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Endocrinology, doi:10.1210/en.2007-0820
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Endocrinology Vol. 149, No. 2 642-650
Copyright © 2008 by The Endocrine Society

Impact of Transgenic Overexpression of SH2-Containing Inositol 5'-Phosphatase 2 on Glucose Metabolism and Insulin Signaling in Mice

Syota Kagawa1, Yoshiyuki Soeda1, Hajime Ishihara, Takeshi Oya, Masakiyo Sasahara, Saori Yaguchi, Ryo Oshita, Tsutomu Wada, Hiroshi Tsuneki and Toshiyasu Sasaoka

Departments of Clinical Pharmacology (S.K., Y.S., S.Y., R.O., T.W., H.T., T.S.), and Pathology (T.O., M.S.), University of Toyama, Toyama 930-0194, Japan; and Sainou Hospital (H.I.), Toyama 930-0887, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka{at}pha.u-toyama.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a 5'-lipid phosphatase hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P3 to PI(3,4)P2 in the regulation of insulin signaling, and is shown to be increased in peripheral tissues of diabetic C57BL/KSJ-db/db mice. To clarify the impact of SHIP2 in the pathogenesis of insulin resistance with type 2 diabetes, we generated transgenic mice overexpressing SHIP2. The body weight of transgenic mice increased by 5.0% (P < 0.05) compared with control wild-type littermates on a normal chow diet, but not on a high-fat diet. Glucose tolerance and insulin sensitivity were mildly but significantly impaired in the transgenic mice only when maintained on the normal chow diet, as shown by 1.2-fold increase in glucose area under the curve over control levels at 9 months old. Insulin-induced phosphorylation of Akt was decreased in the SHIP2-overexpressing fat, skeletal muscle, and liver. In addition, the expression of hepatic mRNAs for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was increased, that for sterol regulatory element-binding protein 1 was unchanged, and that for glucokinase was decreased. Consistently, hepatic glycogen content was reduced in the 9-month-old transgenic mice. Structure and insulin content were histologically normal in the pancreatic islets of transgenic mice. These results indicate that increased abundance of SHIP2 in vivo contributes, at least in part, to the impairment of glucose metabolism and insulin sensitivity on a normal chow diet, possibly by attenuating peripheral insulin signaling and by altering hepatic gene expression for glucose homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A PHOSPHATIDYLINOSITOL (PI) 3-kinase-dependent pathway plays a crucial role in the metabolic action of insulin (1, 2). Insulin induces the activation of the p110 catalytic subunit of PI3-kinase via the tyrosine phosphorylation of insulin receptor substrates (IRSs) and their association with the p85 regulatory subunit (2, 3, 4). PI3-kinase functions as a lipid kinase producing PI(3,4,5)P3 from PI(4,5)P2 in vivo. The PI(3,4,5)P3 acts as a lipid second messenger to activate downstream molecules, including Akt by its phosphorylation at Ser and Thr residues (2, 4, 5, 6). The phosphorylation of Akt is involved in the uptake of glucose by facilitating the translocation of glucose transporter 4 to the plasma membrane in the skeletal muscle and adipose tissue (2, 5, 6). The PI3-kinase/Akt pathway also plays an important role in regulating glucose homeostasis via hepatic gene expression (2). Therefore, it has been speculated that disruptions of the regulation of PI(3,4,5)P3 metabolism are part of the pathogenesis of insulin resistance and type 2 diabetes (4, 5, 7).

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that hydrolyzes PI(3,4,5)P3 to produce PI(3,4)P2, which contributes to the negative regulation of insulin signaling both in vitro and in vivo (8, 9). Overexpression of SHIP2, via the 5'-phosphatase activity, inhibited insulin-induced activation of Akt, glucose uptake, and glycogen synthesis in 3T3-L1 adipocytes and L6 myotubes (10, 11). To date, two kinds of SHIP2 knockout mice have been generated. The first kind exhibited severe hypoglycemia resulting in death within 3 d of birth (12). Heterozygous knockout of the SHIP2 gene was not lethal and enhanced insulin sensitivity without affecting other biological systems. However, it has become apparent that concomitant ablation of the Phox2A gene also occurred in these mice, although the role of Phox2A may be limited in neuronal cell differentiation and negligible in glucose homeostasis (13). Therefore, it is unclear whether the phenotype of the SHIP2 knockout mice is due to deletion of either SHIP2, Phox2A, or both. Recently, a second kind of SHIP2 knockout mouse was reported to be protected from high-fat diet-induced obesity and insulin resistance, although it displayed normal glucose and insulin tolerance on a normal chow diet (9). Interestingly, insulin-induced phosphorylation of Akt was increased in the liver and skeletal muscle, and blood concentrations of triglycerides and nonesterified free fatty acid (NEFA) were decreased in the knockout mice, even when they were fed a normal chow diet. In any case, SHIP2 appears to play an important role in the control of glucose and/or energy homeostasis via the regulation of insulin’s action.

In an animal model of type 2 diabetes with insulin resistance, endogenous SHIP2 expression was enhanced in skeletal muscle and adipose tissue of db/db mice (14). In an analysis with human genomic DNA, Marion et al. (15) found a 16-bp deletion located in the 3'-untranslated region of the SHIP2 gene. This deletion appears to affect the expression of SHIP2 protein because transfection of the mutant resulted in increased promoter activity of SHIP2 gene based on a luciferase assay in L6 myoblasts and HEK293 cells. In addition, some polymorphisms of the SHIP2 gene (Inppl1) are considered to be associated with hypertension, obesity, type 2 diabetes, and metabolic syndrome in European Caucasian populations (16). Furthermore, a SHIP2 gene polymorphism found in the catalytic region in a Japanese cohort may confer protection from insulin resistance (17). Together, alterations of SHIP2 expression and/or enzymatic function appear to have a profound impact on the state of glucose and energy homeostasis during the development of insulin resistance.

