FMS1201D: Transforming Medicine Grant Proposal

Correlation Between Relative Activation of SREBP-1c to Foxa2 with Hepatic Steatosis in NAFLD Patients

Submitted by: Loh Jun Yan (A0115081U) Ng Choon Wee Shawn (A0098718L) Ng Hui Shan (A0099027X) Tan Jenn Sern Gabriel (A0115980A) Wong Wei Sheng, Wilson (A0117983U)

ABSTRACT The relationship between non-alcoholic fatty liver disease (NAFLD) and insulin resistance (IR) is well established. Foxa2 and SREBP-1c are transcription factors downstream of the insulin receptor which are both activated during IR. It is hypothesized that a higher activated SREBP-1c:activated Foxa2 ratio is correlated with a greater degree of hepatic steatosis. A higher ratio in the liver, relative to the adipocytes of peripheral fat, is also expected to correlate with hepatic steatosis by influencing liveradipocyte lipid partitioning. Liver biopsies will be obtained from NAFLD patients co-presenting IR. The level of active Foxa2 and SREBP-1c will be determined using immunohistochemistry and Western blotting, which can be correlated with the degree of hepatic steatosis measured using MRS-PDFF. By correlating the relative activation of SREBP-1c to Foxa2 with hepatic steatosis, the pathogenesis of NAFLD can be better understood and a genetic threshold for NAFLD development may be determined.

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SPECIFIC AIMS AND HYPOTHESIS Although the positive correlation between non-alcoholic fatty liver disease (NAFLD) and insulin resistance (IR) has been established, the mechanisms mediating the two of them are not completely understood. Recently, Foxa2 and SREBP-1 have been identified as transcription factors which link IR and NAFLD together. In a 2008 study by Kohjima et. al., tissue samples from NAFLD patients showed both an increase in activation of Foxa2 and up-regulation of SREBP-1c. This is peculiar, given that both of these transcription factors have opposing functions, with Foxa2 activation leading to an increased fatty acid oxidation through up-regulation of enzymes associated with beta-oxidation of fatty acids, and SREBP-1c leading to up-regulation of genes involved with lipogenesis. In the first phase of our proposal, we intend to investigate the relationship between absolute amounts of activated Foxa2 in conjunction with SREBP-1c in NAFLD liver biopsies and the different grades of steatosis in NAFLD patients. With these values, it is then possible to obtain the activated SREBP1c:activated Foxa2 ratio and compare it with the different grades of steatosis in NAFLD patients. We hypothesize that a higher activated SREBP-1c:activated Foxa2 ratio in NAFLD liver biopsies is correlated with a greater degree of steatosis. SREBP-1c probably dominates Foxa2 in NAFLD pathogenesis, leading to an overall accumulation of lipids in the liver. The second, more interesting phase aims to observe any differences in the activated SREBP1c:activated Foxa2 ratio between hepatocytes and adipocytes. Donnelly et al. (2005) have shown that TAG in liver primarily originates from fatty acids stored in adipocytes in NAFLD. Thus, we hypothesize that a higher ratio in the liver relative to the adipocytes of peripheral fat leads to an increased triacylglycerol (TAG) accumulation in the liver, as observed in steatosis in NAFLD. This could be due to a net shift of TAG from adipocytes to the liver. BACKGROUND AND CLINICAL SIGNIFICANCE Non-alcoholic fatty liver disease (NAFLD) is a global cause of chronic liver disease and is becoming increasingly relevant in Asia (Chitturi, 2004; Farrell, 2003). Since its discovery in 1980 by Ludwig et al., much research has been conducted on its clinical associations and pathogenic mechanisms. Clinically, NAFLD has been recognized as the hepatic manifestation of metabolic syndrome (Ludwig et al., 1980, Chang & Chen, 2011; Chen et al., 2005; Roden, 2006), which typically encompasses insulin resistance, obesity and type 2 diabetes mellitus (Cho, 2011). Owing to lifestyle changes, Asia is facing an increasing prevalence of metabolic syndrome and NAFLD. As insulin resistance is a common hallmark mechanism that underlies these two metabolic diseases, a better understanding of the relationships and mechanisms between insulin resistance and NAFLD would be crucial in tackling the burden of NAFLD in Asia. ! #!

