Inhibition of intestinal Niemann-Pick type C1 Like 1 (NPC1L1) transporter, an essential mediator of intestinal cholesterol absorption, has emerged as an important target for management of hypercholesterolemia. Despite such advances in the therapeutic modalities, reducing plasma cholesterol to low levels in patients with high risk for coronary heart disease remains poor. Therefore, identifying novel mechanisms targeting NPC1L1 is warranted for more effective treatment of hypercholesterolemia. The inhibition of NPC1L1, however, triggers the activation of adaptive cellular pathways such as the Sterol Response Element Binding Protein-2 (SREBP2) that could hinder the efficiency of cholesterol lowering drugs. Suppressing these compensatory mechanisms should also be taken into consideration when designing more effective treatment of hypercholesterolemia. Our efforts are focused at investigating the novel mechanisms involved in the inhibition of NPC1L1 as well as delineating roles of intestinal SREBP2 in body cholesterol homeostasis. Our preliminary data showed that proteasomal and lysosomal-dependent pathways are involved in the normal turnover of NPC1L1 protein. Also, inhibition of MAPK pathway caused a decrease in the cellular level of NPC1L1 by decreasing its protein stability. Our findings showed that DNA methylation is a major epigenetic determinant involved in controlling NPC1L1 expression in intestinal epithelia cells. To understand the role of compensatory pathways, we have recently generated novel transgenic mice with intestine- specific overexpression of constitutively active SREBP2 (designated as ISR2 mice). Our data showed an increase in levels of plasma cholesterol in the LDL and VLDL fractions in ISR2 mice indicating that the activation of intestinal SREBP2 alone is sufficient to increase plasma cholesterol. We hypothesized that inhibition of MAPK pathway decreases NPC1L1 protein stability via proteasomal and/or lysosomal degradation, and that DNA methylation is involved in NPC1L1 gene silencing. We further hypothesize that the increase in intestinal lipid synthesis/absorption and chylomicron production by active intestinal SREBP2 lead to an increase in plasma cholesterol. Our studies are designed to address these hypotheses utilizing both in vitro and in vivo models. Studies designed for Specific Aim 1 will investigate the mechanisms involved in the regulation of NPC1L1 protein stability by the MAPK dependent pathway and determine the structural domains of NPC1L1 involved.
Specific Aim 2 will systematically investigate mechanisms of NPC1L1 gene silencing by DNA methylation. Studies designed for Specific Aim 3 will focus on investigating the compensatory mechanisms underlying hypercholesterolemia in mice with intestine specific-overexpression of active SREBP2 (ISR2 mice). Our proposed studies are important to elucidate the critical role of the intestine in the maintenance of cholesterol homeostasis and to unravel potential targets for more effective treatment of hypercholesterolemia.

Public Health Relevance

Diseases related to high cholesterol in the blood such as diabetes mellitus are common among the VA patients. Diabetic patients have increased risk for coronary heart disease and their plasma cholesterol should be decreased to a substantial low levels (<100mg/dL). Achieving this low target of plasma cholesterol remains challenging. The proposed studies are focused at investigating mechanisms that could effectively decrease intestinal cholesterol absorption and aid in the development of more efficient and superior treatment of high levels of plasma cholesterol. The proposed studies are, therefore, relevant to the VA patients as well as the general patients population.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Gastroenterology (GAST)
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Jesse Brown VA Medical Center
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Kumar, Anoop; Chatterjee, Ishita; Gujral, Tarunmeet et al. (2017) Activation of Nuclear Factor-?B by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3. Gastroenterology 153:1338-1350.e3
Muthusamy, Saminathan; Malhotra, Pooja; Hosameddin, Mobashir et al. (2015) N-glycosylation is essential for ileal ASBT function and protection against proteases. Am J Physiol Cell Physiol 308:C964-71
Priyamvada, Shubha; Anbazhagan, Arivarasu N; Gujral, Tarunmeet et al. (2015) All-trans-retinoic Acid Increases SLC26A3 DRA (Down-regulated in Adenoma) Expression in Intestinal Epithelial Cells via HNF-1?. J Biol Chem 290:15066-77
Nazir, Saad; Kumar, Anoop; Chatterjee, Ishita et al. (2015) Mechanisms of Intestinal Serotonin Transporter (SERT) Upregulation by TGF-?1 Induced Non-Smad Pathways. PLoS One 10:e0120447
Priyamvada, Shubha; Gomes, Rochelle; Gill, Ravinder K et al. (2015) Mechanisms Underlying Dysregulation of Electrolyte Absorption in Inflammatory Bowel Disease-Associated Diarrhea. Inflamm Bowel Dis 21:2926-35
Kumar, Anoop; Hecht, Cameron; Priyamvada, Shubha et al. (2014) Probiotic Bifidobacterium species stimulate human SLC26A3 gene function and expression in intestinal epithelial cells. Am J Physiol Cell Physiol 307:C1084-92
Ma, Ke; Malhotra, Pooja; Soni, Vinay et al. (2014) Overactivation of intestinal SREBP2 in mice increases serum cholesterol. PLoS One 9:e84221
Anbazhagan, Arivarasu N; Priyamvada, Shubha; Kumar, Anoop et al. (2014) Translational repression of SLC26A3 by miR-494 in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 306:G123-31
Malhotra, Pooja; Soni, Vinay; Kumar, Anoop et al. (2014) Epigenetic modulation of intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) gene expression by DNA methylation. J Biol Chem 289:23132-40
Singh, Varsha; Kumar, Anoop; Raheja, Geetu et al. (2014) Lactobacillus acidophilus attenuates downregulation of DRA function and expression in inflammatory models. Am J Physiol Gastrointest Liver Physiol 307:G623-31

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