Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Target audience for our research is the wider biomedical research community including researchers at private and public research organization, hospitals, biotech, and pharmaceutical companies. Research lectures are accessible for professors, postdocs, graduate students and senior undergraduates. Outreach lectures are designed to be accessible for a general college educated audience. Changes/Problems: The grapefruit juice project had been an addition to our oringinal goals. However, due to a lack of funding we will not be able to emediatly continue with this interesting project and instead focus again on the core topics listed for this proposal. What opportunities for training and professional development has the project provided? Graduate students trained: Brittney Bivins, participated in project 2 Hyo Min Park, participated in project 4 Kevin Tharp, participated in project 1 and 3 Graduate student training is being provided through participation in seminars and conferences as well as by direct mentoring by the PI. Postdocs trained: Courtney Anderson, participated in project 1 Postdocs are trained thourgh participation in seminars, attendance of national, and international conferences and through direct mentoring by the PI and his collaborators. How have the results been disseminated to communities of interest? Results from our work have be disseminated to the public through peer-reviewed publications, scientific lectures at meetings, suchas as the Keystone conference, lectures at public outreach events, contribution to publications by UCB CNR, and through our web site. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The main milestones we have been working toward during the reporting period are: 1) Determine the role of CoQ uptake in the function of brown adipose tissue and elucidate the mechanisms of cellular CoQ uptake. Our hypothesis is that CD36 is a CoQ transporter. 2) Identification of the main fatty acid transporters in the islet of Langerhans and creation of loss and gain of function models for these transporters to evaluate their contribution to lipid induced toxicity. 3) Characterization of the anti-obesity and anti-hyperglycemic effects of grapefruit juice. 4) Further testing and evaluation of bioluminescent compounds to image lipid fluxes in live animals with the ultimate goal ofimaging fatty acid and CoQ fluxes in live animals. Overall, our ongoing work has significantly furthered our understanding of the way overnutrition affects health, and has generated novel approaches for the diagnostic and treatment of nutrient related dieseases. 1) CoQ is an important dietary supplement thought to prevent oxidative damage and aging related disorders. However, the molecular mechanisms of its cellular uptake and organismal transport have remained elusive. We have been working toward characterizing a novel role of CoQ in brown adipose tissue and the characterization of how CoQ is carried to and taken up by brown fat. Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells mostly synthesize CoQ endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, the mechanism by which CoQ is taken up by cells and the role of this process in BAT function has not been well studied. During the reporting period, we uncovered that the scavenger receptor CD36 is required for uptake of CoQ by BAT and non-shivering thermogenesis. Mice lacking CD36 display BAT hypertrophy, CoQ deficiency, and defective non-shivering thermogenesis. Surprisingly, in addition to its function in the electron transport chain, CoQ deficiency dramatically impacted Ucp1 expression. Together, these data reveal an important axis linking CD36, cellular CoQ uptake, and UCP1 expression that is able to control BAT function and thermogenesis. Thus, this research could ultimately lead to novel interventions to increase energy output to in the fight against overnutrition and obesity by utilizing dietary factors such as CoQ. 2) Increased plasma free fatty acid levels during obesity can directly affect beta cell function by inhibiting glucose-stimulated insulin secretion and inducing beta cell apoptosis, further contributing to the development of diabetes. Although it has been suggested that fatty acids can alter insulin secretion by binding to cell surface receptors, the lipotoxic effects of fatty acids are thought to be dependent on their entry into the cell. The mechanism of fatty acid transport into beta cells is not well characterized. We found expression of three Fatty Acid Transporters (FATPs) in islets isolated from C57BL/6J mice and MIN6 cells, a mouse islet-derived cell line, via quantitative PCR. We