Research Overview

Novel Cellular Mechanisms for Reducing Hyperlipidemia and nonalcoholic fatty liver disease (NAFLD)

The liver plays a central role in whole-body lipid metabolism by regulating the uptake, synthesis, oxidation and export of fatty acids (FAs) and lipids. Dysfunction of lipid metabolism in liver underlies the development of obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. Although the mechanisms that regulate the hepatic uptake, activation, and metabolism of fatty acids (FAs) are not fully understood, nearly all pathways of FA metabolism require conversion of FAs to acyl-CoAs by acyl-CoA synthetases. Long chain acyl-CoA synthetase (ACSL) is a family of five enzymes (ACSL1, 3, 4, 5 and 6) that catalyze the formation of fatty acyl-CoAs from ATP, CoA, and long chain fatty acids. Within ACSL family members, ACSL4 has unique substrate specificity for arachidonic acid (AA) and hepatic ACSL4 has been demonstrated being abnormally expressed in pathological conditions including hepatocarcinoma and NAFLD. However, to date, no literature reports have clearly defined the specific roles played by ACSL4 in liver lipid metabolism under normal and disease state such as the benign form of NAFLD (steatosis) and the nonalcoholic steatohepatitis (NASH). Recently, we demonstrated that expression and activity of hepatic ACSL4 are upregulated by activators of peroxisome proliferator-activated receptor delta (PPAR D) in vivo and in vitro. Through conducting lipidomic studies we further demonstrated that depletion of ACSL4 in hepatic cells selectively reduced cellular contents of several PC species including PC(18:0/18:1), a critical lipid mediator in PPAR D signaling pathway. we also uncovered a novel substrate-induced posttranslational regulatory mechanism by which AA specifically downregulates ACSL4 protein abundance in hepatic cells by promoting its ubiquitination and proteasomal degradation. Since AA-derived eicosanoids have been implicated in the pathogenesis of NAFLD, the AA-induced ACSL4 degradation could be one contributing factor to the progression of hepatic steatosis to NASH with increased hepatic inflammation caused by imbalance between AA-CoA and unesterified AA that leads to increased production of AA-derived eicosanoids. The overall goals of this project are to fully characterize new regulatory mechanisms that uniquely impact the expression and function of ACSL4 in liver tissue to reach a better understanding of its specific roles in hepatic lipid metabolism under healthy and pathophysiological conditions including hyperlipidemia and NAFLD.

Singh AB, Kan CFK, Kraemer FB, Sobel RA, Liu J. Liver-specific knockdown of long-chain acyl-CoA synthetase 4 reveals its key role in VLDL-TG metabolism and phospholipid synthesis in mice fed a high-fat diet. Am J Physiol Endocrinol Metab. 2019 May 1;316(5):E880-E894.

Singh AB, Dong B, Xu Y, Zhang Y, Liu J. Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Mar;1864(3):358-371.

Singh AB, Liu J. Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated By Peroxisome Proliferator-activated Receptor δ, J Biol Chem. 2016 Dec 2. pii: jbc.M116.743575.

Singh AB, Kan CF, Dong B, Liu J. SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter, J Biol Chem. 2016 Mar 4;291(10):5373-84.

Kan CF*, Singh AB*, Dong B, Shende VR, Liu J.PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro, Biochim Biophys Acta. 2015 May;1851(5):577-87. doi: 10.1016/j.bbalip.2015.01.008.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J. Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation, J Lipid Res. 2014 Aug;55(8):1657-67. doi: 10.1194/jlr.M045971

Shende VR*, Singh AB*, Liu J. A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation, Biochem J. 2015 Dec 15;472(3):275-86.

Dong B, Kan CF, Singh AB, Liu J. High-fructose diet downregulates long-chain acyl-CoA synthetase 3 expression in liver of hamsters via impairing LXR/RXR signaling pathway, J Lipid Res. 2013 May;54(5):1241-54.

