Effect of apolipoprotein A-V on plasma triglyceride, lipoprotein size, and composition in genetically engineered mice

Transgenic (Tg) mice that overexpress the human apolipoprotein A-V gene (APOA5) yet lack an endogenous mouse apoa5 gene (APOA5 Tg mice) were generated. Subsequently, the effect of human apoA-V expression on plasma triglyceride (TG) concentration and lipoprotein and apolipoprotein distribution was determined and compared with that in mice deficient in apoA-V (apoa5(-/-) mice). NMR analysis of plasma lipoproteins revealed that APOA5 Tg mice had a very low VLDL concentration (26.4 +/- 7.7 nmol/dl), whereas VLDL in apoa5(-/-) mice was 18- fold higher (467 +/- 152 nmol/dl). SDS-PAGE analysis of the d < 1.063 g/ml plasma fraction revealed that the apoB-100/apoB-48 ratio was 14-fold higher in APOA5 Tg versus apoa5(-/-) mice and that the apoE/total apoB ratio was 7-fold greater in APOA5 Tg versus apoa5(-/-) mice. It is anticipated that a reduction in apoB-100/apoB-48 ratio as well as that for apoE/apoB would impair the uptake of VLDL and remnants in apoa5(-/-) mice, thereby contributing to increased plasma TG levels. The concentration of apoA-V in APOA5 Tg mice was 12.5 +/- 2.9 microg/ml, which is approximately 50- to 100-fold higher than that reported for normolipidemic humans. ApoA-V was predominantly associated with HDL but was rapidly and efficiently redistributed to apoA- V-deficient VLDL upon incubation. Consistent with findings reported for human subjects, apoA-V concentration was positively correlated with TG levels in normolipidemic APOA5 Tg mice. It is conceivable that, in a situation in which apoA-V is chronically overexpressed, complex interactions among factors regulating TG homeostasis may result in a positive correlation of apoA-V with TG concentrations.     

Apolipoprotein A-V dependent modulation of plasma triacylglycerol: a puzzlement

The discovery of apolipoprotein A-V (apoA-V) in 2001 has raised a number of intriguing questions about its role in lipid transport and triglyceride (TG) homeostasis. Genome wide association studies (GWAS) have consistently identified APOA5 as a contributor to plasma TG levels. Single nucleotide polymorphisms (SNP) within the APOA5 gene locus have been shown to correlate with elevated plasma TG. Furthermore, transgenic and knockout mouse models support the view that apoA-V plays a critical role in maintenance of plasma TG levels. The present review describes recent concepts pertaining to apoA-V SNP analysis and their association with elevated plasma TG. The interaction of apoA-V with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is discussed relative to its postulated role in TG-rich lipoprotein catabolism. The potential role of intracellular apoA-V in regulation of TG homeostasis, as a function of its ability to associate with cytosolic lipid droplets, is reviewed. While some answers are emerging, numerous mysteries remain with regard to this low abundance, yet potent, modulator of TG homeostasis. Given the strong correlation between elevated plasma TG and heart disease, there is great scientific and public interest in deciphering the numerous biological riddles presented by apoA-V. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Evidence for a complex relationship between apoA-V and apoC-III in patients with severe hypertriglyceridemia

The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8-78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG.     

ApoE2-associated hypertriglyceridemia is ameliorated by increased levels of apoA-V but unaffected by apoC-III deficiency

