMetabolism in DKO MiceNext we used real-time PCR to analyze the expression of spe-

cific hepatic genes that regulate metabolism and growth, and

Cell Metabolism


Figure 3. Gene Expression in the Liver of Control and Knockout Mice

(A–C) The normalized expression of liver genes was analyzed in fasted (16 hr) and fed (4 hr) 6-week-old control (CNTR), DKO, LKO1, LKO2, or TKO mice (n = 2–8)

using Affymetrix GeneChips. Liver genes that were changed significantly (FDR < 0.05) were further analyzed for either a positive (+) or negative (�) correlation with

principal component 1 (PC1), PC2, and PC3 using the NIA Array Analysis Tool. Data were presented as the average normalized expression (log2 scale) of gene

clusters positively correlated (C) and negatively correlated (,) with principal component PC1 (A), PC2 (B) or PC3 (C), respectively. The error bars represent the

standard deviation.

(D–F) Gene expression was independently confirmed by real-time PCR in the liver of 6-week-old control (CNTR) and knockout mice (n = 3) fasted for 16 hr (�) or

fasted and then allowed access to food for 4 hr (+). (D) shows expression of genes involved in metabolism, including Gck, Pck1, G6pc, and Cpt1a. (E) shows

expression of genes involved in gene regulation, including Ppargc1a, Fgf21, Srebp1c, and Onecut1. (F) shows expression of genes involved in animal growth,

including Ghr, Igf1, Igfbp1, and Igfals. Data are presented as relative expression of the gene of interest over b-actin (mean ± SEM). * p < 0.05 versus CNTR

mice under the same feeding condition by Student’s t test.

to measure their response to feeding. As previously shown, feed-

ing control mice increased the expression of Gck and decreased

the expression of Pck1, G6pc, and Cpt1a (Badman et al., 2007;

Yoon et al., 2001). Gck mRNA was not detected in fasted or fed

DKO liver and feeding failed to reduce the expression of Pck1

and G6pc in DKO liver; however, Cpt1a responded normally

(Figure 3D). Feeding control mice altered several hepatic regula-

tory factors, including decreased expression of Ppargc1a and

Fgf21 and increased expression of Srebp1c and Onecut1 (Fig-

ure 3E). However, in the DKO liver the average expression of


Cell Metabolism


Figure 4. Deletion of Hepatic Foxo1 (TKO Mice) Reverses the Diabetes and Growth Retardation in DKO Mice

(A and B) Body length and bone mineral density by dual energy X-ray absorptiometry were measured for 3-month-old floxed control (CNTR), DKO, and TKO mice

(n = 4–9). Values were compared by Kruskal-Wallis (K-W) test and Dunn’s procedure with tabled p values for small n. * p < 0.05 versus CNTR. Error bars represent

SEM.

(C) Growth curves of control, DKO, and TKO mice. Data (n = 6–22 per group at each point) were analyzed by ANOVA, and the analysis showed a significant

difference between DKO mice and the other groups with p < 0.05.

(D) Hepatic glycogen content was measured in 6-week-old mice (n = 4–6) that were refed for 4 hr after overnight fasting. The K-W test demonstrated a significant

difference in data location for one or more of the groups (p < 0.01), but post procedures did not identify significant between-group differences. Error bars

represent SEM.

(E) Signal transduction analysis in the liver of 6-week-old control and knockout mice that were either fasted overnight (�) or refed for 4 hr after fasting (+).

(F) Insulin resistance was determined by HOMA2 approximation using fasting blood glucose and insulin data collected from 7- to 8-week-old mice (n = 5–9).

LKOF = Foxo1 liver-specific knockout mice. Data were analyzed by ANOVA and Scheffe postprocedure for comparison of all groups. * p < 0.05 versus all other

groups. Error bars represent SEM.


Cell Metabolism


Srebp1c and Fgf21 decreased but remained sensitive to feeding,

Onecut1 mRNA level was undetected, and Ppargc1a mRNA

level was strongly increased (Figure 3E). Regardless, the aver-

age expression of these genes and sensitivity to feeding was

at least partially restored in the TKO liver (Figures 3D and 3E).