then used immunohistochemistry to localize these transporters in isolated islets. FATP1 was localized to beta cells, FATP4 to alpha cells, and FATP3, to delta. Although the fact that each of these transporters may be playing distinctive roles in their respective cell types is intriguing, we decided to focus on the function of FATP1 in beta cells. To directly assess the impact of FATP1 expression on beta cell deterioration in hyperlipidemia, we isolated islets from FATP-null and wild-type mice, treated them with palmitate for 48 hours ex vivo, and measured apoptosis specifically in the beta cells. Compared to wild-type mice, FATP1 knockout mice fail to induce apoptosis when treated with palmitate. These results suggest that deletion of FATP1 can protect from high fat-induced apoptosis in beta cells, possibly through limiting the entry and accumulation of fatty acids inside the cell. 3) In work carried out in collaboration with Dr. Napoli of our department we have been investigating the health promoting effects of grapefruit juice consumption in the context of diet-induced obesity. To determine the metabolic effects of grapefruit juice (GFJ) consumption we established a model of C57Bl/6 mice that drank 50% sweetened GFJ ad libitum as their sole source of liquid or isocaloric water. We investigated the effects of GFJ on metabolism in response to low and high-fat diets. Liquid and food consumption were similar between the GFJ and control groups, but GFJ supplementation improved metabolic variables in mice fed a high-fat diet. When fed a high-fat diet, mice treated with GFJ had an 18.4% decrease in weight, a 13-17% decrease in blood glucose, a three-fold decrease in serum insulin, a four-fold decrease in HOMA IR, and a 38% decrease in liver triglycerides, compared to controls. Mice fed a low-fat diet that drank GFJ had only a two-fold decrease in fasting insulin and in HOMA IR, but none of the other outcomes observed in mice fed a high-fat diet. GFJ consumption produced a decrease in blood glucose comparable to that of the commonly used anti-diabetic drug metformin. This evidence from a well-controlled animal study should encourage further investigations into the health-benefits of GFJ dietary supplementation, particularly in the context of obesity. Unfortunately, funding for this highly interesting and promising project has seized preventing us from further characterizing the molecular basis of the GFJ effect and from translating our findings into human studies. Since our goals are well aligned with the USDA's mission, feedback and suggestions from the readers of this report would be welcome. 4) Changes in the transport and absorption of lipids are play a key role in the development of many obesity associated disorders as well as malabsorption of dietary nutrients and lipid soluble vitamins in diseases such as Crohn's disease and cystic fibrosis. While animals models of these dietary and genetic disorders exist, there are no easily methods to visualize and quantitate the uptake of these dietary components in a live animal. To address this deficit, we have been working with our collaborator Dr. Dubikovskaya on novel chemical biology approaches to create novel tools for the bioluminescent imaging of dietary molecules such as fatty acids. This has already led to the publication (and patent) in the prior reporting cycle of a method to use in vivo imaging of bioluminescent fatty acids to determine intestinal absorption and uptake by other various tissues. In the current reporting period we have expanded on these studies by generating liver and heart specific luciferase expressing animals to specifically image cardiac and hepatic fatty acid uptake. Preliminary studies have shown that this is indeed possible and that the effects of inhibitors of hepatic fatty acid uptake can be imaged in real time in live animals. An additional problem we have begun to tackle with our collaborators is the lack of suitable bioorthogonal reactions to monitor the uptake and metabolic fate of nutrients. To this end we have reported a novel "split luciferin" approach that enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. This should be highly useful for both bioluminescence and specific molecular targeting in vivo to explore the uptake, distribution, and metabolic fate of different nutrients in live animals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Anderson, C.M. and A. Stahl, SLC27 fatty acid transport proteins. Mol Aspects Med, 2013. 34(2-3): p. 516-28.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Godinat, A., H.M. Park, S.C. Miller, K. Cheng, D. Hanahan, L.E. Sanman, M. Bogyo, A. Yu, G.F. Nikitin, A. Stahl, and E.A.