Recently, we investigated the impact of liver-specific overexpression of SHIP2 on glucose metabolism and insulin signaling by using adenovirus-mediated gene transfer in C57BL/KSJ-db/+m mice (18). Under the conditions, glucose tolerance and insulin sensitivity were impaired through attenuation of insulin-induced phosphorylation of Akt in the liver but not skeletal muscle and adipose tissue. However, the results were not definitive because of the short-term overexpression of SHIP2 in the liver. Because increased levels of SHIP2 protein and/or catalytic function may be involved in the pathogenesis of type 2 diabetes, we generated transgenic mice overexpressing SHIP2 to clarify whether glucose metabolism and insulin signaling are impaired by long-term overexpression of SHIP2 in vivo. In addition, we explored the mechanism underlying the impairment of glucose and energy homeostasis in SHIP2 transgenic mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human regular insulin (Humalin R) was provided by Eli Lilly and Co. (Indianapolis, IN). The polyclonal anti-Ser473 and -Thr308 phospho-specific Akt antibodies were purchased from Cell Signaling Technology (Beverly, MA). The monoclonal anti-Akt1 antibody and the monoclonal antiphosphotyrosine antibody (PY99) were from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-IRS1 antibody was from Upstate Biotechnology (Lake Placid, NY). The polyclonal anti-SHIP2 antibody has been described previously (8). All other reagents were of analytical grade and purchased from Sigma-Aldrich Corp. (St. Louis, MO) or Wako Pure Chemical Industries (Osaka, Japan).

Plasmid construction
The cDNA encoding mouse SHIP2 was amplified based on GenBank/DDBJ/ENBL accession no. AF162781.1, and was subcloned into pGEM-T vector (Promega, Madison, WI). pCAGGS-mouse SHIP2 was constructed by ligating mouse SHIP2 cDNA into the XhoI site located between the CAG promoter and 3'-flanking region of the rabbit β-globin gene of the pCAGGS vector, which was kindly provided by Dr. Jun-ichi Miyazaki (Osaka University, Osaka, Japan) (19). Adequate insertion of the construct was confirmed by direct sequencing, and expression of the mouse SHIP2 protein in Cos1 cells was determined by using anti-SHIP2 antibody (8).

Generation of SHIP2 transgenic mice
A 6.1-kb fragment was obtained by digestion of pCAGGS-mouse SHIP2 with SalI and HindIII, and purified using CsCl2 gradient centrifugation. As shown in Fig. 1Go, the excised fragment was used for microinjection into the pronuclei of fertilized C57BL/6J mouse eggs (20). The microinjection was operated by Oriental Yeast Co., Ltd. (Tokyo, Japan). Founder mice (F0) were mated with C57BL/6J mice to generate F1-F4 mice, males of which were used for characterization in this study. The presence of the transgene was identified by PCR using a tail with the following primers: sense, 5'-CCTTCTTCTTTTTCCTACAGCTCCTGGGCA-3'; and antisense, 5'-AGCAGCGCGGCTCAGGTCACGGTGATACCA-3'. Two lines of transgenic mice were established. All mice were maintained under standard light (12-h light, 12-h dark cycle) and temperature conditions, and provided with food and water ad libitum. A normal chow diet is composed of 12 kcal percent fat (no. 5053; PMI Nutrition International, St. Louis, MO). For the high-fat feeding study, mice were fed a chow with 60 kcal percent fat (D12492; Research Diets Inc., New Brunswick, NJ) for 8 or 12 wk after 4 wk on normal chow. All experimental procedures used in this study were approved by the Committee of Animal Experiments at University of Toyama.


Figure 1
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  		FIG. 1. Generation of transgenic (TG) mice overexpressing SHIP2. A, The CAG-SHIP2-pA transgene cassettes for the generation of SHIP2 transgenic mice. Mouse SHIP2 cDNA was inserted into the XhoI site of the expression vector pCAGGS. CAG and pA indicate the CAG promoter (modified chicken β-actin promoter with CMV-IE enhancer) and the polyadenylation signal, respectively. The arrows on the bar indicate the primers for genotyping. B, Immunoblots showing overexpression of SHIP2 protein in the indicated tissues of 3- to 5-month-old SHIP2 transgenic mice. Results shown are representative of three separate experiments. WT, wild-type littermates.

 
Measurements of serum parameters
Three- to 5-month-old mice were deprived of food overnight, and blood samples were collected from the orbital sinus under anesthesia. After centrifugation, supernatants of the blood samples were subjected to measurements of serum insulin and leptin levels using ELISA kits (Morinaga, Tokyo, Japan). Serum adiponectin levels were also analyzed with an ELISA kit (R&D Systems, Inc., Minneapolis, MN). Serum levels of cholesterol, triglyceride, and NEFA were determined with a colorimetric kit (Wako).

Glucose tolerance and insulin tolerance tests
Glucose and insulin tolerance tests were performed in 3- to 5- and 9-month-old mice. For the glucose tolerance test, mice fasted overnight were injected ip with glucose (2 g/kg body weight). For the insulin tolerance test, human regular insulin (1.5 U/kg body weight) was ip injected into random-fed mice. Blood samples were collected from the tail vein 0, 15, 30, 60, 90, and 120 min after the injection. Blood glucose and serum insulin levels were measured with a FreeStyle Kissei (Kissei Pharmaceutical Co. Ltd., Tokyo, Japan) and the ELISA kit, respectively.

Western blotting
After fasting overnight, 3- to 5-month-old mice were ip injected with human regular insulin (5 U/kg body weight) or saline. The mice were anesthetized and killed 30 min after the injection, and the liver, hindlimb muscle, and epididymal white adipose tissue (WAT) were rapidly isolated. These tissues were homogenized, lysed, and subjected to Western blot analysis according to the protocol as previously described (18). The intensity of the band corresponding to the phosphorylated IRS1 or Akt was normalized to that in wild-type littermate mice treated with insulin in each experiment.

