Synthesis of Novel MicroRNA-30c Analogs to Reduce Apolipoprotein B Secretion in Human Hepatoma Cells.

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2'-O-methyl (2'OMe), 2'-fluoro (2'F), pseudouridine (á´ª), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases. This protocol was validated in: J Biol Chem (2021), DOI: 10.1016/j.jbc.2022.101813.

GLP-2 Dysregulates Hepatic Lipoprotein Metabolism, Inducing Fatty Liver and VLDL Overproduction in Male Hamsters and Mice.

Fundamental complications of insulin resistance and type 2 diabetes include the development of nonalcoholic fatty liver disease and an atherogenic fasting dyslipidemic profile, primarily due to increases in hepatic very-low-density lipoprotein (VLDL) production. Recently, central glucagon-like peptide-2 receptor (GLP2R) signaling has been implicated in regulating hepatic insulin sensitivity; however, its role in hepatic lipid and lipoprotein metabolism is unknown. We investigated the role of glucagon-like peptide-2 (GLP-2) in regulating hepatic lipid and lipoprotein metabolism in Syrian golden hamsters, C57BL/6J mice, and Glp2r-/- mice consuming either a normal chow or high-fat diet (HFD). In the chow-fed hamsters, IP GLP-2 administration significantly increased fasting dyslipidemia, hepatic VLDL production, and the expression of key genes involved in hepatic de novo lipogenesis. In HFD-fed hamsters and chow-fed mice, GLP-2 administration exacerbated or induced hepatic lipid accumulation. HFD-fed Glp2r-/- mice displayed reduced glucose tolerance, VLDL secretion, and microsomal transfer protein lipid transfer activity, as well as exacerbated fatty liver. Thus, we conclude that GLP-2 plays a lipogenic role in the liver by increasing lipogenic gene expression and inducing hepatic steatosis, fasting dyslipidemia, and VLDL overproduction. In contrast, the lack of Glp2r appears to interfere with VLDL secretion, resulting in enhanced hepatic lipid accumulation. These studies have uncovered a role for GLP-2 in maintaining hepatic lipid and lipoprotein homeostasis.

A single-atom 3D sub-attonewton force sensor.

Forces drive all physical interactions. High-sensitivity measurement of the effect of forces enables the quantitative investigation of physical phenomena. Laser-cooled trapped atomic ions are a well-controlled quantum system whose low mass, strong Coulomb interaction, and readily detectable fluorescence signal make them a favorable platform for precision metrology. We demonstrate a three-dimensional sub-attonewton sensitivity force sensor based on a super-resolution imaging of a single trapped ion. The force is detected by measuring the ion's displacement in three dimensions with nanometer precision. Observed sensitivities were 372 ± 9, 347 ± 18, and 808 ± 51 zN/[Formula: see text], corresponding to 24×, 87×, and 21× above the quantum limit. We verified this technique by measuring a 95-zN light pressure force, an important systematic effect in optically based sensors.

Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity.

The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvß3 and αvß5 integrins. Mice deficient in Mfge8 or the αvß3 and αvß5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2-dependent signaling pathway. These data identify a key role for Mfge8 and the αvß3 and αvß5 integrins in regulating enterocyte lipid processing.

Ultrafast, high repetition rate, ultraviolet, fiber-laser-based source: application towards Yb+ fast quantum-logic.

Trapped ions are one of the most promising approaches for the realization of a universal quantum computer. Faster quantum logic gates could dramatically improve the performance of trapped-ion quantum computers, and require the development of suitable high repetition rate pulsed lasers. Here we report on a robust frequency upconverted fiber laser based source, able to deliver 2.5 ps ultraviolet (UV) pulses at a stabilized repetition rate of 300.00000 MHz with an average power of 190 mW. The laser wavelength is resonant with the strong transition in Ytterbium (Yb+) at 369.53 nm and its repetition rate can be scaled up using high harmonic mode locking. We show that our source can produce arbitrary pulse patterns using a programmable pulse pattern generator and fast modulating components. Finally, simulations demonstrate that our laser is capable of performing resonant, temperature-insensitive, two-qubit quantum logic gates on trapped Yb+ ions faster than the trap period and with fidelity above 99%.

Human serum preß1-high density lipoprotein levels are independently and negatively associated with coronary artery diseases.

