Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

BACKGROUND: SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. METHODS: Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. RESULTS: TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. CONCLUSIONS: These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Transcriptomic and Proteomic Analysis Reveals the Potential Role of RBMS1 in Adipogenesis and Adipocyte Metabolism.

Adipocytes play a critical role in maintaining a healthy systemic metabolism by storing and releasing energy in the form of fat and helping to regulate glucose and lipid levels in the body. Adipogenesis is the process through which pre-adipocytes are differentiated into mature adipocytes. It is a complex process involving various transcription factors and signaling pathways. The dysregulation of adipogenesis has been implicated in the development of obesity and metabolic disorders. Therefore, understanding the mechanisms that regulate adipogenesis and the factors that contribute to its dysregulation may provide insights into the prevention and treatment of these conditions. RNA-binding motif single-stranded interacting protein 1 (RBMS1) is a protein that binds to RNA and plays a critical role in various cellular processes such as alternative splicing, mRNA stability, and translation. RBMS1 polymorphism has been shown to be associated with obesity and type 2 diabetes, but the role of RBMS1 in adipose metabolism and adipogenesis is not known. We show that RBMS1 is highly expressed during the early phase of the differentiation of the murine adipocyte cell line 3T3-L1 and is significantly upregulated in the adipose tissue depots and adipocytes of high-fat-fed mice, implying a possible role in adipogenesis and adipose metabolism. Knockdown of RBMS1 in pre-adipocytes impacted the differentiation process and reduced the expression of some of the key adipogenic markers. Transcriptomic and proteomic analysis indicated that RBMS1 depletion affected the expression of several genes involved in major metabolic processes, including carbohydrate and lipid metabolism. Our findings imply that RBMS1 plays an important role in adipocyte metabolism and may offer novel therapeutic opportunity for metabolic disorders such as obesity and type 2 diabetes.

Profiling of G-Protein Coupled Receptors in Adipose Tissue and Differentiating Adipocytes Offers a Translational Resource for Obesity/Metabolic Research.

G protein-coupled receptors (GPCRs) are expressed essentially on all cells, facilitating cellular responses to external stimuli, and are involved in nearly every biological process. Several members of this family play significant roles in the regulation of adipogenesis and adipose metabolism. However, the expression and functional significance of a vast number of GPCRs in adipose tissue are unknown. We used a high-throughput RT-PCR panel to determine the expression of the entire repertoire of non-sensory GPCRs in mouse white, and brown adipose tissue and assess changes in their expression during adipogenic differentiation of murine adipocyte cell line, 3T3-L1. In addition, the expression of GPCRs in subcutaneous adipose tissues from lean, obese, and diabetic human subjects and in adipocytes isolated from regular chow and high-fat fed mice were evaluated by re-analyzing RNA-sequencing data. We detected a total of 292 and 271 GPCRs in mouse white and brown adipose tissue, respectively. There is a significant overlap in the expression of GPCRs between the two adipose tissue depots, but several GPCRs are specifically expressed in one of the two tissue types. Adipogenic differentiation of 3T3-L1 cells had a profound impact on the expression of several GPCRs. RNA sequencing of subcutaneous adipose from healthy human subjects detected 255 GPCRs and obesity significantly changed the expression of several GPCRs in adipose tissue. High-fat diet had a significant impact on adipocyte GPCR expression that was similar to human obesity. Finally, we report several highly expressed GPCRs with no known role in adipose biology whose expression was significantly altered during adipogenic differentiation, and/or in the diseased human subjects. These GPCRs could play an important role in adipose metabolism and serve as a valuable translational resource for obesity and metabolic research.

Association of Advanced Lipoprotein Subpopulation Profiles with Insulin Resistance and Inflammation in Patients with Type 2 Diabetes Mellitus.