Similar results were observed with the GeneChips for these

and other genes (Table S1 and data not shown). Thus, Foxo1

prevented the adaptation of postprandial liver gene expression

in DKO mice, whereas this inhibition was released upon deletion

of Foxo1.

The Effect of Foxo1 upon Genes RegulatingGrowth in DKO MiceGrowth of the DKO mice was retarded prior to the onset of sig-

nificant hyperglycemia, and this growth defect was persistent

throughout the period analyzed (Figures 4C and S1D). While

DKO and TKO mice consumed normal amounts of food and

had a normal body composition at 3 months of age (Figure S3),

the DKO mice were shorter and had lower bone mineral density

and 20% less body mass than the control mice (Figures 4A–4C).

By contrast, TKO mice displayed normal body length, bone min-

eral density, and body mass (Figures 4A–4C). Hepatic genes that

promote somatic growth—including Ghr, Igf1, and Igfals—were

expressed weakly in DKO mice but normally in TKO mice (Fig-

ures 3F and S4). Moreover, genes that inhibit growth, especially

the Foxo1 target Igfbp1, were strongly increased in the liver of

DKO mice and rendered less sensitive to feeding (Figure 3F).

The average expression of other genes associated with organis-

mal growth and survival were also dysregulated in DKO mice but

normalized in the TKO mice (Table S1). Thus constitutively active

hepatic Foxo1 per se was responsible for the observed growth

deficit of DKO mice (Figures 4A–4C).

Nutrient Homeostasis in DKO and TKO MiceTo contrast the response of the DKO and TKO-liver to nutrients,

we investigated the phosphorylation of several signaling proteins

in the liver of fasted (16 hr) and fed (4 hr after the fast) mice. The

phosphorylation of AMPK, a key energy sensor, was not altered

in the liver of fasted or postprandial DKO or TKO mice compared

to the control, suggesting that hepatic energy levels were not

dramatically altered (Figure 4E). Feeding control mice stimulated

the phosphorylation of Akt (S473); however, Akt (T308) and Erk1/

2 phosphorylation were not strongly increased (Figure 4E). Phos-

phorylated Akt (T308 or S473) was undetected in the DKO and

TKO liver, and feeding had no effect upon Erk phosphorylation

(Figure 4E). By comparison, feeding strongly stimulated the

phosphorylation of S6K1 and ribosomal protein S6 in the liver

of control, DKO, and TKO mice, showing that hepatic mTOR

(mammalian target of rapamycin)-mediated nutrient sensing

was at least partially independent of the Irs1/2 / Akt /

Foxo1 pathway (Figure 4E).

The dysregulated circulating concentrations of fasting glu-

cose, insulin, and adiponectin and fed glucose were normalized

in the TKO mice (Figures S2A–S2D). Remarkably, fasting insulin

resistance—estimated by the homeostasis model assessment

(HOMA2)—was also reduced to the normal range in TKO mice

(Figure 4F). TKO mice displayed a better response to injected

insulin than DKO mice, although this did not reach significance

(Figure 4G). Compared to DKO mice, glucose tolerance of TKO

mice was significantly improved and indistinguishable from the

control, whereas mice lacking hepatic Foxo1 alone (LKOF) were

significantly more glucose tolerant than the controls (Figure 4H).

Hepatic glycogen was lower in DKO liver relative to control, but

increased toward the normal range in TKO liver (Figure 4D).

Serum FFA and triglyceride concentrations were low in the

8-week-old DKO and TKO mice compared to the normal serum

concentrations in LKO1 and LKO2 mice (Figures 5A, 5B, S5A,

and S5B). Hepatic triglyceride concentration was not signifi-

cantly changed in 7-week-old DKO or TKO mice (Figure 5E).

However, triglyceride secretion in 3-month-old DKO mice was

decreased more than 60% compared to control mice, whereas

triglyceride secretion was partially restored in TKO mice

(Figure 5F).