BACKGROUND: Serum preß1-high density lipoprotein (preß1-HDL) was defined by two-dimensional non-denaturing linear gel electrophoresis and apolipoprotein A-I immuno-blotting. Serum preß1-HDL seems to play an important role in reverse cholesterol transport, a well-known anti-atherosclerosis process. However, there are still debatable questions for its quantification and coronary artery disease (CAD) relevance. METHODS: We isolated the preß1-HDL using a new native polyacrylamide gel electrophoresis (PAGE) system and lipid pre-staining serum. We established a two-demensional gel electrophoresis system. RESULTS: We measured the preß1-HDL in Tangier disease patients and subjects with cholesterol ester transfer protein (CETP) mutation. The preß1-HDL is clearly separated from lipid-free apoA-I monomer and cannot be converted into other HDL particles under lecithin-cholesterol acyltransferase (LCAT) inhibition. This preß1-HDL is a spheroidal particle with the highest apoA-1/cholesterol ratio and highest density (≥1.21 g/ml), as compared with all other HDLs. Importantly, we found that serum from subjects with Tangier disease or with cholesterol ester transfer protein (CETP) mutation have no detectible preß1-HDL particles. We recruited a total of 102 subjects underwent diagnostic coronary angiography and measured their preß1-HDL levels. Among them, 56 had no stenosis of coronary artery and 46 were diagnosed as CAD, which was predefined as the presence of a luminal diameter stenosis ≥50 % in at least 1 major coronary artery territory. We found that preß1-HDL is independently and negatively associated with the severity of the coronary artery stenosis (Gensini score). CONCLUSION: We established a novel and simple method for human serum preß1-HDL quantification. We found that human lower preß1-HDL is an independent predictor for severer coronary artery stenosis.

Glucagon-Like Peptide 2 (GLP-2) Stimulates Postprandial Chylomicron Production and Postabsorptive Release of Intestinal Triglyceride Storage Pools via Induction of Nitric Oxide Signaling in Male Hamsters and Mice.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

Central Nervous System Regulation of Intestinal Lipoprotein Metabolism by Glucagon-Like Peptide-1 via a Brain-Gut Axis.

OBJECTIVE: Intestinal overproduction of atherogenic chylomicron particles postprandially is an important component of diabetic dyslipidemia in insulin-resistant states. In addition to enhancing insulin secretion, peripheral glucagon-like peptide-1 (GLP-1) receptor stimulation has the added benefit of reducing this chylomicron overproduction in patients with type 2 diabetes mellitus. Given the presence of central GLP-1 receptors and GLP-1-producing neurons, we assessed whether central GLP-1 exerts an integral layer of neuronal control during the production of these potentially atherogenic particles. APPROACH AND RESULTS: Postprandial production of triglyceride-rich lipoproteins was assessed in Syrian hamsters administered a single intracerebroventricular injection of the GLP-1 receptor agonist exendin-4. Intracerebroventricular exendin-4 reduced triglyceride-rich lipoprotein-triglyceride and -apolipoprotein B48 accumulation relative to vehicle-treated controls. This was mirrored by intracerebroventricular MK-0626, an inhibitor of endogenous GLP-1 degradation, and prevented by central exendin9-39, a GLP-1 receptor antagonist. The effects of intracerebroventricular exendin-4 were also lost during peripheral adrenergic receptor and central melanocortin-4 receptor inhibition, achieved using intravenous propranolol and phentolamine and intracerebroventricular HS014, respectively. However, central exendin9-39 did not preclude the effects of peripheral exendin-4 treatment on chylomicron output. CONCLUSIONS: Central GLP-1 is a novel regulator of chylomicron production via melanocortin-4 receptors. Our findings point to the relative importance of central accessibility of GLP-1-based therapies and compel further studies examining the status of this brain-gut axis in the development of diabetic dyslipidemia and chylomicron overproduction.

The impact of phospholipid transfer protein (PLTP) on lipoprotein metabolism.