Plasma lipoproteins exist as several subpopulations with distinct particle number and size that are not fully reflected in the conventional lipid panel. In this study, we sought to quantify lipoprotein subpopulations in patients with type 2 diabetes mellitus (T2DM) to determine whether specific lipoprotein subpopulations are associated with insulin resistance and inflammation markers. The study included 57 patients with T2DM (age, 61.14 ± 9.99 years; HbA1c, 8.66 ± 1.60%; mean body mass index, 35.15 ± 6.65 kg/m2). Plasma lipoprotein particles number and size were determined by nuclear magnetic resonance spectroscopy. Associations of different lipoprotein subpopulations with lipoprotein insulin resistance (LPIR) score and glycoprotein acetylation (GlycA) were assessed using multi-regression analysis. In stepwise regression analysis, VLDL and HDL large particle number and size showed the strongest associations with LPIR (R2 = 0.960; p = 0.0001), whereas the concentrations of the small VLDL and HDL particles were associated with GlycA (R2 = 0.190; p = 0.008 and p = 0.049, respectively). In adjusted multi-regression analysis, small and large VLDL particles and all sizes of lipoproteins independently predicted LPIR, whereas only the number of small LDL particles predicted GlycA. Conventional markers HbA1c and Hs-CRP did not exhibit any significant association with lipoprotein subpopulations. Our data suggest that monitoring insulin resistance-induced changes in lipoprotein subpopulations in T2DM might help to identify novel biomarkers that can be useful for effective clinical intervention.

Association of Serum Zinc and Inflammatory Markers with the Severity of COVID-19 Infection in Adult Patients.

COVID-19 infection can spread in the host body without any adequate immune response. Zinc is an essential trace element with strong immunoregulatory and antiviral properties and its deficiency might lead to inflammation and oxidative stress. The aim of the current study was to determine the association of serum zinc and inflammatory markers with the severity of COVID-19 infection. This was a prospective observational study in which 123 COVID-19-positive adult patients and 48 controls were recruited. The initial comparative analysis was conducted between COVID-19 patients and controls. COVID-19-positive patients were further divided into three different groups (mild, moderate, and severe) based on the severity of COVID-19 infection. COVID-19 patients showed significantly lower serum zinc levels (8.8 ± 2.3 µmol/L) compared to healthy controls (11.9 ± 1.8 µmol/L). There was a negative correlation between serum zinc levels and the severity of COVID-19 infection (r = −0.584, p < 0.0001) and this effect was independent of age (r = −0.361, p < 0.0001). Furthermore, inflammatory markers showed a positive correlation with the severity of COVID-19 infection and a negative correlation with the levels of serum zinc. The study demonstrated an association between COVID-19 infection with low serum zinc levels and elevated inflammatory markers. Further studies to assess the significance of this observation are needed, which may justify zinc supplementation to mitigate the severity of COVID-19 infection.

Differential Regulation of Glucosylceramide Synthesis and Efflux by Golgi and Plasma Membrane Bound ABCC10.

Glucosylceramide (GlcCer) synthesis by the enzyme glucosylceramide synthase (GCS) occurs on the cytosolic leaflet of the Golgi and is the first important step for the synthesis of complex glycosphingolipids (GSLs) that takes place inside the lumen. Apart from serving as a precursor for glycosylation, newly synthesized GlcCer is also transported to the plasma membrane and secreted onto HDL in the circulation. The mechanism by which GlcCer is transported to HDL remains unclear. Recently, we showed that ATP-binding cassette transporter protein C10 (ABCC10) plays an important role in the synthesis and efflux of GlcCer in Huh-7 cells. In this study, we found that treatment of Huh-7 cells with an ABCC10 inhibitor, sorafenib, decreased the synthesis and efflux of GlcCer. However, treatment of cells with cepharanthine reduced only the efflux, but not synthesis, of GlcCer. These results indicate that ABCC10 may regulate the synthesis and efflux of GlcCer differentially in liver cells.

ATP-Binding Cassette Protein ABCC10 Deficiency Prevents Diet-Induced Obesity but Not Atherosclerosis in Mice.

