Hepatic Akt Activation Induces Marked Hypoglycemia,
Hepatomegaly, and Hypertriglyceridemia With Sterol
Regulatory Element Binding Protein Involvement
Hiraku Ono,1 Hitoshi Shimano,2 Hideki Katagiri,3 Naoya Yahagi,4 Hideyuki Sakoda,1 Yukiko Onishi,1Motonobu Anai,1 Takehide Ogihara,3 Midori Fujishiro,4 Amelia Y.I. Viana,5 Yasushi Fukushima,4Miho Abe,4 Nobuhiro Shojima,4 Masatoshi Kikuchi,1 Nobuhiro Yamada,2 Yoshitomo Oka,3 andTomoichiro Asano4

Akt is critical in insulin-induced metabolism of glucose
and lipids. To investigate functions induced by hepatic
Akt activation, a constitutively active Akt, NH2-terminally myristoylation signal–attached Akt (myr-Akt),was overexpressed in the liver by injecting its adenovirus into mice. Hepatic myr-Akt overexpression resulted
in a markedly hypoglycemic, hypoinsulinemic, and hypertriglyceridemic phenotype with fatty liver and hepatomegaly. To elucidate the sterol regulatory elementbinding protein (SREBP)-1c contribution to these phenotypic features, myr-Akt adenovirus was injected into
SREBP-1 knockout mice. myr-Akt overexpression induced hypoglycemia and hepatomegaly with triglyceride
accumulation in SREBP-1 knockout mice to a degree
similar to that in normal mice, whereas myr-Akt–induced hypertriglyceridemia in knockout mice was
milder than that in normal mice. The myr-Akt–inducedchanges in glucokinase, phosphofructokinase, glucose6-phosphatase, and PEPCK expressions were not affected by knocking out SREBP-1, whereas stearoyl-CoA
desaturase 1 induction was completely inhibited inknockout mice. Constitutively active SREBP-1– overexpressing mice had fatty livers without hepatomegaly,
hypoglycemia, or hypertriglyceridemia. Hepatic acetylCoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and glucose-6-phosphate dehydrogenase
expressions were significantly increased by overexpressing SREBP-1, whereas glucokinase, phosphofructokinase, glucose-6-phosphatase, and PEPCK expressions were not or only slightly affected. Thus,
From the 1Institute for Adult Diseases, Asahi Life Foundation, Shinjuku-ku,
Tokyo, Japan; the 2Division of Endocrinology and Metabolism, Department ofInternal Medicine, Institute of Clinical Medicine, University of Tsukuba,
Tsukuba City, Ibaraki, Japan; the 3Division of Molecular Metabolism andDiabetes, Department of Internal Medicine, Tohoku University Graduate
School of Medicine, Aoba-ku, Sendai, Japan; the 4Department of Internal
Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku,
Tokyo, Japan; and the 5Research Center of Health, Physical Fitness andSports, Nagoya University, Chikusa-ku, Nagoya, Japan.
Address correspondence and reprint requests to Tomoichiro Asano, Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113, Japan. E-mail: asano-tky@umin.ac.jp.
Received for publication 14 March 2003 and accepted in revised form 18
September 2003.
ACC, acetyl-CoA carboxylase; FAO, fatty acid oxidation; FAS, fatty acidsynthase; G6Pase, glucose-6-phosphatase; G6PD, glucose-6-phosphate dehydrogenase; GK, glucokinase; GSK-3, glycogen synthase kinase 3; LacZ, E. coli
␤-galactosidase gene; LPL, lipoprotein lipase; myr-Akt, NH2-terminally myristoylation signal–attached Akt; PFK, phosphofructokinase; PK, L-type pyruvatekinase; PPAR␣, peroxisome proliferator–activated receptor-␣; SCD1, stearoylCoA desaturase 1; SREBP, sterol regulatory element binding protein.
© 2003 by the American Diabetes Association.
DIABETES, VOL. 52, DECEMBER 2003

SREBP-1 is not absolutely necessary for the hepatic
Akt-mediated hypoglycemic effect. In contrast, myrAkt–induced hypertriglyceridemia and hepatic triglyceride accumulation are mediated by both Akt-induced
SREBP-1 expression and a mechanism involving fatty
acid synthesis independent of SREBP-1. Diabetes 52:
2905–2913, 2003

