Search

Obesity plays a major role in type II diabetes (T2DM) progression because it applies metabolic and oxidative stress resulting in dysfunctional beta-cells and activation of intra-islet pancreatic stellate cells (PaSCs) which cause islet fibrosis. Administration of antioxidant N-acetyl-L-cysteine (NAC) in vivo improves metabolic outcomes in diet-induced obese diabetic mice, and in vitro inhibits PaSCs activation. However, the effects of NAC on diabetic islets in vivo are unknown. This study examined if dosage and length of NAC treatment in HFD-induced diabetic mice effect metabolic outcomes associated with maintaining healthy beta-cells and quiescent PaSCs, in vivo . Male C57BL/6N mice were fed normal chow (ND) or high-fat (HFD) diet up to 30 weeks. NAC was administered in drinking water to HFD mice in preventative treatment (HFD pNAC ) for 23 weeks or intervention treatment for 10 (HFD iNAC ) or 18 (HFD iNAC+) weeks, respectively. HFD pNAC and HFD iNAC+ , but not HFD iNAC , mice showed significantly improved glucose tolerance and insulin sensitivity. Hyperinsulinemia led by beta-cell overcompensation in HFD mice was significantly rescued in NAC treated mice. A reduction of beta-cell nuclear Pdx-1 localization in HFD mice was significantly improved in NAC treated islets along with significantly reduced beta-cell oxidative stress. HFD-induced intra-islet PaSCs activation, labeled by αSMA, was significantly diminished in NAC treated mice along with lesser intra-islet collagen deposition. This study determined that efficiency of NAC treatment is beneficial at maintaining healthy beta-cells and quiescent intra-islet PaSCs in HFD-induced obese T2DM mouse model. These findings highlight an adjuvant therapeutic potential in NAC for controlling T2DM progression in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schuurman, Wallace, Sahi, Barillaro, Zhang, Rahman, Sawyez, Borradaile and Wang.)

Joint homeostasis failure can result in osteoarthritis (OA). Currently, there are no treatments to alter disease progression in OA, but targeting early changes in cellular behavior has great potential. Recent data show that nuclear receptors contribute to the pathogenesis of OA and could be viable therapeutic targets, but their molecular mechanisms in cartilage are incompletely understood. This study examines global changes in gene expression after treatment with agonists for four nuclear receptor implicated in OA (LXR, PPARδ, PPARγ, and RXR). Murine articular chondrocytes were treated with agonists for LXR, PPARδ, PPARγ, or RXR and underwent microarray, qPCR, and cellular lipid analyses to evaluate changes in gene expression and lipid profile. Immunohistochemistry was conducted to compare two differentially expressed targets (Txnip, Gsta4) in control and cartilage-specific PPARδ knockout mice subjected to surgical destabilization of the medial meniscus (DMM). Nuclear receptor agonists induced different gene expression profiles with many responses affecting lipid metabolism. LXR activation downregulated gene expression of proteases involved in OA, whereas RXR agonism decreased expression of ECM components and increased expression of Mmp13. Functional assays indicate increases in cell triglyceride accumulation after PPARγ, LXR, and RXR agonism but a decrease after PPARδ agonism. PPARδ and RXR downregulate the antioxidant Gsta4, and PPARδ upregulates Txnip. Wild-type, but not PPARδ-deficient mice, display increased staining for Txnip after DMM. Collectively, these data demonstrate that nuclear receptor activation in chondrocytes primarily affects lipid metabolism. In the case of PPARδ, this change might lead to increased oxidative stress, possibly contributing to OA-associated changes., Key Message: Nuclear receptors regulate metabolic genes in chondrocytes. Nuclear receptors affect triglyceride levels. PPARδ mediates regulation of oxidative stress markers. Nuclear receptors are promising therapeutic targets for osteoarthritis.