Northern blot analysis
Three- to 5-month-old mice fasted for 16 h were killed under anesthesia for mRNA extraction, and the liver was rapidly excised and snap frozen in liquid nitrogen. Mice were not fasted for the analysis of sterol regulatory element-binding protein 1 (SREBP1) mRNA. Total RNA was extracted from 50 mg liver specimen using the RNeasy Mini kit (QIAGEN, Valencia, CA). Total RNA (10 µg) in each sample was separated by electrophoresis on a 1.2% formaldehyde denaturing agarose gel and transferred to a Hybond-N+ membrane (GE Healthcare, Buckinghamshire, UK). Northern probes for glucokinase (GK), SREBP1, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) mRNAs were prepared as previously described (21, 22, 23, 24). Hybridization was performed in Rapid-Hyb buffer (GE Healthcare), and the membrane was analyzed using an image analyzer BAS5000 (Fuji Photo Film Co., Ltd., Kanagawa, Japan).

Measurements of hepatic glycogen and triglyceride content
The pieces of liver were isolated from 3- and 9-month-old mice in the random-fed state. Hepatic glycogen content was measured with a Glucose Assay Kit (Sigma-Aldrich), and hepatic triglyceride content was measured using Triglyceride E-Test WAKO (Wako), as previously described (25, 26).

Histological analysis
Liver, WAT, and pancreas isolated from 7- to 8-month-old mice were immediately fixed, sectioned, and stained with hematoxylin and eosin (H&E). For detecting steatosis, the liver was stained with Oil Red O. For staining glycogen, the liver was stained with periodic acid-Schiff after treatment with a 1% celloidin solution for preserving the glycogen. Adipocytes in 5-µm thick sections (H&E staining) were photographed under a light microscope, and the mean size of 200 cells was calculated using a VH analyzer (Keyence, Osaka, Japan).

Immunohistochemistry
Pancreatic sections were prepared using an autostainer system (DakoCytomation, Carpinteria, CA). Thereafter, the sections were incubated with rabbit polyclonal antibodies against glucagon or insulin (N-series, each 1:10; DakoCytomation) for 30 min, and were further incubated with Envision-plus (DakoCytomation) and diaminobenzidine. All sections were counterstained with H&E.

RT-PCR
WAT, intrascapular brown adipose tissue (BAT), and hindlimb muscle were collected from 5- to 7- and 9-month-old mice, and total RNA was extracted using TRIsure (Nippon Genetics, Tokyo, Japan). cDNA synthesis was performed by SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) using 1 µg total RNA as previously described (27). Quantitative real-time PCR was performed with SYBR Premix Ex Taq (Takara Bio, Shiga, Japan), using a Mx3000p Real-Time PCR System according to the manufacturer’s instructions (Stratagene, La Jolla, CA). The primer pairs used were as follows: uncoupling protein (UCP) 1, 5'-TACCAAGCTGTGCGATGT-3' and 5'-AAGCCCAATGATGTTCAGT-3'; UCP2, 5'-GCCCGGGCTGGTGGTGGTC-3' and 5'-CCCCGAAGGCAGAAGTGAAGTGG-3'; and UCP3, 5'-ATCGCCAGGGAGGAAGGA-3' and 5'-GTTGACAATGGCATTTCTTGTGA-3'. The PCR primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from QIAGEN. The relative mRNA expression levels of UCP1, 2, and 3 were calculated as a ratio to those of GAPDH.

Statistical analysis
Data were expressed as means ± SE. The significance of differences between two groups was assessed with the Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of SHIP2 transgenic mice
We generated transgenic mice expressing SHIP2 under the control of the cytomegalovirus-immediate early (CMV-IE) enhancer and a modified chicken β-actin promoter (Fig. 1AGo). Immunoblotting demonstrated that the SHIP2 protein was overexpressed in liver, skeletal muscle, WAT, pancreas, BAT, and brain in SHIP2 transgenic mice (Fig. 1BGo). Table 1Go shows serum parameters of these mice. The fasting plasma insulin level was increased in SHIP2 transgenic mice, whereas fasting glucose, leptin, adiponectin, total cholesterol, triglyceride, and NEFA levels were comparable between the transgenic mice and wild-type littermates.


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  		TABLE 1. Serum parameters in SHIP2 transgenic (TG) mice and wild-type (WT) littermates

 
Body weight, glucose tolerance, and insulin tolerance in SHIP2 transgenic mice
Transgenic mice overexpressing SHIP2 became heavier with age on a normal chow diet, as shown by a 5.0% increase in the body weight at 24 wk old (Fig. 2AGo). In addition, the glucose tolerance test showed that blood glucose concentrations after the ip injection of glucose were higher in SHIP2 transgenic mice than wild-type littermates at 3–5 (Fig. 2BGo) and 9 months old (Fig. 2CGo). The glucose areas under the curves were 14 and 17% higher at respective ages. Serum insulin levels were elevated in SHIP2 transgenic mice compared with those in wild-type littermates at 3–5 (Fig. 2DGo) and 9 months old (Fig. 2EGo), and the increase of serum insulin levels was relatively greater at 30–120 min after the glucose injection in the 9-month-old than 3- to 5-month-old transgenic mice. The insulin tolerance test revealed that blood glucose concentrations at 90 and 120 min after the insulin injection were elevated in SHIP2 transgenic mice compared with those in wild-type littermates at 3–5 (Fig. 2FGo) and 9 months old (Fig. 2GGo). The glucose area under the curve was 15% higher at 9 months old, whereas the difference was not statistically significant at 3–5 months old. However, the profile of body weight was similar between the transgenic mice and wild-type littermates on a high-fat diet (Fig. 3AGo). Although fasting glucose concentrations were elevated in the transgenic mice maintained on the high-fat diet, blood glucose concentrations after glucose loading were not different between SHIP2 transgenic mice and wild-type littermates (Fig. 3BGo). Along this line, blood glucose concentrations after the insulin injection were similar between the transgenic mice and wild-type littermates on the high-fat diet (Fig. 3CGo). Similar results were observed in another line of transgenic mice (data not shown).