Phosphatidylinositol 3-kinase activation via insulin
receptor substrate proteins and downstream Akt
has been shown to play a critical role in insulininduced metabolic actions (1–3). In fat and muscle, the role of Akt has been widely recognized as
mediation of insulin-stimulated glucose uptake (4 – 6).
Indeed, overexpression of constitutively active forms of
Akt is sufficient to induce glucose transport in 3T3-L1
adipocytes and L6 muscle cells (7–9), whereas Akt2deficient mice showed impaired glucose tolerance due to adecrease in insulin-induced glucose uptake in skeletal
muscle and increased hepatic glucose production (10).
On the other hand, in the liver, the rate of glucose
utilization is determined by rate-limiting metabolic enzymes that catalyze glucose to generate triglyceride andglycogen by pathways known as glycolysis, lipogenesis,
and glycogenesis. Moreover, the liver can produce glucose
from lactate, pyruvate, and amino acids via gluconeogenesis and also from glycogen, via glycogenolysis, both ofwhich are also rate limited by key metabolic enzymes at
each step. Therefore, the rates of hepatic glucose input
and output depend on rate-limiting metabolic enzymes,
and insulin is the most important regulator of these
enzymes (11).
Akt is regarded as a key signal molecule mediating themetabolic actions of insulin in muscle, fat, and the liver(2,12). The first aim of this study was to characterize thephenotype induced by the activation of hepatic Akt. For
this purpose, we overexpressed constitutively active Akt
by using adenovirus transfer into the mouse liver. This
allowed investigation of phenotypic features, including
glucose and lipid metabolism. Much remains unclear regarding the pathway from Akt activation to the regulationof the enzymes involved in glucose and lipid metabolism.
To date, it has been reported that Akt phosphorylates andactivates glycogen synthase kinase (GSK)-3 (13,14). Phos2905

SREBP IN AKT-MEDIATED METABOLIC ACTIONSTABLE 1
Sequences of PCR primers for cloning mouse cDNA probes
Probe

Primer 1

Primer 2

Positionin cDNA

ACC
CPT1
CPT2
FAS
G6Pase
G6PD
GK
LCAD
LDLR
LPL
MCAD
PEPCK
PFK
PK
PPAR␣
SCAD
SCD1
VLCAD

5⬘-GACTTCATGAATTTGCTGAT-3⬘
5⬘-GATCTACAATTCCCCTCTGC-3⬘
5⬘-CTAGACACCTCCTGGTCCTA-3⬘
5⬘-GCAGATCCTTTGATGATTCA-3⬘
5⬘-TGCTGCTCACTTTCCCCACCAG-3⬘
5⬘-ACCCTTCATCCTGCGCTGT-3⬘
5⬘-CGACTCTGGGGACCGAAGGCAG-3⬘
5⬘-AATGGGAGAAAGCTGGAGAA-3⬘
5⬘-TCCATCGCAGCTGGGTCTGT-3⬘
5⬘-TGCCACTTCAACCACAGCAG-3⬘
5⬘-CAGAGAGGAGATTATCCCCG-3⬘
5⬘-GACAGCCTGCCCCAGGCAGTGA-3⬘
5⬘-AGGCTCTCGGCTGAACATCA-3⬘
5⬘-ACCGTGAGCCTCCAGAGGCT-3⬘
5⬘-ATGGTGGACACAGAGAGCC-3⬘
5⬘-ATGGGTGAGCTCGGGCTGCT-3⬘
5⬘-ATGCCGGCCCACATGCTCCA-3⬘
5⬘-GCTTACATGCTGAGTGCCAA-3⬘

5⬘-AAGCTGAAAGCTTTCTGTCT-3⬘
5⬘-CTGCATCTGCTGCTCCAGCT-3⬘
5⬘-CAACTTCTGTCTGAGCTCTG-3⬘
5⬘-TTACACCTTGCTCCTTGCTG-3⬘
5⬘-TCTCCAAAGTCCACAGGAGGT-3⬘
5⬘-CGGATGACCAGCTCATTA-3⬘
5⬘-TGATGCGCATCACGTCCTCACT-3⬘
5⬘-TAGGGCATGACAATATCTGA-3⬘
5⬘-TACACTGTGTCACATTGACGC-3⬘
5⬘-TCCCACCAGCTTGGTGTAGC-3⬘
5⬘-TAGCAGTTTGCACCCCTGTA-3⬘
5⬘-CTGGCCACATCTCGAGGGTCAG-3⬘
5⬘-TGAAGGCGTCCCTCCTCGCT-3⬘
5⬘-TGTTGGTATAGCCAGAGCCA-3⬘
5⬘-GAGCTCTCCTCACCGATGGA-3⬘
5⬘-ATCCACTGTTGCTTCTGCTG-3⬘
5⬘-TACTCCAGCTTGGGCGGGGG-3⬘
5⬘-CCTTCAAAGATCCGGAAGAT-3⬘

1640–1809
915–1114
781–980
793–942
601–800
1108–1230
1133–1304
250–448752–951
398–595
171–370
196–395
826–985
1591–1856
439–575
263–462
136–335
1275–1474

CPT, carnitine palmitoyltransferase; LCAD, long-chain acyl-CoA dehydrogenase; LDLR, LDL receptor; MCAD, medium-chain acyl-CoA
dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase; VLCAD, very-long-chain acyl-CoA dehydrogenase.

