European Journal of Pharmacology 616 (2009) 346–352
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo Heather H. Zhou a,1, Chen-Ni Chin b,1, Margaret Wu c, Weihua Ni a, Shuo Quan a, Franklin Liu c, Qing Dallas-Yang c, Kenneth Ellsworth c, Thu Ho b, Aiwu Zhang a, Tajneen Natasha b, Jing Li a, Kevin Chapman a, William Strohl b, Cai Li c, I-Ming Wang d, Joel Berger c, Zhiqiang An b, Bei B. Zhang c, Guoqiang Jiang c,⁎ a
Target Validation, Merck & Co., Inc., Rahway, NJ 07065, United States Biologics Research, Merck & Co., Inc., West Point, PA 19486, United States c Metabolic Disorders – Diabetes, Merck & Co., Inc., Rahway, NJ 07065, United States d Rosetta Inpharmatics, Seattle, WA 98109, United States b
a r t i c l e
i n f o
Article history: Received 30 January 2009 Received in revised form 19 June 2009 Accepted 25 June 2009 Available online 3 July 2009 Keywords: PC-1 ENPP1 (ectonucleotide pyrophosphatase/ phosphodieterase) Diabetes Insulin sensitivity
a b s t r a c t Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had ~ 80% lower hepatic PC-1 mRNA levels, ~ 30% lower ambient fed plasma glucose, ~ 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Plasma cell membrane glycoprotein-1 (PC-1) or ectonucleotide pyrophosphatase/phosphodieterase (ENPP1) is a type 2 transmembrane protein with a large carboxyl terminal extracellular domain, a single transmembrane domain, and a short cytoplasmic amino terminus (Goldfine et al., 1999). PC-1 regulates, among others, insulin signaling and glucose homeostasis in both in vitro and in vivo settings (Abate et al., 2006; Goldfine et al., 1998; Goldfine et al., 1999). Endogenous PC-1 was first found to inhibit insulin signaling in skin fibroblasts from insulin resistant patients (Maddux et al., 1995). Overexpression of PC-1 inhibits insulin signaling in various types of
⁎ Corresponding author. PO Box 2000, RY80N-A26, Merck & Co., Inc., Rahway, NJ 07065, United States. Tel.: +1 732 594 2176; fax: +1 732 594 3570. E-mail address:
[email protected] (G. Jiang). 1 These authors contribute equally to this work. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.06.057
cells in vitro (Belfiore et al., 1996; Kumakura et al., 1998; Maddux and Goldfine, 2000). PC-1 appears to be physically associated with insulin receptor on the cell surface (Maddux and Goldfine, 2000). In addition to inhibiting insulin receptor tyrosine phosphorylation, PC-1 may also affect insulin signaling at post-receptor sites (Kumakura et al., 1998). A role for PC-1 in the derangement of glucose metabolism in vivo has been suggested by several studies. Expression levels of PC-1 have been found to be elevated in insulin resistant rodents and humans (Frittitta et al., 1998; Frittitta et al., 1996; Frittitta et al., 1997; Youngren et al., 1996). Acute hepatic overexpression of PC-1 using adenovirus reduces insulin signaling, increases plasma glucose and insulin levels, and reduces oral glucose tolerance in mice (Dong et al., 2005). Transgenic mice stably overexpressing PC-1 in both liver and muscle show elevated ambient plasma glucose and insulin, reduced oral glucose tolerance, and reduced glucose uptake into muscles (Maddux et al., 2006). Finally, several human genetic studies have suggested
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that the PC-1 Lysine121Glutamine (K121Q) polymorphism is associated with insulin resistance and/or obesity in multiple human populations (Abate et al., 2003; Baratta et al., 2003; Gonzalez-Sanchez et al., 2008; Kubaszek et al., 2003; Matsuoka et al., 2006; Pizzuti et al., 1999; Tasic et al., 2007). However, these findings were not consistently observed in some other populations (Abate et al., 2003; Gonzalez-Sanchez et al., 2003; Jacobsen et al., 2002; Keshavarz et al., 2006; Morrison et al., 2004; Rasmussen et al., 2000). The rare variant Q121 of PC-1 was reported to have a stronger physical interaction with insulin receptor than the more common K121 form of PC-1 in vitro (Costanzo et al., 2001). It was proposed that PC-1 inhibition could represent a potential new approach for type 2 diabetes treatment in humans (Goldfine et al., 1999). While overexpression of PC-1 has been shown to inhibit insulin action both in vitro and in vivo, there is no evidence demonstrating that inhibition of PC-1 improves insulin action either in vitro or in vivo. In the current study, we investigated the biological effects of modulation of PC-1 expression levels. We first observed that overexpression of the full length PC-1 by transient transfection largely reduced insulin-stimulated insulin receptor phosphorylation in HEK293 cells stably expressing recombinant human insulin receptor (HEK293.IR cells). In contrast, knocking down PC-1 expression by transient transfection with siRNA promoted insulin-stimulated Akt phosphorylation in HuH7 cells. Finally, downregulation of PC-1 expression in the liver using adenovirus expressing PC-1shRNA reduced ambient and fasting plasma glucose levels and improved glucose tolerance in db/db mice. These results represent the first report that suppression of PC-1 expression improves insulin action in vitro and in vivo.
