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Akt1/PKBα Is Required for Normal Growth but Dispensable for Maintenance of Glucose Homeostasis in Mice

2001; Elsevier BV; Volume: 276; Issue: 42 Linguagem: Inglês

10.1074/jbc.c100462200

1083-351X

Han Cho, Joanne L. Thorvaldsen, Qingwei Chu, Fei Feng, Morris J. Birnbaum,

FOXO transcription factor regulation

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBβ, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science292, 1728–1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBα, display a conspicuous impairment in organismal growth. Akt1−/− mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBβ null mice, Akt1/PKBα-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 andAkt2 genes with respect to organismal growth and insulin-regulated glucose metabolism. The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBβ, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science292, 1728–1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBα, display a conspicuous impairment in organismal growth. Akt1−/− mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBβ null mice, Akt1/PKBα-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 andAkt2 genes with respect to organismal growth and insulin-regulated glucose metabolism. protein kinase B phosphatidylinositol 3-kinase polymerase chain reaction embryonic stem insulin receptor substrate insulin-like growth factor kilobase Recent genetic analyses have emphasized the evolutionary conservation of insulin signaling as a generalized organismal response to nutritional abundance. Nonetheless, much uncertainty remains concerning how this signaling pathway diverges to allow independent regulation of such disparate biological outputs as metabolism, aging, and growth. The serine-threonine kinase Akt, also known as protein kinase B (PKB),1 represents an important mediator of insulin action in worms and flies. InCaenorhabditis elegans, mutations in Akt result in alterations in development and aging whereas in flies, Akt/PKB, as well as other components of the insulin signaling pathway, has been implicated as critical regulators of organism growth and longevity (1Bohni R. Riesgo-Escovar J. Oldham S. Brogiolo W. Stocker H. Andruss B.F. Beckingham K. Hafen E. Cell. 1999; 97: 865-875Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 2Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (317) Google Scholar, 3Scanga S.E. Ruel L. Binari R.C. Snow B. Stambolic V. Bouchard D. Peters M. Calvieri B. Mak T.W. Woodgett J.R. Manoukian A.S. Oncogene. 2000; 19: 3971-3977Crossref PubMed Scopus (150) Google Scholar, 4Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (565) Google Scholar, 5Tatar M. Kopelman A. Epstein D. Tu M.P. Yin C.M. Garofalo R.S. Science. 2001; 292: 107-110Crossref PubMed Scopus (1296) Google Scholar, 6Clancy D.J. Gems D. Harshman L.G. Oldham S. Stocker H. Hafen E. Leevers S.J. Partridge L. Science. 2001; 292: 104-106Crossref PubMed Scopus (1171) Google Scholar). In rodents and humans, the three isoforms Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, which share a high degree of sequence homology, are encoded by distinct genes (7Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (973) Google Scholar, 8Murthy S.S. Tosolini A. Taguchi T. Testa J.R. Cytogenet. Cell Genet. 2000; 88: 38-40Crossref PubMed Scopus (46) Google Scholar). Preliminary analyses of the three gene products support the notion that these isoforms have similar biochemical characteristics (7Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (973) Google Scholar). In mice, one of these Akt/PKB family members, Akt2/PKBβ, has been shown to be required for insulin to maintain normal glucose homeostasis (9Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw 3rd, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1568) Google Scholar). In the absence of Akt2/PKBβ, insulin-stimulated glucose uptake in muscle and fat was significantly reduced in association with reduction in whole body glucose disposal. However, the blockade in glucose uptake in response to insulin was incomplete, raising the possibility other PI 3-kinase-dependent effectors, including other Akt isoforms, might also signal to metabolism outputs. To determine whether the highly related Akt1/PKBα is also required for insulin-regulated glucose homeostasis in mice, we disrupted the c-Akt gene (hereafter referred as Akt1), which encodes Akt1/PKBα. To map the Akt1locus and derive DNA fragments for homologous recombination, we screened a mouse