Figure 2
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  		FIG. 2. Body weight, glucose tolerance, and insulin tolerance in SHIP2 transgenic mice on a normal chow diet. A, Body weight of male SHIP2 transgenic mice (TG) and wild-type littermates (WT) on a normal chow diet. B–E, Blood glucose levels (B and C) and serum insulin levels (D and E) during glucose tolerance tests in 3- to 5- (B and D) and 9-month-old (C and E) transgenic mice and wild-type littermates. The mice were fasted for 16 h and then injected ip with glucose (2 g/kg body weight). F and G, Blood glucose levels during insulin tolerance tests in 3- to 5- (F) and 9-month-old (G) transgenic mice and wild-type littermates. The mice fed ad libitum were injected ip with insulin (1.5 U/kg body weight). Blood glucose and serum insulin levels were measured at the indicated times after glucose or insulin injection. Results are means ± SE. *, P < 0.05; and {dagger}, P = 0.0612 vs. the corresponding value in wild-type littermates.

 

Figure 3
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  		FIG. 3. Body weight, glucose tolerance, and insulin tolerance in SHIP2 transgenic mice on a high-fat diet. A, Body weight of male SHIP2 transgenic mice (TG) and wild-type littermates (WT) on a high-fat diet. B, For glucose tolerance tests, 3-month-old transgenic mice and wild-type littermates maintained on a high-fat diet were fasted for 16 h and then injected ip with glucose (2 g/kg body weight). C, For insulin tolerance tests, 3-month-old transgenic mice and wild-type littermates maintained on a high-fat diet were injected ip with insulin (1.5 U/kg body weight) in the random-fed state. Blood glucose levels were measured at the indicated times after glucose or insulin injection. Results are means ± SE. *, P < 0.05 vs. the corresponding value in wild-type littermates.

 
Impairment of insulin signaling in SHIP2 transgenic mice
To investigate the mechanism underlying the impaired glucose tolerance and decreased insulin sensitivity in SHIP2 transgenic mice, we examined the effect of SHIP2 overexpression on the early step of insulin signaling. Insulin-induced tyrosine phosphorylation of IRS1 was not altered in the WAT (Fig. 4AGo) and skeletal muscle (Fig. 4BGo), whereas it was decreased by 40.1 ± 5.4% in the liver (Fig. 4CGo) of SHIP2 transgenic mice. Akt is a key molecule downstream of PI3-kinase, which mediates the metabolic action of insulin (2). Insulin-induced phosphorylation of Akt at Ser473 was decreased by 37.0 ± 9.7%, 63.8 ± 5.7%, and 69.1 ± 8.0% in the WAT (Fig. 4DGo), skeletal muscle (Fig. 4EGo), and liver (Fig. 4FGo), respectively, of SHIP2 transgenic mice. Similar results were obtained for the phosphorylation of Akt at Thr308 (data not shown). The amount of IRS1 and Akt was not altered in the WAT, skeletal muscle, and liver of SHIP2 transgenic mice compared with wild-type littermates (Fig. 4Go). In addition, the amount of glucose transporter 4 protein expressed in the WAT was similar between SHIP2 transgenic mice and wild-type littermates (data not shown).


Figure 4
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  		FIG. 4. Insulin-induced phosphorylation of IRS1 and Akt in SHIP2 transgenic mice. Male SHIP2 transgenic mice (TG) and wild-type littermates (WT) starved for 16 h were injected ip with insulin (5 U/kg body weight). After 30 min, the WAT (A and D), skeletal muscle (B and E), and liver (C and F) were excised and lysed. A–C, The lysates were immunoprecipitated with anti-IRS1 antibody. The precipitates or whole cell lysates were subjected to immunoblot analysis with antiphosphotyrosine antibody and anti-IRS1 antibody. D–F, The lysates were subjected to immunoblot analysis with anti-Ser473-phospho-specific Akt antibody and anti-Akt1 antibody. Results are representative of three separate experiments and shown as means ± SE. *, P < 0.05 vs. the level of IRS1 or Akt phosphorylation in wild-type littermates.

 
Hepatic gene expression in SHIP2 transgenic mice and wild-type littermates
The liver is one of the most crucial organs for the in vivo control of glucose homeostasis (28, 29). Because changes in hepatic gene expression are involved in the regulation of gluconeogenesis, glycolysis, and fat synthesis (30), we conducted a Northern blot analysis to investigate the hepatic gene expression in SHIP2 transgenic mice. The level of G6Pase mRNA (Fig. 5AGo) and PEPCK mRNA (Fig. 5BGo) was increased by 39.9 ± 11.4%, and 39.1 ± 9.0%, respectively. On the other hand, the level of SREBP1 mRNA (Fig. 5CGo) was not altered, and that of GK mRNA (Fig. 5DGo) was decreased by 9.7 ± 2.7% in the transgenic mice. The abundance of 18S rRNA (Fig. 5EGo) was equivalent among the tissue samples to ensure that the same amount of total RNA was used.


Figure 5
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  		FIG. 5. Hepatic gene expression in SHIP2 transgenic mice. Total RNA isolated from the liver of male SHIP2 transgenic mice (TG) and wild-type littermates (WT) was subjected to Northern blotting with probes for G6Pase (A), PEPCK (B), SREBP1 (C), and GK (D) mRNAs and 18S rRNA (E). Results are means ± SE. *, P < 0.05 vs. the level of expression in wild-type littermates.

 
Hepatic glycogen and lipid contents in SHIP2 transgenic mice
Glycogen synthesis is mainly regulated by GK and glycogen synthase, a downstream target of Akt (31, 32). Because insulin-induced phosphorylation of Akt was impaired and expression of mRNA for GK was decreased in the liver of SHIP2 transgenic mice, we investigated whether in vivo overexpression of SHIP2 affects hepatic glycogen synthesis in random-fed mice. A difference in hepatic glycogen content was not seen in the 3-month-old mice (Fig. 6AGo). In contrast, hepatic glycogen content was significantly reduced in the 9-month-old transgenic mice compared with their wild-type littermates (Fig. 6BGo). Histological analysis also revealed a decreased hepatic glycogen content in the 9-month-old SHIP2 transgenic mice. On the other hand, content of muscle glycogen was not different between SHIP2 transgenic mice and wild-type littermates either at 3 or 9 months old (data not shown).