Dubikovskaya, A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of
Protease Activity in Living Mice. ACS Chem Biol, 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Dubikovskaya, E., Chudnovskiy, R., Karateev, G., Park, H. M., and A. Stahl, Measurement of Long-Chain Fatty Acid
Uptake into Adipocytes. Methods in Enzymology, in press.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The main output has been activities to conduct and analyze experiments aimed at furthering our understanding of the molecular mechanisms underlying obesity related disorders. Cells use fatty acids as an energy source, for intracellular signaling, for anchoring proteins to the plasma membrane, and for membrane biosynthesis. However, increased levels of intracellular fatty acid can cause lipotoxicity and moreover have been linked to the development of several prevalent diseases including type-2 diabetes, hepatosteatosis, and cardiovascular disorders. Our goal is to understand on a molecular level how fatty acids enter cells, how this process is regulated, and if it can be utilized as a target for the treatment of lipid-related disorders. We have also expanded research into the preclinical imaging of fatty acid fluxes using novel imaging reagents that couple bioluminescent signal generation to fatty acid uptake. Another new area of research during this reporting period has been the unraveling of factors that limit the channeling of fatty acids into the mitochondria of brown adipose tissue where they can be oxidized during thermogenesis. These results may open new avenues for the treatment of obesity related disorders. Results from this research have been disseminated to the public through peer-reviewed publications, scientific lectures at meetings, lectures at public outreach events such as the UC Berkeley homecoming, and through our web site. PARTICIPANTS: Participants Individuals that received salary support from this project in 2011: None This project has provided training and assistance with professional development to: Postdoctoral Fellows: Courtney Anderson Biao Nie Graduated Students: Hyo-Min Park Brittney Bivins Kevin Tharp Research Specialist: Kevin Tharp Rost Chudnovsky Undergraduate Research Volunteers: Heather Tran SuJin Song Yuli Chen Katherine Cole Cina Poursheikhani Kimia Etemadi Robin Lai David Yun Gabriela Diaz Karen Chan Stephanie Ng Diana Viet Heon Kang Collaborators/Partner organizations: Martin Brand, The Buck Institute for Ageing Kevin Healy, UCB Carolyn Bertozzi, UCB Elena Dubikovskaya, University of Lausanne TARGET AUDIENCES: Target audience for our research is the wider biomedical research community including researchers at private and public research organization, hospitals, biotech, and pharmaceutical companies. Research lectures are accessible for professors, postdocs, graduate students and senior undergraduates. Outreach lectures are designed to be accessible for a general college educated audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During the 2012 reporting period we have focused on three areas of research. 1) Characterization of FATP5 as a DCA transporter: Our previous studies have shown that some bile acids, particularly DCA and UDCA, inhibit FATP5 mediate fatty acid uptake by isolated hepatocytes as well as in in vivo models. However, the mechanism of inhibition has remained elusive. To this end, we have been testing the hypothesis that FATP5 could act as a high affinity transporter for DCA. Using radiolabeled DCA we were indeed able to show that FATP5 behaves more like a classical bile acid transporter. Current studies are under way to determine the exact substrate specificity of bile acid uptake, which we hypothesis will correlate with potency of FFA uptake inhibition. 2) Imaging of hepatic fatty acid uptake: Manipulation of fatty acid fluxes in model organisms has been key to both testing hypothesis regarding the genesis of hepatic steatosis as well as to the development of new treatment approaches based on FATP inhibitors. However, until recently it has been virtually impossible to observe fatty acid fluxes, such as intestinal or hepatic fatty acid uptake, directly in live animal. To address this problem we have synthesized, together with our collaborators in the chemistry department at UC Berkeley, a bioluminescent fatty acid, which, thanks to an activatable linker, will generate luciferin only upon cellular uptake. We generated a liver-specific luciferin-expressing animal, which will generate light exclusively from hepatocytes following luciferin injections. Importantly, using the bioluminescent fatty acid probe, we were able to image the inhibitory effect of DCA on hepatic fatty acid uptake in vivo in real-time. Quantitative data from the bioluminescent approach taken at peak photon flux time for control animals showed a high agreement with a previously published invasive method based on the injection and hepatic extraction of a bodipy-fatty acid. 3) Prevention of gallstone disease: We previously observed that loss of FATP2 prevents gallstone formation in animals fed a lithogenenic diet. We found that FATP5 is also expressed by gallbladder epithelial cells and using FATP5KO animals we now are able to show that these animals are also protected from gallstone formation when fed a lithogenic diet. Interestingly, this was not due to a change in the cholesterol saturation index of the bile. Instead, protection from gallstone formation correlated with accumulation of triglycerides in the gallbladder wall. Feeding animals a lithogenic diet greatly increased TAG content in the gallbladder tissues. However, loss of FATP5 protect animals from this effect based on whole tissue TAG analysis as well as 3D reconstructions of lipid droplets in gallbladder sections. Importantly, TAG accumulation in the gallbladder has been linked to decreased gallbladder contractility leading to stagnant bile and increased gallstones. We were able to demonstrate a greatly decreased ejection volume following a lithogenic diet. Impressively, contractility was restored back to untreated levels in FATP5KO animals.
