Human Cholesteryl Ester Transfer Protein Research Articles

Atherosclerosis: an overview of mouse models and a detailed methodology to quantify lesions in the aortic root.

Cardiovascular disease, the primary cause of human mortality globally, is predominantly caused by a progressive disorder known as atherosclerosis. Atherosclerosis refers to the process of accumulation of cholesterol-enriched lipoproteins and the concomitant initiation of inflammatory processes in the arterial wall, including the recruitment of immune cells. This leads to the formation of atherosclerotic plaques, initially causing a thickening of the arterial wall and narrowing of arteries. However, as plaque formation progresses, atherosclerotic plaques may become unstable and rupture, leading to a blood clot that blocks the affected artery or travels through the blood to block blood flow elsewhere. In the early 1990s, emerging gene editing methods enabled the development of apolipoprotein E knockout (Apoe-/- ) and low-density lipoprotein receptor knockout (Ldlr-/- ) mice. These mice have been instrumental in unraveling the complex pathogenesis of atherosclerosis. Around the same time, human APOE*3-Leiden transgenic mice were generated, which were more recently cross-bred with human cholesteryl ester transfer protein (CETP) transgenic mice to generate APOE*3-Leiden.CETP mice. This model appears to closely mimic human lipoprotein metabolism and responds to classic lipid-lowering interventions due to an intact ApoE-LDLR pathway of lipoprotein remnant clearance. In this review, we describe the role of lipid metabolism and inflammation in atherosclerosis development and highlight the characteristics of the frequently used animal models to study atherosclerosis, with a focus on mouse models, discussing their advantages and limitations. Moreover, we present a detailed methodology to quantify atherosclerotic lesion area within the aortic root region of the murine heart, as well as details required for scoring atherosclerotic lesion severity based on guidelines of the American Heart Association adapted for mice.

CETP expression ameliorates endothelial function in female mice through estrogen receptor-α and endothelial nitric oxide synthase pathway.

Endothelial dysfunction is an early manifestation of atherosclerosis. The cholesteryl ester transfer protein (CETP) has been considered pro-atherogenic by reducing plasma HDL levels. However, CETP may exhibit cell- or tissue-specific effects. We have previously reported that male mice expressing the human CETP gene show impaired endothelium-mediated vascular relaxation associated with oxidative stress. Although sexual dimorphisms on the metabolic role of CETP has been proposed, possible sex differences in the vascular effects of CETP were not previously studied. Thus, here we investigated the endothelial function of female CETP transgenic mice as compared to non-transgenic controls (NTg). Aortas from CETP females presented preserved endothelium-dependent relaxation to acetylcholine and an endothelium-dependent reduction of phenylephrine-induced contraction. eNOS phosphorylation (Ser1177) and calcium-induced NO levels were enhanced, while reactive oxygen species (ROS) production and NOX2 and SOD2 expression were reduced in the CETP female aortas. Furthermore, CETP females exhibited increased aortic relaxation to 17β-estradiol (E2) and upregulation of heat shock protein 90 (HSP90) and caveolin-1, proteins that stabilize estrogen receptor (ER) in the caveolae. Indeed, CETP females showed an increased E2-induced relaxation in a manner sensitive to ERα and HSP90 inhibitors methylpiperidinopyrazole (MPP) and geldanamycin, respectively. MPP also impaired the relaxation response to acetylcholine in CETP but not in NTg females. Altogether, the study indicates that CETP expression ameliorates anti-contractile endothelial effect and relaxation to E2 in females. This was associated with less ROS production, and increased eNOS-NO and E2-ERα pathways. These results highlight the need for considering sex-specific effects of CETP on cardiovascular risk.

Adcy9 Gene Inactivation Improves Cardiac Function After Myocardial Infarction in Mice.

