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)

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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 liver-

adipocyte 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 SREBP-

1c: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 SREBP-

1c: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.

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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).

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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 SREBP-

1c and, quantify the bands with IMAGEQUANT.

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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

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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 $239.00

Fasting Enzyme-Linked Immunoassay

2. Insulin Human ELISA Kit (1 x 96 tests) Abcam ab100578 $605 1 $605.00

3. Nunc-Immuno 12 microplate washer

VWR 470175 $1442.50 1 $1442.50

4. VWR microcentrifuge tubes, graduated, natural (0.5mL) VWR 89000-010 $73 2 $146.00

5. VWR microcentrifuge tubes, graduated, natural (2.0mL) VWR 20170-170 $70.0 2 $140.00

6. VWR Disposable serological pipettes (10mL)

VWR 89130-898 $137.40 1 137.40

7. Plastic reagent reservoirs Thermo Scientific 370906 $308.65 2 617.30

Fasting Glucose Enzyme Colorimetric method

8. Glucose Colorimetric Assay Kit II (100 assays)

BioVision K686-100 $422.30 1 $422.30

Magnetic Resonance Spectroscopy - measured Proton Density Fat Fraction (MRS-PDFF)

9. Philips Achieva 1.5T A-series Magnetic Resonance Spectroscope

NUS Centre of Imaging Research

- - - -

Immunohistochemistry - Paraffin test

10. Anti-FOXA2 Antibody [clone 2F12] IHC-plus (50 ug)

LifeSpan BioSciences, Inc

LS-B5423 $712 1 $712

11. Anti-SREBP1 antibody (100 ug) Abcam ab93638 $454 1 $454

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12. Goat Anti-Mouse IgG H&L (Alexa Fluor 647) (500 ug)

Abcam ab150115 $139 1 $139

13. Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (500 ug)

Abcam ab150077 $139 1 $139

14. DAPI Solution (1 mL) Thermo Scientific 62248 $139 2 $278

15. Xylene (500 mL) Sigma-Aldrich 534056 $59.60 1 $59.60

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

Fisher Scientific BP2818-4 $306.95 1

$306.95

Western Blot

17. Homogenizer - - - - -

18. Anti-FOXA2 antibody (ab5074) (100 g)

abcam ab5074 $375.00 1 $375.00

19. Anti-SREBP1 antibody [2A4] (ab3259) (500 L)

abcam ab3259

$400.00 1 $400.00

Nonidet-P40 (NP40) buffer

20. Sodium Chloride 150mM Sigma Aldrich S6546-1L $97.90 1 $97.90

21. 0.1% Triton X-100 Sigma Aldrich T9284-100ML $132.00 1

$132.00

22. Trizma hydrochloride pH

8.0 50 mM Sigma Aldrich

T5941-500G $288.00 1

$288.00

23. Protease inhibitors Sigma Aldrich

P8340-1ML $154.50 1

$154.50

Running buffer

24. Tris base 25 mM Fisher scientific BP1521 $154.60 1

$154.60

25. Glycine 190 mM Sigma Aldrich G8898-1KG $173.00 1

$173.00

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26. 0.1% SDS Teknova S0180 $50.40 1 $50.40

Transfer buffer

27. Methanol 20% (1 L) Sigma Aldrich

34860-1L-R $107.50 1

$107.50

28. SDS Gel Preparation Kit

Sigma Aldrich

08091-1KT-F $609.00 3

$1827.00

29. Sigma-Aldrich MSMINIDUO horizontal gel electrophoresis system

Sigma Aldrich

EP1101-1EA $606.00 3

$1827.00

30. Criterion Cell and PowerPac Basic Power Supply Bio-Rad - - - -

31. PVDF Transfer Membrane, 0.45m, 26.5cm x 3.75m Thermo Scientific 88518 $323.00 1

$323.00

32.

Fisher BioReagents Bovine Serum Albumin, Fraction V, Heat Shock Treated

Fisher Scientific

BP1600-100 $232.50 1

$232.50

Miscellaneous

33. Eppendorf Research plus pipette, variable volume (20 - 200 L)

Sigma Aldrich

Z683817-1EA $497.00 10

$4970.00

34. Eppendorf Research Plus Single Channel Pipette, Fixed Volume (1,000 L)

Eppendorf EPPR4428 $222.04 5 $1110.2

35. BRAND Transferpette pipette, digital adjustable 12 -channel (20-200 L)

Sigma Aldrich

Z328219-1EA $1240.00 5

$6200.0

36. Eppendorf epT.I.P.S. box (20-300 L)

Sigma Aldrich

Z640239-96EA $55.50 10

$555.00

37. Pipette tips (1000L) VWR 83007-376 $345.56 1 $345.56

38. Gloves, Goggles, Plastic transfer pipettes etc - - - - -

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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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Chen, S. H., He, F., Zhou, H. L., Wu, H. R., Xia, C., & Li, Y. M. (2001). Relationship between

nonalcoholic fatty liver disease and metabolic syndrome. J Dig Dis, 12(2), 125-30.

Chitturi, S., Farrell, G. C., & George J. (2004). Nonalcoholic steatohepatitis in the Asia-Pacific region:

future shock? J Gastroenterol Hepatol, 19(4), 368-374.

Cho, L. W. (2011). Metabolic Syndrome. Singapore Med J, 52, 779-785

Donnelly, K. L., Smith, K. I., Schwarzenberg, S. J., Jessurun, J., Boldt, M.D. & Parks, E. J. (2005).

Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic

fatty liver disease. J Clin Invest, 115(5), 13431351. doi:10.1172/JCI23621

Dowman J. K., Tomlinson J. W., & Newsome P. N (2010). Pathogenesis of non-alcoholic fatty liver

disease. QJM. 103, 7183.

Farrell, G. C. (2003). Nonalcohlic steatohepatitis: What is it, and why is it important in the Asia-Pacific

region? J Gastroenterol Hepatol, 18(2), 124-138.

Hilden, M., Christoffersen, P., Juhl, E., & Dalgaard, J. B. (1977). Liver histology in a ''normal''

population: examinations of 503 consecutive fatal traffic casualties. Scand J Gastroenterol,

12(5), 593597.

Horton, J. D., Goldstein, J. L., & Brown, M. S. (2002). SREBPs: activators of the complete program of

cholesterol and fatty acid synthesis in the liver. The Journal of Clinical Investigation, 109(9),

1125-1131.

Kohjima, M., Higuchi, N., Kato, M., Kotoh, K., Yoshimoto, T., Fujino, T., Nakamuta, M. (2008).

SREBP-1c, regulated by the insulin and AMPK signaling pathways, plays a role in

nonalcoholic fatty liver disease. International journal of molecular medicine, 21(4), 50711.

Ludwig, J., Viggiano, T. R., McGill, D. B., & Oh, B. J. (1980). Nonalcoholic steatohepatitis: Mayo

Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc, 55(7), 434-438.

Marchesini, G., Brizi, M., Bianchi, G., Tomassetti, S., Bugianesi, E., Lenzi, M., Melchionda N.

(2001). Nonalcoholic fatty liver disease: A feature of the metabolic syndrome. Diabetes, 50,

18441850.

Matsuzaka, T., Shimano, H., Yahagi, N., Amemiya-Kudo, M., Okazaki, H., Tamura, Y., Iizuka, Y.,

Ohashi, K., Tomita, S., Sekiya, M., Hasty, A., Nakagawa, Y., Sone, H., Toyoshima, H.,

Ishibashi, S., Osuga, J., & Yamada, N. (2004). Insulin-independent induction of sterol

regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice.

Diabetes, 53(3), 5609.

12

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.