genomic BAC library by the polymerase chain reaction (PCR). The targeting vector was constructed by inserting a left arm fragment, which included exons 2 and 3, into KpnI andXbaI sites and a right arm fragment, which extended from within exon 8 to downstream of exon 11, into the XhoI site of pPNT (10Tybulewicz V.L. Crawford C.E. Jackson P.K. Bronson R.T. Mulligan R.C. Cell. 1991; 65: 1153-1163Abstract Full Text PDF PubMed Scopus (1195) Google Scholar). After transfection of the targeting vector into E14 embryonic stem (ES) cells, G418- and ganciclovir-resistant colonies were screened for homologous recombination by Southern blot analysis. ES cells carrying a recombinant allele were injected into C57BL/6 blastocysts, which were subsequently implanted into pseudopregnant CD-1 foster mothers. Resulting chimeric males were mated with C57BL/6 females to assess germ line transmission as determined by the birth ofagouti pups, which were screened for the targeted allele. For genotyping by PCR, the following primers were used in a single reaction: 851, 5′-AGCTCTTCTTCCACCTGTCTC-3′; 852, 5′-GCTCCATAAGCACACCTTCAGG-3′; 853, 5′-GTGGATGTGGAATGTGTGCGAG-3′. For genotyping by Southern blotting, a PCR-amplified fragment (∼400 base pairs) corresponding to sequence upstream of the left homologous recombination region (Fig. 1) was random labeled with [32P]dCTP. After timed matings of heterozygous Akt1 parents, embryos were harvested at 13.5 days postcoitus. Embryos were dissected to remove the head and the visceral organs and were then finely minced and trypsinized before being plated in the presence of 10% fetal bovine serum in Dulbecco's modified Eagle's medium. Glucometer Elite (Bayer) was used to determine glucose concentration from whole blood collected from the transversely sectioned tip of mouse tails. Sera were separated from whole blood for the determination of circulating insulin and free fatty acid concentration. For insulin levels, rat insulin enzyme-linked immunosorbent assay was performed by the Radioimmunoassay Core Facility at the Penn Center for Diabetes. NEFA C kit (Wako) was used to determine free fatty acid levels. The targeting strategy for disruption of the Akt1 gene consisted of replacement of the coding exons 4, 5, 6, and 7 and the 5′ portion of exon 8 with the neomycin resistance gene (Fig.1a). Exon 5 encodes the lysine residue necessary for the catalytic activity of Akt1/PKBα. The genotype of the ES cells and animals were easily distinguished by Southern blotting or PCR (Fig. 1, b and c). Mouse embryonic fibroblasts heterozygous and homozygous for the targeted allele were isolated and examined for the presence of Akt1/PKBα mRNA and protein by Northern and Western blot, respectively. BothAkt1 mRNA and protein were undetectable in mouse embryonic fibroblasts in which both alleles were targeted (Fig. 1,d and e). Thus, the targeted disruption resulted in a functionally null allele. Furthermore, we could not detect any compensatory increase in the expression of Akt2 orAkt3 in the Akt1−/− mouse embryonic fibroblasts, as assessed by Northern blot analysis (not shown). When mice heterozygous for the targeted Akt1 allele were mated inter se, fewer than expectedAkt1−/− mice were observed 2–3 weeks after birth (Table I). In contrast,Akt1−/− mice appeared with the expected mendelian frequency when 13.5-day-old embryos were examined. Thus, loss of expression of Akt1 resulted in partial lethality occurring some time between midembryonic development and the time of weaning. Careful monitoring of a small number of litters revealed that a significant number of Akt1−/− pups died within the first 3 days of birth, suggesting that the lethality may have occurred during the early neonatal period (data not shown). In all cases, the surviving Akt1−/− pups continued to grow into adulthood and were fertile.Table IObserved genotypic distribution of offsprings from heterozygous matings+/++/−−/−P14-P211463308725.93%58.61%15.45%E13.522542123.68%55.67%21.65% Open table in a new tab The surviving Akt1−/− animals were distinguishable from wild-type animals because of their smaller size. Examination of mice at birth revealed an ∼20% reduction in body weight in Akt1/PKBα-deficient mice compared with wild-type mice (Fig.2a), suggesting that reduction in size occurs during embryonic development. The decrease in body weight was evident throughout postnatal development regardless of sex and persisted into adulthood (Fig. 2b). At 14 months of age, wild type male mice were 37.7 ± 2.2 g whereasAkt1−/− male mice were 27.7 ± 