Excess plasma lipid levels are a risk factor for various cardiometabolic disorders. Studies have shown that improving dyslipidemia lowers the progression of these disorders. In this study, we investigated the role of ATP-binding cassette transporter C10 (ABCC10) in regulating lipid metabolism. Our data indicate that deletion of the Abcc10 gene in male mice results in lower plasma and intestinal triglycerides by around 38% and 36%, respectively. Furthermore, deletion of ABCC10 ameliorates diet-induced obesity in mice and leads to a better response during insulin and glucose tolerance tests. Unexpectedly, ABCC10 deficiency does not affect triglyceride levels or atherosclerosis in ApoE-deficient mice. In addition, our studies demonstrate low oleate uptake by enterocytes (~25-30%) and less absorption (~37%) of triglycerides in the small intestine of ABCC10 knockout mice. Deletion of the Abcc10 gene also alters several lipid metabolism genes in the intestine, suggesting that ABCC10 regulates dietary fat absorption, which may contribute to diet-induced obesity in mice.

ATP-Binding Cassette Transporter Family C Protein 10 Participates in the Synthesis and Efflux of Hexosylceramides in Liver Cells.

In addition to sphingomyelin and ceramide, sugar derivatives of ceramides, hexosylceramides (HexCer) are the major circulating sphingolipids. We have shown that silencing of ABCA1 transmembrane protein function for instance in cases of loss of function of ABCA1 gene results in low levels of HDL as well as a concomitant reduction in plasma HexCer levels. However, proteins involved in hepatic synthesis and egress of HexCer from cells is not well known although ABCA1 seems to be indirectly controlling the HexCer plasma levels by supporting HDL synthesis. In this study, we hypothesized that protein(s) other than ABCA1 are involved in the transport of HexCer to HDL. Using an unbiased knockdown approach, we found that ATP-binding cassette transporter protein C10 (ABCC10) participates in the synthesis of HexCer and thereby affects egress to HDL in human hepatoma Huh-7 cells. Furthermore, livers from ABCC10 deficient mice had significantly lower levels of HexCer compared to wild type livers. These studies suggest that ABCC10 partakes in modulating the synthesis and subsequent efflux of HexCer to HDL in liver cells.

Reduction in Insulin Mediated ERK Phosphorylation by Palmitate in Liver Cells Is Independent of Fatty Acid Induced ER Stress.

Saturated free fatty acids (FFAs) such as palmitate in the circulation are known to cause endoplasmic reticulum (ER) stress and insulin resistance in peripheral tissues. In addition to protein kinase B (AKT) signaling, extracellular signal-regulated kinase (ERK) has been implicated in the development of insulin resistance. However, there are conflicting data regarding role of ERK signaling in ER stress-induced insulin resistance. In this study, we investigated the effects of ER stress on insulin resistance and ERK phosphorylation in Huh-7 cells and evaluated how oleate prevents palmitate-mediated ER stress. Treatment with insulin resulted in an increase of 38-45% in the uptake of glucose in control cells compared to non-insulin-treated control cells, along with an increase in the phosphorylation of AKT and ERK. We found that treatment with palmitate increased the expression of ER stress genes, including the splicing of X box binding protein 1 (XBP1) mRNA. At the same time, we observed a decrease in insulin-mediated uptake of glucose and ERK phosphorylation in Huh-7 cells, without any change in AKT phosphorylation. Supplementation of oleate along with palmitate mitigated the palmitate-induced ER stress but did not affect insulin-mediated glucose uptake or ERK phosphorylation. The findings of this study suggest that palmitate reduces insulin-mediated ERK phosphorylation in liver cells and this effect is independent of fatty-acid-induced ER stress.

Microsomal triglyceride transfer protein-mediated transfer of ß-carotene from donor to acceptor vesicles in vitro.