phorylated GSK-3 then induces the activation of glycogensynthase. Akt is thus involved in one mechanism of
insulin-induced activation of glycogen synthase. In addition, recently, Akt was reported (15) to increase theexpression of sterol regulatory element binding protein(SREBP)-1, and some previous reports (16 –24) have suggested an important role of SREBP-1 not only in lipid
metabolism but also in glucose metabolism. Thus, we
endeavored to elucidate the contributions of SREBP-1 tohepatic Akt-induced phenotypes by utilizing SREBPknockout mice and hepatic SREBP-1a– and -1c– overexpressing mice. Comparing the mice overexpressing active
Akt and SREBP-1 and the SREBP-1 knockout mice overexpressing active Akt revealed the contribution of
SREBP-1 to various Akt-induced phenotypes and alteredgene expressions. These results contribute to understanding the molecular mechanisms underlying insulin-activating Akt-induced in vivo phenotypes.
RESEARCH DESIGN AND METHODS
Adenoviruses, animals, and measurements of serum/hepatic components. Full-length mouse Akt cDNA (25) was NH2-terminally tagged with thesequence corresponding to myristoylation signal peptide as previously described (26). Adenoviruses expressing ␤-galactosidase (i.e., the E. coli ␤-galactosidase gene [LacZ]) and NH2-terminally myristoylation signal–attachedAkt (myr-Akt) were created as already described (27). Adenoviruses expressing constitutively active human SREBP-1a (amino acids 1– 460) and constitutively active SREBP-1c (1– 436) were constructed using an Ad Easy kit(Quantum Biotechnology). Male C57BL/6 mice (8 –10 weeks of age) or male
SREBP-1 knockout mice (28) (15 weeks of age) were injected, via the tail vein,with adenovirus at a dose of 2.5 ⫻ 107 plaque-forming units/g body wt. Animals
were fasted for 14 h (experiments in Figs. 1 and 2) or 24 h (experiments in
Figs. 3– 6) before blood sampling and killed. Blood glucose was measured witha portable blood glucose monitor, Glutest-Ace (Sanwa Kagaku Kenkyusho,
Nagoya Japan). The plasma insulin level was determined with a radioimmunoassay kit (Pharmacia, Tokyo, Japan). Serum triglyceride and cholesterol
were assayed as previously described (29,30). Hepatic total lipid was extracted as described using the Folch method (31), washed with saline andwater, dried, and resuspended in Krebs-Ringer buffer containing 3% BSA.
Triglyceride content in this resuspension was assayed as previously described(29). Hepatic glycogen content was measured as previously described (32). All
2906

animal studies were conducted according to the Japanese guidelines for thecare and use of experimental animals.
Preparation of riboprobes for RNase protection assays. Riboprobes ofmouse SREBP-1a and SREBP-1c for RNase protection assays were described
previously (33). Riboprobes of other enzymes were amplified from mouseembryonic cDNA using the PCR primers described in Table 1. PCR products
were subcloned into pCR2.1 (Invitrogen), pBlueScript (Stratagene), or pCMVScript (Stratagene), and all sequences were certified using a CEQ-2000
sequencer (Beckman).
RNA preparation, Northern blotting, and RNase protection assay. Total
hepatic RNA was isolated using Trizol reagent (Isogen; Nippon Gene, Tokyo,
Japan). RNA concentrations were estimated based on absorbance at 260 nm.
Northern blotting of SREBP-1 was conducted by running 20-␮g RNA samples
on a 1% agarose gel containing formaldehyde and transferred to a nylon
membrane. The 47-mer oligo-DNA probe of SREBP-1 (5⬘-ATGTAGTCGATG
GCCTTGCGCAAGACAGCAGATTTATTCAGCTTTGC-3⬘), which is the same inhumans and mice, was labeled with [␥-32P]ATP using the DNA 5⬘ End-Labeling
System (Promega) and purified with a ProbeQuant G-50 Micro Column(Amersham). The membranes were hybridized with 106 cpm/ml of theradiolabeled probe in Ultra-hyb Buffer (Ambion) at 42°C overnight andwashed according to the manufacturer’s instructions. Blots were exposedusing a Molecular Imager GS-525 (Biorad). RNase protection assays werecarried out according to the manufacturer’s instructions (RPA III kit; Ambion,
Austin, TX). RNA samples (10 ␮g) from each mouse were hybridized with the
riboprobes. After treatment with RNase, the intensities of the resultant bands
were determined using the Molecular Imager GS-525.
Immunoblotting. For immunoblotting of SREBP proteins, nuclear extracts
from mouse livers were prepared as described previously (34). Aliquots ofnuclear proteins (20 ␮g) were subjected to SDS-PAGE. Immunoblot analyses
were performed using the ECL Western Blotting Detection System kit(Amersham Pharmacia Biotech). The primary antibodies for SREBPs were aspreviously described (24,28). For the detection of tissue Akt, each tissue
sample was homogenized in 10 volumes of lysis buffer (20 mmol/l Tris, pH 7.5,
150 mmol/l NaCl, 1 mmol/l EDTA, 1% Nonidet P-40, and 1 mmol/l phenylmethylsulfonyl fluoride) and centrifuged. Protein concentrations of the collectedsupernatant were assayed using a bicinchoninic acid assay kit (Pierce), andsamples were adjusted to 3 mg/ml. Forty-five micrograms of each tissue
specimen were subjected to SDS-PAGE. The polyclonal antibody for Akt was
raised in rabbits using the keyhole limpet hemocyanin– conjugated COOHterminal 17 amino acids of Akt.
Akt activity assay. Akt activity in the liver was assayed using an Akt Kinase
Assay kit (Cell Signaling Technology). Hepatic tissue was homogenized in a
10-fold volume of lysis buffer, and 200 ␮l of the lysate were incubated with 10
␮l of immobilized Akt monoclonal antibody for 2 h. Washing and kinase
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FIG. 1. Phenotypes of mice injected with LacZ or myr-Akt adenovirus. C57BL/6 male mice were injected with 2.5 ⴛ 107 plaque-forming units/g bodyweight of adenovirus-containing ␤-galactosidase (LZ) or myr-Akt construct via the tail vein. Blood sampling and killing were carried out 4 days
after the injections, following 14 h of fasting. A: Western blotting of various tissues lysates. Each tissue (45 ␮g) was electrophoresed andimmunoblotted with anti-Akt antibody. Odd numbered lanes represent tissues from LacZ-injected mice, and even numbered lanes represent thosefrom myr-Akt–injected mice. B: Abdominal cavity. C: Sudan III staining of the liver. D: Akt kinase assay of livers. E: Body and liver weights, blood
glucose, plasma insulin concentration, triglyceride, total cholesterol, and hepatic glycogen content. Results represent means ⴞ SE of three mice.
FBS, fasting blood sugar.