Apolipoprotein E2 (apoE2)-associated hyperlipidemia is characterized by a disturbed clearance of apoE2-enriched VLDL remnants. Because excess apoE2 inhibits LPL-mediated triglyceride (TG) hydrolysis in vitro, we investigated whether direct or indirect stimulation of LPL activity in vivo reduces the apoE2-associated hypertriglyceridemia. Here, we studied the role of LPL and two potent modifiers, the LPL inhibitor apoC-III and the LPL activator apoA-V, in APOE2-knockin (APOE2) mice. Injection of heparin in APOE2 mice reduced plasma TG by 53% and plasma total cholesterol (TC) by 18%. Adenovirus-mediated overexpression of LPL reduced plasma TG by 85% and TC by 40%. Both experiments indicate that the TG in apoE2-enriched particles is a suitable substrate for LPL. Indirect activation of LPL activity via deletion of Apoc3 in APOE2 mice did not affect plasma TG levels, whereas overexpression of Apoa5 in APOE2 mice did reduce plasma TG by 81% and plasma TC by 41%. In conclusion, the hypertriglyceridemia in APOE2 mice can be ameliorated by the direct activation of LPL activity. Indirect activation of LPL via overexpression of apoA-V does, whereas deletion of apoC-III does not, affect the plasma TGs in APOE2 mice. These data indicate that changes in apoA-V levels have a dominant effect over changes in apoC-III levels in the improvement of APOE2-associated hypertriglyceridemia.     

Apolipoprotein A5, a unique modulator of fasting and postprandial triglycerides

The discovery of apolipoprotein A5 (APOA5) in 2001 has raised a number of intriguing questions about its role in lipid transport and triglyceride (TG) homeostasis. Genome-wide association studies have consistently identified APOA5 as a regulator of plasma TG levels, which is further supported by studies in transgenic and knockout mouse models. The present review describes recent concepts pertaining to the roles of APOA5 in TG metabolism as related to the vascular compartment, liver, adipose tissue and the gut. Recent evidence indicates that APOA5 may also affect postprandial TG metabolism through influencing chylomicron formation and transport by the intestine into the intestinal lymph. While substantial evidence supports the notion that APOA5 plays both extracellular and intracellular roles in TG homeostasis, mysteries remain on how this low-abundance, liver-derived protein may modulate TG homeostasis, including via the gut. Given the strong correlation between elevated plasma TG and cardiometabolic diseases, there is great scientific and public interest in understanding the intriguing mysteries presented by APOA5.     

Effect of a short- and long-term treatment with Ginkgo biloba extract on amyloid precursor protein levels in a transgenic mouse model relevant to Alzheimer's disease

Several clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer’s disease (AD). The aim of the present long-term feeding trial was to study the impact of dietary EGb761 on Amyloid precursor protein (APP) metabolism in mice transgenic for human APP (Tg2576). Tg2576 mice were fed diets with and without EGb761 (300 mg/kg diet) for 1 and 16 months, respectively. Long-term treatment (16 months) with EGb761 significantly lowered human APP protein levels by ∼50% as compared to controls in the cortex but not in the hippocampus. However, APP levels were not affected by EGb761 in young mice. Current data indicate that APP seems to be an important molecular target of EGb761 in relation to the duration of the Ginkgo biloba treatment and/or the age of the animals. Potential neuroprotective properties of EGb761 may be, at least partly, related to its APP lowering activity.     

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.     

Bile acids override steatosis in farnesoid X receptor deficient mice in a model of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, and the pathogenesis is still not well known. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily and plays an essential role in maintaining bile acid and lipid homeostasis. In this study, we study the role of FXR in the pathogenesis of NFALD. We found that FXR deficient (FXR^−/−) mice fed methionine- and choline-deficient (MCD) diet had higher serum ALT and AST activities and lower hepatic triglyceride levels than wild-type (WT) mice fed MCD diet. Expression of genes involved in inflammation (VCAM-1) and fibrosis (α-SMA) was increased in FXR^−/− mice fed MCD diet (FXR^−/−/MCD) compared to WT mice fed MCD diet (WT/MCD). Although MCD diet significantly induced hepatic fibrosis in terms of liver histology, FXR^−/−/MCD mice showed less degree of hepatic steatosis than WT/MCD mice. Moreover, FXR deficiency synergistically potentiated the elevation effects of MCD diet on serum and hepatic bile acids levels. The super-physiological concentrations of hepatic bile acids in FXR^−/−/MCD mice inhibited the expression of genes involved in fatty acid uptake and triglyceride accumulation, which may be an explanation for less steatosis in FXR^−/−/MCD mice in contrast to WT/MCD mice. These results suggest that hepatic bile acids accumulation could override simple steatosis in hepatic injury during the progression of NAFLD and further emphasize the role of FXR in maintaining hepatic bile acid homeostasis in liver disorders and in hepatic protection.     