2.0 g. A role for Akt in the determination of cell and compartment size has been established in Drosophila melanogaster, but the present data provide the first indication as to the importance of this kinase in this regard to growth of a mammalian organism (2Verdu J. Buratovich M.A. Wilder E.L. Birnbaum M.J. Nat. Cell Biol. 1999; 1: 500-506Crossref PubMed Scopus (317) Google Scholar). In the fruit fly, the role of Akt in cell growth is relevant to its position in the insulin signal transduction pathway, as genetic manipulation of the fly insulin receptor, IRS ortholog Chico, or PI 3-kinase also results in similar alterations in growth (1Bohni R. Riesgo-Escovar J. Oldham S. Brogiolo W. Stocker H. Andruss B.F. Beckingham K. Hafen E. Cell. 1999; 97: 865-875Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 11Chen C. Jack J. Garofalo R.S. Endocrinology. 1996; 137: 846-856Crossref PubMed Scopus (287) Google Scholar, 12Weinkove D. Neufeld T.P. Twardzik T. Waterfield M.D. Leevers S.J. Curr. Biol. 1999; 9: 1019-1029Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Experiments with mice in which signaling through the IGF-1 receptor has been reduced also led to reduction in body weight, although the relative contributions of cell size and cell number have not been established (13Baker J. Liu J.P. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 73-82Abstract Full Text PDF PubMed Scopus (2111) Google Scholar, 14Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2635) Google Scholar, 15DeChiara T.M. Efstratiadis A. Robertson E.J. Nature. 1990; 345: 78-80Crossref PubMed Scopus (1460) Google Scholar). Consistent with these data, mice rendered null for IRS-1 or IRS-2, two scaffolding proteins that serve as crucial substrates for the IGF-1 and insulin receptors, also demonstrate defects in growth (16Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nat. Genet. 1999; 23: 32-40Crossref PubMed Scopus (486) Google Scholar, 17Araki E. Lipes M.A. Patti M.E. Bruning J.C. Haag 3rd., B. Johnson R.S. Kahn C.R. Nature. 1994; 372: 186-190Crossref PubMed Scopus (1121) Google Scholar, 18Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. et al.Nature. 1994; 372: 182-186Crossref PubMed Scopus (924) Google Scholar). Because a conserved signaling pathway exists in which insulin or IGF-1 stimulates Akt activity via docking of PI 3-kinase to a tyrosine-phosphorylated IRS-1 or IRS-1, it is likely that these signaling proteins also represent intermediates in a pathway regulating cell and organismal growth. Interestingly, p70 S6 kinase, which is also activated by insulin but whose precise relationship with Akt remains unclear, also appears to be important for normal growth of both flies and mice (19Shima H. Pende M. Chen Y. Fumagalli S. Thomas G. Kozma S.C. EMBO J. 1998; 17: 6649-6659Crossref PubMed Google Scholar,20Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (629) Google Scholar). Recently, we have shown that Akt2/PKBβ is critical to the normal control of glucose homeostasis by insulin (9Cho H. Mu J. Kim J.K. Thorvaldsen J.L. Chu Q. Crenshaw 3rd, E.B. Kaestner K.H. Bartolomei M.S. Shulman G.I. Birnbaum M.J. Science. 2001; 292: 1728-1731Crossref PubMed Scopus (1568) Google Scholar). Thus, it was important to also examine the contribution of Akt1/PKBα to metabolism in vivo. As an initial step, we assessed adult mice for an alteration in whole body glucose metabolism by measuring the concentration of blood glucose. As shown in Table II, there was no change in blood glucose under either random-fed conditions or following a 15 h-fast. As a more sensitive measure of insulin resistance, we also measured circulating insulin levels, which also were unchanged in the Akt1−/− mice (Table II). Circulating free fatty acid levels at fed or fasting states were also indistinguishable between the Akt1−/− and wild-type mice, suggesting that lipid metabolism was unaffected by removal of Akt1/PKBα.Table IIConcentrations of circulating metabolites and insulinMalesFemales+/+−/−+/+−/−Fasting glucose (mg/dl)91.7 ± 2.19 (10)91.4 ± 7.6 (9)98.1 ± 3.1 (14)90.3 ± 3.9 (14)Fed glucose (mg/dl)160.7 ± 9.7 (7)180.9 ± 8.3 (7)145.75 ± 2.6 (12)153.1 ± 6.0 (12)Fasting insulin (ng/ml)0.30 ± 0.059 (10)0.28 ± 0.048 (9)0.28 ± 0.022 (13)0.27 ± 0.019 (14)Fasting FFA (meq/liter)0.94 ± 0.076 (8)0.93 ± 0.069 (7)NDNDFed FFA (meq/liter)0.14 ± 0.034 (8)0.14 ± 0.023 (7)NDNDValues are the mean ± S.D. obtained from 4–5-month-old mice. Values in parentheses denote number of mice tested. ND refers to values not determined. No comparisons between sex-matched