reaction with GSK-3 fusion protein were done according to the manufacturer’s
instructions.
Quantification of the results. Results are expressed as means ⫾ SE, and thesignificance was assessed using one-way ANOVA and unpaired Student’s ttests.

RESULTS

Overexpression of constitutively active Akt caused
marked hepatomegaly, fatty liver, hypertriglyceridemia, and hypoglycemia. Four days after the intravenous
viral injections, the tissue distribution of overexpressed
myr-Akt was investigated by immunoblotting with anti-Akt
antibody. Results showed that myr-Akt was highly selectively expressed in the liver and undetectable in othertissues (Fig. 1A). Endogenous Akt expressions did not
differ between LacZ and myr-Akt mice in most tissues, but
in fat, endogenous Akt was slightly decreased by theoverexpression of myr-Akt. Overexpression of myr-Akt
DIABETES, VOL. 52, DECEMBER 2003

resulted in markedly higher kinase activity than endogenous Akt in control mice, as demonstrated in the experiment using GSK-3 as a substrate for the Akt kinase (Fig.
1D).
The livers of constitutively active Akt (myr-Akt)–injected mice were whitish and markedly enlarged as compared with those of control mice (Fig. 1B). Liver weight in
myr-Akt–injected mice was ⬃2.5-fold greater than that of
the control LacZ-injected mice, without a significant difference in overall body weight (Fig. 1E). Sudan III staining
showed fine- to medium-sized lipid droplets to have accumulated diffusely in the livers of myr-Akt–injected mice,indicating fatty liver (Fig. 1C). Supratesticular fat did not
differ significantly in size between control and myr-Akt–
injected mice (data not shown).
Serum parameters and hepatic glycogen contents arepresented in Fig. 1E. Serum glucose levels of myr-Akt–
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SREBP IN AKT-MEDIATED METABOLIC ACTIONS

FIG. 2. Detection of SREBP from the liver. A: RNase protection
assay for detecting all SREBP-1 subtypes. A 10-␮g aliquot of liver
total RNA from each of three mice was hybridized with riboprobesof SREBP-1a or -1c. B: Western blotting of liver nuclear extract.
Aliquots of hepatic nuclear extract were subjected to SDS-PAGE
and immunoblotted with anti–SREBP-1 or anti–SREBP-2 antibodies.

injected mice were significantly reduced (40 ⫾ 5 and 60 ⫾8 mg/dl in the fasted and ad libitum states, respectively),
whereas those of the controls were 110 ⫾ 19 and 121 ⫾ 22
mg/dl, respectively. Fasting plasma insulin levels of myrAkt–injected and control mice were 0.7 ⫾ 0.5 and 4.8 ⫾ 1.4
␮IU/ml, respectively. Serum total cholesterol in the fasted
state was slightly but significantly higher in myr-Akt–
injected mice than in controls. Serum triglyceride in thefasted state was markedly increased, by 4.7-fold, in myrAkt–injected mice. Hepatic glycogen content was also
markedly increased in the myr-Akt– expressing liver.
Induction of SREBP-1c in myr-Akt– overexpressing
liver. SREBP-1c has been attracting attention because it isinduced by insulin stimulation (20,35,36) and then functions as a transcription factor that induces several ratelimiting lipogenic enzymes in the liver (16,22,24,37,38).
Recent reports (15,18) have described insulin-induced
SREBP-1c induction as being mediated through the phosphatidylinositol 3-kinase pathway, and it was suggested(15) that Akt is also involved in this induction of SREBP1c. Thus, we examined the effect of myr-Akt overexpression on SREBP-1c expression in the liver. RNase
protection assay revealed an increase in SREBP-1c mRNA
with no significant change in the SREBP-1a mRNA level(Fig. 2A). Immunoblotting of hepatic nuclear extracts
revealed that the nuclear form of SREBP-1 from the
myr-Akt– expressing liver to be increased while that of
SREBP-2 was not affected (Fig. 2B). These results are ingood agreement with those of a previous report (15).
To reveal the contribution of increased SREBP-1 expression to the myr-Akt–induced phenotypes, two models
were utilized. In one model, wherein SREBP-1 knockout
mice received a myr-Akt–adenoviral injection, we observed the phenotypes that myr-Akt induced independently of SREBP-1 induction. The other, overexpression of
constitutively active types of SREBP-1 in the mouse liver,allowed the phenotypes resulting from SREBP-1 induction
to be assessed. Figure 3 shows the Northern blotting ofliver RNA from six types of mice: 1) normal mice expressing control LacZ, 2) normal mice expressing myr-Akt, 3)
SREBP-1 knockout mice expressing control LacZ, 4)
SREBP-1-knockout mice expressing myr-Akt, 5) normal
mice expressing constitutively active SREBP-1a, and 6)
normal mice expressing constitutively active SREBP-1c.
Induction of SREBP-1 by myr-Akt overexpression (compare lane 1 with lane 2 in Fig. 3) was not observed in
SREBP-1 knockout mice (lanes 3 and 4). On the other
hand, mRNAs of overexpressed nuclear forms of
SREBP-1a and -1c, which are known to function as constitutively active SREBP-1s (38), were observed to becomparable to or stronger dense bands (lanes 5 and 6)(Fig. 3) than that of myr-Akt–induced SREBP-1 (lane 2).
Moreover, endogenous SREBP-1 mRNA was induced by2908