Apolipoprotein A-V is a potential target for treating coronary artery disease: evidence from genetic and metabolomic analyses

Triglyceride (TG)-lowering LPL variants in combination with genetic LDL-C-lowering variants are associated with reduced risk of coronary artery disease (CAD). Genetic variation in the APOA5 gene encoding apolipoprotein A-V also strongly affects TG levels, but the potential clinical impact and underlying mechanisms are yet to be resolved. Here, we aimed to study the effects of APOA5 genetic variation on CAD risk and plasma lipoproteins through factorial genetic association analyses. Using data from 309,780 European-ancestry participants from the UK Biobank, we evaluated the effects of lower TG levels as a result of genetic variation in APOA5 and/or LPL on CAD risk with or without a background of reduced LDL-C. Next, we compared lower TG levels via APOA5 and LPL variation with over 100 lipoprotein measurements in a combined sample from the Netherlands Epidemiology of Obesity study (N = 4,838) and the Oxford Biobank (N = 6,999). We found that lower TG levels due to combined APOA5 and LPL variation and genetically-influenced lower LDL-C levels afforded the largest reduction in CAD risk (odds ratio: 0.78 (0.73–0.82)). Compared to patients with genetically-influenced lower TG via LPL, genetically-influenced lower TG via APOA5 had similar and independent, but notably larger, effects on the lipoprotein profile. Our results suggest that lower TG levels as a result of APOA5 variation have strong beneficial effects on CAD risk and the lipoprotein profile, which suggest apo A-V may be a potential novel therapeutic target for CAD prevention.     

An apolipoprotein A-V gene SNP is associated with marked hypertriglyceridemia among Asian-American patients

Apolipoprotein A-V (apoA-V) is an important regulator of plasma levels of triglyceride (TG) in mice. In humans, APOA5 genetic variation is associated with TG in several populations. In this study, we determined the effects of the p.185Gly>Cys (c.553G>T; rs2075291) polymorphism on plasma TG levels in subjects of Chinese ancestry living in the United States and in a group of non-Chinese Asian ancestry. The frequency of the less common cysteine allele was 4-fold higher (15.1% vs. 3.7%) in Chinese high-TG subjects compared with a low-TG group (Chi-square = 20.2; P < 0.0001), corresponding with a 4.45 times higher risk of hypertriglyceridemia (95% confidence interval, 2.18-9.07; P < 0.001). These results were replicated in the non-Chinese Asians. Heterozygosity was associated, in the high-TG group, with a doubling of TG (P < 0.001), mainly VLDL TG (P = 0.014). All eleven TT homozygotes had severe hypertriglyceridemia, with mean TG of 2,292 +/- 447 mg/dl. Compared with controls, carriers of the T allele had lower postheparin lipoprotein lipase activity but not hepatic lipase activity. In Asian populations, this common polymorphism can lead to profound adverse effects on lipoprotein profiles, with homozygosity accounting for a significant number of cases of severe hypertriglyceridemia. This specific apoA-V variant has a pronounced effect on TG metabolism, the mechanism of which remains to be elucidated.     