Publications
- Jeppesen, J., Jordy, A.B., Sjoberg, K.A., Fullekrug, J., Stahl, A., Nybo, L., and Kiens, B. (2012). Enhanced Fatty Acid Oxidation and FATP4 Protein Expression after Endurance Exercise Training in Human Skeletal Muscle. PLoS One 7, e29391.
- Abbasi, A., Thamotharan, M., Shin, B.C., Jordan, M.C., Roos, K.P., Stahl, A., and Devaskar, S.U. (2012). Myocardial Macro-Nutrient Transporter Adaptations in the Adult Pre-gestational Female Intra-uterine and Postnatal Growth Restricted Offspring. Am J Physiol Endocrinol Metab.
- Nie, B., Park, H.M., Kazantzis, M., Lin, M., Henkin, A., Ng, S., Song, S., Chen, Y., Tran, H., Lai, R., Her, C., Maher, J.J., Forman, B.M., and Stahl, A. (2012). Specific bile acids inhibit hepatic fatty acid uptake. Hepatology, 2012 Apr 24. doi: 10.1002/hep.25797. [Epub ahead of print].
- Poreba, M.A., Dong, C.X., Li, S.K., Stahl, A., Miner, J.H., and Brubaker, P.L. (2012). Role of fatty acid transport protein 4 in oleic acid-induced glucagon-like peptide-1 secretion from murine intestinal L cells. American journal of physiology. Endocrinology and metabolism, doi:10.1152/ajpendo.00116.
- Henkin, A.H., Cohen, A.S., Dubikovskaya, E.A., Park, H.M., Nikitin, G.F., Auzias, M.G., Kazantzis, M., Bertozzi, C.R., and Stahl, A. (2012). Real time non-invasive imaging of fatty acid uptake in vivo. ACS Chemical Biology, 2012 Aug 28. [Epub ahead of print]
- Anderson, A. and Stahl, A. SLC27 fatty acid transport proteins. (2013) Molecular Aspects of Medicine, issue 34, 2013. NIHMS 394098 [Epub ahead of print].
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The main output has been activities to conduct and analyze experiments aimed at furthering our understanding of the molecular mechanisms underlying obesity related disorders. Cells use fatty acids as an energy source, for intracellular signaling, for anchoring proteins to the plasma membrane, and for membrane biosynthesis. However, increased levels of intracellular fatty acid can cause lipotoxicity and moreover have been linked to the development of several prevalent diseases including type-2 diabetes, hepatosteatosis, and cardiovascular disorders. Our goal is to understand on a molecular level how fatty acids enter cells, how this process is regulated, and if it can be utilized as a target for the treatment of lipid-related disorders. We have also expanded research into the preclinical imaging of fatty acid fluxes using novel imaging reagents that couple bioluminescent signal generation to fatty acid uptake. Another new area of research during this reporting period has been the unraveling of factors that limit the channeling of fatty acids into the mitochondria of brown adipose tissue where they can be oxidized during thermogenesis. These results may open new avenues for the treatment of obesity related disorders. Results from this research have been disseminated to the public through peer-reviewed publications, scientific lectures at meetings, lectures at public outreach events such as the UC Berkeley homecoming, and through our web site. PARTICIPANTS: Individuals that received salary support from this project in 2011: None This project has provided training and assistance with professional development to: Postdoctoral Fellows: Courtney Anderson Melissa Kazantzis Biao Nie Graduated Students: Hyo-Min Park Brittney Bivins Allison McQueen Research Specialist: Kevin Tharp Rost Chudnovsky Undergraduate Research Volunteers: Heather Tran SuJin Song Yuli Chen Katherine Cole Cina Poursheikhani Kimia Etemadi Robin Lai David Yun Gabriela Diaz Karen Chan Stephanie Ng Diana Viet Heon Kang Collaborators/Partner organizations: Martin Brand, The Buck Institute for Ageing Kevin Healy, UCB Carolyn Bertozzi, UCB TARGET AUDIENCES: Target audience for our research is the wider biomedical research community including researchers at private