Source: UNIVERSITY OF NEBRASKA submitted to NRP
ACTIVATION OF THE DSRNA ACTIVATED PROTEIN KINASE PKR BY MICRORNAS IN THE PATHOGENESIS OF HEPATIC STEATOSIS AND HYPERLIPIDEMIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1002056
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jan 10, 2014
Project End Date
Nov 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Nutritional & Health Sciences
Non Technical Summary
Non-alcoholic fatty liver disease (NAFLD) affects more than 20 percent of the U.S. population, a prevalence attributed to the national increase in obesity and diabetes. Human subjects with hepatic steatosis have a higher prevalence of cardiovascular disease (CVD). The currently accepted theory for the development of NAFLD is the "two-hit" hypothesis; the "first hit" mainly consists of lipid accumulation in hepatocytes, followed by inflammation and hepatocellular injury in a "second hit" mediated by reactive oxygen species and proinflammatory cytokines and transcription factors. While this is a useful conceptual framework, the exact cellular and molecular mechanisms leading to NAFLD are not fully understood. The dsRNA-activated protein kinase R (PKR) was originally identified as a key component in the innate immunity response. Recent studies have further developed this concept by demonstrating the essential role of PKR in the initiation of metabolic inflammation and the activation of the inflammatory complex under metabolic stress. However, knowledge of the underlying molecular mechanism for the activation of PKR by nutrient factors is limited. Therefore, the goal of this project is to test the hypothesis that microRNAs whose expression is induced by lipid overload (e.g. through high fat diet) translocate into mitochondria to initiate inflammatory response and activate PKR kinase. Activated PKR is then proposed to link cellular stress signaling from mitochondria to the endoplasmic reticulum (ER), activates hepatic lipid synthesis by enhancing the expression of enzymes involved in de novo lipogenesis, leading to hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). This hypothesis will be tested by three specific aims: (1) Determine the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress. Specific microRNAs (e.g. microRNA-378/378*, microRNA-455) play an important role in energy metabolism and the development of metabolic syndrome. A nucleus-encoded microRNA has been shown to translocate into mitochondria and regulate mitochondrial function in cardiac myocytes. Hence, the impacts of a high-fat diet on overall microRNAs expression and profile of mitochondrial translocated-microRNAs in hepatocytes will be determined by microRNA array. Biomarkers of mitochondrial stress will also be analyzed. (2) Determine whether high-fat-diet-induced microRNAs act as activators of PKR and the role of PKR in mediating mitochondrial and ER stress under lipid overload. To this end, induction of mitochondria and ER stress will be investigated in PKR-null mice and microRNA-378/378*-null mice fed a high-fat diet. (3) Determine the effects of PKR on cAMP responsive binding protein (CREB) signaling cascade and consequences on hepatic lipid metabolism via CREBH-Insig-2a signaling. CREBH-null mice will be infected with an adenovirus overexpressing Insig-2a with the intent to rescue these mice from high-fat-diet-induced NAFLD.The proposed research has the potential to advance our understanding of the pathogenesis of obesity and NAFLD. This would be the first example of a kinase being activated by small nucleotides, microRNAs, in response to excessive nutrition supply. Supported by this project, the investigator's long-term goal is to discover novel pathophysiological mechanisms underpinning lipid-induced metabolic diseases, especially obesity, hepatic steatosis, and diabetes. Because microRNAs, PKR and CREBH are potential therapeutic targets, demonstrating a mechanistic link among microRNAs, PKR and CREBH would provide novel insights into NAFLD etiology and potentially lead to innovative nutrient-based therapeutic strategies.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70270101010100%
Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
7010 - Biological Cell Systems;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
The overarching goal of this project is to discover the mechanism for the activation of the dsRNA activated protein kinase PKR upon nutrient surplus and its attribution to the development of high-fat-diet-induced hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). We are guided by the centralhypothesis that activation of the dsRNA activated protein kinase PKR by microRNAs conveys cellular stress signals from mitochondria to the endoplasmic reticulum (ER) and disturbs hepatic lipid metabolism, leading to the development of hyperlipidemia and NAFLD. the proposed hypothesis will be tested by three specific objectives.Objective 1: determines the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress. This objective tests the hypothesis that microRNAs whose increased expression is induced by high-fat-diet are able to translocate into hepatic mitochondria to initiate the inflammatory response.Objective 2: determines whether high-fat-diet -induced microRNAs act as activators of PKR and the activated PKR in mediating cellular stress under metabolic stress conditions. This objective will explore a potentially novel mechanism by which overexpression of microRNAs induced by high-fat diet binds to PKR dsRNA binding domains and induces PKR activation.Objective 3: determines the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling. This objective will test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue.