Figure 6
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  		FIG. 6. Hepatic glycogen and lipid content in SHIP2 transgenic mice. Hepatic glycogen and lipid levels were measured in male SHIP2 transgenic mice (TG) and wild-type littermates (WT) at 3 (A and C) and 9 months old (B and D) in the random-fed state. A and B, Graphs show hepatic glycogen content quantified by measuring the amount of glucose derived from glycogen. Photographs show sections of liver with periodic acid-Schiff staining, which indicates glycogen-positive cells as purple. C and D, Graphs show hepatic triglyceride content determined by measuring chromatic intensity. Photographs show sections of liver with Oil Red O staining, which indicates fat-positive cells as red. Results are means ± SE. *, P < 0.05 vs. the amount of hepatic glycogen in wild-type littermates. Scale bar, 100 µm.

 
Insulin signaling in the liver also plays an important role in the regulation of lipogenesis (33). Therefore, we next investigated the accumulation of lipid in the liver of the random-fed transgenic mice. Consistent with the unaltered expression of mRNA for SREBP1, which is crucial for lipogenesis, hepatic triglyceride content was not changed in the 3- (Fig. 6CGo) or 9-month-old (Fig. 6DGo) transgenic mice. Similarly, the number of fat-positive cells determined by Oil Red O staining in the liver of SHIP2 transgenic mice was similar to that in the wild-type littermates (Fig. 6Go, C and D).

Morphology of adipocytes and pancreas in SHIP2 transgenic mice
SHIP2 knockout mice were resistant to high-fat diet-induced obesity (9). Therefore, we investigated the change in adiposity in SHIP2 transgenic mice. The weight of epididymal WAT was not significantly different between the transgenic mice and wild-type littermates (Fig. 7AGo). Based on a histological analysis with H&E staining, the mean size of adipocytes did not differ between the transgenic mice and wild-type littermates (Fig. 7Go, B and C).


Figure 7
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  		FIG. 7. Morphology of adipocytes in SHIP2 transgenic mice. A, Weight of epididymal fat pad from 7- to 8-month-old male SHIP2 transgenic mice (TG) and wild-type littermates (WT). B and C, WAT sections from epididymal fat of 7- to 8-month-old SHIP2 transgenic mice and wild-type littermates were stained with H&E (B), and the averaged size was calculated from 200 fat cells (C). Results are means ± SE. Scale bar, 100 µm.

 
The morphology and insulin content of pancreas were not altered in SHIP2 knockout mice (12). To investigate further the possible impact of SHIP2 in the pancreas, we examined the number and size of pancreatic {alpha} and β-cells. We performed H&E staining, and immunohistochemistry with antibodies against glucagon (an {alpha}-cell-specific marker) and insulin (a β-cell-specific marker) in pancreatic sections of SHIP2 transgenic mice and wild-type littermates. The results showed that the morphology of pancreatic {alpha} and β-cells in the transgenic mice was indistinguishable from that in the wild-type littermates (the results are listed in supplemental Fig. 1, which is published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Expression of mRNA for UCPs in SHIP2 transgenic mice
UCPs are known to mediate energy metabolism in the WAT, BAT, and skeletal muscle (34, 35, 36, 37). SHIP2 knockout mice were protected from gains of body weight, at least in part, due to an increase in the level of UCP3 mRNA in the skeletal muscle (9). Because SHIP2 transgenic mice exhibited an increase in body weight compared with wild-type littermates, we investigated whether transgenic overexpression of SHIP2 affects mRNA expression for UCPs in the WAT, BAT, and skeletal muscles by using the quantitative real-time PCR method. Expression of UCP1 mRNA was markedly reduced in the WAT (Fig. 8BGo), but not BAT (Fig. 8AGo), of 5- to 7-month-old SHIP2 transgenic mice, compared with that of wild-type littermates. Transgenic overexpression of SHIP2 did not affect the expression of UCP2 mRNA in the BAT (Fig. 8CGo) or WAT (Fig. 8DGo). The amounts of UCP3 mRNA in the BAT (Fig. 8EGo) and skeletal muscles (Fig. 8FGo) of SHIP2 transgenic mice were comparable to those in the wild-type littermates. Similar results were obtained in 9-month-old SHIP2 transgenic mice and wild-type littermates (data not shown).


Figure 8
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  		FIG. 8. Expression of mRNA for UCPs in SHIP2 transgenic mice. The amount of mRNA for UCPs was quantified by real-time PCR in BAT, WAT, and skeletal muscle of 5- to 7-month-old male SHIP2 transgenic mice (TG) and wild-type littermates (WT). Expression of mRNA for UCP1 (A and B), UCP2 (C and D), and UCP3 (E and F) was analyzed in BAT (A, C, and E), WAT (B and D), and skeletal muscle (F) from the mice. Results are means ± SE. *, P < 0.05 vs. the amount of mRNA for UCPs in wild-type littermates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several critical nodes have been identified in metabolic insulin signaling pathways (38). Lipid phosphatases, especially phosphatase and tensin homolog deleted on chromosome 10 and SHIP2, are physiologically important negative regulators of the PI3-kinase node at the level of PI(3,4,5)P3 (13, 39). Furthermore, SHIP2 is considered more specific to insulin signaling in target tissues than phosphatase and tensin homolog deleted on chromosome 10, which was originally identified as a tumor suppressor (13). In fact, evidence from animal studies and human genetic studies suggests that the attenuation of insulin signaling by excessive SHIP2 is involved in insulin resistance as a cause of type 2 diabetes (14, 15, 17). Therefore, it is important to clarify the impact of an excess of SHIP2 in vivo on glucose metabolism and insulin tolerance in the pathogenesis and development of type 2 diabetes.