the expression of constitutively active SREBP-1 as a resultof positive feedback regulation of SREBP-1 (24).
Phenotypes of hepatic myr-Akt– expressing SREBPknockout mice and hepatic constitutively active
SREBP-1– expressing mice. The phenotypes of six typesof mice are summarized in Fig. 4. Hepatomegaly was
similar in myr-Akt– expressing livers, irrespective of thepresence or absence of the SREBP-1 gene, whereas
SREBP-1 overexpression induced whitish fatty liver withno change in hepatic weight (Fig. 4A). Hepatic triglycerides accumulated in response to overexpression of either
myr-Akt or SREBP-1, but it should be noted that myr-Akt
overexpression induced marked hepatic triglyceride accumulation also in the SREBP knockout mice (Fig. 4B).
The hypoglycemic effect induced by hepatic myr-Akt
overexpression was also induced in the SREBP knockout
mice and was similar to that observed in normal mice (Fig.
4C). In contrast, no significant hypoglycemia occurred inassociation with active SREBP-1 overexpression. Serumtriglyceride elevation with myr-Akt overexpression wasmarked as mentioned before, whereas the increase inserum triglyceride with active SREBP-1 overexpression
was minimal (Fig. 4D). The serum triglyceride concentration in SREBP-1 knockout mice was very low. Interestingly, however, myr-Akt overexpression had a significant
serum triglyceride elevating effect in the SREBP-1 knockout mice.
Regulation of mRNA levels of glycolytic and gluconeogenic rate-limiting enzymes by Akt and

FIG. 3. Northern blotting of liver RNA from six types of mice. Six typesof mice were created, and 4 days later, 24-h fasted mice were killed
after blood sampling. The six types were 1) C57BL/6 mice with LacZ
adenovirus injection (LacZ), 2) C57BL/6 mice with myr-Akt adenovirus
injection, 3) SREBP-1 knockout mice with LacZ adenovirus injection(SREBP1-KO-LacZ), 4) SREBP-1 knockout mice with myr-Akt adenovirus injection (SREBP1-KO-myrAkt), 5) C57BL/6 mice with constitutively active SREBP-1a adenovirus injection (SREBP1a), and 6)
C57BL/6 mice with constitutively active SREBP-1c adenovirus injection (SREBP1c). Three mice of each type were created in cases 1, 2, 5,
and 6. For 3 and 4, only two mice each were available, so two hepatic
lobes from each mouse were excised, such that four total lobes for eachtype were manipulated independently to isolate RNA. Aliquots of RNA(20 ␮g) were electrophoresed, transferred, and blotted with a 5ⴕlabeled oligo-DNA probe detecting SREBP-1 as described in RESEARCHDESIGN AND METHODS. Representative data from each type of mouse areshown. Constitutively active SREBP-1 is a nuclear form whose mRNA isshorter than the endogenous precursor mRNA, which appears below
the endogenous SREBP-1.
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FIG. 4. Physiological and biochemical data of six types ofmice. As described in the legend of Fig. 3, all three mice ofeach type were injected with LacZ (LZ), myr-Akt (mAkt),SREBP-1a (BP1a) or SREBP-1c (BP1c) adenovirus, orboth. SREBP-1 knockout mice were injected with LacZ or
myr-Akt adenovirus. Four days after injection, mice were
fasted for 24 h, blood samples were taken, and then mice
were killed. Graphs show the means ⴞ SE of liver weight/
body weight ratios (A), hepatic triglyceride content (B),fasting blood glucose (C), and serum triglyceride level(D). FBS, fasting blood sugar; KO, knockout; TG, triglyceride.