and public research organization, hospitals, biotech, and pharmaceutical companies. Research lectures are accessible for professors, postdocs, graduate students and senior undergraduates. Outreach lectures are designed to be accessible for a general college educated audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During the 2011 reporting period we have made good progress with our studies focused on the role of hepatic fatty acid transporters in the development of hepatic steatosis, a disorder that now affects millions of Americans. Since genetic manipulation of hepatic fatty acid transporters (FATP2 and FATP5) indicated that their inhibition may have positive effects on diet induced hepatic lipid accumulation and insulin sensitivity we developed a high-througput compatible assay to screen for small molecular inhibitors of FATP2 and -5. During proof-of-concept screens we made the surprising finding that certain bile acids can inhibit FATP5. Bile acids are known to play important roles as detergents in the absorption of hydrophobic nutrients and as signaling molecules in the regulation of metabolism. We tested the novel hypothesis that naturally occurring bile acids interfere with protein-mediated hepatic long chain free fatty acid (LCFA) uptake using stable cell lines expressing fatty acid transporters as well as primary hepatocytes from mouse and human livers which were incubated with primary and secondary bile acids to determine their effects on LCFA uptake rates. Also, mice were treated with secondary bile acids in vivo to assess their ability to inhibit diet-induced hepatic triglyceride accumulation. We found that Ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) are potent inhibitors of the liver-specific fatty acid transport protein 5 (FATP5). Both UDCA and DCA were able to inhibit LCFA uptake by primary hepatocytes in a FATP5-dependent manner. When mice were fed a high-fat diet, UDCA and DCA inhibited the accumulation of hepatic triglycerides by more than 50%. Thus, the data demonstrate a novel role for specific secondary bile acids in the regulation of hepatic LCFA uptake. The results illuminate a previously unappreciated means by which secondary bile acids can impact liver metabolism, and highlight a unique hepatoprotective function of UDCA that could inform its future clinical use. In a second project we have focused on the development of novel preclinical imaging teachnologies in a collaboration with the Bertozzi group at UCB. Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin-sensitivity. To enable non-invasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used as a valuable tool to localize and quantitate in real-time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic physiological processes and pathological alterations in multiple disease models.
Publications
- Falcon, A., Doege, H., Fluitt, A., Tsang, B., Watson, N., Kay, M.A., and Stahl, A. FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase. Am J Physiol Endocrinol Metab. Sep;299(3):E384-93, 2010.
- Henkin, A. H., Ortegon, A. M., Cho, S., Shen, W. J., Falcon, A., Kraemer, F. B., Lee, S. J., and Stahl, A. Evidence for protein-mediated fatty acid efflux by adipocytes. (2011) Acta Physiol (Oxf). In press.
- Kazantzis, M., and Stahl, A. Fatty acid transport proteins, Implications in physiology and disease. (2011) Biochim Biophys Acta. In press.
- Mishima, T., Miner, H.M., Morizane, M., Stahl, A., Sadovsky, Y. The Expression and Function of Fatty Acid Transport Protein-2 and -4 in the Murine Placenta. PLoS ONE 6(10): e25865. doi:10.1371/journal.pone.0025865, 2011.
- Jeppesen, J., Jordy, A.B., Sjoberg, K.A., Fullekrug, J., Stahl, A., Nybo, L., and Kiens, B. Enhanced Fatty Acid Oxidation and FATP4 Protein Expression after Endurance Exercise Training in Human Skeletal Muscle. PLoS One 7, e29391, 2012.