Total serum cholesterol concentrations were also low in the

8-week-old DKO and TKO mice compared to the normal choles-

terol levels in LKO1 and LKO2 mice (Figures 5C and S5C). To fur-

ther examine cholesterol distribution and changes with time, we

analyzed total serum cholesterol, high-density cholesterol (HDL),

and low-and very low-density cholesterol (LDL and VLDL) in the

4-month-old control, DKO, and TKO mice. LDL-/VLDL-choles-

terol concentrations were normal in DKO and TKO mice; how-

ever, HDL-cholesterol concentrations were significantly lower

in the DKO and TKO mice compared to the control mice

(Figure 5D). It is noteworthy that the serum HDL-cholesterol con-

centrations were increased by 52% DKO: 24.6 mg/dl; TKO: 37.3

mg/dl (p < 0.05) in the TKO mice relative to the DKO mice (Fig-

ure 5D). Interestingly, the protein levels of ApoA-I, ApoB48/100,

and ApoE were indistinguishable between the DKO and TKO

mice and the control mice (Figure 5H). However, the expression

of genes determined on Affymetrix GeneChips involving choles-

terol homeostasis (mainly cholesterol biosynthesis) and lipid

synthesis were generally lower and not responsive to feeding

in DKO liver compared to the control liver (Figure 5G). By con-

trast, expression of the same set of genes was largely normalized

in the TKO liver, which was consistent with the improvement in

serum HDL-cholesterol levels and triglyceride secretion in the

TKO mice (Figures 5D, 5F, and 5G).

DISCUSSION

Our results suggest that Foxo1 is a dominant regulator of hepatic

gene expression that is ordinarily inactivated through the Irs1 or

Irs2 branch of the insulin signaling system. Without Irs1 and Irs2

(DKO mice), the ordinary transition of liver gene expression from

the fasted to fed state is inhibited, resulting in glucose intoler-

ance and diabetes. Acute or chronic inactivation of Foxo1 in

DKO mice releases this inhibition and allows hepatic gene ex-

pression and metabolism to adapt more normally to the nutrient

status. This finding is consistent with the important role of dFoxo

in Drosophila, which is involved in nutrient response and

(G and H) Insulin (G) and glucose tolerance tests (H) were performed in 6- to 8-week-old liver-specific knockout (n = 8–13) and pooled floxed control (n = 26) mice.

Individual areas under curves were analyzed by ANOVA; groups that share a vertical bar at the final time point did not significantly differ. All other between-group

comparisons in (G and H) were significant with p < 0.05. Error bars represent SEM.


Cell Metabolism


Figure 5. Lipid Parameters and Gene Expression in DKO and TKO Mice

(A–C) Serum free fatty acids (FFA) and triglyceride and cholesterol levels (mean ± SEM) were measured after blood samples were collected from overnight fasted

8-week-old control, DKO, and TKO-mice (n = 6–10), respectively. Bars indicate interquartile ranges (first to third quartile), and horizontal lines inside the bars

represent median values. Data were analyzed by ANOVA for comparison of all groups. * p < 0.05 versus CNTR.

(D) Serum total cholesterol, HDL-cholesterol, and LDL/VLDL-cholesterol levels were measured in the overnight fasted 4-month-old control, DKO, and TKO mice

(n = 6–8) using a commercial assay kit. * p < 0.05 between CNTR and DKO or TKO mice. Error bars represent SEM.

(E) Hepatic triglyceride content was determined in 7-week-old mice (n = 3–15) that were fasted overnight. Bars indicate interquartile ranges (first to third quartile),

and horizontal lines inside the bars represent median values. The K-W statistic analysis did not reach significance.

(F) Triglyceride secretion was analyzed in 3-month-old control, DKO, and TKO mice (n = 6–8) that were fasted for 4 hr and then injected with Triton WR1339.