SREBP-1 activations. Glucokinase (GK), phosphofructokinase-1 (PFK), and L-type pyruvate kinase (PK) are therate-limiting enzymes determining hepatic glycolytic activity, whereas glucose-6-phosphatase (G6Pase) and PEPCK
are hepatic gluconeogenic rate-limiting key enzymes.
Their mRNA levels were measured by RNase protection
assay.
Overexpression of myr-Akt markedly upregulated GK inboth normal and SREBP-1 knockout mouse liver (Fig. 5A).
Active-type SREBP-1s slightly but not significantly increased GK mRNA. Myr-Akt overexpression also increased PFK mRNA regardless of the presence of
SREBP-1, whereas overexpression of SREBP had no effecton PFK mRNA expression (Fig. 5B). The PK mRNA levelalso did not differ significantly from overexpression of
myr-Akt or SREBP (Fig. 5C).
The mRNAs of the two important rate-limiting enzymes
regulating gluconeogenesis, G6Pase and PEPCK, weresignificantly downregulated by overexpression of myr-Akt,which was similar to the observations in SREBP-1 knockout mice. In contrast, overexpression of the active-type
SREBP-1 had no significant effect on G6Pase and PEPCK
mRNA levels (Fig. 5D and E).
Regulation of mRNA levels of rate-limiting lipogenic
enzymes by Akt and SREBP-1 activations. Figure 6
shows the effects of myr-Akt and SREBP-1 overexpressions on rate-limiting lipogenic enzymes. Myr-Akt overexpression increased fatty acid synthase (FAS) mRNAslightly (Fig. 6A) and stearoyl-CoA desaturase 1 (SCD1)
markedly (Fig. 6B), whereas amounts of acetyl-CoA carboxylase (ACC) and glucose-6-phosphate dehydrogenase(G6PD) mRNAs did not change significantly (Fig. 6C and
D). In contrast, all four enzymes were induced by constiDIABETES, VOL. 52, DECEMBER 2003

tutively active SREBP-1a or -1c overexpression, and theeffect of SREBP-1a was stronger than that of SREBP-1c.
Myr-Akt–induced SCD1 mRNA induction was completely
abolished in SREBP-1 knockout mice. As for ACC, FAS,
and G6PD, the regulatory effect of Akt activation appears
to be relatively weak compared with that of SREBP-1.
Effects on hepatic fatty acid oxidation and triglyceride utilization in tissues by adenoviral myr-Akt expression. To investigate whether the effects of myr-Akt on
triglyceride metabolism are due to altered lipid breakdown, we measured mRNA levels of key hepatic proteins
involved in fatty acid oxidation (FAO) (Table 2). In the
myr-Akt– expressing liver, peroxisome proliferator–activated receptor (PPAR)␣ and FAO enzymes tended todecrease as compared with the control liver, but that effectwas not significant. In contrast, when myr-Akt was expressed in the SREBP-1 knockout mouse liver, myr-Akt
significantly increased PPAR␣ and FAO enzymes.
To investigate whether the discrepancy in serum versus
hepatic triglyceride levels is a result of myr-Akt–induced
transcriptional changes in the LDL receptor or lipoprotein
lipase (LPL), the mRNA levels of the LDL receptor in theliver and of the LDL receptor and LPL in fat and muscle
were measured (Table 3). In the liver, myr-Akt upregulated
the LDL receptor similarly in normal and SREBP-1 knockout mice. In fat and muscle, neither LPL nor LDL receptor
mRNA levels differed significantly among the six types ofmice studied.
DISCUSSION

Akt reportedly plays key roles in various cellular functions, including glucose transport, glycogen synthesis,2909

SREBP IN AKT-MEDIATED METABOLIC ACTIONS

FIG. 5. RNase protection assays of key glycolytic/
gluconeogenic enzymes. Three independent samples
from three C57BL/6 mice that had been injected with
LacZ (LZ), myr-Akt (mAkt), SREBP-1a (BP1a), or
SREBP-1c (BP1c) adenovirus or four independent(two lobes from each mouse) samples from two
SREBP-1 knockout (KO) mice that had been injectedwith LacZ or myr-Akt adenovirus were prepared. A
10-␮g aliquot of total hepatic RNA was hybridized
with riboprobes of each enzyme. The results represent the means ⴞ SE of three or four samples. The
scale of the graphs represents the fold increase in thevalue in LacZ adenovirus–injected C57BL/6 mice.