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expression using an antiserum against 6Xhistidine tag (Santa Cruz Biotechnology). 2.3. siRNA knockdown and insulin-stimulated Akt phosphorylation in HuH7 cells SmartPool PC-1 siRNA (Dharmacon, M-003809-01) and control non-targeting siRNA (NT siRNA) (Dharmacon, D-001206-12-20) were purchased from Dharmacon. HuH7 cells were maintained in DMEM High Glucose supplemented with 10% fetal calm serum, and 100 units/ml of both penicillin and streptomycin. For experiments, the cells were seeded into 6-well plates in growth medium 24 h prior to transient transfection. The cells were transiently transfected with either the control NT or PC-1 siRNA using Lipofectamine 2000 Reagent as described above. For each well, 7.5 μl siRNA at 20 um was combined with 242.75 μl of OptiMEM to make solution A. 5 μl of Lipofectamine 2000 was added to 245 μl of OptiMEM to make solution B. After mixing, the transfection mix was then added to wells containing 500 μl growth media. After overnight incubation, the media was aspirated off and changed to DMEM High Glucose + 10% fetal calf serum. After another overnight incubation, a set of the cells were incubated with serum free medium for 3 h, treated with insulin (Sigma) for 30 min, and lysed for measurement of Akt phosphorylation using the Bio-Plex Phosphoprotein Detection Kit per manufacturer's instruction (BioRad). The protein lysate was also used for western blot analysis to determine knock-down efficiency of PC-1 protein using an antiserum against PC-1 (Santa Cruz). Total RNA was extracted from the second set of cells for real time PCR (Taqman) analysis to determine knock-down efficiency of PC-1 mRNA. The Taqman probe for PC-1 was purchased from Applied Biosystem.
2. Materials and methods 2.4. PC-1shRNA plasmid generation and validation 2.1. Construction of PC-1 expression vector Human PC-1 DNA was purchased from OriGene Technologies, Inc., and was subcloned into the expression vector pCDNA3.1.hygro for cell surface expression with a 6XHistidine-tag at the C-terminus. 2.2. Transient overexpression and insulin-stimulated insulin receptor tyrosine phosphorylation in cells HEK293 cells stably expressing recombinant human insulin receptor (HEK.293.IR cells) were maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, 1× nonessential amino acids, 100 units/ml of both penicillin and streptomycin, and 1 mg/ml G418. For experiments, the cells were seeded into 6-well plates in growth medium 24 h prior to transient transfection. The cells were transiently transfected with either a control empty vector (pcDNA3.1hygro) or PC-1 expression vector using Lipofectamine 2000 Reagent (Invitrogen). Briefly, for each well, 4 μg of plasmid DNA was combined with 250 μl of OptiMEM to make solution A. 10 μl of Lipofectamine 2000 was added to 250 μl of OptiMEM to make solution B. After a 5 min incubation at room temperature, Solutions A and B were combined, and allowed to incubate for 20 min at room temperature. The resulting transfection mix was added at the ratio of 1:5 transfection mix:media into wells. After overnight incubation, the transfection mix was aspirated off and changed to regular growth media. After another overnight incubation, the growth media was aspirated off, the cells were washed 2 times with DMEM + 0.1% BSA, and incubated in DMEM + 0.1% BSA for 3 h. The cells were then treated with vehicle or insulin (Sigma) for 30 min. Treatments were terminated by washing the cells 3 times with ice-cold PBS. Cells were then lysed and levels of total insulin receptor protein, insulin receptor phosphorylated at tyrosine 972 (IR pY972) were measured using ELISA kits (Invitrogen) per manufacturer's instructions. The lysate was also subjected to western blot analysis to determine PC-1