Serum triglyceride concentrations were measured at 0, 1, and 2 hr after the injection. At the 0 time point, the serum triglyceride levels in all groups of mice

were below 10 mg/dl. * p < 0.05 between CNTR and DKO or TKO mice. Error bars represent SEM.

(G) Normalized expression of top 10% significantly changed genes involved in cholesterol homeostasis and lipid synthesis was analyzed by Ingenuity Pathway

Analysis software. Bars indicate interquartile ranges (first to third quartile) of normalized expression values and horizontal lines inside the bars represent median

expression values. The gene set includes Abca1 (ATP-binding cassette, sub-family A [ABC1], member 1), Acyl (ATP citrate lyase), Cyp51a1 (cytochrome P450,

family 51, subfamily A, polypeptide 1), Dhcr24 (24-dehydrocholesterol reductase), Dhcr7 (7-dehydrocholesterol reductase), Fasn (fatty acid synthase), Fdft1 (far-

nesyl-diphosphate farnesyltransferase 1), Fdps (farnesyl diphosphate synthase), Gck (Glucokinase), Hmgcr (3-hydroxy-3-methylglutaryl-coenzyme A reductase),


Cell Metabolism


metabolic adaptation through regulation of numerous biological

processes (Gershman et al., 2007).

Our results do not support the conclusion that Foxo1 is prefer-

entially regulated through the Irs2 branch of the insulin signaling

cascade, as previously suggested (Wolfrum et al., 2004). Both

Irs1 and Irs2 suffice for phosphorylation of Foxo1, and addition

of either as an adenovirus to DKO liver restores Foxo1 phosphor-

ylation and rescues hyperglycemia. Our results do not confirm

a predominant role for Irs2 in lipid metabolism, as previously sug-

gested (Taniguchi et al., 2005). Glucose tolerance in LKO2 mice

is largely indistinguishable from that of controls, at least at the

age analyzed in this report, whereas it is mildly but significantly

impaired in LKO1 mice. Thus Irs1 appears to have a greater con-

tribution toward glucose homeostasis than Irs2. Hepatic Irs1 ex-

pression is generally stable, whereas Irs2 expression rises during

fasting and falls after feeding, which might explain the mild dys-

regulation of hepatic metabolism in the absence of Irs2. Never-

theless, a uniquely dynamic interaction between Irs2 and IR

suggests that Irs2 might play a more important role when cellular

ATP concentrations are high (Wu et al., 2008).

Hepatic gene expression is dramatically altered by fasting and

feeding owing to the effects of insulin during the postprandial

state and the action of counterregulatory hormones including

glucagon and glucocorticoids during fasting. Many hepatic

genes are dysregulated in DKO mice, confirming that insulin sig-

naling plays a critical role; however, upon deletion of Foxo1 in the

TKO mice, gene expression is largely normalized in both the

fasted and postprandial states. Thus, the inactivation of Foxo1

is essential before hepatic gene expression can respond to other

signals. Although Foxo1 can be regulated by many mechanisms,

its inactivation in postprandial liver appears to be under the dom-

inant control of the Irs1/2 / Akt cascade (Figure 6). Insulin fails

in DKO mice to stimulate hepatic Akt (T308) phosphorylation,

which is required to activate Akt. Consequently, Foxo1 is not

phosphorylated in the DKO mice during intravenous insulin injec-

tions or by feeding.