It has been reported that phospholipid transfer protein (PLTP) is an independent risk factor for human coronary artery disease. In mouse models, it has been demonstrated that PLTP overexpression induces atherosclerosis, while its deficiency reduces it. PLTP is considered a promising target for pharmacological intervention to treat atherosclerosis. However, we must still answer a number of questions before its pharmaceutical potential can be fully explored. In this review, we summarized the recent progresses made in the PLTP research field and focused on its effect on apoB-containing- triglyceride-rich particle and HDL metabolism.

Mg deficiency results in modulation of serum lipids, glutathione, and NO synthase isozyme activation in cardiovascular tissues: relevance to de novo synthesis of ceramide, serum Mg and atherogenesis.

The present work tested the hypothesis that short-term (S-T) dietary deficiency of magnesium (Mg) (21 days) in rats would: 1) result in reduction in serum(s) sphingomyelin (SM) and changes in several blood lipids, HDL-cholesterol (HDL-C) and phosphatidylcholine (PC) concomitant with elevations in s cholesterol (chol), s LDL+VLDL and trigycerides (TG), as well as reduction in the PC/cholesterol ratio; 2) lead to oxidative stress, characterized by reductions in glutathione (glut) content in the various chambers of the heart and activation of e-NOS and n-NOS in the atria, ventricles and aortic smooth muscle (ASM); 3) produce early cardiac damage characterized by leakage of creatine kinase (CK) and lactic dehydrogenase (LDH); and 4) demonstrate that these pathophysiological changes are a result of profound reductions in s ionized Mg (Mg(2+)) and activation of the SM-ceramide pathway. In addition, we hypothesized that: 1) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg(2+) would lead to de novo synthesis of ceramide and activation of NO synthase with reduction in glut, both of which would be attenuated by inhibition of sphingomyelinase (SMase) and serine palmitoyl CoA transferase (SPT); and 2) low levels of Mg(2+)added to the drinking water would either prevent or ameliorate these manifestations. Our data indicate that S-T Mg deficiency resulted in reductions in s Mg(2+), SM, PC, HDL-C and the PC/chol ratio concomitant with decreases in tissue levels of glut, leakage of cardiac CK and LDH, as well as activation of e-NOS and n-NOS in all chambers of the heart and ASM. The greater the reduction in s Mg(2+), the greater the effects on all parameters analyzed; very significant correlations to levels of s SM and Mg(2+) were found with all of the serum and tissue biochemical -molecular analytes measured. Our experiments also showed that VSMCs exposed to low Mg(2+)resulted in activation of NO synthase, loss of glut and de novo synthesis of ceramide which were attenuated by inhibitors of SMase and SPT. Low levels of drinking water Mg(2+)(e.g., 15 ppm) were cardio- and vascular protective. We believe these new findings support our concept of an important role for the SM-ceramide pathway in the manifestations of Mg deficiency and atherogenesis.

Hepatic steatosis in hepatitis C is a storage disease due to HCV interaction with microsomal triglyceride transfer protein (MTP).

Liver steatosis is a frequent histological feature in patients chronically infected with hepatitis C virus (HCV). The relationship between HCV and hepatic steatosis seems to be the result of both epigenetic and genetic factors. In vivo and in vitro studies have shown that HCV can alter intrahepatic lipid metabolism by affecting lipid synthesis, oxidative stress, lipid peroxidation, insulin resistance and the assembly and secretion of VLDL. Many studies suggest that HCV-related steatosis might be the result of a direct interaction between the virus and MTP. It has been demonstrated that MTP is critical for the secretion of HCV particles and that inhibition of its lipid transfer activity reduces HCV production. However, higher degrees of hepatic steatosis were found in chronic hepatitis C patients carrying the T allele of MTP -493G/T polymorphism that seems to be associated with increased MTP transcription. We propose here that liver steatosis in hepatitis C could be a storage disease induced by the effects of the virus and of its proteins on the intracellular lipid machinery and on MTP. Available data support the hypothesis that HCV may modulate MTP expression and activity through a number of mechanisms such as inhibition of its activity and transcriptional control. Initial up regulation could favour propagation of HCV while down regulation in chronic phase could cause impairment of triglyceride secretion and excessive lipid accumulation, with abnormal lipid droplets facilitating the "storage" of virus particles for persistent infection.

Phosphatase and tensin homolog (PTEN) regulates hepatic lipogenesis, microsomal triglyceride transfer protein, and the secretion of apolipoprotein B-containing lipoproteins.

Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.