Dietary ß-carotene is the most abundant vitamin A precursor. Once absorbed by the enterocytes, the provitamin A carotenoid can either be cleaved into retinoids (vitamin A and its derivatives) or incorporated in its intact form within chylomicrons to be distributed throughout the body for utilization and/or storage by other tissues. From the liver, together with endogenous lipids, intact ß-carotene can also be incorporated within very low-density lipoprotein/low-density lipoprotein (VLDL/LDL) for transport to other tissues and organs. Microsomal triglyceride transfer protein (MTP) is a key regulator of lipoprotein biosynthesis in intestine and liver as it facilitates the incorporation of dietary and endogenous lipids into nascent lipoproteins. MTP is also critical for transferring ß-carotene into lipoprotein particles for secretion. Here, we present an in vitro method to assess the transfer of ß-carotene by MTP from donor to acceptor vesicles. This transfer can be assessed by precipitating donor vesicles and measuring amounts of ß-carotene transferred to acceptor vesicles. The levels of transferred ß-carotene are quantified by HPLC analysis and intrinsic fluorescence of ß-carotene. This chapter demonstrates the feasibility of this method which is also useful to study the role of MTP for incorporation of other carotenoids that are known to be carried within VLDL/LDL and chylomicrons for organ distribution.

Lipid Raft Integrity and Cellular Cholesterol Homeostasis Are Critical for SARS-CoV-2 Entry into Cells.

Lipid rafts in cell plasma membranes play a critical role in the life cycle of many viruses. However, the involvement of membrane cholesterol-rich lipid rafts in the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into target cells is not well known. In this study, we investigated whether the presence of cholesterol-rich microdomains is required for the entry of SARS-CoV-2 into host cells. Our results show that depletion of cholesterol in the rafts by methyl-beta-cyclodextrin (MßCD) treatment impaired the expression of the cell surface receptor angiotensin-converting enzyme 2 (ACE2), resulting in a significant increase in SARS-CoV-2 entry into cells. The effects exerted by MßCD could be substantially reversed by exogenous cholesterol replenishment. In contrast, disturbance of intracellular cholesterol homeostasis by statins or siRNA knockdown of key genes involved in the cholesterol biosynthesis and transport pathways reduced SARS-CoV-2 entry into cells. Our study also reveals that SREBP2-mediated cholesterol biosynthesis is involved in the process of SARS-CoV-2 entry in target cells. These results suggest that the host membrane cholesterol-enriched lipid rafts and cellular cholesterol homeostasis are essential for SARS-CoV-2 entry into cells. Pharmacological manipulation of intracellular cholesterol might provide new therapeutic strategies to alleviate SARS-CoV-2 entry into cells.

ATP-binding cassette protein ABCA7 deficiency impairs sphingomyelin synthesis, cognitive discrimination, and synaptic plasticity in the entorhinal cortex.

Sphingomyelin (SM) is an abundant plasma membrane and plasma lipoprotein sphingolipid. We previously reported that ATP-binding cassette family A protein 1 (ABCA1) deficiency in humans and mice decreases plasma SM levels. However, overexpression, induction, downregulation, inhibition, and knockdown of ABCA1 in human hepatoma Huh7 cells did not decrease SM efflux. Using unbiased siRNA screening, here, we identified that ABCA7 plays a role in the biosynthesis and efflux of SM without affecting cellular uptake and metabolism. Since loss of function mutations in the ABCA7 gene exhibit strong associations with late-onset Alzheimer's disease across racial groups, we also studied the effects of ABCA7 deficiency in the mouse brain. Brains of ABCA7-deficient (KO) mice, compared with WT, had significantly lower levels of several SM species with long chain fatty acids. In addition, we observed that older KO mice exhibited behavioral deficits in cognitive discrimination in the active place avoidance task. Next, we performed synaptic transmission studies in brain slices obtained from older mice. We found anomalies in synaptic plasticity at the intracortical synapse in layer II/III of the lateral entorhinal cortex but not in the hippocampal CA3-CA1 synapses in KO mice. These synaptic abnormalities in KO brain slices were rescued with extracellular SM supplementation but not by supplementation with phosphatidylcholine. Taken together, these studies identify a role of ABCA7 in brain SM metabolism and the importance of SM in synaptic plasticity and cognition, as well as provide a possible explanation for the association between ABCA7 and late-onset Alzheimer's disease.