- Abbasi, A., Thamotharan, M., Shin, B.C., Jordan, M.C., Roos, K.P., Stahl, A., and Devaskar, S.U. Myocardial Macro-Nutrient Transporter Adaptations in the Adult Pre-gestational Female Intra-uterine and Postnatal Growth Restricted Offspring. Am J Physiol Endocrinol Metab, 2012.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The main output has been activities to conduct and analyze experiments aimed at furthering our understanding of the molecular mechanisms underlying obesity related disorders. Cells use fatty acids as an energy source, for intracellular signaling, for anchoring proteins to the plasma membrane, and for membrane biosynthesis. However, increased levels of intracellular fatty acid can cause lipotoxicity and moreover have been linked to the development of several prevalent diseases including type-2 diabetes, hepatosteatosis, and cardiovascular disorders. Our goal is to understand on a molecular level how fatty acids enter cells, how this process is regulated, and if it can be utilized as a target for the treatment of lipid-related disorders. Results from this research have been disseminated to the public through peer-reviewed publications, scientific lectures at meetings, lectures at public outreach events such as the UC Berkeley homecoming, and through our web site. PARTICIPANTS: Participants Individuals that received salary support from this project in 2010: None This project has provided training and assistance with professional development to: Postdoctoral Fellows: Alaric Falcon Melissa Kazantzis Biao Nie Postdoctoral Research Volunteers: Punita Bhasin Graduated Students: Diana Athonvarangkul Hyo-Min Park International visiting Graduate Student: Sungyun Cho (Korea University) Research Specialist: Amy Fluitt Undergraduate Research Volunteers: Heather Tran SuJin Song Yuli Chen Katherine Cole Cina Poursheikhani Kimia Etemadi Robin Lai David Yun Gabriela Diaz Karen Chan Stephanie Ng Diana Viet Heon Kang Collaborators/Partner organizations: Martin Brand, The Buck Institute for Ageing Sung-Joon Lee, Korea University Gerald Shulman, Yale University TARGET AUDIENCES: Target audience for our research is the wider biomedical research community including researchers at private and public research organization, hospitals, biotech, and pharmaceutical companies. Research lectures are accessible for professors, postdocs, graduate students and senior undergraduates. Outreach lectures are designed to be accessible for a general college educated audience PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
One central area of progress during the 2010 report period has been non-alcoholic fatty liver disease (hepatic steatosis). We focused on the contribution of fatty acid uptake and metabolism to the development and progression of steatosis. Rather than solely relying on conventional knockout models to target a multitude of NASH genes, we developed adeno-associated viral vectors for the stable, liver-specific, and non-toxic expression of shRNAs in collaboration with Dr. Mark Kay. Using this approach, my laboratory demonstrated achieving specific, non-toxic, and persistent knockdown of FATP5 and -2 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. We showed that knockdown of wither FATP5 or -2 also reversed established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. We concluded that continued activity of hepatic FATP5 and FATP2 are required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease. This work lead to publications in JBC and the American Journal of Physiology. Based on the hypothesis that FATPs constitute targets for therapeutic intervention, we have worked to develop small molecule inhibitors of FATPs by developing a high-throughput compatible fatty acid uptake assay using a fluorescently-labeled fatty acid and a non-toxic cell impermeable quenching agent. This combination allows fatty acid transport to be measured in real time using fluorescent plate readers or standard fluorescent microscopy. With this assay, my laboratory can faithfully reproduce known differentiation-and hormone-induced changes in fatty acid uptake by 3T3-L1 cells and determine fatty acid uptake kinetics with previously unobtainable temporal resolution. With this robust assay in hand, we are initiating collaboration with commercial and academic institutions, as well as with NIH's Molecular Libraries Screening Centers Network to discover FATP specific inhibitors. We also have begun a rational screening approach for natural inhibitors of hepatic fatty acid transporters with a particularly focus on bile acids. Ultimately, the aim is to translate novel insights into new diagnostic and treatment options for human diseases such as diabetes, steatosis, and cancer. The HTS assay technology was published in the Journal of Lipid Research and in the Journal of Biomolecular Screening. A manuscript detailing our findings that bile acids inhibit hepatic fatty acid uptake is currently in preparation.