Insulin is also known to promote the phosphorylation of Akt at

its hydrophobic C-terminal motif (S473), which contributes to

substrate recognition (Manning and Cantley, 2007). Unlike the

Akt (T308) phosphorylation, intravenous insulin injections stimu-

late Akt (S473) phosphorylation in DKO mice to about 30% of the

normal response; however, Akt (S473) phosphorylation is barely

detected in the TKO mice. Although the insulin receptor is func-

tional in the DKO and TKO mice, the mechanism coupling it to

Akt (S473) phosphorylation in DKO mice is unclear. One possibil-

ity is that the TSC1-TSC2 complex that is normally inhibited by

insulin signaling may have a positive effect on Akt (S473) phos-

phorylation (Huang et al., 2008). Another possibility is that a con-

stitutive Foxo1 in DKO liver might promote Akt (S473) phosphor-

ylation by modulating expression of Akt regulators such as Trib3

that binds to the Akt catalytic site (Matsumoto et al., 2006); how-

ever, the Affymetrix GeneChip data are consistent with this

hypothesis, as the mRNA concentration of Trib3 was strongly

increased in the DKO liver compared to the control or TKO liver

(Table S1). Regardless of the mechanism, Akt (S473) phosphor-

ylation alone does not sufficiently inactivate Foxo1 because only

Foxo1 (T24) phosphorylation was abolished in mTORC2-defi-

cient cells (Guertin et al., 2006; Jacinto et al., 2006), and it has

been suggested that Foxo1 (S256) phosphorylation plays

a ‘‘gatekeeper’’ role in the subsequent phosphorylation of other

Thr/Ser residues in Foxo1 by insulin (Barthel et al., 2005).

Hepatic deficiency of Irs1 and Irs2 leads to a significant growth

retardation in both prediabetic and diabetic DKO mice. Consis-

tent with this phenotype, the expression of several hepatic genes

that promote organismal growth (Igf1, Igfals, and Ghr) were sig-

nificantly decreased in DKO mice, whereas Igfbp1 that inacti-

vates circulating Igf1 was increased (Le et al., 2001). The deletion

of hepatic Foxo1 (TKO mice) restores normal expression of these

genes, and the TKO mice grow to a normal size. Thus, postpran-

dial inactivation of Foxo1 by insulin is essential for the usual sys-

temic effect of growth hormone and hormonal Igf1 upon body

growth (Figure 6).

Numerous gluconeogenic or regulatory genes, including

Ppargc1a, Pck1, and G6pc, are not suppressed in postprandial

DKO liver, but regain some sensitivity to feeding in TKO liver.

Such genes are likely to be positively regulated by Foxo1, as pre-

viously suggested (Altomonte et al., 2003; Daitoku et al., 2003;

Matsumoto et al., 2007; Nakae et al., 2001; Puigserver et al.,

2003; Zhang et al., 2006), or inaccessible to negative regulation

when Foxo1 is active. Similar to our results, the elevated expres-

sion of gluconeogenic genes in Insr+/� mice was normalized

upon deletion of one allele of Foxo1 (Nakae et al., 2002).

Onecut1 (also known as Hnf6) expression is decreased in DKO

liver and restored in TKO liver, suggesting that it might be regu-

lated by Foxo1; however, it does not contain a consensus FOXO-

binding site in its proximal promoter. It might be regulated indi-

rectly through Ghr / Jak2 signaling since expression of Ghr

also increases in the TKO liver (Figure 6) (Lahuna et al., 2000).

Other genes including Gck show a clear loss of feeding-induced

transcription in DKO liver, which is consistent with the finding

that hepatic Gck is suppressed by a constitutively active trans-

genic Foxo1 (Zhang et al., 2006). Although Gck does not contain

a FOXO core recognition motif, its expression is partially restored

in the TKO liver. The deletion of Foxo1 might indirectly promote

Gck expression through Onecut1 (Lannoy et al., 2002). Onecut1

also induces expression of Foxa2, which may promote glycoly-

sis, fatty acid oxidation, and ketogenesis, and inhibit glucocorti-

coid-induced Pck1 expression in liver (Figure 6) (Pierreux et al.,

1999; Rausa et al., 1997; Wolfrum et al., 2004). However, a signif-

icant change in Foxa2 expression was not detected by the Affy-

metrix GeneChip analysis.