NAFLD is characterized by imbalances in fatty acid delivery, production and disposal (Browning, & Horton, 2004) leading to hepatic triacylglyceride accumulation (Dowman et al., 2010). As aforementioned, the link between insulin resistance and NAFLD is well established, and insulin resistance is sine quo non with NAFLD pathogenesis (Varman & Gerald, 2012; Utzschneider & Kahn, 2006; Marchesini et al., 2001; Bugianesi et al., 2005). Forkhead box protein A2 (Foxa2) and sterol regulatory element-binding protein-1c (SREBP-1c) are two proteins that play important roles in the mechanisms of both insulin resistance and NAFLD. Foxa2 is a transcriptional factor that is involved in the mechanisms underlying insulin resistance and NAFLD. It is encoded by the Foxa2 gene and is involved in the maintenance of glucose and lipid homeostasis in the liver, primarily as a positive regulator of fatty acid oxidation (Kohjima et al., 2008). Foxa2 is in turn negatively regulated by insulin receptor substrates (IRSs) (Kohjima et al., 2008). In a normal individual, upon binding of insulin to its receptor, phosphorylation of IRSs cause the activation of the phosphatidylinositol 3-kinase (PI3K)- AKT signalling cascade that results in the phosphorylation of Foxa2. This phosphorylation results in the nuclear exclusion of Foxa2, hence causing the transcriptional inactivation of Foxa2 regulated gene expression (Wolfrum et al., 2003), and therefore decreased expression of beta oxidation genes and increased fat accumulation. In a state of insulin resistance in humans, a change occurs in the expression of the two predominant IRSs in the liver, IRS1 and IRS2 (Biddinger & Kahn, 2006; Kohjima et al., 2008). Expression of IRS2 is lowered, resulting in the reduction in activation of the PI3K-AKT pathway and a reduction in phosphorylation of Foxa2, thereby leading to the translocation of Foxa2 into the nucleus and activation of Foxa2 regulated beta oxidation genes. Likewise, in NAFLD patients, the decreased expression of IRS-2 has been shown to lead to activation of Foxa2 and the upregulation of fatty acid oxidation (Kohjima et al., 2008). SREBP-1c is another transcriptional factor that is associated in the mechanisms underlying both insulin resistance and NAFLD. SREBP-1c is the dominant isoform of SREBP present in the liver and adipose tissues (Biddinger & Kahn, 2006). They are largely responsible for the mediation of insulins effects on de novo lipogenesis by the positive regulation of genes encoding for the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) (Biddinger & Kahn, 2006; Shimano, 2006; Horton et al., 2002). SREBP-1c has been shown to be positively regulated with IRS-1 (Kohjima et al., 2008). Insulin activates the hepatic expression of SREBP-1c thereby stimulating lipogenesis (Matsuzaka et al., 2004; Osborne, 2000). In insulin resistance, increased expression of IRS-1 results in upregulation of SREBP-1c and increase in lipogenesis, while in NAFLD, aberrant insulin signaling via IRS-1 in NAFLD results in the upregulation of SREBP-1c, thereby causing increased lipogenesis in the hepatocytes and irregular fat metabolism (Kohjima et al., 2008). ! $!

Based on the above discussion, insulin resistance, through activation of Foxa2 and upregulation of SREBP-1c, appears to have engendered opposing pathways in lipid metabolism - an increase in fatty acid oxidation and an increase in lipogenesis. Through our research, we aim to investigate the relative activation of SREBP-1c to Foxa2 by insulin resistance, and its resultant effect on NAFLD. METHODS/APPROACH The study involves a total of 40 Asian patients, ranging from 20 70 years old. This includes a group of 20 obese patients diagnosed with non-alcoholic fatty liver disease (NAFLD) and insulin resistance (IR), and a control group of 20 healthy individuals who are not diagnosed with NAFLD and IR. The bioelectrical impedance analysis method will be used to evaluate the body fat percentage to identify obese individuals (Hilden, Christoffersen, Juhl & Dalgaard, 1977). In order for patients to be considered as suffering from NAFLD, they should not consume more than 20g of alcohol per day and should not present with any form of cirrhosis, fibrosis, or non-specific hepatitis (Vilar et al., 2013). IR is characterized by estimating the insulin sensitivity using the Homeostasis Assessment Model Insulin
Resistance (HOMA-IR). This involves taking the fasting insulin (mUI/L), to be obtained through