A 19 nucleotide short hairpin PC-1 RNA (PC-1shRNA) sequence was designed to specifically target mouse PC-1 using a Rosetta proprietary algorithm. The shRNA was synthesized as a pair of complementary antiparallel oligonucleotides with a loop sequence (ttcaagaga) and BamHI and HindIII overhangs (Forward: gatccccGGATTCTACCAGATATCTAttcaagagaTAGATATCTGGTAGAATCCtttttgga; Reverse: agcttccaaaaaGGATTCTACCAGATATCTAtctcttgaaTAGATATCTGGTAGAATCCggg). The forward and reverse oligonucleotides were annealed and ligated into a Gateway entry vector pENTR1A (Invitrogen) containing the human H1 promoter. To determine the efficacy with which PC1shRNA suppresses PC-1 expression, the pENTR1A-PC-1shRNA plasmid was transfected into NIH3T3 cells (ATCC) that express mouse PC-1 endogenously using Nucleofector (Lonza) per manufacturer's instructions. An empty pENTR1A vector containing a H1 promoter and termination sequence was used as the negative control. Transfected cells were harvested at 72 h post initiation of transfection, and total RNA was prepared for Taqman analysis using a primer and probe set for PC-1 purchased from Applied Biosystems. Recombinant adenovirus preparation and in vitro knock-down confirmation — For the production of the recombinant adenoviral vector expressing PC-1shRNA (Ad-PC-1shRNA), PC-1shRNA cassette in pENTR1A-PC-1shRNA was recombined into Gateway-based pAdBlock-iT DEST vector (Invitrogen). Ad-PC-1shRNA was produced in HEK293 cells and purified by two rounds of CsCl density gradient ultracentrifugation. The purified virus was desalted by dialysis and concentrated over CentriPrepYM-50 column before use. To test the efficacy of Ad-PC-1shRNA, primary hepatocytes were isolated from C57BL6 mice, transduced with Ad-PC-1shRNA at different multiplicities of infection (MOIs) for 2 h, and harvested at 72 h post transduction. The empty vector adenovirus containing H1 promoter and termination sequence was used as the negative control. Real time PCR analysis was performed and knock-down efficiency was determined.
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chloroform, and then subjected to centrifugation. The aqueous phase was mixed with 0.6 volume of 100% ethanol, and then passed through a RNeasy 96 well plate (Qiagen). RNase-Free DNase I (Qiagen) in RDD buffer (80 μl) was added to each of the wells. After 5 min incubation at room temperature, the plate was centrifuged, washed subsequently with buffer RW1 and RPE, and eluted with 50 μl of RNase-free water. The synthesis of cDNA from RNA and the quantitative amplification of target cDNAs by Taqman PCR were performed using reagent kits in the ABI PRISM 7700 sequence detection system per manufacturer's instruction (PE Applied Biosystems). Expression levels of interested genes were normalized to that of 18S ribosomal RNA in the same sample. 2.7. Western blot analysis Cell lysates were resuspended in SDS-loading buffer (Invitrogen) and separated in precast 4–20% gradient NuPAGE SDS-PAGE gels
Fig. 1. Overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells. HEK293 cells used in the experiment stably express recombinant human insulin receptor. A) Shown are western blots of the lysates from cells transfected with an empty vector (lane 1) or the PC-1 expression vector (lane 2). B). Results from ELISA analysis of lysates from cells transfected with empty vector or PC-1 expression vector without insulin treatment (basal) or with 10 nM insulin treatment for 30 min. The experiments were performed three times with similar results. IR: insulin receptor.