Effects of apoA-V on HDL and VLDL metabolism in APOC3 transgenic mice

Apolipoprotein A-V (apoA-V) and apoC-III are exchangeable constituents of VLDL and HDL. ApoA-V counteracts the effect of apoC-III on triglyceride (TG) metabolism with poorly defined mechanisms. To better understand the effects of apoA-V on TG and cholesterol metabolism, we delivered apoA-V cDNA into livers of hypertriglyceridemic APOC3 transgenic mice by adenovirus-mediated gene transfer. In response to hepatic apoA-V production, plasma TG levels were reduced significantly as a result of enhanced VLDL catabolism without alternations in VLDL production. This effect was associated with reduced apoC-III content in VLDL. Increased apoA-V production also resulted in decreased apoC-III and increased apoA-I content in HDL. Furthermore, apoA-V-enriched HDL was associated with enhanced LCAT activity and increased cholesterol efflux. This effect, along with apoE enrichment in HDL, contributed to HDL core expansion and alpha-HDL formation, accounting for significant increases in both the number and size of HDL particles. As a result, apoA-V-treated APOC3 transgenic mice exhibited decreased VLDL-cholesterol and increased HDL-cholesterol levels. ApoA-V-mediated reduction of apoC-III content in VLDL represents an important mechanism by which apoA-V acts to ameliorate hypertriglyceridemia in adult APOC3 transgenic mice. In addition, increased apoA-V levels accounted for cholesterol redistribution from VLDL to larger HDL particles. These data suggest that in addition to its TG-lowering effect, apoA-V plays a significant role in modulating HDL maturation and cholesterol metabolism.     

Altered apolipoprotein A-V expression during the acute phase response is independent of plasma triglyceride levels in mice and humans

Plasma triglyceride (TG) levels are altered during the acute phase response (APR). Plasma levels of the recently discovered apolipoprotein A-V (apoA-V) are inversely associated with plasma TG. The aim of this study was to investigate the change of apoA-V plasma levels and hepatic apoA-V expression during the APR in relation to plasma TG. During human APR plasma apoA-V was decreased as were plasma TG (each P<0.01). Also early in the course of the murine APR plasma apoA-V levels and hepatic apoA-V expression were decreased and changed in the same direction as plasma TG. Treatment of HepG2 cells with TNF-alpha and IL-1beta decreased apoA-V mRNA levels early by 42% and 55%, respectively (each P<0.001). However, in promoter/reporter assays the human apoA-V promoter was unresponsive to proinflammatory cytokines. Instead, we demonstrate that a significant decrease in apoA-V mRNA stability in response to treatment with TNF-alpha and IL-1beta is the underlying basis of decreased apoA-V expression during the APR (P<0.05). These data demonstrate that (i) apoA-V expression decreases early during the APR due to changes in mRNA stability, and (ii) during the APR apoA-V is not inversely related to plasma TG levels in mice and humans, thereby identifying a relevant pathophysiological setting, in which the previously reported close inverse association between these parameters does not hold true.     

Inhibition of hepatic damage and liver fibrosis by brain natriuretic peptide

Anti-fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl₄)-induced liver fibrosis model using BNP-transgenic (Tg) and wild-type (WT) mice. Twice-a-week intraperitoneal injections of CCl₄ for 8 weeks resulted in massive liver fibrosis, augmented transforming growth factor (TGF)-β₁ and type I procollagen α₁ chain (Col1a1) mRNA expression, and the hepatic stellate cell (HSC) activation in WT mice, all of which were significantly suppressed in Tg mice. These observations indicate that BNP inhibits liver fibrosis by attenuating the activation of HSCs.     

CPAP enhances and maintains chronic inflammation in hepatocytes to promote hepatocarcinogenesis