Project Methods
The following experimental strategies and approaches will be applied to address the central hypothesis and three objectives we proposed in this project.Objective 1. Determine the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress.1.1.1 Determine the lipid induced-microRNA profile by microRNA array and translocation of microRNAs from cytoplasm into mitochondria upon exposure to HFD. In vitro Study. McA cells, a rat hepatoma cell line will be treated with the saturated fatty acid palmitic acid (PA) and monounsaturated fatty acid oleate (OA). Total microRNA will be extracted from the treated cells with the microRNANeasy Transcription Kit (Qiagen) and then be subjected to analyze the microRNA profile via illumina-based deep sequencing. Three replicates will be sequenced for each treatment. Illumina sequencing will be performed at the UNL Genomic Core Facility. Data obtained from this assay will provide an overall profile of lipid-induced liver microRNAs.In vivo study. To further investigate the impact of lipids on microRNA expression in vivo, we will conduct an in vivo time course study in mice fed a high-fat-diet (HFD). Determination of the diet composition and feeding time course is based on our previous experiments and literature. It has been shown that a HFD containing 45% of calories from fat (Open Source Diets) induces mitochondrial dysfunction, obesity and hepatic steatosis in C57BL/6 male mice after 8 weeks of feeding, while 12-20 weeks of high-fat feeding causes ER stress. Thus, we expect that feeding the mice with the same composition of HFD in a time-course manner will allow us to dynamically observe expression of microRNAs and their kinetic impacts on the homeostasis of cellular organelles, such as induction of mitochondrial stress. Therefore, four groups of C57BL/6 male mice (n=8/group) at the age of 12 weeks (purchased from Jackson's laboratory) will be subjected to a control diet (CD) or HFD diet containing 45% of calories from fat (Open Source Diets) for 8 weeks and 12 weeks, respectively. Blood samples will be collected weekly during the feeding trial and analyzed for plasma lipoprotein profile via fast protein liquid chromatography (FPLC) and lipid content via gas chromatography (GC) (Agilent Technologies). The lipid profile will include triglyceride, total cholesterol, VLDL-cholesterol and HDL-cholesterol. At the end of the feeding periods, we will euthanize the mice to collect liver tissues to extract total microRNAs and to compare the difference between HFD and CD. The isolated microRNAs will further be subjected to microRNA array to compare the hepatic microRNA expression profiles induced by CD and HFD. We will further determine whether HFD induces translocation of microRNAs, including micro-R378/378* and microRNA-455-3p, from cytoplasm into mitochondria using the same approach described in in vitro study of this Section.1.1.2 Determine the impact of lipid induced-microRNAs on mitochondrial energy metabolism and inflammatory response.In vitro study. To examine whether microRNAs induced by HFD (HFD-microRNAs) have different impacts on mitochondrial energy metabolism and inflammatory response than microRNAs isolated from livers of mice fed control diet (CD-microRNAs), 0.5 mg of CD-microRNAs and HFD-microRNAs will be transfected into McA cells (1x105) using lipofectamine 2000 (Invitrogen). Forty-eight hours post-transfection, the cells will be subjected to the assay described immediately below to examine the impacts of CD-microRNA vs. HFD-microRNAs on mitochondrial functional activities. · Analyze fatty acid metabolism rate in isolated mitochondria.·Determine the concentrations of ROS in mitochondria and ER in cells transfected withmicroRNAs.·Examine the activation of stress markers in mitochondria.In vivo study. To determine the impact of individual microRNAs on mitochondrial function in vivo, we will employ a microRNA-378/378*-null mouse model from Dr. Eric Olson at the University of Texas Southwestern Medical Center in Dallas (letter attached). Four groups of WT littermates or microRNA-378/378*-null male mice (n=8/group) at the age of 12 weeks will be subjected to either a CD or HFD for 8 and 12 weeks, respectively. Plasma will be collected weekly during the feeding trial to measure lipid contents. At the end of the feeding period, liver tissues will be isolated and subjected to tests for mitochondrial function as proposed above in this Section. Upon comparing the differences between WT and micorRNA-378/378*-null mice, we will be able to determine the impact of microRNA-378/378* on mitochondrial energy metabolism and inflammatory response in vivo.Objective 2. Determines whether high-fat-diet-induced microRNAs act as activators of PKR and the role of the activated PKR in mediating cellular stress under metabolic stress conditions.2.1.1 Determine whether HFD-induced microRNAs are able to activate PKR. ·Determine PKR activation by PKR kinase assay, phospho-specific antibody and 35S labeling assay.·Examine activation of PKR in vivo using microRNA-378/378*-null mice.2.1.2 Characterize the involvement of PKR in mediating stress signal between mitochondria and the ER.· In vivo study on the impact of PKR activity on mitochondria and ER by using PKR-null mice.Objective 3: Determine the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling.3.1.1 Examine whether PKR suppresses CREBH activity by modulating the phosphorylated status of CREBH.· In vitro kinase assay to determine whether PKR is able to phosphorylate CREBH.· In vivo study to confirm the regulatory effect of PKR on CREBH by applying the CREBH-null mice.3.1.2 Determine the role of CREBH on hepatic triglyceride synthesis through transcriptional regulation of Insig-2a.·In vitro study to establish Insig-2 as a target gene of CREBH via chromatin immunoprecipitation (ChIP).·In vivo study to rescue CREBH-null mice from hypertriglyceridemia by overexpressing Insig-2a in the CREBH-null mice.·In vivo study to protect CREBH-null mice from HFD-induced NAFLD by expressing Insig-2a.Statistical analyses and sample Size Calculation. For in vitro experiments, data will be analyzed using ANOVA or independent t tests. For diet study at a time course, group differences (control vs. HFD) at different time points will be determined by two-way ANOVA. Statistical significance will be set at P<0.05. In animal experiments, a sample size of eight mice per group will have 80% power to detect a change in means of at least 25%, assuming the coefficient of variation is 20%. When available, variability reported in previous studies, including our own was used for calculation and in absence thereof, a coefficient of variation of 20% was assumed. Binary outcome variables data will be analyzed using the two-tailed Student's t-test. One-way ANOVA will be used for comparison of more than two treatments. Data will be presented as least-square means and SE of the mean. Probability values <0.05 will be considered statistically significant.

