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1000.Full-Text.Pdf Original Article Restoration of Glucokinase Expression in the Liver Normalizes Postprandial Glucose Disposal in Mice With Hepatic Deficiency of PDK1 Yasuo Okamoto,1 Wataru Ogawa,1 Akihiko Nishizawa,1 Hiroshi Inoue,1 Kiyoshi Teshigawara,1 Shinichi Kinoshita,1 Yasushi Matsuki,2 Eijiro Watanabe,2 Ryuji Hiramatsu,2 Hiroshi Sakaue,1 Tetsuo Noda,3 and Masato Kasuga1 Phosphoinositide-dependent kinase-1 (PDK1) is implicated Mice in which hepatic PI3K activity is inhibited by a in the metabolic effects of insulin as a key mediator of dominant negative form of the enzyme (3) or those lacking phosphoinositide 3-kinase–dependent signaling. Here we regulatory subunits of PI3K in the liver (4) manifest show that mice with liver-specific PDK1 deficiency mani- multiple defects in the metabolic actions of insulin and fest various defects in the metabolic actions of insulin in phenotypes that resemble type 2 diabetes. Moreover, the the liver as well as a type 2 diabetes–like phenotype metabolic changes apparent in these animals are similar to characterized by marked hyperinsulinemia and postpran- those observed in mice with liver-specific knockout of the dial hyperglycemia. The hepatic abundance of glucokinase, insulin receptor (LIRKO) (5), confirming the notion that an important determinant of glucose flux and glucose- evoked signaling in hepatocytes, was substantially reduced PI3K-dependent signaling plays a central role in insulin in these mice. Restoration of hepatic glucokinase expres- action in the liver. sion, with the use of an adenoviral vector, induced insulin- Downstream of PI3K, 3-phosphoinositide–dependent ki- like effects in the liver and almost completely normalized nase-1 (PDK1) and its activation of members of the AGC the fasting hyperinsulinemia and postprandial hyperglyce- family of protein kinases are implicated in the effects of mia in these animals. These results indicate that, if the insulin (6). Mora et al. (7) recently generated mice with hepatic abundance of glucokinase is maintained, ingested liver-specific deficiency of PDK1 by breeding animals that glucose is normally disposed of even in the absence of harbor “floxed” Pdk1 alleles with those that express Cre acute activation of proximal insulin signaling, such as the under the control of the albumin gene promoter with activation of Akt, in the liver. Diabetes 56:1000–1009, 2007 ␣-fetoprotein gene enhancer (Alfp-Cre). The activation of several signaling molecules, regulation of genes involved in nutrient metabolism, and induction of glycogen accu- mulation by insulin were affected in the liver of these mice. he liver is one of the most important target However, these animals also developed prominent edema organs of insulin and thus plays an essential role and liver damage, and they died between 4 and 16 weeks in nutrient metabolism (1,2). Such effects of of age (7). The metabolic role of hepatic PDK1 in living Tinsulin in the liver are mediated by a phospho- animals has thus not been sufficiently investigated with inositide 3-kinase (PI3K)-dependent signaling pathway. these animals. We have now generated mice with liver-specific PDK1 deficiency achieved by the excision of floxed alleles of From the 1Department of Clinical Molecular Medicine, Division of Diabetes and Digestive and Kidney Diseases, Kobe University Graduate School of Pdk1 with Cre expressed under the control of the albumin Medicine, Kobe, Japan; the 2Genomics Science Laboratories, Dainippon gene promoter (Alb-Cre). Unlike the mice with liver- Sumitomo Pharmaceuticals, Takarazuka, Japan; and the 3Department of Cell specific PDK1 deficiency previously described (7), the Biology, Japanese Foundation for Cancer Research (JFCR), Cancer Institute, Tokyo, Japan. mice characterized in the present study exhibited neither Address correspondence and reprint requests to Wataru Ogawa, Depart- liver damage nor premature death. We were thus able to ment of Clinical Molecular Medicine, Division of Diabetes and Digestive and scrutinize the role of PDK1 in nutrient metabolism in Kidney Diseases, Kobe University Graduate School of Medicine, Kobe, Japan 657-0011. E-mail: [email protected]. normally developed animals and show that hepatic defi- Received for publication 19 September 2006 and accepted in revised form ciency of PDK1 in mice results in various defects in the 17 January 2007. regulation of nutrient metabolism, among which a reduc- Published ahead of print at http://diabetes.diabetesjournals.org on 31 Jan- tion in the abundance of glucokinase is largely responsible uary 2007. DOI: 10.2337/db06-1322. ALT, alanine aminotransferase; AxCAGck, adenoviral vector-encoding rat for an impairment of postprandial glucose disposal. glucokinase; ERK, extracellular signal–related kinase; FAS, fatty acid syn- thase; G6P, glucose 6-phosphate; G6PC, glucose-6-phosphatase; GS, glycogen synthase; IGFBP1, IGF-1 binding protein; IRS, insulin receptor substrate; P13K, phosphoinositide 3-kinase; PCK-1, phosphoenolpyruvate carboxyki- RESEARCH DESIGN AND METHODS nase; PDK1, phosphoinositide-dependent kinase-1; PFU, plaque-forming