Chronic and persistent inflammation is a well-known carcinogenesis promoter. Hepatocellular carcinoma (HCC) is one of the most common inflammation-associated cancers; most HCCs arise in the setting of chronic inflammation and hepatic injury. Both NF-κB and STAT3 are important regulators of inflammation. Centrosomal P4.1-associated protein (CPAP), a centrosomal protein that participates primarily in centrosome functions, is overexpressed in HCC and can increase TNF-α-mediated NF-κB activation and IL-6-induced STAT3 activation. A transgenic (Tg) mouse model with hepatocyte-specific CPAP expression was established to investigate the physiological role of CPAP in hepatocarcinogenesis. Obvious inflammatory cell accumulation and fatty change were observed in the livers of CPAP Tg mice. The alanine aminotransferase (ALT) level and the expression levels of inflammatory genes, such as IL-6, IL-1β and TNF-α, were higher in CPAP Tg mice than in wild type (WT) mice. High-dose/short-term treatment with diethylnitrosamine (DEN) increased the ALT level, proinflammatory gene expression levels, and STAT3 and NF-κB activation in CPAP Tg mice; low-dose/long-term DEN treatment induced more severe liver tumor formation in CPAP Tg mice than in WT mice. CPAP can increase the expression of chemokine (C-C motif) ligand 16 (CCL-16), an important chemotactic cytokine, in human hepatocytes. CCL-16 expression is positively correlated with CPAP and TNF-α mRNA expression in the peritumoral part of HCC. In summary, these results suggest that CPAP may promote hepatocarcinogenesis through enhancing the inflammation pathway via increasing the expression of CCL-16.Subject terms: Liver cancer, Tumour immunology     

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

The Na⁺-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (∼ 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (∼ 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (∼ 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at ∼ 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 °C or 37 °C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.     

Efficient lowering of triglyceride levels in mice by human apoAV protein variants associated with hypertriglyceridemia

Variation in the apolipoprotein A5 (APOA5) gene has consistently been associated with increased plasma triglyceride (TG) levels in epidemiological studies. In vivo functionality of these variations, however, has thus far not been tested. Using adenoviral over-expression, we evaluated plasma expression levels and TG-lowering efficacies of wild-type human apoAV, two human apoAV variants associated with increased TG (S19W, G185C) and one variant (Q341H) that is predicted to have altered protein function. Injection of mice with adenovirus encoding wild-type or mutant apoAV resulted in an identical dose-dependent elevation of human apoAV levels in plasma. The increase in apoAV levels resulted in pronounced lowering of plasma TG levels at two viral dosages. Unexpectedly, the TG-lowering efficacy of all three apoAV variants was similar to wild-type apoAV. In addition, no effect on TG-hydrolysis-related plasma parameters (free fatty acids, glycerol and post-heparin lipoprotein lipase activity) was apparent upon expression of all apoAV variants. In conclusion, our data indicate that despite their association with hypertriglyceridemia and/or predicted protein dysfunction, the 19W, 185C and 341H apoAV variants are equally effective in reducing plasma TG levels in mice.     

Rare APOA5 promoter variants associated with paradoxical HDL cholesterol decrease in response to fenofibric acid therapy

Individuals with mixed dyslipidemia, including high triglycerides (TGs) and low high density lipoprotein cholesterol (HDL-C), have increased risk for coronary events. We examined the effect of rare genetic variants in the APOA5 gene region on plasma HDL-C, apolipoprotein A-I (apoA-I), and TG response to fenofibric acid monotherapy and in combination with statins. The APOA5 gene region was sequenced in 1,612 individuals with mixed dyslipidemia in a randomized trial of fenofibric acid alone and in combination with statins. Student''s t-test and rare variant burden tests were used to examine plasma HDL-C, apoA-I, and TG response. Rare APOA5 promoter region variants were associated with decreased HDL-C and apoA-I levels in response to fenofibric acid therapy; rare missense variants were associated with increased TG response to combination therapy. Further study is needed to examine the effect of these rare variants on coronary outcomes in this population in response to fenofibric acid monotherapy or combined with statins     

Absence of adipose differentiation related protein upregulates hepatic VLDL secretion,relieves hepatosteatosis,and improves whole body insulin resistance in leptin-deficient mice

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep^ob/ob mice. We bred Adfp^−/− mice with Lep^ob/ob mice to create Lep^ob/ob/Adfp^−/− and Lep^ob/ob/Adfp^+/+ mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep^ob/ob/Adfp^+/+ mice, Lep^ob/ob/Adfp^−/− mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep^ob/ob/Adfp^−/− mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep^ob/ob/Adfp^−/− mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.