Igf1 and Igfals expression might also be regulated by Foxo1

(directly or indirectly) in addition to known regulation by Stat5b

(Figure 6) (Le et al., 2001). Together, these results reveal that in-

sulin can play a permissive role in the regulation of endocrine

growth through Foxo1-regulated gene expression of Ghr, Igf1,

Idi1 (isopentenyl-diphosphate delta isomerase 1), Ldlr (low density lipoprotein receptor), Mvk (mevalonate kinase), Nsdhl (NAD[P] dependent steroid dehydro-

genase-like), Nudt7 (nudix [nucleoside diphosphate linked moiety X]-type motif 7), Pcsk9 (proprotein convertase subtilisin/kexin type 9), Sc4mol (sterol-C4-

methyl oxidase-like), Sqle (squalene epoxidase), and Thrsp (thyroid hormone responsive SPOT14 homolog). * p < 0.05 between DKO and CNTR or TKO-mice

(n = 2–8).

(H) Apolipoprotein levels were analyzed by immunoblotting analysis of fasted serum from control, DKO, and TKO mice (n = 2) using specific antibodies.


Cell Metabolism


Figure 6. A Working Model for an Integrative Role of Foxo1 in

the Insulin and Growth-Hormone Signaling Pathways

Insulin can inhibit Foxo1 transcriptional activity through Irs1/2 /

PI3K / Akt pathway to phosphorylate Foxo1. As a transcription fac-

tor, Foxo1 can activate one set of genes including Ppargc1a, Pck1,

G6pc, and Igfbp1 and directly or indirectly suppress another set of

genes, including Ghr, Igf1, Onecut1, and Gck. Through regulation of

Ghr gene expression, Foxo1 also influences expression of growth

hormone-regulated genes including Igf1, Igfals, and Onecut1. More-

over, in response to insulin and growth-hormone signals, Onecut1

can impact on nutrient metabolism through activation of Foxa2 and

Gck gene expression and suppression of Pck1 gene expression.

Fonts in bold indicate protein molecules, and fonts in italic indicate

mRNA molecules. Arrows indicate activation, and blunted lines repre-

sent inhibition. Solid lines or arrows indicate reported links in the liter-

ature, and dotted lines or arrows indicate implicated links from this

current study.

Igfbp1, and Igfals in coordination with growth hormone and nu-

trients (Figure 6). On the other hand, growth hormone also inte-

grates with insulin to regulate metabolism, possibly through

Onecut1-mediated gene expression of Foxa2, Gck, and Pck1

(Figure 6) (Lannoy et al., 2002; Pierreux et al., 1999; Rausa

et al., 1997).

Similar to mice lacking hepatic insulin receptors (LIRKO) (Mi-

chael et al., 2000), DKO mice have reduced circulating free fatty

acid and triglyceride concentrations compared to controls. This

effect is consistent with a decreased triglyceride secretion in

DKO mice, as suggested previously for the LIRKO mice (Bid-

dinger et al., 2008). However, both DKO and LIRKO mice have

nearly normal hepatic triglyceride contents although Srebp-1c

expression is decreased in the liver of DKO mice and LIRKO

mice (Biddinger et al., 2008). DKO mice also have decreased se-

rum cholesterol concentrations at 2 and 4 months of age, and the

deletion of Foxo1 partially increases HDL-cholesterol concentra-

tions in TKO mice. This response is consistent with the normal-

ized expression of cholesterol biosynthesis genes in TKO liver.

However, there are no significant changes in ApoB48/100,

ApoA-I, or ApoE in DKO mice compared to control mice,

whereas ApoB48/100 protein levels were increased in the LIRKO

mice (Biddinger et al., 2008). Whether hepatic insulin resistance

increases susceptibility to atherosclerosis in DKO mice needs

further investigation. Overall, these results suggest that choles-

terol homeostasis is also regulated by other insulin-dependent

processes in addition to inactivation of Foxo1 (Gibbons, 2003;

Horton et al., 2002).