enzyme-immuno-assay technique, multiplied by fasting glucose (mmol/L), to be measured by enzymatic colorimetric method, divided by 22.5. Liver and subcutaneous adipose tissue biopsies will be obtained from 20 patients with asymptomatic cholelithiasis during programmed laparoscopic cholecystectomy. These biopsies have to be confirmed to be healthy in order to be considered under the control group (Moya et al., 2013). Liver and subcutaneous adipose tissues biopsies were also extracted from NAFLD and IR patients undergoing gastric bypass surgery (Westerbacka et al., 2007). To quantify the fat content in the liver to determine the variations in lipid accumulation, the Philips Achieva 1.5T A-Series Magnetic Resonance Spectroscopy-measured Proton Density Fat Fraction (MRS-PDFF) can be used (Noureddin et al., 2013). The amount of Foxa2 and SREBP-1c will be determined in the nucleus and cytoplasm of both hepatocytes and adipocytes using two methods immunohistochemistry and western blot. In the immunohistochemical analysis, mouse anti-human FOXA2 primary antibody (LS-B5423) and rabbit anti-human SREBP1 primary antibody (ab93638) will be used to bind to the Foxa2 and SREBP-1c in the nucleus and cytoplasm. The DAPI nuclear counterstain will be used to define the nucleus to confirm the translocation of the transcription factors to the nucleus. The absolute value of intensity in the nucleus and cytoplasm can then be quantified with the help of the Lucia G and ImageJ software. The second method involves performing western blot on the cytoplasmic contents and nuclear contents to isolate cytoplasmic and nuclear Foxa2 and SREBP1c and, quantify the bands with IMAGEQUANT. ! %!

To analyse the differential activation of Foxa2 in varying levels of lipid accumulation, the percentage of Foxa2 in the nucleus out of the total number in the cell (nucleus and cytoplasm) can be used. A higher percentage activation will indicate a higher activity of Foxa2. A similar analysis can be done for SREBP-1c. Following this, we will express SREBP-1c and Foxa2 as a ratio to observe their relative activation with respect to the different grades of NAFLD in patients. We hypothesize that a higher ratio of SREBP-1c to Foxa2 should be observed as the severity of NAFLD progresses. If such a ratio exists, we will then proceed to compare the relative activation of SREBP-1c to Foxa2 between hepatocytes and adipocytes. The percentage activation of Foxa2 or SREBP-1c or, the relative activation of these two transcription factors can be compared with the varying levels of lipid accumulation using the Linear Regression model on SPSS. The correlation between the two variables can then be quantified by obtaining the ! value in the linear equation of y = " + !x + #. The significance of the different grades of NAFLD to the percentage activation or relative activation can be obtained using hypothesis testing, where a p-value of <0.05 will be considered significant. EVALUATION If the results show that there is a statistically significant correlation of the activated Foxa2:SREBP-1c ratio to severity of hepatic lipid accumulation, it provides justification for us to look at the varying ratios between adipocytes and hepatocytes and hence bring us insights as to how this ratio might lead to the relative partitioning of lipids in the adipocytes and hepatocytes. However, even if the data goes contrary to our hypothesis, this could hint at other possible pathways affecting liver lipid accumulation, underlying the complexity of metabolic diseases. The first phase of our study will provide us with a basis of understanding of the relative levels of activated Foxa2 and SREBP-1c in relation to TAG levels in the liver, hence opening doors to possible therapeutic interventions. This is crucial as Foxa2 and SREBP-1c regulate many metabolic genes essential for survival, hence a more moderate method of fine-tuning regulation of these proteins should be adopted instead of methods that might drastically affect expression of many other genes such as the knockout of gene etc. The second phase of our study would provide us insight as to how differential activation/expression ratio of this 2 proteins could confer a relative protective mechanism against these excess fatty acids, thereby presenting novel therapeutic targets to help ameliorate the detrimental accumulation of TAG in liver.

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BUDGET No. Name Company Catalog Number Cost per item Quantity Required Total Cost

Bioelectrical Impedance Analysis 1. 3M Red Dot Foam Monitoring Electrodes 3M 2560 $239.00 1 $239.0 0

Fasting Enzyme-Linked Immunoassay 2. Insulin Human ELISA Kit (1 x 96 tests) Nunc-Immuno 12 microplate washer VWR microcentrifuge tubes, graduated, natural (0.5mL) VWR microcentrifuge tubes, graduated, natural (2.0mL) VWR Disposable serological pipettes (10mL) Abcam ab100578 $605 1 $605.0 0 $1442. 50 $146.0 0 $140.0 0 137.40

3.

VWR

470175

$1442.50 1

4. 5.

VWR VWR

89000-010 $73 20170-170 $70.0

2 2

6.

VWR Thermo Scientific

89130-898 $137.40

7.