Post-transcriptional Regulation of Liver LDL Receptor Expression by Berberine

The expression level of the liver low density lipoprotein receptor (LDLR) is one of the most important regulators of human plasma LDL cholesterol (LDL-c). Increased hepatic LDLR expression results in an improved clearance of LDL-c from circulation, hence directly reducing the risk of coronary heart disease. Currently, in the research area of LDLR, the transcriptional regulation of the LDLR gene has been extensively investigated and most of the pharmaceutical interventions are aimed to increase LDLR gene transcription, although indirectly, in order to lower plasma LDL-c. However, the posttranscriptional modulations of LDLR are much understudied and the mechanisms controlling LDLR mRNA turnover are largely unknown. The overall objectives of this project are to fully elucidate the molecular and cellular mechanisms by which BBR regulates LDL mRNA stability in cell culture and in animal models.

we have generated a transgenic mouse model that express luciferase LDL-receptor 3′UTR (Luc-UTR) transgene under the control of mouse albumin promoter which directs the transgene expression in liver tissue specifically. We show that treating Alb-Luc-UTR mice with Berberine, natural cholesterol lowering drug led to significant increases in hepatic bioluminescence signals, and LDL-receptor mRNA levels in treated mice. These effects were accompanied by specific reductions of mRNA decay-promoting factor heterogeneous nuclear ribonucleoprotein D (hnRNPD) in liver of BBR-treated mice,

We further investigated that the depletion of hnRNPD in liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDL-receptor expression in hyperlipidemic mice. Additional studies of gene knockdown in Alb-Luc-UTR transgenic mice provide strong evidence supporting the essential role of LDLR 3′UTR in hnRNPD-mediated LDL-receptor mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDL-receptor expression via inducing hnRNPD to accelerate LDL-receptor mRNA degradation.

Singh AB, Liu J. Berberine decreases plasma triglyceride levels and upregulates hepatic TRIB1 in LDLR wild type mice and in LDLR deficient mice. Sci Rep. 2019 Oct 30;9(1):15641.

Singh AB, Kan CF, Shende V, Dong B, Liu J. A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression, J Lipid Res. 2014 Jul;55(7):1397-407. doi: 10.1194/jlr.M049429. PubMed PMID: 24792925.

Singh AB, Li H, Kan CF, Dong B, Nicolls MR, Liu J. The critical role of mRNA destabilizing protein heterogeneous nuclear ribonucleoprotein d in 3' untranslated region-mediated decay of low-density lipoprotein receptor mRNA in liver tissue, Arterioscler Thromb Vasc Biol. 2014 Jan;34(1):8-16.

Dong B, Singh AB, Azhar S, Seidah NG, Liu J. High-fructose feeding promotes accelerated degradation of hepatic LDL receptor and hypercholesterolemia in hamsters via elevated circulating PCSK9 levels, Atherosclerosis. 2015 Apr;239(2):364-74.

Shende VR, Wu M, Singh AB, Dong B, Kan CF, Liu J. Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice, J Lipid Res. 2015 Apr;56(4):801-9.

Dong B, Li H, Singh AB, Cao A, Liu J. Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway, J Biol Chem. 2015 Feb 13;290(7):4047-58.

Dong B, Singh AB, Fung C, Kan K, Liu J. CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism, Atherosclerosis. 2014 Aug;235(2):449-62.

Role of Retinoid Mediated Signaling in Diabetes and Cardiac Remodeling

Diabetes mellitus (DM) is of major epidemiological importance, accounting for a high incidence of heart failure in these patients. Hyperglycemia, as an independent risk factor, directly causes cardiac damage and leads to diabetic cardiomyopathy. Apart from the mechanisms of DM-induced cardiac remodeling, little is known about the transition from compensated cardiomyopathy to heart failure. It has been shown that DM also affects the metabolic availability of vitamin A. Activation of RXR-mediated signaling improves insulin resistance in type 2 DM, indicating that RA signaling is involved in the development of DM. We propose using in vitro cultured neonatal cardiac myocytes and fibroblasts and in vivo Zucker Diabetic Fatty rats, to determine the effect of DM on the expression/activation of RA signaling, address the molecular mechanisms of RA-mediated signaling in DM-induced cardiac remodeling. Identifying the specific molecular mechanisms of RA signaling, involved in DM-mediated cellular effects, may provide an alternative approach for developing improved therapies for patients with DM and related cardiac complications.