In summary, we find that diabetes from a nearly complete loss

of hepatic insulin signaling through the Irs1 and Irs2 branches of

the pathway is alleviated by inactivation of Foxo1. Although other

factors including Torc2 and Foxa2 play an important role in

hepatic gene expression (Dentin et al., 2007; Wolfrum et al.,

2004; Zhang et al., 2005), transcriptomic analysis suggests a

majority of insulin-regulated genes are directly or indirectly con-

trolled by the PI3K / Akt / Foxo1 branch of insulin signaling

pathway. Thus, drugs that interfere specifically with hepatic

Foxo1 function are expected to have substantial effects upon


dysregulated metabolism and might be of benefit for the treat-

ment of diabetes, as previously suggested (Matsumoto et al.,

2007). Finally, hepatic Foxo1 attenuates somatic growth by

suppressing genes in the growth hormone signaling cascade,

revealing how daily food intake can coordinate the systemic

response to growth hormone.

EXPERIMENTAL PROCEDURES

Animals

All animal experiments were performed according to procedures approved by

the Children’s Hospital Boston Institutional Animal Care and Use Committee.

The floxed Irs2 and floxed Foxo1 mice were generated as previously reported

(Lin et al., 2004; Paik et al., 2007). To generate liver-specific knockout mice,

floxed Irs1, Irs2, and Foxo1 mice were crossed with albumin-Cre mice (The

Jackson Laboratory). The LKO1, LKO2, DKO, and their corresponding control

floxed mice were maintained on a C57/BL6 and 129Sv mixed genetic back-

ground; the LKOF, TKO, and their corresponding floxed mice were maintained

on a C57/BL6, 129Sv, and FVB mixed background. The procedure for gener-

ation of floxed Irs1 mice is described below.

The Irs1 targeting construct (Figure S1A) was based on the pPNT vector

modified to contain two loxP sites flanking a neo selectable marker; the neo

cassette was immediately preceded by unique NotI and XhoI sites and imme-

diately followed by a unique BamHI site. The right arm of the construct was in-

serted at the BamHI site and consisted of the first 3 kb of genomic Irs1 DNA

immediately 30 of the stop codon. A unique NheI site�1.6 kb 50 of the Irs1 start

codon served as the site of insertion for a third (distal) loxP site, destroying the

NheI and creating a new BamHI site. The targeting construct was linearized for

transfection into R1 ES cells. Following transfection, potential homologous re-

combinant ES cells were double selected in G418 and gancyclovir and

screened by sequential Southern blotting of KpnI- and BamHI-digested geno-

mic DNA with probes K and B (Figure S1A).

A single homologous recombinant ES cell clone containing the distal

loxP was transfected with a Cre-expressing plasmid. The resulting colonies

were screened with primers c2 (50-CAGCAATGAGGGCAACTCCCCAAGA

CGCTCCA) and rev1 (50-AGAGAGAAGCCCTTCTGTGGCTGCTCCAAACA

CA) to identify partial recombinants lacking the neo cassette but retaining

Irs1 flanked by loxP sites (floxed Irs1 allele, Irs1L). Several nonunique floxed

Irs1 ES subclones were injected separately into C57BL/6 blastocysts to gen-

erate highly chimeric mice containing cells heterozygous for the Irs1L alleles.

The germlines of these mice were sampled by repeated matings to C57BL/6

females to generate agouti pups heterozygous for the Irs1L alleles.

Cell Metabolism


Adenoviruses

Adenoviruses carrying either Irs1, Irs2, and GFP coding sequences, or Foxo1

siRNA or GFP siRNA were delivered to 12-week-old male mice by tail vein in-

jection at 6.5 3 107 viral particles per gram body weight. Mice were monitored

for blood glucose levels at day 0 (preinjection), 2, 3, and 4 (postinjection).

Foxo1 shRNA was constructed using the sense sequence 50-gagcgtgccctactt-

caag-30, short linker, and the corresponding antisense of the above sequence.

Blood Chemistry and Metabolic Analysis

Serum samples were collected from mice that were either overnight fasted or

fed ad libitum, and were analyzed for insulin, free fatty acids, cholesterol, tri-

glycerides, and adiponectin using commercial kits. Blood glucose levels

were measured using a portable glucometer (Bayer). For glucose tolerance

tests, mice were fasted overnight and injected with 2 g/kg body weight D-glu-

cose intraperitoneally as previously described (Dong et al., 2006). For insulin

tolerance tests, mice were fasted for 3 hr and injected intraperitoneally with

1 U/kg body weight humulin R (Lilly), and blood glucose levels were measured

at indicated time points.