Plastic reagent reservoirs

370906

$308.65

617.30

Fasting Glucose Enzyme Colorimetric method 8. Glucose Colorimetric Assay Kit II (100 assays) BioVision K686-100 $422.30 1 $422.3 0

Magnetic Resonance Spectroscopy - measured Proton Density Fat Fraction (MRS-PDFF) 9. Philips Achieva 1.5T Aseries Magnetic Resonance Spectroscope NUS Centre of Imaging Research -

Immunohistochemistry - Paraffin test 10. Anti-FOXA2 Antibody [clone 2F12] IHC-plus (50 ug) Anti-SREBP1 antibody (100 ug) LifeSpan BioSciences LS-B5423 , Inc Abcam ab93638 $712 1 $712

11. !

$454

$454 '!

12.

Goat Anti-Mouse IgG H&L (Alexa Fluor 647) (500 ug) Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (500 ug) DAPI Solution (1 mL) Xylene (500 mL)

Abcam

ab150115

$139

$139

13.

Abcam Thermo Scientific SigmaAldrich

ab150077

$139

$139

14. 15.

62248 534056

$139 $59.60

2 1

$278 $59.60 $306.9 5

16.

Ethanol, Absolute (200 Fisher Proof), Molecular Biology Scientific Grade, Fisher BioReagents

BP2818-4

$306.95

Western Blot 17. 18. Homogenizer Anti-FOXA2 antibody (ab5074) (100 $g) Anti-SREBP1 antibody [2A4] (ab3259) (500 $L) abcam ab5074 $375.00 1 $375.0 0 $400.0 0

19.

abcam

ab3259

$400.00

Nonidet-P40 (NP40) buffer 20. 21. 22. 23. Sodium Chloride 150mM 0.1% Triton X-100 Trizma hydrochloride pH 8.0 50 mM Protease inhibitors Sigma Aldrich Sigma Aldrich Sigma Aldrich Sigma Aldrich S6546-1L T9284100ML T5941500G P83401ML $97.90 $132.00 $288.00 $154.50 1 1 1 1 $97.90 $132.0 0 $288.0 0 $154.5 0

Running buffer 24. 25. ! Tris base 25 mM Glycine 190 mM Fisher scientific Sigma Aldrich BP1521 G88981KG $154.60 $173.00 1 1 $154.6 0 $173.0 0 (!

26.

0.1% SDS

Teknova

S0180

$50.40

$50.40

Transfer buffer 27. Methanol 20% (1 L) SDS Gel Preparation Kit Sigma-Aldrich MSMINIDUO horizontal gel electrophoresis system Criterion Cell and PowerPac Basic Power Supply PVDF Transfer Membrane, 0.45$m, 26.5cm x 3.75m Fisher BioReagents Bovine Serum Albumin, Fraction V, Heat Shock Treated Sigma Aldrich Sigma Aldrich Sigma Aldrich Bio-Rad Thermo Scientific Fisher Scientific 34860-1LR 080911KT-F EP11011EA 88518 $107.50 1 $107.5 0 $1827. 00 $1827. 00 $323.0 0 $232.5 0

28.

$609.00

29.

$606.00 $323.00

3 1

30. 31.

32.

BP1600100

$232.50

Miscellaneous Eppendorf Research plus pipette, variable volume (20 - 200 L) Eppendorf Research Plus Single Channel Pipette, Fixed Volume (1,000 $L) BRAND Transferpette pipette, digital adjustable 12 -channel (20-200 L) Eppendorf epT.I.P.S. box (20-300 L) Pipette tips (1000L) Gloves, Goggles, Plastic transfer pipettes etc Sigma Aldrich Z6838171EA $4970. 00

33.

$497.00

10

34.

Eppendorf

EPPR4428 $222.04

$1110. 2

35.

Sigma Aldrich Sigma Aldrich VWR -

Z3282191EA Z64023996EA

$1240.00 5

$6200. 0 $555.0 0 $345.5 6 -

36.

$55.50

10

37. 38.