In the present study, we investigated for the first time the long-term influences of excessive SHIP2 in vivo, by generating SHIP2 transgenic mice, and found that whole-body glucose homeostasis is impaired under such conditions. The transgenic mice exhibited significant gains of body weight, and impairment of glucose tolerance and insulin sensitivity compared with control wild-type littermates. All these changes are greater than those observed in db/+m mice with liver-specific transient overexpression of SHIP2 using adenovirus-mediated gene transfer (18), suggesting that chronic overexpression of SHIP2 in multiple tissues in addition to the liver causes further changes in metabolism. Thus, increased abundance of SHIP2 appears to be an exacerbating factor for obesity and impaired glucose metabolism in mice fed the normal chow diet. Interestingly, on the high-fat diet, the gain of body weight, glucose tolerance, and insulin tolerance were comparable between the transgenic mice and wild-type littermates. Overexpression of SHIP2 does not appear to make the mice more insulin resistant on a high-fat diet. Alternatively, the effect of transgenic SHIP2 overexpression may be maximally masked by other factors elicited by high-fat feeding. The impact of environmental load, including a high-fat diet, on body weight and glucose tolerance appears to differ depending on the increase or decrease in the amount of SHIP2. In this regard, the second-reported SHIP2 knockout mice were protected from high-fat diet-induced obesity and insulin resistance, though glucose and insulin tolerance were unaltered in these mice when they were fed a normal chow diet (9). However, the involvement of SHIP2 in the biosynthesis and secretion of insulin can be excluded by the absence of any changes in islet structure, and insulin and glucagon contents in SHIP2 transgenic mice. Similarly, experiments with SHIP2 heterozygous knockout mice revealed an unaltered secretion of insulin and morphology of islets based on a histological analysis of the pancreas (12).

Insulin-induced activation of Akt via its phosphorylation appears to be crucial in the regulation of glucose metabolism (2, 5, 6). Insulin-induced phosphorylation of Akt was decreased in the adipose tissue, skeletal muscle, and liver of SHIP2 transgenic mice, as expected from the elevated 5'-phosphatase activity. Therefore, it is most likely that the impairment of glucose and energy homeostasis in the transgenic mice is caused by an attenuation of insulin signaling through the PI3-kinase/Akt pathway. In addition, insulin-induced tyrosine phosphorylation of IRS1 was unexpectedly decreased in the liver, whereas it was unaltered in the adipose tissue and skeletal muscle of the SHIP2 transgenic mice. The decrease does not appear to be due to increased serine phosphorylation of IRS1 and its subsequent proteasomal degradation because the total amount of IRS1 was unaltered in the transgenic mice. Although the precise mechanism by which the molecule upstream of PI3-kinase is affected remains unclear, the decreased phosphorylation of IRS1 observed in the liver of SHIP2 transgenic mice might be due to chronic effects of excess SHIP2 because the transient overexpression of SHIP2 by adenovirus-mediated gene transfer did not affect insulin-induced tyrosine phosphorylation of IRS1 in the liver (18).

Insulin regulates glucose and lipid metabolism in the liver by controlling hepatic gene expression (2, 33). It is known that the mRNA expression for G6Pase, PEPCK, and SREBP1 is increased, whereas that for GK is decreased in the liver in cases of type 2 diabetes (18, 40, 41). The present results showed that the abundance of G6Pase and PEPCK mRNAs was increased, that of SREBP1 mRNA was unaltered, and that of GK mRNA was decreased, in the liver of SHIP2 transgenic mice. Similarly, hepatic glycogen content was decreased in 9-month-old, but not in 3-month-old, SHIP2 transgenic mice, whereas hepatic triglyceride content was not altered in either 3- or 9-month-old mice. Indeed, these results are in good agreement with our previous report showing that hepatic expression of G6Pase and PEPCK mRNAs was increased in mice with a liver-specific transient expression of SHIP2 (18). Thus, the increase in SHIP2 specifically affects glucose metabolism rather than lipid metabolism in the liver.

Gain of body weight on the normal chow diet in SHIP2 transgenic mice does not appear to be caused by the increase of fat weight and WAT area because these in the epididymal fat pads were similar between SHIP2 transgenic mice and wild-type littermates. Unexpectedly, we found that the transgenic overexpression of SHIP2 caused a decrease in the level of UCP1 in WAT, but not in BAT. Because UCP1 involves heat production mainly in BAT as a key step of energy expenditure (42), functional significance of the observed decrease in UCP1 expression in WAT remains unknown. On the other hand, SHIP2 knockout mice were reported to be protected from obesity when maintained on a high-fat diet mainly via an up-regulation of UCP3 expression in skeletal muscles (9). Thus, the change in the level of SHIP2 expression appears to have a different impact on the expression of UCPs in different tissues.

An increasing amount of evidence indicates that the hypothalamic actions of insulin are important in maintaining whole-body energy balance by regulating food intake, energy expenditure, and hepatic glucose production (43). Therefore, neuron-specific insulin receptor knockout mice exhibited increased food intake and moderate diet-dependent obesity (44). In the present study, we detected that the level of SHIP2 was also elevated in the hypothalamus of SHIP2 transgenic mice. However, the amount of food intake under ad libitum feeding conditions was not apparently different between SHIP2 transgenic mice and wild-type mice, either on the normal chow or high-fat diet (data not shown). Regardless of the fact, we cannot exclude the possibility that the excessive SHIP2 affects the hypothalamic actions of insulin regulating glucose homeostasis and energy expenditure, and that accumulation of subtle daily changes in food intake leads to the mild weight gain in the transgenic mice. Further studies would be required to clarify the role of SHIP2 in hypothalamic functions.

In conclusion, we provide direct evidence that a chronic excess of SHIP2 causes attenuation of peripheral insulin signaling in target tissues and associated changes in hepatic gene expression for glucose homeostasis, resulting in impairment of glucose metabolism and insulin sensitivity. Therefore, preserving normal levels of SHIP2 activity may be valuable, at least in part, to protect against the development of glucose intolerance and insulin resistance.