Progress 01/10/14 to 11/30/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? N/A

Publications


    Progress 10/01/16 to 09/30/17

    Outputs
    Target Audience:1) The nutritional research community 2) Trainees (e.g., graduate students and postdoctoral fellows) in Su lab 3) Citizens of USA and the rest of the world Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Two graduate students and one postdoctoral fellow were involved in this project last year. They are guided and trained by the project director on their research projects and the molecule biology research skills. One graduate student completed his Master degree in December, 2017. 2) The students and postdoctoral fellows in the PD's lab were also encouraged to present their research results on the international conferences, Experimental Biology conference-2017 (Chicago, USA), Arterioscler Thromb Vasc Biol (ATVB) Scientific Session-2017 (Minneapolis, USA), Obesity-Summit -2017 (Toronto Canada), and Nebraska Gateway to Netrigenomics-2017at UNL. 3) All the trainees in Su lab, two postdocs and one graduate student, have publications on high profile scientific journals, including articles in Arterioscler Thromb Vasc Biol, J Nutr Biochem, J Mol Med and Mol Nutr Food Res. How have the results been disseminated to communities of interest?The research results have been presented on three international scientific conferences and a research symposium. They are also published as four peer-reviewed articles in high impact professional journals. These research findings will advance our understanding of the pathogenesis of diabetes and fatty liver disease. It will also promote UNL's reputation in the field of biomedical research and further strengthen research ties between UNL and other institutions because of research collaborations and scientific exchange with other research institutes. What do you plan to do during the next reporting period to accomplish the goals?The next funding cycle will be the last year of this project. We will complete the unfinished experiments in three Objectives. For Objective 1 and 2, we will develop an in vitro kinase assay to determine the direct role of microRNAs in the activation of PKR. We will also complete the publication of a manuscript that summarizes the novel findings in these two objectives. For Objective 3, we are currently investigating the impact of CREBH on microRNA143/145 expression and their association with the development of inflammatory bowel disease and liver fibrosis. This study will be continued in 2018.

    Impacts
    What was accomplished under these goals? Objective 1: determines the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress. This objective tests the hypothesis that microRNAs whose increased expression is induced by high-fat-diet are able to translocate into hepatic mitochondria to initiate the inflammatory response. During this funding period, we further tested the hypothesis that microRNA-378 can targetmitochondrial signaling and induce insulin resistance by investigating the function of PPARa, a key transcription factor involved in mitochondrial fatty acid beta-oxidation. We generated a luciferase construct bearing the 3'-UTR of PPARa, tested the luciferase activity in the presence or absence of microRNA-378 mimics and demonstrated that PPARa is a true target of microRNA-378. These research results have been presented in "the Nebraska Gateway to Netrigenomics" at UNL in 2017 and the Arterioscler Thromb Vasc Biol (ATVB) Scientific Session, Minneapolis, USA. A manuscript summarized the research finding is currently under preparation. Objective 2: determines whether high-fat-diet -induced microRNAs act as activators of PKR and the activated PKR in mediating cellular stress under metabolic stress conditions. This objective will explore a potentially novel mechanism by which overexpression of microRNAs induced by high-fat diet binds to PKR dsRNA binding domains and induces PKR activation. Since we have demonstrated that microRNA-378 was able to directly interact and activate PKR in the previous funding cycle, we further used nanoparticles carrying a microRNA-378 inhibitor (anti-microRNA-378) or control microRNA (scramble) to treat the fructose-fed mice. Our results showed that reduced microRNA-378 by inhibitors alleviated activation of PKRand the associated metabolic inflammation induced by high-fructose diet. These results were presented in the international conference of Obesity-Summit -2017 in Toronto Canada and Experimental Biology conference-2017, Chicago, USA. We further used nanoparticles carrying microRNA-29b1 mimics to treat high-fat-fed mice for determining the protective effect of miR-29b1 in non-alcoholic fatty liver disease and liver fibrosis. Results from this study have been served as preliminary data for an R21 grant application submitted to NIH in 2017. A peer-reviewed article in which we proposed that, for the first time, a set of microRNAs (e.g., microRNA-122, -155, -34a and -29) assemble as a regulatory network that regulate hepatic lipid metabolism and the hedgehog pathway and is involved in the progressive liver injury from non-alcoholic fatty liver disease to fibrosis. This article has been published in a very high impact journal, Adv. Drug Deliv. Rev. Objective 3: determines the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling. This objective will test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue. In objective 3, we further demonstrated that bioactive compounds in the Nebraska-grown fenugreek seed activated the CREBH-Insig signaling pathway which in turn inhibited hepatic de novo lipogenesis, improved mitochondrial and endoplasmic reticulum stress and improved insulin sensitivity in the fructose-induced diabetic mice. This study revealed a novel mechanistic pathway which may land supports for the development of Nebraska-grown fenugreek seed as a nutritional supplement for the prevention and treatment of diabetes. Results of this study have been published in the journal of Mol Nutr Food Res.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sud N., Zhang H., Pan K., Cheng X., Cui J., and Su Q. (Senior author) (2017) Aberrant expression of microRNA induced by high fructose diet: Implications in the pathogenesis of hyperlipidemia and hepatic insulin resistance. J Nutr Biochem (Impact factor: 4.7). 2017 May; 43:125-131. Epub 2017 Feb 20. PMID: 28284064.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Song Y., Zhao M., Shen J., Khound R., Zhang K., and Su Q. (Senior author) (2017). CREBH mediates metabolic inflammatory signaling to overproduction of VLDL in metabolic syndrome. J Mol Med (Impact factor: 4.9). 2017 Apr 28. doi: 10.1007/s00109-017-1534-4. [Epub ahead of print]. PMID: 28455595.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Khound, R., Taher, J., Baker C., and Su, Q. (Senior author) (2017). GLP-1 Elicits an Intrinsic Gut-Liver Metabolic Signal to Ameliorate Diet-Induced VLDL Overproduction and Insulin Resistance.. Arterioscler Thromb Vasc Biol (ATVB) (Impact factor: 6.6), 2017;37:2252-2259.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Khound R., Shen J., Song Y., Santra D., and Su Q. (Senior author) (2017). Phytoceuticals in fenugreek ameliorate VLDL overproduction and insulin resistance via the Insig signaling pathway. Mol Nutr Food Res (Impact factor: 4.32) 2017 Dec 6. doi: 10.1002/mnfr.201700541. [Epub ahead of print].
    • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Effects of Vagotomy and Fenugreek on Hyperlipidemia and Insulin Resistance


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:1) Supervising the research activities of 2 graduate students and 1 summer student (undergraduate student). 2) Teaching two courses, NUTR-896 and NUTR-250 which involved 6 graduate students and 30undergraduate students. 3) Disseminating the research work from this project in An international conference (ATVB-2016) that hadabout 1,500 attendees. Three research symposiums at UNL with about 200 attendees in total. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Two graduate students were involved in this project last year. They are guided and trained by the project director on their research projects and the molecule biology research skills. One graduate student has completed his Master degree in July, 2016. 2)The students and postdoctoral fellows in the PD's lab were also encouraged to participate presentations in the international conference (ATVB-2016), Nebraska Gateway to Netrigenomics and the "Nebraska Center for the Prevention of Obesity Diseases 2nd Annual Symposium at UNL." at UNL (2016). 3) All the lab members (two postdocs and two graduate students) have publications on high profile scientific journals. How have the results been disseminated to communities of interest?The research results have been presented on an international scientific conference and published as three manuscripts in high impact journals. These research findings have advanced our understanding of the pathogenesis of obesity and fatty liver disease. It will also promote UNL's reputation in the field of biomedical research and further strengthen research ties between UNL and other institutions because of the research collaborations with other research institutes. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to work on all three Objectives. For Objective 1, we have identified that the mRNA expression of liver X receptor (LXR) is significantly increased in the microRNA-378 knock mice. Since LXR is a transcription factor that regulates hepatic lipid metabolism and inflammatory signaling, we will further explore the role of LXR in ameliorating metabolic inflammation induced by lipogenic diet in the next reporting period. For Objective 2, we will develop an in vitro kinase assay to determine the role of microRNAs in the activation of PKR. We are expecting a publication will be generated from the research results collected from Objectives 1 and 2. For Objective 3, we will characterize a novel role of CREBH in metabolic inflammation and inflammatory bowel disease. This research will advance our knowledge of the pathogenesis of metabolic syndrome and inflammatory bowel disease.

    Impacts
    What was accomplished under these goals? Objective 1: determines the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress. This objective tests the hypothesis that microRNAs whose increased expression is induced by high-fat-diet are able to translocate into hepatic mitochondria to initiate the inflammatory response. During this funding period, we used bioinformatics tools to analysis the data set of miRNA deep sequences of liver tissues from high fructose fed mice. We identified ten microRNAs assembled as a regulatory network which targets multiple key genes that are involved in lipid and carbohydrate metabolism. We further verified the mRNA expression of major targets of these microRNAs by quantitative-PCR. Using a mouse model depleted the microRNA-378 genetically, we demonstrated that depletion of microRNA-378 protects mice from high fructose diet induced mitochondrial stress and metabolic inflammatory response. The research results have been summarized as two manuscripts. One has completed the revision and resubmitted back to the Journal of Nutritional Biochemistry currently. The other manuscript is currently under preparation. Objective 2: determines whether high-fat-diet -induced microRNAs act as activators of PKR and the activated PKR in mediating cellular stress under metabolic stress conditions. This objective will explore a potentially novel mechanism by which overexpression of microRNAs induced by high-fat diet binds to PKR dsRNA binding domains and induces PKR activation. We have made significant progress on Objective 2 during this funding period, we found that microRNA-378 directly interacted with and activate the protein kinase PKR. Inhibition of PKR activation restored mitochondrial oxidative capacity and protected cells from mitochondrial stress. This novel finding demonstrated that, in addition to regulate their target genes on the mRNA level, microRNA is able to directly interact with RNA binding protein kinase which greatly broadens the physiological activity of microRNAs. The research results have been presented in the "Nebraska Center for the Prevention of Obesity Diseases 2nd Annual Symposium at UNL." Objective 3: determines the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling. This objective will test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue. Continuing with our work in the previous funding period, we further demonstrated that activation of CREBH mediated the metabolic inflammatory signaling to hepatic overproduction of very low density lipoprotein. This finding provides mechanistic insight into the molecular mechanism of hyperlipidemia in metabolic syndrome. A research article on this topic has been submitted to the Journal of Molecular Medicine which has completed the reviewed and is currently under revision for resubmission.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Sud N, Rutledge AC, Pan K, and Su Q. (2016) Activation of the dsRNA-Activated Protein Kinase PKR in Mitochondrial Dysfunction and Inflammatory Stress in Metabolic Syndrome. Curr Pharm Des. 2016 Feb 2. [Epub ahead of print] PMID:26831644.
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang H., Zhao M., Sud N., Christine P., Shen J., Song Y., Pashaj A., Zhang K., Carr T., and Su Q.(2016). Glucagon regulates hepatic lipid metabolism via cAMP and Insig-2 signaling: implication for the pathogenesis of hypertriglyceridemia and hepatic steatosis Scientific Reports. 2016; 6:32246. PMID: 27582413.
    • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Neetu Sud N., Zhang H., Pan K., Cui J., and Su Q.(2016) Aberrant expression of microRNA induced by high fructose diet: Implications in the pathogenesis of hyperlipidemia and hepatic insulin resistance (completed revision and resubmitted).
    • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Song Y., Zhao M., Shen J., Khound R., Zhang K., and Su Q. (Senior author) (2016). CREBH mediates metabolic inflammatory signaling to overproduction of VLDL in metabolic syndrome (Under review).
    • Type: Journal Articles Status: Under Review Year Published: 2016 Citation: Khound R., Song Y. and Su Q. (2016). GLP-1 mediates the intrinsic gut-liver regulatory signals in anti-VLDL overproduction and insulin resistance (Under review).