Molecular Mechanisms of Sphingolipid Transport on Plasma Lipoproteins.

Sphingolipids are biomolecules with diverse physiological functions in signaling as well as plasma membrane structure. They are associated with either cellular membranes or plasma lipoproteins and any changes in their levels may contribute to certain metabolic diseases. Sphingolipids are evenly distributed in lipoproteins and may be used as prognostic and diagnostic markers. Mechanisms involved in the transport of sphingolipids have been recently explored and here we discuss the most recent advances in the molecular mechanisms of sphingolipids transport by lipoproteins. It has been shown that microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter family A protein 1 (ABCA1) play an important role in plasma sphingolipid homeostasis. However, the exact mechanisms are not well known. Though much research has already been done to emphasize the impact of sphingolipids changes in many pathological disorders, understanding mechanisms by which circulating lipoproteins assist in transporting sphingolipids may provide novel information that may help in devising strategies to therapeutically target these pathways to treat various metabolic disorders.

Modulation of ATP8B1 Gene Expression in Colorectal Cancer Cells Suggest its Role as a Tumor Suppressor.

AIM: The study aims to understand the role of tumor suppressor genes in colorectal cancer initiation and progression. BACKGROUND: Sporadic colorectal cancer (CRC) develops through distinct molecular events. Loss of the 18q chromosome is a conspicuous event in the progression of adenoma to carcinoma. There is limited information regarding the molecular effectors of this event. Earlier, we had reported ATP8B1 as a novel gene associated with CRC. ATP8B1 belongs to the family of P-type ATPases (P4 ATPase) that primarily function to facilitate the translocation of phospholipids. OBJECTIVE: In this study, we attempt to implicate the ATP8B1 gene located on chromosome 18q as a tumor suppressor gene. METHODS: Cells culture, Patient data analysis, Generation of stable ATP8B1 overexpressing SW480 cell line, Preparation of viral particles, Cell Transduction, Generation of stable ATP8B1 knockdown HT29 cell line with CRISPR/Cas9, Generation of stable ATP8B1 knockdown HT29 cell line with shRNA, Quantification of ATP8B1 gene expression, Real-time cell proliferation and migration assays, Cell proliferation assay, Cell migration assay, Protein isolation and western blotting, Endpoint cell viability assay, Uptake and efflux of sphingolipid, Statistical and computational analyses. RESULTS: We studied indigenous patient data and confirmed the reduced expression of ATP8B1 in tumor samples. CRC cell lines were engineered with reduced and enhanced levels of ATP8B1, which provided a tool to study its role in cancer progression. Forced reduction of ATP8B1 expression either by CRISPR/Cas9 or shRNA was associated with increased growth and proliferation of CRC cell line - HT29. In contrast, overexpression of ATP8B1 resulted in reduced growth and proliferation of SW480 cell lines. We generated a network of genes that are downstream of ATP8B1. Further, we provide the predicted effect of modulation of ATP8B1 levels on this network and the possible effect on fatty acid metabolism-related genes. CONCLUSION: Tumor suppressor gene (ATP8B1) located on chromosome 18q could be responsible in the progression of colorectal cancer. Knocking down of this gene causes an increased rate of cell proliferation and reduced cell death, suggesting its role as a tumor suppressor. Increasing the expression of this gene in colorectal cancer cells slowed down their growth and increased cell death. These evidences suggest the role of ATP8B1 as a tumor suppressor gene.

Diet-induced differential effects on plasma lipids secretion by the inositol-requiring transmembrane kinase/endoribonuclease 1α.