DNA synthesis, antiapoptotic activity, and cell proliferation. Thus, the role of Akt is not limited to the metabolic
actions of insulin, and intensive studies have focused on
Akt in various fields of cell biology, tissue development,
and so on. Several reports have described tissue-specificfindings in mice transgenic for Akt. The heart (39 – 41) andpancreas (42,43), in which an active mutant of Akt wasoverexpressed, showed marked tissue enlargement.
To study the role of hepatic Akt, constitutively active Akt
was overexpressed selectively in the liver, and the resultant phenotype was investigated. We cannot rule out thepossibility that myr-Akt stimulates some signals that endogenous Akt does not; however, because Akt is phosphorylated, translocated to the membrane fraction, andthen transmits the signal(s), the overexpression of myrAkt rather than wild-type Akt is more physiological, al2910

though other molecules such as atypical PKC are also
likely to transmit signals.
We found that hepatic overexpression of constitutively
active Akt led to a phenotype characterized by marked
hepatomegaly, hypoglycemia with hypoinsulinemia, andhypertriglyceridemia with fatty liver. Hepatomegaly islikely to result from cell proliferation via Akt’s oncogenic
activity and the accumulation of lipid and glycogen. It wasalso demonstrated that constitutively active Akt mimics
the effects of insulin in the liver, hypoglycemia, hepatic
glycogen accumulation, transcriptional upregulation ofGK, and downregulation of G6Pase and PEPCK (11,44).
We speculate that, based on the observation that myr-Akt
is sufficient for producing a hypoglycemic effect, theagents activating Akt could be novel antidiabetic drugs
even if they exert their effects only in the liver. However,
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FIG. 6. RNase protection assays of key lipogenic enzymes
and G6PD. Three independent samples from three C57BL/6
mice that had been injected with LacZ (LZ), myr-Akt(mAkt), SREBP-1a (BP1a), or SREBP-1c (BP1c) adenovirus
or four independent (two lobes from each mouse) samples
from two SREBP-1 knockout (KO) mice that had been
injected with LacZ or myr-Akt adenovirus were prepared. A
10-␮g aliquot of total hepatic RNA was hybridized withriboprobes of each enzyme. The results represent themeans ⴞ SE of three or four samples. The scale of the graphs
represents the fold increase in the value in LacZ adenovirus–injected C57BL/6 mice.

it is possible that if such a drug was to act continuously or
too strongly, undesirable side effects, such as hypertriglyceridemia and hepatomegaly, might develop.
It was recently hypothesized that SREBP-1, the expression of which is increased by Akt activation (15), contributes to the regulation of hepatic glucose-metabolizing
enzymes that function to produce hypoglycemic effects.
SREBP-1c reportedly enhances transcriptions of GK(19,21) and PK (20) and also inhibits transcription of
PEPCK (19,22), suggesting that SREBP-1c is a key factor inhepatic hypoglycemic function (19). However, other reports suggested that expressions of G6Pase and PEPCK
are regulated by Forkhead transcription factor (FKHR)(45,46) or GSK-3 (47), which are directly phosphorylated
by Akt and inactivated (13,14,48,49).

Our results using myr-Akt expressing SREBP-1 knockout mice clearly demonstrated that SREBP-1 is not necessary for Akt-mediated induction of GK and inhibition of
G6Pase and PEPCK transcriptions. As for PK, several
reports (50 –52) have shown that it is not insulin but rather
glucose that regulates its transcription. This is consistent
with our observation that myr-Akt overexpression has noeffect.
The lipogenic/FAO enzyme and triglyceride results arecomplicated but interesting. Among the lipogenic enzymes, SCD1 is markedly upregulated by myr-Akt overexpression in normal mice but not in SREBP-1 knockout
mice, suggesting that transcriptional regulation of SCD1
by Akt is mediated entirely via the SREBP-1 pathway orthat SREBP-1 acts as a strict permissive factor for SCD1TABLE 2
RNase protection assay of PPAR␣ and fatty acid oxidative enzymes

PPAR␣
CPT1
CPT2
VLCAD
LCAD
MCAD
SCAD

LacZ

myr-Akt

Knockout
LacZ

Knockout
myr-Akt

SREBP-1a

SREBP-1c

1.00 ⫾ 0.05
1.00 ⫾ 0.11
1.00 ⫾ 0.14
1.00 ⫾ 0.06
1.00 ⫾ 0.04
1.00 ⫾ 0.06
1.00 ⫾ 0.10

0.80 ⫾ 0.05
0.71 ⫾ 0.07
0.90 ⫾ 0.03
1.08 ⫾ 0.13
0.81 ⫾ 0.02
0.91 ⫾ 0.11
1.00 ⫾ 0.05

0.23 ⫾ 0.04*
0.54 ⫾ 0.03㛳
0.56 ⫾ 0.00§
0.71 ⫾ 0.05㛳
0.56 ⫾ 0.11§
0.48 ⫾ 0.18㛳
0.69 ⫾ 0.02†

0.52 ⫾ 0.03†‡
0.78 ⫾ 0.22
0.98 ⫾ 0.16¶
1.08 ⫾ 0.21¶
0.71 ⫾ 0.12㛳
0.59 ⫾ 0.13
1.00 ⫾ 0.06‡

0.52 ⫾ 0.07†
0.59 ⫾ 0.15
0.74 ⫾ 0.10
0.76 ⫾ 0.08
0.79 ⫾ 0.13
0.45 ⫾ 0.07§
0.84 ⫾ 0.07

0.61 ⫾ 0.13§
0.71 ⫾ 0.14㛳
0.61 ⫾ 0.11§
0.75 ⫾ 0.08
0.69 ⫾ 0.13㛳
0.48 ⫾ 0.13㛳
0.85 ⫾ 0.07