    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shen J, Tong X, Sud N, Maldonado-Gomez M, Khound R., Song Y., Walter J and Su Q. (2016). LDL receptor signaling mediates the triglyceride-lowering action of Akkermansia muciniphila in genetic induced hyperlipidemia. Arterioscler Thromb Vasc Biol. 2016 Jul; 36(7):1448-56. doi: 10.1161/ATVBAHA.116.307597. Epub 2016 May 26. PMID: 27230129
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shen J, Tong X, Sud N, Maldonado-Gomez M, Walter J and Su Q. (2016). LDL receptor signaling mediates the triglyceride-lowering action of Akkermansia muciniphila in genetic induced hyperlipidemia. Arterioscler Thromb Vasc Biol (ATVB) Scientific Session. Omni Nashville, Tenn, USA.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shen, J, Pan, K, Santa, D and Su Q.(2016) Fenugreek Seeds Inhibit Hepatic VLDL Overproduction by Attenuating Metabolic Inflammatory Stress. Arterioscler Thromb Vasc Biol (ATVB) Scientific Session. Omni Nashville, Tenn, USA.
    • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Song Y., Cheng, X., Sud N. Shen. J. and Su Q.(2016). Increased expression of microRNA-378 induces metabolic inflammation and cellular stress signaling. Nebraska Center for the Prevention of Obesity Diseases 2nd Annual Symposium at UNL, Lincoln, Nebraska, USA.


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience:1) Supervising the research activities of 2 graduate students and 1 summer student (undergraduate student). 2) Teaching three courses, NUTR-896, NUTR-921 and NUTR-250 which involved 16 graduate students and 32 undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1) Two graduate students were involved in this project last year. They are guided and trained by their supervisor on the research projectsand the molecule biologyresearch skills.One graduate studenthas completed his Master degree in June, 2015 (co-supervisor with a professor in China). 2)The students and postdoctoral fellowin the lab were also encouraged to participate presentations in the Nebraska Gateway to Netrigenomics and NPOD ResearchRetreat organized by the Department of Nutrition and Health Sciences 2015. How have the results been disseminated to communities of interest?The researchresults have been presented on two international scientific conferences and published as threemanuscripts in high impact journals. These research findings will advance our understanding of the pathogenesis of hyperlipidemia and diabetes. It will also promote UNL's reputation in the field and further strengthen research tiesbetween UNL and other institutions as there are ongoing research collaborations with other research institutes. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will continue to work on all three Objectives. For Objective 1, we are generating a liver specific knockout of microRNA-378 to further explore the association between microRNA-378 with the metabolic inflammation. If successful, this study will unveil anovel mechanism for metabolic diseases. For Objective 2, we will continue to determine the role of microRNAs in the activation of PKR by in vitro kinase assay. Determination of microRNAs can directly activate PKR has significantly physiological relevance as it will greatly broadenthe function of PKR in cellular activity and the related metabolic diseases.We plan to have one publication on the research results collected from Objective 2. For Objective 3, We will continue to characterize the role of CREBH in metabolic inflammation and its association with PKR. This study will establish a novel signaling pathway in regulating metabolic inflammation. We plan to present the research work in an international conference and has one publication on this objective.

    Impacts
    What was accomplished under these goals? The research results obtainedfrom thisHatch project have the followingimpacts: (i) advance our knowledge of the etiology of lipogenic diet inducedobesity and diabetes;(ii) can be used tomodify current dietary recommendations for the purpose ofimproving human nutrition; (iii) to raise the awareness of the publics forhealthy food consuming; (iv) provide rationale for theindustries to produce beneficial gut bacteria as probiotics and promote human health; (v) provide evidence for the design of nutritional supplements tospecifically target microRNAs andPKR in the prevention and treatment of obesity and fatty liver disease. Objective 1:determines the role ofhigh-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress.This objective tests the hypothesis that microRNAs whose increased expression is induced by high-fat-diet are able to translocate into hepatic mitochondria to initiate the inflammatory response. During this funding period, we conducted a microRNA expression profile in the livers of high fructose fed mice by miRNA deep sequencing. Using bioinformatics tools to analysis the data, we identified four major cellular proteins that play key roles in fructose induced obesity and insulin resistance.We further used mice which are deleted in the geneexpresses microRNA-378 to study the underlying molecular mechanism for the association between high fructose diet, microRNAs and type-2 diabetes. Given the reputation of the principle investigator (PI) in the field of microRNA and obesity study, the PI was invited by the journal of Drug Development Research to contribute an review article which overviews the role of non-coding RNAs and lipid metabolism. This article has been published in thejournal ofDrug Development Researchin 2015. Objective 2:determineswhether high-fat-diet -induced microRNAs act as activators of PKR and the activated PKR in mediating cellular stress under metabolic stress conditions.This objective will explore a potentially novel mechanism by which overexpression of microRNAs induced by high-fat diet binds to PKR dsRNA binding domains and induces PKR activation. In the past one year, we have made significant progress on Objective 2, we found that high fructose diet was able to activate the protein kinase PKR.The activated PKR regulated the cAMP signaling to induce liver lipid synthesis which contributes to the fructose-induced hyperlipidemia. The research results were presented in the IDeA Conference (North Dakota, USA, 2015). The results have also been summarized as a research article andpublished in the journal ofCurrent Pharmaceutical Designin 2015. Objective 3:determines the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling.This objective will test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue. Continuing with our work in the previous funding period, we further demonstrated that impairment of CREBH resulted in hyperactivation of lipid synthesis and fatty liver disease. A chemical compound, lipoic acid, can activate CREBH and lower blood triglycerides. More important, we found that a beneficial gut bacterial strain can regulate liver lipid metabolism and prevent hyperlipidemia induced by genetic depletion of CREBH. These novel findings havebeen presented on the Experimental Biology conference (Boston, Massachusetts, 2015)and a research articleon this topic has been submitted to the journal ofArterioscler Thromb Vasc Biolwhich is currently under revision for resubmission.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tong X, Christian P, Zhao M, Wang H, Moreau M and Su Q.(2015). Activation of hepatic CREBH and Insig signaling in the anti-hypertriglyceridemic mechanism of R-?-lipoic acid. Journal of Nutritional Biochemistry. 2015 Sep;26(9):921-8 doi: 10.1016/j.jnutbio.2015.03.011. PMID: 26007286.