2.5. In vivo experiments All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories. Male db/db mice (Lepr−/−) were purchased from Jackson laboratory, and housed eight per cage in temperature-, humidity-, and lightcontrolled rooms (21–23 °C, 47–65%, 12-h light/dark cycle) with ad libitum access to water and food (Diet 5008, Purina). At the beginning of the experiment, db/db mice were ~8 weeks old. Db/db mice were grouped based on average body weight and ambient glucose level in blood drawn from tail vein prior to the study. Ad-PC-1shRNA or control viruses were administrated by tail vein injection with 3 × 109 plaqueforming units (PFU) per mouse in 100 μl physiological saline. Blood was collected by tail vein bleeding from db/db mice 7 days post injection to determine ambient glucose and insulin levels. Oral glucose tolerance assay was performed in 4-h fasted db/db mice after 2 g/kg of dextrose challenge by oral gavage. Blood was collected from tail vein at the indicated time points pre- and post-challenge for glucose measurement. Plasma insulin was measured using Ultra-sensitive Insulin Elisa Kit (ALPCO), and glucose was measured using Autokit Glucose (Wako Diagnostics). At the end of the OGTT, the animals were sacrificed; blood was collected by cardiac puncturing for measurement of plasma adiponectin, triglyceride, free fatty acid, and total cholesterol levels. Livers were harvested for subsequent Taqman analysis to determine knock-down efficiency. 2.6. Taqman analysis Cells or tissues were lysed in Ultraspec™ Total RNA Isolation Reagent (Biotecx Laboratories, Inc.). The lysates were mixed with 0.2 volume of
Fig. 2. Silencing of PC-1 expression in HuH7 hepatoma cells improves insulin signaling. HuH7 cells were transiently transfected with a control non-targeting siRNA (NT siRNA) or a siRNA against PC-1 (PC-1 siRNA). A) Shown are results of Taqman analysis of cells at 72 h post initiation of transfection with the siRNAs. The expression levels of PC-1 in these cells were normalized to the expression levels of actin in the same samples. B) Shown are western blots of the lysates from cells at 72 h post initiation of transfection with the siRNAs. A total of 40 μg of total protein from the lysates was loaded on each lane. C) Results from ELISA analysis of lysates from cells at 72 h post initiation of transfection. The cells were treated with either vehicle or 0.3 to 30 nM insulin for 30 min. The experiments were performed three times with similar results.
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cells. ELISA analysis showed that, in HEK293.IR cells transfected with control empty vector, insulin treatment increased insulin receptor tyrosine phosphorylation by approximately 14-fold (Fig. 1B). In HEK293.IR cells transfected with the PC-1 expression vector, insulin treatment increased insulin receptor tyrosine phosphorylation by only approximately 7-fold (Fig. 1B). Thus, overexpression of PC-1 elicited a ~50% reduction in insulin-stimulated insulin receptor tyrosine phosphorylation. These results are consistent with the previous findings that overexpression of PC-1 inhibits insulin-dependent insulin receptor activation in cultured cells.
3.2. PC-1 siRNA reduces PC-1 expression at both mRNA and protein levels and increases insulin signaling in HuH7 cells
Fig. 3. PC-1shRNA efficiently diminishes PC-1 expression in mouse primary hepatocytes in vitro. A) NIH 3T3 cells were transiently transfected with the empty plasma pENTR1A or the PC-1shRNA expression vector pENTR1A.PC-1shRNA. Shown are results from Taqman analysis on the cells at 72 h post initiation of transduction. B) Primary mouse hepatocytes were infected with empty adenovirus (Ad-Control) or PC-1shRNA adenovirus (Ad-PC-1shRNA) at 1 and 10 MOI (multiplicity of infection). Shown are results from Taqman analysis on the cells at 72 h post initiation of transduction. The expression levels of PC-1 in these cells in both Fig A and B were normalized to the expression levels of actin in the same samples.
A relatively high level of endogenous PC-1 mRNA and protein is detectable in human hepatoma HuH7 cells (data not shown), suggesting that HuH7 cells could be used as a cellular model to determine whether suppression of PC-1 expression will improve insulin action. Accordingly, siRNA was employed to silence the expression of PC-1 in HuH7 cells and insulin signaling was measured. In comparison with HuH7 cells transfected with the control non-targeting siRNA (NT siRNA), HuH7 cells transfected with PC-1 siRNA have 90% lower levels of PC-1 mRNA (Fig. 2A). In addition, while endogenous PC-1 protein was readily detectable in HuH7 cells transfected with the control NT siRNA by western blot, it was undetectable in HuH7 cells transfected with the PC-1 siRNA (Fig. 2B). Results from ELISA analysis showed that the level of basal Akt phosphorylation in HuH7 cells transfected with the control NT siRNA was similar to that in HuH7 cells transfected with the PC-1 siRNA (Fig. 2C). In contrast, while insulin induced Akt phosphorylation in a dose dependent fashion in HuH7 cells transfected with both control NT siRNA and PC-1 siRNA, the magnitude of induction in cells transfected with PC-1 siRNA was significantly greater than that in cells transfected with NT siRNA. Taken together, these results demonstrated that PC-1 siRNA effectively silenced the expression of endogenous PC-1 in the HuH7 cells at both the mRNA and protein levels, and that suppression of PC-1 expression improves insulin receptor signaling in vitro.