For VLDL triglyceride secretion analysis, mice were fasted for 4 hr during

daytime and injected with 500 mg/Kg body weight Triton WR1339 via tail

vein as previously described (Shachter et al., 1996). Blood samples were col-

lected at 0, 1, and 2 hr after the injections and then analyzed for triglyceride

concentrations using a commercial kit (Wako).

Hepatic Glycogen and Lipid Content Analysis

Glycogen content in liver was analyzed in 4 hr refed mice after overnight fast as

previously described (Dong et al., 2006). Hepatic triglyceride content was an-

alyzed in overnight fasted mice as previously described (Dong et al., 2006).

Protein Analysis

For insulin signaling analysis, mice were fasted overnight and anesthetized

with Avertin (200 mg/kg body weight). A dose of 5 units of insulin was injected

via vena cava. Liver, muscle, fat, and brain tissues were harvested after 4 min

of stimulation. Tissue lysates were prepared as previously described (Dong

et al., 2006). Equal amounts of tissue lysates were resolved by SDS-PAGE

and transferred to nitrocellulose for immunoblotting analysis using specific an-

tibodies. For quantitative analysis, enhanced chemiluminescence (ECL) sig-

nals on immunoblots were analyzed by Kodak Molecular Imaging Software.

Affymetrix GeneChip Analysis

Sixteen control mice (four Irs1L/L, four Irs2L/L, four Irs1L/L::Irs2L/L, and four

Irs1L/L::Irs2L/L::FoxOL/L) and sixteen liver-specific knockout mice (four LKO1,

four LKO2, four DKO, and four TKO) at 6 weeks of age were fasted for 16 hr;

and half of the mice from each genotype were sacrificed for tissue collection

(fasted groups) and the other half were fed with regular chow for the next 4

hr and then sacrificed for tissue collection (fed groups). Total RNA was isolated

from the liver tissue using TRIzol (Invitrogen), and 15 mg of each RNA sample

was used for microarray analysis on the Affymetrix MOE430 2.0 GeneChips

by the Harvard Biopolymer facility. Data analysis was detailed in the Supple-

mental Experimental Procedures for Affymetrix GeneChip Analysis available

with this article online.

Quantitative Real-Time PCR Analysis

mRNA levels of genes analyzed in the experiments were quantified by real-

time PCR. The procedure was essentially same as previously described

(Dong et al., 2006). The relative levels of gene expression were calculated by

normalizing to the internal control b-actin.

Statistical Analysis

Data are presented as mean ± SEM. For all tests, a = 0.05; downward adjust-

ment of a (a*) for error control of multiple comparisons was made as appropri-

ate. Significant differences, if any, are described in the figure legends.

ACCESSION NUMBERS

Microarray data were archived to the ArrayExpress database in a MIAME

compliant format under accession number E-MEXP-1649.

SUPPLEMENTAL DATA

Supplemental Data include five figures, two tables, Supplemental Experimental

Procedures, and Supplemental References and can be found with this article

online at http://www.cellmetabolism.org/cgi/content/full/8/1/65/DC1/.

ACKNOWLEDGMENTS

The authors want to thank Dr. Ji-Hye Paik for the help in transferring Foxo1

floxed mice. We also would like to thank Mr. Omer Hancer and Dr. Nancy J.

Fidyk for the help in image processing and Dr. Florian Muller for critically read-

ing this manuscript. This work was funded by the Howard Hughes Medical

Institute and US National Institutes of Health grants DK038712 (M.F.W.),

DK055326 (M.F.W.), and K99DK077505 (X.C.D.).

Received: March 11, 2008

Revised: May 16, 2008

Accepted: June 6, 2008

Published: July 1, 2008

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