A vital role of vascular smooth muscle cells (SMCs) is to stabilize the artery wall by elaborating fibrils of type I collagen. This is especially important in atherosclerotic lesions. However, SMCs in these lesions can be laden with lipids and the impact of this modification on collagen fibril formation is unknown. To address this, we converted human vascular SMCs to a foam cell state by incubating them with either LDL or VLDL. Biochemical markers of a SMC phenotype were preserved. However, microscopic tracking revealed a profound perturbation in the ability of the cells to assemble collagen fibrils, reducing assembly by up to 79%. This dysfunction was mirrored by an inability of smooth muscle foam cells to assemble fibronectin. Lipid-loaded SMCs did not display a generalized defect in the actin cytoskeleton and the formation of vinculin-containing focal adhesion complexes was preserved. However, lipid-loaded SMCs were unable to assemble fibrillar adhesion complexes and clustering of tensin and alpha5beta1 integrin was disordered. Moreover, phosphorylation of tensin, required for fibrillar adhesion complex formation, was suppressed by up to 57%, with a concomitant decrease in activation of Src and FAK and restriction of activated Src to the cell edges. Forced activation of Src-FAK signaling in lipid-engorged SMCs rescued both fibrillar adhesion formation and fibrillogenesis. We conclude that lipid accumulation by SMCs disables the machinery for collagen and fibronectin assembly. This previously unknown relationship between atherogenic lipids and integrin-based signaling could underlie plaque vulnerability.

Objective: Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, has pleiotropic effects, including regulation of macrophage differentiation and lipid homeostasis. The PPARgamma ligands, thiazolidinediones (TZDs), attenuate atherosclerosis in mice by uncertain mechanisms. The objective of this study was to determine whether activation of PPARgamma or its obligate heterodimer, retinoid X receptor (RXR), modulates macrophage foam cell formation induced by oxidized (ox) lipoproteins., Methods and Results: Incubation of THP-1 macrophages with oxHTG-VLDL, oxREM, or oxLDL increased cellular cholesteryl ester over 6-fold. Preincubation with the TZD, ciglitazone, the RXR-specific ligand, 9-cis retinoic acid (9cRA) or the combination reduced CE mass accumulation by up to 65%. Ciglitazone and 9cRA increased CD36 mRNA (up to 4-fold); however, uptake of [125I]oxLDL was only modestly enhanced (up to 1.8-fold) becaues of a concomitant PPARgamma:RXR-induced decrease in SRAI/II activity (up to 40%). This suggested that PPARgamma:RXR activation inhibited cholesteryl ester accumulation by enhancing cholesterol efflux. Ciglitazone and 9cRA were found to increase the expression of ATP-binding cassette proteins A1 and G1, resulting in enhanced cholesterol efflux to lipoprotein-deficient serum, apoAI and HDL3., Conclusions: PPARgamma and/or RXR activation inhibit foam cell formation through enhanced cholesterol efflux despite increased oxLDL uptake. These observations explain the reduced atherosclerosis in TZD-treated mice and may extend the therapeutic implications of these ligands.

Transforming growth factor beta1 (TGF-beta1) is secreted by various cells, including macrophages, smooth muscle cells, and endothelial cells. TGF-beta1 is present in atherosclerotic lesions, but its role in regulating macrophage foam cell formation is not understood. Hypertriglyceridemic very low density lipoprotein (VLDL) remnants (VLDL-REMs) in their native or oxidized form will induce cholesteryl ester (CE) and triglyceride (TG) accumulation in macrophages. Therefore, we examined whether TGF-beta1 can modulate the macrophage uptake of native or oxidized VLDL-REMs (oxVLDL-REMs). Incubation of J774A.1 macrophages with VLDL-REMs and oxVLDL-REMs compared with control cells increased cellular CE (13- and 21-fold, respectively) and TG mass (21-and 18-fold, respectively). Preincubation with TGF-beta1 before incubation with VLDL-REMs or oxVLDL-REMs significantly decreased CE (73% and 54%, respectively) and TG mass (42% and 41%, respectively). TGF-beta1 inhibited the activity and expression of 2 key components needed for VLDL-REM uptake: lipoprotein lipase and low density lipoprotein receptor. TGF-beta1 inhibited CE mass induced by oxVLDL-REMs in part by decreasing the expression of scavenger receptor type AI/II and CD36. Furthermore, TGF-beta1 enhanced cholesterol efflux through upregulation of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Thus, TGF-beta1 inhibits macrophage foam cell formation induced by VLDL-REMs or oxVLDL-REMs, which suggests an antiatherogenic role for this cytokine.