Intestinal and hepatic lipid metabolism plays an essential role in regulating plasma lipid levels. These lipids are mobilized on apolipoprotein B (apoB)-containing lipoproteins and their plasma homeostasis is maintained by balancing production and catabolism. Microsomal triglyceride transfer protein (MTP) which is expressed mainly in the intestine and liver plays an essential role in regulating the assembly and secretion of apoB-lipoproteins. Any imbalance in the production or clearance of lipoproteins leads to hyperlipidemia which is a major risk factor for atherosclerosis, obesity, diabetes, and metabolic syndrome. Here, we identify a new role of inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in the regulation of plasma lipids. We generated intestine specific IRE1α knockout mice to study whether intestinal IRE1α regulates plasma lipids by modulating intestinal lipid absorption. Intestine specific deletion of Ire1a gene in mice fed chow diet, significantly reduced plasma cholesterol and triglycerides by 29% and 43% in Ire1a-⁣/- mice (P < 0.01 & P < 0.001, respectively). These changes were not associated with any alteration of MTP activity nor its mRNA expression. On the other hand, Western diet increased plasma triglyceride by 37% (P < 0.01) without affecting total plasma cholesterol in Ire1a-⁣/- mice. Interestingly, this effect was associated with a significant increase in the intestinal MTP activity and its mRNA expression (25%, P < 0.01 and 70%, P < 0.05, respectively). Collectively, our findings reveal key role of intestinal IRE1α in the regulation of plasma lipids that may provide a therapeutic target for disorders of lipid metabolism.

Microsomal Triglyceride Transfer Protein: From Lipid Metabolism to Metabolic Diseases.

Microsomal triglyceride transfer protein (MTP) was first identified as an endoplasmic reticulum (ER) resident protein that helps in the transfer of neutral lipids to nascent apolipoprotein B (apoB). Its critical role in the assembly and secretion of apoB-containing lipoproteins was identified in abetalipoproteinemia patients who have mutations in MTP and completely lack apoB-containing lipoproteins in the circulation. It has been established now that MTP not only is involved in the transfer of neutral lipids but also plays a role in cholesterol ester and cluster of differentiation 1d (CD1d) biosynthesis. Besides neutral lipids, MTP may also help in the transfer of sphingolipids such as ceramides and sphingomyelin to the apoB-containing lipoproteins. MTP is a multifunctional protein, and its deregulation during pathophysiological conditions gives rise to different metabolic conditions. This book chapter discusses the physiological role and regulation of MTP to maintain the homeostasis of lipids and lipoproteins. It also reviews the regulation of MTP during certain pathophysiological conditions and provides a brief overview of therapeutic interventions that can be possibly used to target its activity or expression to alleviate some of these metabolic diseases.

Leptin-mediated differential regulation of microsomal triglyceride transfer protein in the intestine and liver affects plasma lipids.

The hormone leptin regulates fat storage and metabolism by signaling through the brain and peripheral tissues. Lipids delivered to peripheral tissues originate mostly from the intestine and liver via synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. An intracellular chaperone, microsomal triglyceride transfer protein (MTP), is required for the biosynthesis of these lipoproteins, and its regulation determines fat mobilization to different tissues. Using cell culture and animal models, here we sought to identify the effects of leptin on MTP expression in the intestine and liver. Leptin decreased MTP expression in differentiated intestinal Caco-2 cells, but increased expression in hepatic Huh7 cells. Similarly, acute and chronic leptin treatment of chow diet-fed WT mice decreased MTP expression in the intestine, increased it in the liver, and lowered plasma triglyceride levels. These leptin effects required the presence of leptin receptors (LEPRs). Further experiments also suggested that leptin interacted with long-form LEPR (ObRb), highly expressed in the intestine, to down-regulate MTP. In contrast, in the liver, leptin interacted with short-form LEPR (ObRa) to increase MTP expression. Mechanistic experiments disclosed that leptin activates signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) signaling pathways in intestinal and hepatic cells, respectively, and thereby regulates divergent MTP expression. Our results also indicated that leptin-mediated MTP regulation in the intestine affects plasma lipid levels. In summary, our findings suggest that leptin regulates MTP expression differentially by engaging with different LEPR types and activating distinct signaling pathways in intestinal and hepatic cells.