Recently our research group has reported that decreased expression of RARα and RXRα has an important role in high glucose (HG)-induced cardiomyocyte apoptosis. However, the regulatory mechanisms of HG effects on RARα and RXRα remain unclear. Using neonatal cardiomyocytes, we have shown that the HG-induced cardiomyocyte apoptosis was potentiated by activation of JNK, and prevented by ATRA and inhibition of JNK. Silencing the expression of RARα and RXRα activated the JNK pathway. In conclusion, HG-induced oxidative stress and activation of the JNK pathway negatively regulated expression/activation of RAR and RXR. The impaired RAR/RXR signaling and oxidative stress/JNK pathway forms a vicious circle, which significantly contributes to hyperglycemia induced cardiomyocyte apoptosis.

Guleria RS, Singh AB, Nizamutdinova IT, Souslova T, Kendall JA Jr, Baker KM, Pan J. Activation of retinoid receptor-mediated signaling ameliorates diabetes-induced cardiac dysfunction in Zucker diabetic rats, J Mol Cell Cardiol. 2013 Apr;57:106-18.

Nizamutdinova IT, Guleria RS, Singh AB, Kendall JA Jr, Baker KM, Pan J. Retinoic acid protects cardiomyocytes from high glucose-induced apoptosis through inhibition of NF-κB signaling pathway J Cell Physiol. 2013 Feb;228(2):380-92.

Singh AB, Guleria RS, Nizamutdinova IT, Baker KM, Pan J. High glucose-induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling. J Cell Physiol. 2012 Jun;227(6):2632-44. doi: 10.1002/jcp.23005.

Discovery and development of novel antidiabetic molecules and natural products validation for the prevention of life style disorders

Diabetes mellitus is a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action or both. Several classes of drugs are currently available for the management of disease; however they have certain limitations, such as adverse side effect and high rates of secondary failure. As such, there are increasing research efforts to identify novel targets and new chemical entities to combat this problem. In this direction, our research team is involved in the discovery, development and validation of new antidiabetic agents from natural as well as synthetic sources. We have established in vitro cell based assay system and various validated in vivo rodent models for the screening, validation and development of lead antidiabetic molecules.

Singh AB, Singh N, Akanksha, Jayendra, Maurya R, Srivastava AK. Coagulanolide modulates hepatic glucose metabolism in C57BL/KsJ-db/db mice, Hum Exp Toxicol. 2012 Oct;31(10):1056-65. doi: 10.1177/0960327112438289. PubMed PMID: 23060434.

Singh AB, Khaliq T, Chaturvedi JP, Narender T, Srivastava AK. Anti-diabetic and anti-oxidative effects of 4-hydroxypipecolic acid in C57BL/KsJ-db/db mice, Hum Exp Toxicol. 2012 Jan;31(1):57-65. doi: 10.1177/0960327111407227. PubMed PMID: 21653626.

Singh AB, Tamarkar AK, Narender T, Srivastava AK. Antihyperglycaemic effect of an unusual amino acid (4-hydroxyisoleucine) in C57BL/KsJ-db/db mice, Nat Prod Res. 2010 Feb;24(3):258-65. doi: 10.1080/14786410902836693. PubMed PMID: 20140804

Singh AB, Yadav DK, Maurya R, Srivastava AK. Antihyperglycaemic activity of alpha-amyrin acetate in rats and db/db mice, Nat Prod Res. 2009;23(9):876-82. doi: 10.1080/14786410802420416. PubMed PMID: 19488928.

Tamrakar AK, Singh AB, Srivastava AK. db/+ Mice as an alternate model in antidiabetic drug discovery research, Arch Med Res. 2009 Feb;40(2):73-8. doi: 10.1016/j.arcmed.2008.12.001. PubMed PMID: 19237015.

Singh AB, Chaturvedi JP, Narender T, Srivastava AK. Preliminary studies on the hypoglycemic effect of Peganum harmala L. Seeds ethanol extract on normal and streptozotocin induced diabetic rats, Indian J Clin Biochem. 2008 Oct;23(4):391-3. doi: 10.1007/s12291-008-0086-3. PubMed PMID: 23105794; PubMed Central PMCID: PMC3453127.