Hepatic lipase-deficient subjects in the Ontario kindred are compound heterozygotes for hepatic lipase mutations (Ser267-->Phe and Thr383-->Met). Cholesteryl ester-rich beta-very-low-density lipoprotein (beta-VLDL) accumulates in plasma and such subjects have premature atherosclerosis. To determine a possible mechanism, we hypothesized that hepatic lipase-deficient beta-VLDL, homozygous for apolipoprotein (apo) E3, would cause cholesteryl ester accumulation and foam cell formation in macrophages. beta-VLDL and pre-beta-VLDL were isolated by Pevikon electrophoresis and incubated with J774 macrophages, cells that do not secrete apoE. beta-VLDL increased cellular cholesteryl ester content 13-fold, whereas pre-beta-VLDL increased cholesteryl ester sevenfold. beta-VLDL increased acyl CoA:cholesterol acyltransferase activity fourfold (measured as [14C]oleate incorporation into cholesteryl ester). Preincubation of hepatic lipase-deficient beta-VLDL with the anti-apoE monoclonal antibody 1D7, which inhibits binding of apoE to low-density lipoprotein receptors, inhibited cellular cholesteryl ester accumulation by 75%, whereas the anti-apoB blocking monoclonal antibody 5E11 failed to inhibit cellular cholesteryl ester accumulation. In contrast to hepatic lipase deficiency, beta-VLDL from type III subjects (E2/E2) failed to increase cellular cholesteryl ester or acyl CoA:cholesterol acyltransferase more than 1.5-fold. Thus, hepatic lipase-deficient beta-VLDL readily induces cholesteryl ester accumulation in J774 macrophages, a process mediated by functional apoE3. This may explain the premature atherosclerosis observed in this kindred.

The present investigations have examined the mechanism(s) whereby Sf 60-400 very low density lipoproteins (VLDL) from Type IV hypertriglyceridemic subjects cause cholesteryl ester and triglyceride accumulation in J774 macrophages. Both apolipoprotein (apo) E-poor and apoE-rich Type IV VLDL subfractions, isolated by heparin-Sepharose chromatography, were capable of enhancing cellular cholesterol and triglyceride content. The apoE-rich fraction was significantly more effective at inducing cholesterol esterification (P < 0.05) and accumulation of esterified cholesterol (P < 0.05), whereas both subfractions caused similar increases in cellular triglyceride content. Thus, the amount of VLDL-associated apoE determined the extent to which Type IV VLDL loaded J774 cells with cholesterol but not triglyceride. Two VLDL subfractions, Sf 60-400 and Sf 20-60, isolated from Type III subjects homozygous for apoE2, caused little or no effect on cellular esterified cholesterol content, whereas both fractions induced the same degree of cellular triglyceride accumulation as Type IV VLDL. Type IV VLDL-induced cholesteryl ester accumulation was blocked by an anti-apoE monoclonal antibody, known to block the binding of apoE to the LDL receptor; however, the increase in cellular triglyceride was unaffected. Therefore, VLDL-induced triglyceride accumulation in this cell line can occur without apoE-mediated uptake of intact VLDL particles. The addition of heparin to J774 cells resulted in a 6-fold increase in lipoprotein lipase (LPL) activity in the media, and significantly enhanced the ability of Type IV VLDL to induce cellular triglyceride accumulation (P < 0.01), but significantly decreased cellular cholesteryl ester content (P < 0.025). Finally, Sf 60-400 VLDL from two subjects homozygous for apoC-II deficiency failed to increase cellular lipid content. However, the addition of exogenous apoC-II to C-II-deficient VLDL resulted in significant increases of both triglyceride and esterified cholesterol in J774 cells. In the presence of apoC-II, the anti-apoE monoclonal antibody blocked the cellular cholesteryl ester increase induced by C-II-deficient VLDL, but had no effect on the increase in cellular triglyceride. Collectively, these experiments demonstrate that extracellular lipolysis of Sf 60-400 VLDL by LPL is required for cholesteryl ester and triglyceride accumulation in J774 macrophages. After interaction with cellular LPL, VLDL triglycerides are hydrolyzed. The resulting free fatty acids are readily taken up by the macrophage, and re-esterified into triglyceride. Lipolysis proceeds until apoE epitopes are exposed, allowing the triglyceride-depleted remnant, containing all the cholesteryl ester, to be taken up via an apoE-mediated process.