Publications
- Zhou, W., Madrid, P., Fluitt, A., Stahl, A., and Xie, X.S. Development and Validation of a High-Throughput Screening Assay for Human Long-Chain Fatty Acid Transport Proteins 4 and 5. J Biomol Screen. 2010 Jun;15(5):488-97. http://jbx.sagepub.com/cgi/rapidpdf/1087057110369700v1
- Falcon, A., Doege, H., Fluitt, A., Tsang, B., Watson, N., Kay, M.A., and Stahl, A. FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase. Am J Physiol Endocrinol Metab. 2010 Jun 8. [Epub ahead of print]. http://ajpendo.physiology.org/cgi/reprint/299/3/E384
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Outputs The main output has been activities to conduct and analyze experiments aimed at furthering our understanding of the molecular events connecting cellular and organismal fatty acid transport with the development of obesity related disorders. Cells use fatty acids as an energy source, for intracellular signaling, for anchoring proteins to the plasma membrane, and for membrane biosynthesis. However, increased levels of intracellular fatty acid can cause lipotoxicity and moreover have been linked to the development of several prevalent diseases including type-2 diabetes, hepatosteatosis, and cardiovascular disorders. Our goal is to understand on a molecular level how fatty acids enter cells, how this process is regulated, and if it can be utilized as a target for the treatment of lipid-related disorders. Results from this research have been disseminated to the public through peer reviewed publications and public lectures. PARTICIPANTS: Participants Individuals that received salary support from this project: Andreas Stahl, PI This project has provided training and assistance with professional development to: Postdoctoral Fellows: Alaric Falcon Melissa Kazantzis Biao Nie Postdoctoral Research Volunteers: Punita Bhasin Graduated Students: Diana Athonvarangkul Dee Grigsby International visiting Graduate Student: Sungyun Cho (Korea University) Research Specialist: Amy Fluitt Undergraduate Research Volunteers: Richard Wu Carolyn Lam Angela Yu Jessica Archambault Kathleen Heng Jeany Ng Peter Vuong Daniel Park Diana Viet Heon Kang Gabriela Diaz David Yun Collaborators/Partner organizations: Martin Brand, The Buck Institute for Ageing Sung-Joon Lee, Korea University Gerald Shulman, Yale University TARGET AUDIENCES: Target Audience Target audience for our research is the wider biomedical research community including researchers at private and public research organization, hospitals, biotech, and pharmaceutical companies. Research lectures are accessible for professors, postdocs, graduate students and senior undergraduates. Outreach lectures are designed to be accessible for a general college educated audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Outcomes / Impacts Research results have originated from all 4 main objectives, however, major progress has been made primarily toward goal 2 during the reporting period which we will be focus on in this report. Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition we initially utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic Fatty Acid Transport Protein 5 in vivo prior or post to establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrated the ability to achieve specific, non-toxic, and persistent knockdown of Fatty Acid Transport Protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of Fatty Acid Transport Protein 5 was also able to reverse already established non-alcoholic fatty liver disease resulting in significantly improved whole-body glucose homeostasis. Thus continued activity of hepatic Fatty Acid Transport Protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease. During this report period we have expanded these studies to the second major hepatic fatty acid transporter, FATP2. Fatty Acid Transport Protein (FATP) 2 is a member of the FATP family of fatty acid uptake mediators and has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). During 2009 we address whether FATP2 is (a) a peroxisomal enzyme, (b) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or (c) a multifunctional protein. We found that in mouse livers only a minor fraction of FATP2 localizes to peroxisomes where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2 while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Using AAV8 based shRNA expression vectors, liver-specific FATP2 knockdown was achieved and significantly reduced hepatosteatosis in the face of continued high-fat feeding concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to both peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 a potential novel target for the treatment of non-alcoholic fatty liver disease. These findings were submitted for publication during the 2009 report period but were only excepted for publication in 2010 and will therefore be listed in the next report.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: It is too soon to report progress. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Too soon to report progress.
Publications
- No publications reported this period
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