    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Sud N, Taher J, and Su Q. (2015) MicroRNAs and Noncoding RNAs in Hepatic Lipid and Lipoprotein Metabolism: Potential Therapeutic Targets of Metabolic Disorders. Drug Development Research. 2015. Aug; 76: 318327 doi: 10.1002/ddr.21269. PMID: 26286650
    • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Sud N, Rutledge AC, Pan K, and Su Q.(2015) Activation of the dsRNA-Activated Protein Kinase PKR in Mitochondrial Dysfunction and Inflammatory Stress in Metabolic Syndrome. Current Pharmaceutical Design. (Invited review, submitted)
    • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Tong X, Shen J, Sud N, Maldonado-Gomez M, Walter J and Su Q.(2015). LDL receptor signaling mediates the triglyceride-lowering action of Akkermansia muciniphila in genetic induced hyperlipidemia. (Submitted to Arterioscler Thromb Vasc Biol and under revision).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: The cAMP Responsive Element Binding Protein H Links Metabolic Inflammation with Hyperlipidemia in Hepatic Insulin Resistance Miaoyun Zhao, Xuedong Tong and Qiaozhu Su. Insulin resistant is commonly associated with both increased circulating levels of tumor necrosis factor-alpha (TNFalpha)and hepatic overproduction of very low density lipoproteins (VLDL). The cAMP responsive binding protein H (CREBH) is a transcription factor whose expression is induced by proinflammatory cytokines. In this study, our objective was to investigate the role of CREBH in mediating lipogenic diet induced hyperlipoproteinemia in the insulin resistance rodent models. By using the wild type and CREBH-null mouse models combined with fatty acid gavage and TNF-alpha treatment, we demonstrated that depletion of CREBH reduced both mRNA and protein expression of apolipoprotein B (apoB), a key structure protein in VLDL particle. In vitro, transient expression of CREBH cDNAs in McA cells induced significant increase of apoB mRNA and protein expression and VLDL secretion which indicated the positive regulatory impact of CREBH on apoB biosynthesis. Furthermore, treating McA cells with TNF-alpha activated CREBH and increased apoB protein level. Challenging the CREBH-null mice with a dose of long chain fatty acid through gavage or TNF-alpha treatment failed to stimulate apoB expression and the subsequent VLDL secretion which resulted in hepatic steatosis. Insulin treatment synergized the effect of CREBH on VLDL secretion which may be a contributing factor in insulin resistance induced hyperlipidemia. In conclusion,these data demonstrated that CREBH is the molecular link that mediates inflammatory cytokine signaling to overproduction of VLDL in hepatic insulin resistance. This novel finding provides new mechanistic insight in inflammatory cytokine induced hyperlipidemia in metabolic diseases
    • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: MicroRNAs induced by lipogenic diet in lipoprotein metabolism and hepatic insulin resistance. IDeA Conference. North Dakota, USA (2015) Q. Su Substantial evidence has demonstrated the important role of microRNAs in energy metabolism and their participation in excessive caloric intake-induced metabolic syndrome. The consequences of the aberrant expression of microRNAs, however, have not been well explored. The present study sought to unveil the potential association between aberrant expression of microRNAs induced by high-fructose diet in the initiation of cellular stress signaling and their contribution to the onset of dyslipidemia and hepatic insulin resistance. By applying nutrition manipulated animal models, the high-fructose fed-mouse and hamster as well as a genetic intervention mouse model whose cAMP responsive element binding protein H (CREBH) gene is depleted, we were able to demonstrate that high-fructose diet significantly inhibited the expression of 10 hepatic microRNAs that are associated with genes involved in mitochondrial b-oxidation and insulin signaling. These phenotypes were closely associated with disruption of mitochondrial membrane integrity and the induction of oxidative stress. In vitro, delivering the microRNAs isolated from the livers of fructose fed-hamsters into a rat hepatoma cell line, McA-7777 cells, induced activation of mitochondrial stress. Fructose feeding further induced activation of the dsRNA-activated protein kinase PKR, a key molecule in metabolic inflammatory response. The active kinase then inhibited the cAMP signaling pathway, leading to increase hepatic lipid synthesis, systemic hyperlipidemia and insulin resistance. Our finding unveils a novel role for microRNAs in maintaining metabolic homeostasis of intracellular stress-responding machinery (e.g. mitochondria). It further provides evidence for exploring microRNAs as pharmaceutical targets for the prevention and treatment of metabolic syndrome derived from lipogenic diets.


    Progress 01/10/14 to 09/30/14

    Outputs
    Target Audience: Target audiences in this project include formal classroom instruction, laboratory instruction andscientific conferences. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students were involved in this project.They have been guided by their supervisor andtrained bythe mentorand a postdoc in the lab for the ability to initiate/complete a research project and the necessary molecular biological skills for theirresearch projects.One of the graduate studnets has completed her Master degree inDecember, 2014. Three students in the lab were also encouraged to participate and gave poster presentations in the Nebraska Gateway to NetrigenomicsRetreat organized by the Department of Nutrition andHealth Sciencesin May, 2014. How have the results been disseminated to communities of interest? The researd results have been presented on four international scientific conferences and published as two manuscripts in high quality journals. These research findindswill advance ourunderstanding of the pathogenesis of hyperlipidemia and non-acolholic fatty liver disease. It will alsoenhance UNL's recognition by peers in our fieldand further strengthen research ties between UNL and other institutions as there are ongoing research collaborations withother research institutes. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, we will work on Objective 2 to etermines whether high-fat-diet -induced microRNAs act as activators of PKR and the activated PKR in mediating cellular stress under metabolic stress conditions. This objective will explore a potentially novel mechanism by which overexpression of microRNAs induced by high-fat diet binds to PKR dsRNA binding domains and induces PKR activation. We will further continue theresearch workin Objective 3 to test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue.