Lipoprotein kinetic studies have demonstrated that a large proportion of Sf 60-400 very low density lipoprotein (VLDL) is cleared directly from the circulation in Type IV hypertriglyceridemic subjects, at an unknown tissue site. The present studies were designed to investigate the role of hepatocytes in this process and to define the conditions, whereby Type IV Sf 60-400 VLDL would induce lipid accumulation in HepG2 cells. Type IV VLDL (Sf 60-400) failed to augment the total cholesterol, esterified cholesterol, or triglyceride content of HepG2 cells following 24-h incubations. Coincubation of bovine milk lipoprotein lipase (LPL) and Type IV VLDL with HepG2 cells induced a 3-fold increment in cellular esterified cholesterol mass (p less than 0.005) and a 7-fold increase in cellular triglyceride mass (p less than 0.005), compared to VLDL alone. The increased cellular lipid mass was associated with increased oleate incorporation into cellular cholesterol esters and triglycerides. Exogenous LPL hydrolyzed 76% of the VLDL triglyceride over 24 h. LPL action on Type IV VLDL was sufficient to promote cellular uptake of these lipoproteins, while elevated media-free fatty acid levels were not. Although HepG2 cells secrete apolipoprotein (apo) E, we assessed the role of VLDL-associated apoE in the lipid accumulation induced by VLDL plus LPL. ApoE-rich and apoE-poor Type IV VLDL subfractions induced similar increments in cellular esterified cholesterol in the presence of LPL, despite a 4-fold difference in apoE content. Sf 60-400 VLDL, from subjects homozygous for the defective apoE2, plus LPL, behaved identically to Type IV VLDL plus LPL. Type IV VLDL plus LPL, preincubated with anti-apoE (1D7) and apoB (5E11) monoclonal antibodies, known to block the binding of apoE and -B, respectively, to the LDL receptor failed to block lipid accumulation. In contrast, apoE-poor Type IV VLDL, apoE2 VLDL, and VLDL plus 1D7 were taken up poorly by J774 cells, cells that secrete LPL, but not apoE. These studies suggest that lipolytic remodeling of large Type IV VLDL by LPL is a prerequisite for their uptake by HepG2 cells and that HepG2 cell-secreted apoE rather than VLDL-associated apoE is the ligand involved in uptake.

The capacity of human triglyceride-rich lipoproteins to induce cholesterol accumulation in the murine J774 macrophage cell line was investigated with large very low density lipoprotein (VLDL, Sf 60-400) obtained from subjects with type III, IV, and V hyperlipoproteinemias. After incubation for 24 hours, VLDLs from type IV and type V subjects were similar in their ability to raise cellular cholesterol deposition threefold to fourfold and cellular triglyceride 16-fold. The increase in cholesterol was entirely due to the dramatic increase in cholesterol ester, from less than 1 to greater than 50 micrograms/mg cell protein. Total cholesterol accumulation was fourfold to fivefold greater than the cholesterol accumulation observed for VLDL or low density lipoprotein (LDL) from normal subjects. Cholesterol esterification (acyl coenzyme A: cholesterol acyltransferase [ACAT] activity) paralleled the rate of cholesterol accumulation in these cells. Treating the macrophages with the ACAT inhibitor 58035, which is known to downregulate the LDL receptor in these cells, diminished cholesterol accumulation by 40% for type IV VLDL and by 23% for normal LDL. Since hypertriglyceridemic VLDL carries excess apoprotein (apo) E molecules, we investigated the role of normal and abnormal apo E. An anti-apo E monoclonal antibody, known to block the binding of apo E to the LDL receptor, blocked type IV VLDL-induced cholesterol ester accumulation by approximately 70%. In contrast to type IV subjects, VLDL from type III subjects (homozygous for apo E2) when incubated with J774 macrophages (which do not secrete apo E) caused only a modest 1.5-2-fold increase in cellular cholesterol. Pre-beta- and beta-migrating VLDL subfractions from type III subjects were equally ineffective in causing cholesterol accumulation. By contrast, beta-VLDL from cholesterol-fed rabbits caused a sevenfold to eightfold increase in cellular cholesterol content. These results indicate that triglyceride-rich lipoproteins from type IV and type V subjects can cause substantial cholesterol ester accumulation and enhanced cholesterol esterification in J774 cells. The lower cholesterol accumulation with type IV VLDL in the presence of apo E antibodies and VLDL from type III subjects demonstrates the importance of functional apo E in this process.