Interplay between ß-carotene and lipoprotein metabolism at the maternal-fetal barrier.

Vitamin A is an essential nutrient, critical for proper embryonic development in mammals. Both embryonic vitamin A-deficiency or -excess lead to congenital malformations or lethality in mammals, including humans. This is due to the defective transcriptional action of retinoic acid, the active form of vitamin A, that regulates in a spatial- and temporal-dependent manner the expression of genes essential for organogenesis. Thus, an adequate supply of vitamin A from the maternal circulation is vital for normal mammalian fetal development. Provitamin A carotenoids circulate in the maternal bloodstream and are available to the embryo. Of all the dietary carotenoids, ß-carotene is the main vitamin A precursor, contributing at least 30% of the vitamin A intake in the industrialized countries and often constituting the sole source of retinoids (vitamin A and its derivatives) in the developing world. In humans, up to 40% of the absorbed dietary ß-carotene is incorporated in its intact form in chylomicrons for distribution to other organs within the body, including the developing tissues. Here, it can serve as a source of vitamin A upon conversion into apocarotenoids by its cleavage enzymes. Given that ß-carotene is carried in the bloodstream by lipoproteins, and that the placenta acquires, assembles and secretes lipoproteins, it is becoming evident that the maternal-fetal transfer of ß-carotene relies on lipoprotein metabolism. Here, we will explore the current knowledge about this important biological process, the cross-talk between carotenoid and lipid metabolism in the context of the maternal-fetal transfer of this provitamin A precursor, and the mechanisms whereby ß-carotene is metabolized by the developing tissues. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

ATP binding cassette family A protein 1 determines hexosylceramide and sphingomyelin levels in human and mouse plasma.

Sphingolipids, including ceramide, SM, and hexosylceramide (HxCer), are carried in the plasma by lipoproteins. They are possible markers of metabolic diseases, but little is known about their control. We previously showed that microsomal triglyceride transfer protein (MTP) is critical to determine plasma ceramide and SM, but not HxCer, levels. In human plasma and mouse models, we examined possible HxCer-modulating pathways, including the role of ABCA1 in determining sphingolipid plasma concentrations. Compared with control samples, plasma from patients with Tangier disease (deficient in ABCA1) had significantly lower HxCer (-69%) and SM (-40%) levels. Similarly, mice deficient in hepatic and intestinal ABCA1 had significantly reduced HxCer (-79%) and SM (-85%) levels. Tissue-specific ablation studies revealed that hepatic ABCA1 determines plasma HxCer and SM levels; that ablation of MTP and ABCA1 in the liver and intestine reduces plasma HxCer, SM, and ceramide levels; and that hepatic and intestinal MTP contribute to plasma ceramide levels, whereas only hepatic MTP modulates plasma SM levels. These results identify the contribution of ABCA1 to plasma SM and HxCer levels and suggest that MTP and ABCA1 are critical determinants of plasma sphingolipid levels.

Regulation of Sphingolipid Metabolism by MicroRNAs: A Potential Approach to Alleviate Atherosclerosis.

The rapidly expanding field of bioactive lipids is exemplified by the many sphingolipids, which are structurally and functionally diverse molecules with significant physiologic functions. These sphingolipids are main constituents of cellular membranes and have been found associated with plasma lipoproteins, and their concentrations are altered in several metabolic disorders such as atherosclerosis, obesity, and diabetes. Understanding the mechanisms that regulate their biosynthesis and secretion may provide novel information that might be amenable to therapeutic targeting in the treatment of these diseases. Several sphingolipid synthesis genes have been targeted as potential therapeutics for atherosclerosis. In recent years, significant progress has been made in studying the role of microRNAs (miRNAs) in lipid metabolism. However, little effort has been made to investigate their role in sphingolipid metabolism. Sphingolipid biosynthetic pathways involve various enzymes that lead to the formation of several key molecules implicated in atherosclerosis, and the identification of miRNAs that regulate these enzymes could help us to understand these complex pathways better and may prove beneficial in alleviating atherosclerosis.