   Acknowledgments
 
We thank Drs. Tamio Noguchi (Nagoya University, Nagoya, Japan), Hitoshi Shimano (Tsukuba University, Ibaragi, Japan), Daryl K. Granner (Vanderbilt University Medical Center, Nashville, TN), Hiromu Nakajima (Osaka Medical Center for Cancer and Cardiovascular Disease, Osaka, Japan), and Wataru Ogawa (Kobe University, Kobe, Japan) for kindly providing cDNA probes used for Northern blot analyses. We also thank Dr. Satoshi Otake (Oriental Yeast Co., Itd, Tokyo, Japan) for valuable technical advice, and Mrs. Tadashi Hoshino, Sunao Murata, and Yoshinori Ichihara (University of Toyama, Toyama, Japan) for assistance in the maintenance of mice, and Drs. Kazuhito Fukui, Ikuko Kimura, and Masashi Kobayashi (University of Toyama, Toyama, Japan) for critical suggestions regarding the study.


   Footnotes
 
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T.S.), the Naito Foundation (to T.S.), and the Sasakawa Scientific Research Grant (to S.K. and Y.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 26, 2007

1 S.K. and Y.S. contributed equally to this study. Back

Abbreviations: BAT, Brown adipose tissue; CMV-IE, cytomegalovirus-immediate early; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GK, glucokinase; G6Pase, glucose-6-phosphatase; H&E, hematoxylin and eosin; IRS, insulin receptor substrate; NEFA, nonesterified free fatty acid; PEPCK, phosphoenolpyruvate carboxykinase; PI, phosphatidylinositol; SHIP2, SH2-containing inositol 5'-phosphatase 2; SREBP1, sterol regulatory element-binding protein 1; UCP, uncoupling protein; WAT, white adipose tissue.

Received June 19, 2007.