units; Mice and adenoviral vectors. The study was approved by the Animal PGC1␣, peroxisome proliferator–activated receptor-␥ coactivator 1␣; PK, Experimentation Committee of Kobe University. Mice that harbor a floxed pyruvate kinase insulin receptor substrate; SCD-1, stearoyl-CoA desaturase-1; Pdk1 allele (Pdk1flox/ϩ mice) (8) or a transgene for Alb-Cre (9) were previously SREBP, sterol regulatory element–binding protein. described. A cDNA-encoding rat glucokinase was synthesized with PCR, and © 2007 by the American Diabetes Association. an adenoviral vector containing this cDNA (AxCAGck) was generated as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance described (10). Mice were injected via the tail vein with adenoviral vectors, with 18 U.S.C. Section 1734 solely to indicate this fact. and experiments were performed 4 days after injection. 1000 DIABETES, VOL. 56, APRIL 2007 Y. OKAMOTO AND ASSOCIATES FIG. 1. PDK1 expression, growth rate, liver and fat mass, and hepatic glycogen content in L-Pdk1KO mice. A: Immunoblot analysis of PDK1 in various tissues of L-Pdk1KO (KO) and Pdk1flox/flox (F/F) mice. Data are representative of at least three independent experiments. BAT, brown mice. Data are means ؎ SEM. C and (9 ؍ and Pdk1flox/flox (n (13 ؍ adipose tissue; WAT, white adipose tissue. B: Growth curves for L-Pdk1KO (n of L-Pdk1KO and Pdk1flox/flox mice either in the randomly fed state of after food (5–3 ؍ and hepatic glycogen content (n (8–6 ؍ D: Liver mass (n deprivation for 16 h. Glycogen content is expressed as milligrams per gram of liver tissue. Data are means ؎ SEM. *P < 0.05, **P < 0.01 (Student’s t test) vs. corresponding value for Pdk1flox/flox mice. E: Mass of subcutaneous white adipose tissue and interscapular brown adipose tissue of P < 0.01 (Student’s t test) vs. corresponding value** .(4 ؍ L-Pdk1KO and Pdk1flox/flox mice in the randomly fed state. Data are means ؎ SEM (n for Pdk1flox/flox mice. Immunoblot analysis and assay of kinase activities. For insulin or TACTATCTACTCA-3Ј and 5Ј-GCCAAGAAACAACAGTTAG-3Ј. The primers for glucose treatment, mice deprived of food for 16 h were injected intraperito- the catalytic subunit of glucose-6-phosphatase (G6PC) (8), phosphoenolpyru- neally with human regular insulin (5 units/kg) or glucose (2 g/kg) and were vate carboxykinase (PCK-1) (14), peroxisome proliferator–activated recep- then killed after 5 or 30 min, respectively. Total homogenates were prepared tor–␥ coactivator 1␣ (PGC1␣) and 36B4 (15), and sterol regulatory element– from the liver and subjected to immunoblot analysis or to immunoprecipita- binding protein (SREBP)1c and SREBP1a (16) were as described. tion. Antibodies to PDK1 were kindly provided by F. Liu (University of Texas, Statistical analysis. Data are presented as means Ϯ SEM and were com- Health Science Center, San Antonio, TX) (11). All other antibodies used for pared between or among groups by Student’s t test or ANOVA. immunoblot analysis were commercially available, and the information of the antibodies will be provided on request. PI3K activity (3) and the activities of Akt and p70 S6K (12,13) in immunoprecipitates were assayed as described. RESULTS Glucose and insulin tolerance tests and refeeding. For the glucose tolerance test, mice deprived of food for 16 h were loaded intraperitoneally Generation of liver-specific PDK1-deficient mice. We flox/ϩ with glucose (2 g/kg). Glycogen and glucose 6-phosphate (G6P) contents as bred Pdk1 mice (8) with Alb-Cre mice (9) and then well as glycogen synthase (GS) activity were determined as described (3) with bred the resulting Pdk1flox/ϩ/Alb-Cre offspring with total liver homogenates prepared from mice killed 30 min after the initiation Pdk1flox/ϩ mice. The genotypes of animals born from the of a glucose tolerance test. For the insulin tolerance test, nonfasted mice were latter cross conformed approximately to the expected injected intraperitoneally with human regular insulin (0.75 units/kg). For assay ratio (data not shown). Given that the abundance of PDK1 of glucose concentration and hepatic gene expression during refeeding, mice ϩ/ϩ were deprived of food for 24 h and then fed with a high-carbohydrate chow in major organs including the liver was similar in Pdk1 flox/flox containing 70% sucrose. mice and Pdk1 mice (data not shown), and that Hepatic gene expression. Hepatic gene expression was evaluated by reverse Alb-Cre mice did not exhibit metabolic abnormalities (9), transcription and real-time PCR analysis with 36B4 mRNA as the invariant we performed subsequent experiments with Pdk1flox/flox/ control, as described (3). The primers used were as follows: glucokinase, Alb-Cre (L-Pdk1KO) mice and Pdk1flox/flox mice generated 5Ј-CCCTGAGTGGCTTACAGTTCGK-3Ј and 5Ј-ACGGATGTGAGTGTTGAAGC- flox/flox flox/flox 3Ј; pyruvate kinase (PK), 5Ј-GAGAACCATGAAGGCGTGAA-3Ј and 5Ј-CCGC by crossing Pdk1 /Alb-Cre mice with Pdk1 CCGAGTTGGTC-3Ј; insulin receptor substrate (IRS)-2, 5Ј-GGAGAACCCAG mice; the offspring of such breeding were born in a ACCCTAAGCTACT-3Ј and 5Ј-GATGCCTTTGAGGCCTTCAC-3Ј; glucose Mendelian ratio (data not shown).
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