(Invitrogen). The proteins were then transferred to PVDF membrane, and probed with alkaline phosphatase-conjugated anti-His antibody (Invitrogen). Detection was performed with ECF Western Blotting Kit (Amersham Pharmacia Biotech) by scanning with a Storm® gel and blot imaging system (Molecular Dynamics) per manufacturer's recommendation. 2.8. Statistical analysis Student's t-test was used throughout this manuscript for both in vitro and in vivo studies. *, ** and ***: p b 0.05, 0.01 and 0.001. 3. Results 3.1. Overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells To confirm previous reports that PC-1 overexpression inhibits insulin signaling, we transiently transfected HEK293 cells stably expressing recombinant human insulin receptor (HEK293.IR cells) with the expression vector for the wild-type full length PC-1 or a control empty vector (pcDNA3.1.hygro). As expected, western blot analysis showed that PC-1 antiserum recognized a ~ 130 kDa band that was present in cells transfected with PC-1 expression vector (Fig. 1A, lane 2) but not in cells transfected with a control vector (Fig. 1A lane 1), confirming transient expression of the recombinant PC-1 in these
Fig. 4. PC-1shRNA efficiently silences hepatic PC-1 expression in db/db mice. Db/db mice were distributed into two groups with similar mean plasma glucose levels at the beginning of the experiment. One group of mice was injected with Ad-Control while the other group was injected with Ad-PC-1shRNA. At day 7 after infection, the animals were sacrificed and livers were harvested. Shown are results from Taqman analysis of the liver. The expression levels of both PC-1 (A) and ACC1 (B) were normalized to the expression levels of β-actin in the same samples.
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3.3. PC-1shRNA efficiently suppresses PC-1 expression in primary hepatocytes in vitro
3.4. PC-1shRNA adenovirus reduces hepatic PC-1 expression and improves glucose metabolism in db/db mice
A 19 nucleotide short hairpin RNA (PC-1shRNA) against mouse PC-1 was designed and cloned into expression vector pENTR1A (pENTR1A. PC-1shRNA). To confirm the ability of PC-1shRNA to silence murine PC-1 expression, plasmid of pENTR1A.PC-1shRNA and the control empty vector pENTR1A were transiently transfected into murine NIH3T3 cells that express PC-1 endogenously. Taqman analysis showed that, in comparison with cells transfected with the empty vector pENTR1A, those transfected with pENTR1A.PC-1shRNA demonstrated ~75% lower levels of PC-1 mRNA (Fig. 3A). These results suggest that PC-1shRNA can effectively silence PC-1 expression in NIH3T3 cells. In order to produce adenovirus expressing PC-1shRNA for in vivo silencing, the PC-1shRNA cassette in pENTR1A.PC-1shRNA was subcloned into an adenoviral destination vector to generate a recombinant adenovirus expressing PC-1shRNA (Ad-PC-1shRNA). To confirm the efficacy of silencing, Ad-PC-1shRNA as well as a control empty virus was transduced into primary mouse hepatocytes isolated from C57BL6 mice (Fig. 3B). Taqman analysis showed that cells transduced with Ad-PC-1shRNA had ~ 90% lower levels of PC-1 mRNA than cells transduced with empty control virus at both 1 and 10 multiplicity of infection (MOI). These results indicate that Ad-PC1shRNA virus was effective in silencing PC-1 expression in the mouse hepatocytes.