83007-376 $345.56 -

1 -

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In the calculation for the budget, basic lab equipment like goggles, gloves, protective wear was omitted. Also, the study will be conducted in a lab providing facilities for MRS-PDFF (as procurement of the spectrometer will be too expensive and out of the budget, hence the cost for MRS is not included). Based on the above estimates on the prices of supplies, the cost of all materials required to run the necessary experiments to collect our data will sum up to approximately 23,334.21 dollars, well within the budget of 30,000 dollars. This will provide comfortable room for additional spending should supplies run low. The estimates are based on a 5-man research team and their required supplies. All pipette tips and microcentrifuge tubes are bought in sets of 1000. ROLE OF TEAM MEMBERS The project began with several meetings where all five members gathered and contributed to the discussions to the best of their abilities. The initial research focus was proposed by Wilson Wong and Shawn Ng, which was then later built upon by the rest of the team. To compile and write this proposal, the five of us focused on separate sections but maintained close communications with each other to clarify any doubts and provide suggestions. It is important to note that despite the splitting of workload, every part of this research and proposal was discussed and agreed on by the entire team. The specific aims and hypothesis was written by Shawn and, the background and clinical significance was concisely compiled by Wilson and Jun Yan. The bulk of the research methods and approach was done by Jaslyn and the final evaluation was collated by Shawn. Gabriel worked closely with Jaslyn to identify the materials required for the methods that were described in order to source for the quotation of the various items in the budget section. All in all, the team worked together to contribute to this research and proposal. REFERENCES CITED Biddinger, S. B., & Kahn, C. R. (2006). From mice to men: insights into the insulin resistance syndromes. Annual review of physiology, 68, 12358. Browning, J. D., & Horton, J. D. (2004). Molecular mediators of hepatic steatosis and liver injury. J Clin Invest., 114(2), 147-52. Bugianesi, E., Gastaldelli, A., Vanni, E., Gambino, R., Cassader, M., Baldi, S., Rizzetto M. (2005). Insulin resistance in non-diabetic patients with non-alcoholic fatty liver disease: Sites and mechanisms. Diabetologia, 48, 634642. Chang, T. Y., & Chen, J. D. (2011). Fatty Liver and Metabolic Syndrome in nonabdominally obese Taiwanese adults. Asia Pac J Public Health, 24(3), 472-479. ! *+!

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Moya, M., Benet, M., Guzmn, C., Tolosa, L., Garca-Monzn, C., Pareja, E., Jover, R. (2012). Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver. PLoS One, 7(1), e30014. doi:10.1371/journal.pone.0030014 Noureddin, M., Lam, J., Peterson, M. R., Middleton, M., Hamilton, G., Le, T. A., Loomba, R. (2013). Utility of magnetic resonance imaging versus histology for quantifying changes in liver fat in nonalcoholic fatty liver disease trials. Hepatology, 58(6), 1930-40. doi:10.1002/hep.26455 Osborne, T. F. (2000). Sterol regulatory element-binding proteins (SREBPs): key regulators of nutritional homeostasis and insulin action. The Journal of biological chemistry, 275(42), 3237982. doi:10.1074/jbc.R000017200 Roden, M. (2006). Mechanisms of Disease: hepatic steatosis in type 2 diabetes-- pathogenesis and clinical relevance. Nat Clin Pract Endocrinol Metab; 2(6), 335-48. Shimano, H. (2001). Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes. Prog Lipid Res 40(6), 439-452. Taniguchi, C. M., Ueki, K., & Kahn, C. R. (2005). Complementary roles of IRS-1 and IRS-2 in the hepatic regulation of metabolism, The journal of clinical investigation, 115(3), 718-727. Utzschneider, K.M., & Kahn, S.E. (2006). Review: The role of insulin resistance in nonalcoholic fatty liver disease. J. Clin. Endocrinol. Metab., 91, 47534761. Varman, T. S., & Gerald, I.S. (2012). Integrating Mechanisms for Insulin Resistance: Common Threads and Missing Links. Cell., 148(5), 852871. Vilar, C.P., Cotrim, H. P., Florentino, G. S., Barreto, C. P., Florentino, A.V., Bragagnoli, G., & Schwingel, P. A. (2013). Association between nonalcoholic fatty liver disease and coronary artery disease. Rev Assoc Med Bras 59(3), 290-7. doi:10.1016/j.ramb.2012.11.006 Westerbacka, J., Kolak, M., Kiviluoto, T., Arkkila, P., Sirn, J., Hamsten, A., Yki-Jrvinen, H. (2007). Genes involved in fatty acid partitioning and binding, lipolysis, monocyte/macrophage recruitment, and inflammation are overexpressed in the human fatty liver of insulin-resistant subjects. Diabetes, 56(11), 2759-65. Wolfrum, C., Besser, D., Luca, E., & Stoffel, M. (2003). Insulin regulates the activity of forkhead transcription factor Hnf-3beta/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization. Proceedings of the National Academy of Sciences of the United States of America, 100(20), 116249.

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