    Impacts
    What was accomplished under these goals? Information resulting from the Hatch projectcould be used to (i) modify current dietary recommendations for the purpose of improving human nutrition; (ii) impact public health by raising the awareness of healthy food consuming; (iii) affect the agricultural industries in Nebraska by encouraging more consuming of dry beans in the daily diets as the omega fatty acids in the dry beans have triglyceride lowing effects; yield novel insights in the association of nutritional signals and metabolic disorders, such as obesity, fatty live diseases and diabetes; (iv) provide evidence for the design of nutritional supplements to specifically target microRNAs, PKR, CREBH and Insig-2a in prevention and treatment of obesity and fatty liver disease. Objective 1: determines the role of high-fat-diet-induced microRNAs in the initiation of mitochondrial inflammatory stress. This objective tests the hypothesis that microRNAs whose increased expression is induced by high-fat-diet are able to translocate into hepatic mitochondria to initiate the inflammatory response. During this funding period, we have found that lipogenic diet (high fructose diet)altered expression of microRNAprofile in the liver of fructose-fed hamsters. Delivering these microRNAs into arat liver cell line induced mitochondrial and ER stress which implicates thatinduced changes on microRNA expression is one of the etiologies in the lipogenic diet induced metabolic disorders. This novel finding has been presented on the Obesity & Weight Management International Conference. San Francisco, USA (2014). This finding may also provide rationalefor thedevelopment ofnutritional supplements to specifically target microRNA expressionin prevention and treatment of obesity and fatty liver disease. Objective 3: determines the impact of PKR on the cAMP responsive binding protein H (CREBH) signaling cascade and hepatic lipid metabolism via CREBH-Insig-2a signaling. This objective will test the hypothesis that PKR activation negatively regulates hepatocyte specific cAMP responsive binding protein (CREBH) through induced phosphorylation of CREBH on an inhibitory seine/threonine residue. In the past one year, our research has established tInsig-2a as atarget gene ofCREBH.The CREBH-Insig-2a pathwaynegatively regulates hepatic lipid metabolism.We further demonstrated that a bioactive molecular lipoic acid exerts its triglyceride lowering effect through the mediation of CREBH and Insig-2a. These findings has been presented on the scientific conference of ATVB-2014 and Gordon conference-2014.Theresults have also been summerized as a manuscript and submitted to Journalof Nutritional Biochemistry which is currently under review.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Christian P. and Su Q.(2014). MicroRNA regulation of mitochondrial and ER stress signaling pathways: implications for lipoprotein metabolism in metabolic syndrome. Am J Physiol Endocrinol Metab. 2014 Sep 2: PMID: 25184990
    • Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Tong, X., Moreau, R., Su, Q.(2014). Activation of hepatic CREBH-Insig-2a signaling in the triglyceride lowing action of (R)-?-lipoic acid (submitted to JNB and under review).
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lipoprotein metabolism, mitochondrial and ER stress in the pathogenesis of metabolic syndrome Presenter: Qiaozhu Su Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE, 68583, USA. Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload-induced ER stress has been implicated in the development of insulin resistance. Emerging evidence further demonstrates a close interplay between disturbances in mitochondrial function and ER homeostasis in the development of metabolic syndrome. The present investigation sought to advance our understanding of the mechanistic links among dysregulation of lipoprotein metabolism, mitochondrial dysfunction and ER stress in the onset of hepatic steatosis and insulin resistance. By applying genetic engineering animal models, such as the peroxisome proliferator-activated receptor alpha (PPAR?) null mice and the human apolipoprotein B100 (apoB100) transgenic mice as well as the nutrition manipulated animal models, including high-fat-fructose-cholesterol diet-induced insulin-resistance rodent models, we have successfully unveiled a novel mechanistic link between PPAR? signaling, cellular stress response and the pathogenesis of chronic metabolic disorders. Specifically, we found that genetic depletion of PPAR? compromised the functional activity of metabolic enzymes involved in fatty acid ?-oxidation, reduced mitochondrial membrane potential and induced mitochondrial unfolding protein response (UPR) in fructose-fed rats. Inhibition of PPAR? signaling further enhanced expression of apoB100 on both transcriptional and translational levels. Elevation of hepatic apoB100 was found to be closely associated with the induction of ER stress which subsequently inhibited the kinase activity of glycogen synthase kinase 3 (GSK-3) and glycogen synthase (GS) by activating c-Jun N-terminal kinase (JNK). This action was associated with the accumulation of lipid droplets in hepatocytes and the suppression insulin signaling cascade, suggesting that dysregulation of apoB100 metabolism could be a causal factor in the induction of hepatic steatosis and insulin resistance. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further demonstrated the link between excessive apoB100 expression and hepatic ER stress. Mechanistic study revealed that reduced activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA), an enzyme involved in maintaining ER calcium homeostasis, was the underlying molecular mechanism in apoB-induced ER stress. Silencing the overproduction of apoB100 by RNA interference (siRNA) was able to rescue cells from ER stress. Restoration of PPAR? activity by a PPAR? agonist recovered the metabolic homeostasis of mitochondria and ER, improved hepatic steatosis and insulin sensitivity. This finding unveils a novel role of PPAR? in relaying stress signals between hepatic subcellular stress-responding machinery (e.g. mitochondria and ER). It further delineates the underlying molecular mechanism in the pathogenesis of hepatic steatosis and insulin resistance upon metabolic stress.