To investigate whether PC-1 suppression improves insulin sensitivity in vivo, Ad-PC-1shRNA or the control viruses were administrated into 8 week old db/db mice by tail vein injection. At day 7 post injection, blood was collected in the morning for measurement of fed glucose and insulin, and the animals were fasted for 4 h for oral glucose tolerance test. At the end of the oral glucose tolerance test, liver was collected for Taqman analysis. In comparison with the livers of db/db mice treated with the empty control virus, livers of db/db mice treated with Ad-PC-1shRNA had ~ 81% lower levels of PC-1 mRNA (Fig. 4A) but similar levels of acetylcoenzyme A carboxylase 1 A (ACC1) (Fig. 4B). These results demonstrated that PC-1shRNA effectively silenced hepatic PC-1 expression in the db/db mice at 7 days after a single injection of the adenovirus. Blood chemistry analysis showed a 31% reduction of fed glucose and 24% decrease of 4 h fasting glucose from db/db mice treated with Ad-PC-1shRNA compared with the control mice treated with Ad-control (Fig. 5A, B). A single treatment of Ad-PC-1shRNA did not cause any statistically significant changes in serum insulin levels in both fed and fasting states (Fig. 5C, D). Ad-PC-1shRNA treated db/db mice exhibited an increase in their ability to clear glucose from blood after a glucose challenge. The area under the curve of the
Fig. 5. In vivo knockdown of PC-1 with shRNA improves insulin sensitivity in db/db mice. Db/db mice were treated with adenovirus as described in Fig. 4. A and F) Results from OGTT test expressed in term of circulating glucose levels (E) or area under the curve (AUC) (F). AUC was calculated as the entire area under the curve (starting from zero glucose).
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graph for glucose vs. time was significantly less (30%) for Ad-PC1shRNA treated mice compared with Ad-control treated mice (Fig. 5E and F). Ad-PC-1shRNA treatment did not significantly affect body weight, food intake, plasma lactate, adiponectin, triglyceride, free fatty acid or total cholesterol levels (data not shown). Taken together, the results that Ad-PC-1shRNA treatment lowered blood glucose levels without affecting blood insulin levels demonstrated that suppression of hepatic PC-1 improves insulin sensitivity in db/db mice.
4. Discussion In this study, we demonstrated that transient overexpression of PC-1 wild type protein inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells stably expressing recombinant human insulin receptor. We subsequently showed that, in HuH7 cells that express high level of endogenous PC-1, transient transfection of PC-1 siRNA effectively silenced PC-1 expression at both mRNA and protein levels and significantly increased insulinstimulated but not basal Akt phosphorylation. Finally, we observed that adenovirus expressing PC-1shRNA specifically silenced hepatic PC-1 expression and lowered blood glucose levels without affecting blood insulin levels in db/db mice. To our knowledge, these results represent the first evidence that suppression of PC-1 expression improves insulin sensitivity in vitro and in vivo. Insulin resistance has been reported in both mice transduced with PC-1 adenovirus as well as transgenic mice overexpressing PC-1 in both liver and muscle (Dong et al., 2005; Maddux et al., 2006). While PC-1 is expressed abundantly in the liver, it has been reported to be expressed in muscle, adipose, epididymis, plasma cells, placenta, kidney, and other tissues (Goldfine et al., 1999). Given that it is well established that adenovirus predominantly transduces liver (Ragozin et al., 2005; Zinn et al., 1998) and thus gene silencing by shRNA adenovirus mainly occurs in liver (Rippmann et al., 2009), the results from the current studies suggest that suppression of hepatic PC-1 is sufficient to lead to certain degree of improvement in insulin sensitivity in vivo. This suggests that it may be beneficial to selectively target hepatic PC-1 for insulin sensitization since PC-1 is known to play an important role in extracellular calcification in both rodents and humans (Ruf et al., 2005; Rutsch et al., 2001; Suk et al., 2005). What remains to be seen however is whether additional suppression of PC-1 expression in other tissues will further improve insulin sensitivity in vivo. While it was reported that both transient adenovirus-mediated and permanent transgenic overexpression of PC-1 resulted in insulin resistance in mice (Dong et al., 2005; Maddux et al., 2006), it has not been reported whether PC-1 overexpression results in alternations of other metabolic parameters which are often associated with metabolic syndrome. In the current study, we demonstrated that silencing of PC-1 in db/db mice improves insulin sensitivity without affecting body weight, food intake, plasma lactate, adiponectin, triglyceride, free fatty acid and total cholesterol levels. The limited effects may be due to decreased PC-1 expression only in liver and in short time frame. PC-1 genetic polymorphism has been reported to be associated with other aspects of metabolic syndromes in addition to hyperglycemia in some studies. Therefore, more research is needed to understand the full impact of chronic suppression of PC-1 expression in multiple insulin-responsive tissues on metabolic syndromes in both rodents and human in addition to glucose control.
Acknowledgements We would like to thank our colleagues Chris Nunes and Bo Wang for performing in vivo injection of adenovirus.
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