Accepted for publication November 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stumvoll M, Goldstein BJ, van Haeften TW 2005 Type 2 diabetes: principles of pathogenesis and therapy. Lancet 365:1333–1346[CrossRef][Medline]
  2. Saltiel AR, Kahn CR 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799–806[CrossRef][Medline]
  3. Sesti G, Federici M, Hribal ML, Lauro D, Sbraccia P, Lauro R 2001 Defects of the insulin receptor substrate (IRS) system in human metabolic disorders. FASEB J 15:2099–2111[Abstract/Free Full Text]
  4. Czech MP, Corvera S 1999 Signaling mechanisms that regulate glucose transport. J Biol Chem 274:1865–1868[Free Full Text]
  5. Rameh LE, Cantley LC 1999 The role of phosphoinositide 3-kinase lipid products in cell function. J Biol Chem 274:8347–8350[Free Full Text]
  6. Vanhaesebroeck B, Alessi DR 2000 The PI3K-PDK1 connection: more than just a road to PKB. Biochem J 346:561–576[CrossRef][Medline]
  7. Jiang G, Zhang BB 2002 PI 3-kinase and its up- and down-stream modulators as potential targets for the treatment of type II diabetes. Front Biosci 7:d903–d907
  8. Ishihara H, Sasaoka T, Hori H, Wada T, Hirai H, Haruta T, Langlois WJ, Kobayashi M 1999 Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling. Biochem Biophys Res Commun 260:265–272[CrossRef][Medline]
  9. Sleeman MW, Wortley KE, Lai KMV, Gowen LC, Kintner J, Kline WO, Garcia K, Stitt TN, Yancopoulos GD, Wiegand SJ, Glass DJ 2005 Absence of the lipid phosphatase SHIP2 confers resistance to dietary obesity. Nat Med 11:199–205[CrossRef][Medline]
  10. Wada T, Sasaoka T, Funaki M, Hori H, Murakami S, Ishiki M, Haruta T, Asano T, Ogawa W, Ishihara H, Kobayashi M 2001 Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity. Mol Cell Biol 21:1633–1646[Abstract/Free Full Text]
  11. Sasaoka T, Hori H, Wada T, Ishiki M, Haruta T, Ishihara H, Kobayashi M 2001 SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes. Diabetologia 44:1258–1267[CrossRef][Medline]
  12. Clement S, Krause U, Desmedt F, Tanti JF, Behrends J, Pesesse X, Sasaki T, Penninger J, Doherty M, Malaisse W, Dumont JE, Le Marchand-Brustel Y, Erneux C, Hue L, Schurmans S 2001 The lipid phosphatase SHIP2 controls insulin sensitivity. Nature 409:92–97[CrossRef][Medline]
  13. Sasaoka T, Wada T, Tsuneki H 2006 Lipid phosphatases as a possible therapeutic target in cases of type 2 diabetes and obesity. Pharmacol Ther 112:799–809[CrossRef][Medline]
  14. Hori H, Sasaoka T, Ishihara H, Wada T, Murakami S, Ishiki M, Kobayashi M 2002 Association of SH2-containing inositol phosphatase 2 with the insulin resistance of diabetic db/db mice. Diabetes 51:2387–2394[Abstract/Free Full Text]
  15. Marion E, Kaisaki PJ, Pouillon V, Gueydan C, Levy JC, Bodson A, Krzentowski G, Daubresse J-C, Mockel J, Behrends J, Servais G, Szpirer C, Kruys V, Gauguier D, Schurmans S 2002 The gene INPPL1, encoding the lipid phosphatase SHIP2, is a candidate for type 2 diabetes in rat and man. Diabetes 51:2012–2017
  16. Kaisaki PJ, Delépine M, Woon PY, Sebag-Montefiore L, Wilder SP, Menzel S, Vionnet N, Marion E, Riveline J-P, Charpentier G, Schurmans S, Levy JC, Lathrop M, Farrall M, Gauguier D 2004 Polymorphisms in type II SH2 domain-containing inositol 5-phosphatase (INPPL1, SHIP2) are associated with physiological abnormalities of the metabolic syndrome. Diabetes 53:1900–1904[Abstract/Free Full Text]
  17. Kagawa S, Sasaoka T, Yaguchi S, Ishihara H, Tsuneki H, Murakami S, Fukui K, Wada T, Kobayashi S, Kimura I, Kobayashi M 2005 Impact of src homology 2-containing inositol 5'-phosphatase 2 gene polymorphisms detected in a Japanese population on insulin signaling. J Clin Endocrinol Metab 90:2911–2919[Abstract/Free Full Text]
  18. Fukui K, Wada T, Kagawa S, Nagira K, Ikubo M, Ishihara H, Kobayashi M, Sasaoka T 2005 Impact of the liver-specific expression of SHIP2 (SH2-containing inositol 5'-phosphatase 2) on insulin signaling and glucose metabolism in mice. Diabetes 54:1958–1967[Abstract/Free Full Text]
  19. Niwa H, Yamamura K, Miyazaki J 1991 Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199[CrossRef][Medline]
  20. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH 1980 Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci USA 77:7380–7384[Abstract/Free Full Text]
  21. Noguchi T, Matsuda T, Tomari Y, Yamada K, Imai E, Wang Z, Ikeda H, Tanaka T 1993 The regulation of gene expression by insulin is differentially impaired in the liver of the genetically obese-hyperglycemic Wistar fatty rat. FEBS Lett 328:145–148[CrossRef][Medline]
  22. Shimano H, Horton JD, Hammer RE, Shimomura I, Brown MS, Goldstein JL 1996 Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a. J Clin Invest 98:1575–1584[Medline]
  23. Beale EG, Chrapkiewicz NB, Scoble HA, Metz RJ, Quick DP, Noble RL, Donelson JE, Biemann K, Granner DK 1985 Rat hepatic cytosolic phosphoenolpyruvate carboxykinase (GTP): structures of the protein, messenger RNA, and gene. J Biol Chem 260:10748–10760[Abstract/Free Full Text]
  24. Shingu R, Nakajima H, Horikawa Y, Hamaguchi T, Yamasaki T, Miyagawa J, Namba M, Hanafusa T, Matsuzawa Y 1996 Expression and distribution of glucose-6-phosphatase catalytic subunit messenger RNA and its changes in the diabetic state. Res Commun Mol Pathol Pharmacol 93:13–24[Medline]
  25. Shen W, Scearce LM, Brestelli JE, Sund NJ, Kaestner KH 2001 Foxa3 (hepatocyte nuclear factor 3{gamma}) is required for the regulation of hepatic GLUT2 expression and the maintenance of glucose homeostasis during a prolonged fast. J Biol Chem 276:42812–42817[Abstract/Free Full Text]
  26. Bligh EG, Dyer WJ 1959 A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917[Medline]
  27. Fukuchi M, Tabuchi A, Tsuda M 2004 Activity-dependent transcriptional activation and mRNA stabilization for cumulative expression of pituitary adenylate cyclase-activating polypeptide mRNA controlled by calcium and cAMP signals in neurons. J Biol Chem 279:47856–47865[Abstract/Free Full Text]
  28. Michael MD, Kulkarni RN, Postic C, Previs SF, Shulman GI, Magnuson MA, Kahn CR 2000 Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell 6:87–97[CrossRef][Medline]
  29. Rutter GA 2000 Diabetes: the importance of the liver. Curr Biol 10:R736–R738
  30. Barthel A, Schmoll D 2003 Novel concepts in insulin regulation of hepatic gluconeogenesis. Am J Physiol Endocrinol Metab 285:E685–E692
  31. Harndahl L, Schmoll D, Herling AW, Agius L 2006 The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism. FEBS J 273:336–346[CrossRef][Medline]
  32. Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, Kadowaki T 1998 Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273:5315–5322[Abstract/Free Full Text]
  33. Postic C, Dentin R, Girard J 2004 Role of the liver in the control of carbohydrate and lipid homeostasis. Diabetes Metab 30:398–408[Medline]
  34. Cannon B, Nedergaard J 2004 Brown adipose tissue: function and physiological significance. Physiol Rev 84:277–359[Abstract/Free Full Text]
  35. Kovacs P, Ma L, Hanson RL, Franks P, Stumvoll M, Bogardus C, Baier LJ 2005 Genetic variation in UCP2 (uncoupling protein-2) is associated with energy metabolism in Pima Indians. Diabetologia 48:2292–2295[CrossRef][Medline]
  36. Saleh MC, Wheeler MB, Chan CB 2002 Uncoupling protein-2: evidence for its function as a metabolic regulator. Diabetologia 45:174–187[CrossRef][Medline]
  37. Clapham JC, Arch JRS, Chapman H, Haynes A, Lister C, Moore GBT, Piercy V, Carter SA, Lehner I, Smith SA, Beeley LJ, Godden RJ, Herrity N, Skehel M, Changani KK, Hockings PD, Reid DG, Squires SM, Hatcher J, Trail B, Latcham J, Rastan S, Harper AJ, Cadenas S, Buckingham JA, Brand MD, Abuin A 2000 Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean. Nature 406:415–418[CrossRef][Medline]
  38. Taniguchi CM, Emanuelli B, Kahn CR 2006 Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96[CrossRef][Medline]
  39. Lazar DF, Saltiel AR 2006 Lipid phosphatases as drug discovery targets for type 2 diabetes. Nat Rev Drug Discov 5:333–342[CrossRef][Medline]
  40. Froguel P, Vaxillaire M, Sun F, Velho G, Zouali H, Butel MO, Lesage S, Vionnet N, Clement K, Fougerousse F, Tanizawa Y, Weissenbach J, Beckmann JS, Lathrop GM, Passa P, Permutt MA, Cohen D 1992 Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus. Nature 356:162–164[CrossRef][Medline]
  41. Barzilai N, Rossetti L 1993 Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin. J Biol Chem 268:25019–25025[Abstract/Free Full Text]
  42. Argyropoulos G, Harper M-E 2002 Uncoupling proteins and thermoregulation. J Appl Physiol 92:2187–2198[Abstract/Free Full Text]
  43. Schwartz MW, Porte Jr D 2005 Diabetes, obesity, and the brain. Science 307:375–379[Abstract/Free Full Text]
  44. Bruning JC, Gautam D, Burks DJ, Gillette J, Schubert M, Orban PC, Klein R, Krone W, Muller-Wieland D, Kahn CR 2000 Role of brain insulin receptor in control of body weight and reproduction. Science 289:2122–2125[Abstract/Free Full Text]




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