Data are means ⫾ SE of three to four samples. A 10-␮g aliquot of total hepatic RNA was hybridized with riboprobes for each enzyme. The
scale represents the fold increase in the value in LacZ adenovirus–injected C57BL/6 mice. CPT, carnitine palmitoyltransferase; LCAD,
long-chain acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; SCAD, short-chain acyl-CoA dehydrogenase; VLCAD,
very-long-chain acyl-CoA dehydrogenase. *P ⬍ 0.001 vs. LacZ; †P ⬍ 0.01 vs. LacZ; ‡P ⬍ 0.01 vs. knockout LacZ; §P ⬍ 0.05 vs. LacZ; 㛳P ⬍ 0.05
vs. knockout LacZ; ¶P ⬍ 0.1 vs. LacZ.
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SREBP IN AKT-MEDIATED METABOLIC ACTIONSTABLE 3
RNase protection assay of LDL receptor and LPL

Liver LDL receptor
Muscle LDL receptor
Fat LDL receptor
Muscle LPL
Fat LPL

LacZ

myr-Akt

Knockout
LacZ

Knockout
myr-Akt

SREBP-1a

SREBP-1c

1.00 ⫾ 0.15
1.00 ⫾ 0.10
1.00 ⫾ 0.16
1.00 ⫾ 0.12
1.00 ⫾ 0.45

1.81 ⫾ 0.20*
0.91 ⫾ 0.21
1.10 ⫾ 0.08
1.07 ⫾ 0.03
0.88 ⫾ 0.21

0.47 ⫾ 0.07†
1.25 ⫾ 0.22
1.04 ⫾ 0.22
1.14 ⫾ 0.21
1.35 ⫾ 0.33

2.30 ⫾ 0.30‡
1.09 ⫾ 0.22
1.06 ⫾ 0.32
0.93 ⫾ 0.26
0.94 ⫾ 0.16

2.15 ⫾ 0.50†
1.12 ⫾ 0.06
1.01 ⫾ 0.07
1.01 ⫾ 0.05
1.24 ⫾ 0.34

0.75 ⫾ 0.05
1.10 ⫾ 0.28
1.10 ⫾ 0.20
1.01 ⫾ 0.15
1.26 ⫾ 0.16

Data are means ⫾ SE of three to four samples. A 10-␮g aliquot of total hepatic RNA was hybridized with riboprobes for each enzyme. The
scale represents the fold increase in the value in LacZ adenovirus–injected C57BL/6 mice. *P ⬍ 0.05 vs. LacZ; †P ⬍ 0.01 vs. LacZ; ‡P ⬍ 0.01
vs. knockout LacZ.

gene expression. Alternatively, it is also possible that basal
levels of SREBP induce the expressions of some key genes
or transcription factors that mediate the effect of Akt. In
contrast, FAS and G6PD were upregulated by myr-Akt inboth normal and SREBP-1 knockout mice to a similar
degree, indicating that Akt increases their expressions via
a pathway independent of SREBP-1. Indeed, even in
SREBP knockout mice, hepatic triglyceride content wassignificantly increased by myr-Akt overexpression. The
regulations of PPAR␣ and FAO enzymes are complex, but
the observation that myr-Akt does not decrease themRNAs of FAO enzymes in SREBP-1 knockout micesuggests that hepatic triglyceride accumulation is not due
to a decrease in FAO. Taken together, these findings
strongly suggest that an SREBP-1–independent lipogenic
pathway exists downstream from Akt.
Another interesting finding is the effect on serum triglycerides. Although SREBP-1 overexpressions resulted inlipid accumulation in the liver to a degree similar to thatobtained with myr-Akt, only myr-Akt produced hypertriglyceridemia. In this study, myr-Akt and SREBP-1ashowed comparable upregulatory effects on the hepatic
LDL receptor. Furthermore, fat and muscle displayed nosignificant changes in the mRNA expression levels of the
LDL receptor or LPL when myr-Akt adenovirus was injected. In myr-Akt– expressing mice, both hepatic andserum triglyceride levels increased, although in SREBP1a– expressing mice, only the hepatic triglyceride level
increased. These results cannot be explained by modified
LDL receptor or LPL gene expressions in the liver, fat, or
muscle. Thus, we speculate that triglyceride secretionfrom the liver is increased in the myr-Akt– expressing
mice, but further study is needed to clarify this issue.
Our observations can be summarized as follows. 1)
Hepatic overexpression of Akt led to a phenotype characterized by marked hepatomegaly, hypoglycemia with hypoinsulinemia, and hypertriglyceridemia with fatty liver.
2) The hypoglycemic effect induced by Akt is associatedwith upregulation of glycogen synthesis, downregulationsof PEPCK and G6Pase, and upregulations of GK, PFK, and
FAS. Furthermore, SREBP-1 is not necessary for the
Akt-mediated hypoglycemic effect. 3) Lipogenic enzymes
are partially regulated by SREBP-1, but an SREBP-1–
independent lipogenic pathway involving transcriptional
regulations of FAS and G6PD may exist in the liver
downstream from Akt activation.
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