genotypes revealed any statistically significant difference (p < 0.05) byt-test. Open table in a new tab Values are the mean ± S.D. obtained from 4–5-month-old mice. Values in parentheses denote number of mice tested. ND refers to values not determined. No comparisons between sex-matched genotypes revealed any statistically significant difference (p < 0.05) byt-test. As a further evaluation of glucose metabolism, we challenged the mice with exogenous glucose and measured circulating levels of the sugar during the ensuing 2 h. The Akt1−/− mice responded as well as the control mice to the glucose load, indicating normal glucose tolerance (Fig.3a). To more directly ascertain insulin responsiveness, the hormone was injected, and the resultant change in blood glucose was measured. Again, theAkt1−/− mice cleared glucose from circulation as efficiently as wild-type control mice (Fig. 3b). These analyses of whole body glucose metabolism indicate that Akt1/PKBα, in marked contrast to Akt2/PKBβ, is not a major effector for insulin-regulated glucose homeostasis. Although the precise distribution of the Akt isoforms among different organs remains somewhat controversial, abundant evidence exists that Akt1/PKBα is expressed in classical insulin target tissues such as liver, muscle, and adipocytes (21Cross D.A. Watt P.W. Shaw M. van der Kaay J. Downes C.P. Holder J.C. Cohen P. FEBS Lett. 1997; 406: 211-215Crossref PubMed Scopus (194) Google Scholar, 22Walker K.S. Deak M. Paterson A. Hudson K. Cohen P. Alessi D.R. Biochem. J. 1998; 331: 299-308Crossref PubMed Scopus (245) Google Scholar, 23Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell. Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 24Summers S.A. Whiteman E.L. Cho H. Lipfert L. Birnbaum M.J. J. Biol. Chem. 1999; 274: 23858-23867Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). For this reason, it is surprising that mice rendered deficient in Akt1/PKBα demonstrate normal glucose homeostasis, at least as ascertained by glucose and insulin tolerance tests. One possibility is that, despite the wide distribution for the expression of Akt1, other isoforms such as Akt2/PKBβ predominate in insulin-responsive tissues, and thus the mouse is relatively tolerant to the loss of Akt1/PKBα in terms of metabolic regulation. The alternative, and in many ways more attractive hypothesis, is that the different Akt isoforms signal to different targets, because of either intrinsic preferences in substrates or localization at distinct intracellular sites. Whatever the mechanism, these data demonstrate quite clearly that genetically Akt1and Akt2 display unique phenotypes, despite the highly conserved protein products they encode. Akt1 is an important regulator of organismal growth, whereas Akt2 is integral to metabolic regulation. It is interesting that this divergence parallels that of IGF-1 and insulin, in which the former serves to control primarily mammalian growth, whereas the latter exists as the most important regulator of metabolism. Nonetheless, in most model systems, both peptides appear quite capable of activating Akt1/PKBα, and thus there is no obvious indication that IGF-1 or insulin would selectively activate distinct Akt isoforms in vivo (25Park B.C. Kido Y. Accili D. Biochemistry. 1999; 38: 7517-7523Crossref PubMed Scopus (43) Google Scholar, 26Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2564) Google Scholar). In summary, despite remarkable conservation in primary coding sequence and protein sequence, Akt1 and Akt2 serve distinct functions in the mouse as indicated by the phenotypes of mice deficient in expression of each of the two isoforms. Akt1 is most important to the growth of the organism both in uteroand after birth, whereas Akt2 is critical to insulin-dependent control of carbohydrate metabolism. TheAkt1 knockout phenotype, in which mice are reduced in size at birth and remain small throughout life, is reminiscent of rodent models with altered expression in proximal components of insulin and IGF signaling and suggest that these hormones control growth through Akt1/PKBα (14Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2635) Google Scholar, 16Withers D.J. Burks D.J. Towery H.H. Altamuro S.L. Flint C.L. White M.F. Nat. Genet. 1999; 23: 32-40Crossref PubMed Scopus (486) Google Scholar). We thank Dr. Jean Richa at the Transgenic and Chimeric Mouse Facility at the University of Pennsylvania for the generation of chimeric mice and Dr. Heather Collins at the Radioimmunoassay Core Facility for assay of serum insulin.