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Insulin induced gene-2a links CREBH signaling to hepatic lipid metabolism in the pathogenesis of hepatic steatosis and hypertriglyceridemia Hai Wang, Miaoyun Zhao, Patricia Christian and Qiaozhu Su The Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0806, USA. Insulin induced gene-2 (Insig-2) is an ER-resident protein that is involved in regulating the activation of SREBP, the master transcription factor that regulates hepatic de novo lipogenesis. Here we report that cAMP-responsive element-binding protein H (CREBH) inhibits hepatic fatty acid synthesis by modulating the expression of Insig-2a, a liver specific isoform of Insig-2. In the liver tissues of CREBH-null mice, we found that genetic depletion of CREBH specifically downregulated expression of Insign-2a mRNA, but not Insig-1, Insig-2b or SCAP, which resulted in the subsequent activation of SREBP-1c and SREBP-2. This regulation specifically occurred in hepatocytes, where both CREBH and Insig-2a are highly expressed. Mechanistic study revealed that exogenous expression of CREBH WT cDNA in a rat hepatoma cell line, McA cells, specifically induced mRNA expression of Insig-2a but not Insig-2b or Insig-1. In contrast, expression of a dominant negative form of CREBH inhibited transcription of Insig-2a mRNA. Analyzing the promoter and the enhancer sites within the Insig-2 gene identified two CRE-binding elements located proximally to the non-coding exon-1 of Insig-2. These two CRE-binding elements are evolutionally conserved among three species: human, rat and mouse. The functional association of CREBH with the putative CREBH-binding elements within the enhancer site was confirmed by a Chromatin Immunoprecipitation (ChIP) assay, which showed that CREBH interacts with the CRE-binding elements. Fasting and re-feeding, which activates and inactivates CREBH respectively, was able to specifically enhance or suppress mRNA expression of Insig-2a but not Insig-2b, Insig-1 or SCAP in the livers of wild type littermates and in an in vitro hepatocyte cell line. In contrast, fasting failed to stimulate Insig-2a mRNA expression in CREBH-null mice, which, upon re-feeding, subsequently resulted in profound activation of SREBP-1c, SREBP-2 and their downstream target genes involved in lipid synthesis, including fasn, acc and scd-1, leading to hepatic steatosis and systemic hypertriglyceridemia. This finding unveils a novel physiological phenomenon at the transition stage of fasting and re-feeding where activated CREBH inhibits fatty acid synthesis in order to maximize the substrate-supply for gluconeogenesis. This novel finding may also provide rationale for pharmaceutical design that specifically targets CREBH and Insig-2a in the treatment of hypertriglyceridemia and hepatic steatosis.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Title: Lipogenic diet-induced alteration of microRNAs in hepatic stress signaling: implications for hepatic lipoprotein metabolism and insulin resistance Presenter: Qiaozhu Su Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE, 68583, USA. Phone number: 402-472-4038 Emerging evidence has demonstrated the important role of microRNAs in energy metabolism and their participation in excessive caloric intake-induced metabolic syndrome. The consequences of the altered microRNAs, however, have not been well explored. The present study sought to advance our understanding of the potential association between reduced expression of microRNAs induced by high-fructose diet in the initiation of cellular stress signaling and their contribution to the onset of dyslipidemia and hepatic insulin resistance. By applying nutrition manipulated animal models, the high-fructose fed-rat and hamster models, and human apolipoprotein B100 (apoB100) transgenic mice, we were able to demonstrate that high-fructose diet significantly reduced expression of 13 hepatic microRNAs that are associated with genes involved in inflammatory cell differentiation and lipid signaling, including microRNA-15 and microRNA-378/378*. These phenotypes were closely associated with disruption of mitochondrial membrane integrity, induction of oxidative stress and activation of mitochondrial unfolding protein response (UPR). In vitro, delivering the microRNAs isolated from the livers of fructose fed-hamsters into McA-7777 cells, a rat hepatoma cell line, induced activation of mitochondrial UPR. Further investigating the lipid and lipoprotein metabolic profiles of the fructose-fed rats, we noticed that the reduced microRNA expression was associated with overproduction of hepatic apoB100, increased secretion of hepatic VLDL-apoB, and the development of hepatic steatosis and insulin resistance. Our finding unveils a novel role for microRNAs in maintaining metabolic homeostasis of intracellular stress-responding machinery (e.g. mitochondria). It also provides evidence for exploring microRNAs as pharmaceutical targets for the prevention and treatment of metabolic syndrome derived from nutrientsurplus.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Activation of hepatic CREBH-Insig-2a signaling in the triglyceride lowering mechanism of R-alpha-lipoic acid Xuedong Tong, Regis Moreau and *Qiaozhu Su Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE USA 68583-0806 Abstract: Activation of the sterol regulatory element-binding proteins (SREBPs), a step regulated by a cluster of ER-resident proteins, Insig-1, Insig-2 and SCAP, is rate limiting in hepatic de novo lipogenesis. We previously reported that feeding R-alpha-lipoic acid (LA) to ZDF (fa/fa) rats improves severe hypertriglyceridemia and lowers abdominal fat mass by inhibiting expression of genes involved in hepatic long-chain fatty acids and triacylglycerol syntheses. In this study, we characterized a novel mechanism of action of LA that explains its triacylglycerol lowering properties. Dietary LA activates liver specific transcription factor cAMP responsive element binding protein H (CREBH), which in turn enhances transcription and translation of Insig-1 and Insig-2. Chromatin immunoprecipitation (ChIP) assay demonstrated interaction between CREBH and the promoter of Insig-2 but not Insig-1. The increased abundance of Insig-1 and Insig-2 proteins contributes to sequester SREBP-1c and SREBP-2 in the ER and prevents their translocation to the Golgi apparatus where they would become activated. As a consequence, mRNA expression of genes involved in fatty acid and cholesterol synthesis, including FASN, ACC, SCD-1, HMGCR and LDL receptor, were significantly decreased in LA-fed animals versus pair-fed controls. Concomitantly, the assembly and secretion of very-low-density lipoproteins (VLDL) by primary hepatocytes were suppressed in the LA-fed ZDF rats as indicated by the decrease in VLDL-associated apolipoprotein B and apolipoprotein E. In vitro, treating a rat McA-RH7777 hepatoma cells with LA (200 ??) activated CREBH, induced expression of Insig-1 and Insig-2, and hindered the palmitic acid-induced synthesis of triacylglycerol. This study provides new mechanistic insight into the triacylglycerol lowering properties of LA and supports the therapeutic potential of LA against hypertriglyceridemia.