Polymorphisms in the adenylate cyclase 9 (ADCY9) gene influence the benefits of the cholesteryl ester transfer protein (CETP) modulator dalcetrapib on cardiovascular events after acute coronary syndrome. We hypothesized that Adcy9 inactivation could improve cardiac function and remodelling following myocardial infarction (MI) in absence of CETP activity. Wild-type (WT) and Adcy9-inactivated (Adcy9Gt/Gt) male mice, transgenic or not for human CETP (tgCETP+/-), were subjected to MI by permanent left anterior descending coronary artery ligation and studied for 4 weeks. Left ventricular (LV) function was assessed by echocardiography at baseline, 1, and 4 weeks after MI. At sacrifice, blood, spleen and bone marrow cells were collected for flow cytometry analysis, and hearts were harvested for histologic analyses. All mice developed LV hypertrophy, dilation, and systolic dysfunction, but Adcy9Gt/Gt mice exhibited reduced pathologic LV remodelling and better LV function compared with WT mice. There were no differences between tgCETP+/- and Adcy9Gt/Gt tgCETP+/- mice, which both exhibited intermediate responses. Histologic analyses showed smaller cardiomyocyte size, reduced infarct size, and preserved myocardial capillary density in the infarct border zone in Adcy9Gt/Gt vs WT mice. Count of bone marrow T cells and B cells were significantly increased in Adcy9Gt/Gt mice compared with the other genotypes. Adcy9 inactivation reduced infarct size, pathologic remodelling, and cardiac dysfunction. These changes were accompanied by preserved myocardial capillary density and increased adaptive immune response. Most of the benefits of Adcy9 inactivation were only observed in the absence of CETP.

Structure of New Ferroverdins Recruiting Unconventional Ferrous Iron Chelating Agents

Ferroverdins are ferrous iron (Fe2+)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe2+, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe2+-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.

Abstract 118: Cholesteryl Ester Transfer Protein Overrides The Protective Effect Of APOA1 On Arterial APOB Lipoprotein Trapping And Atherosclerosis Progression In A Mouse Model Of Type 1 Diabetes

Despite the benefits of LDL-cholesterol-reducing medications, patients with type 1 diabetes (T1D) continue to have an increased risk of incident CVD. Preclinical and clinical observations indicate that triglyceride-rich lipoproteins (TRLs) and their remnants may contribute significantly to CVD risk in T1D. Recently, we have shown that diabetes increases levels of atherogenic TRL remnants and that improvement of remnant clearance halts atherosclerosis in a mouse model of T1D. Cholesteryl ester transfer protein (CETP), which is normally absent in mice, links metabolism of TRLs to that of HDL by mediating the transfer of cholesteryl ester from HDL to VLDL and other APOB-containing lipoproteins in exchange for triglycerides. We hypothesized that CETP impairs the atheroprotective effects of APOA1 by altering TRL and HDL metabolism in diabetes. To test this hypothesis, we developed a virally-induced T1D mouse model expressing the human APOA1 transgene ( APOA1 Tg , Ldlr –/– ). Cohorts of these mice were injected with a liver-targeted adeno-associated virus to express human CETP, and the effects on lipoproteins, plasma apolipoproteins, HDL particle concentrations (HDL-P), and atherosclerosis in the presence and absence of diabetes were determined. This mouse model exhibits an HDL profile similar to humans, with small, medium and large HDL-P, whereas wildtype ( Ldlr -/- ) littermates have only one sized HDL-P. Expression of the APOA1 transgene significantly suppressed diabetes-accelerated necrotic core expansion and accumulation of APOB and APOE in the aortic sinus (compared to wildtype Ldlr -/- mice), while CETP impaired the protective effect of APOA1 on these parameters in diabetic APOA1 Tg , Ldlr –/– mice (n= 22-30, p < 0.05). Further characterization of plasma lipoproteins and apolipoproteins by targeted mass spectrometry, FPLC and differential ion mobility analysis indicated that CETP leads to smaller TRL remnant particles, increases triglyceride content in HDL, lower levels of plasma APOA1 and reduction in large HDL-P under diabetic conditions (n= 8-10, p < 0.05). Our results are consistent with the proposal that inhibition of CETP might be cardioprotective in the setting of T1D and highlight the close links between TRL and HDL metabolism in diabetes.

Expressing the Human Cholesteryl Ester Transfer Protein Minigene Improves Diet-Induced Fatty Liver and Insulin Resistance in Female Mice.

Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with 3H-glucose tracer techniques. While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPARα target genes in fatty acid β-oxidation in the liver. The upregulated fatty acid β-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.

Human cholesteryl ester transfer protein lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

Inhibition of Cholesteryl Ester Transfer Protein Preserves High-Density Lipoprotein Cholesterol and Improves Survival in Sepsis.

The high-density lipoprotein hypothesis of atherosclerosis has been challenged by clinical trials of cholesteryl ester transfer protein (CETP) inhibitors, which failed to show significant reductions in cardiovascular events. Plasma levels of high-density lipoprotein cholesterol (HDL-C) decline drastically during sepsis, and this phenomenon is explained, in part, by the activity of CETP, a major determinant of plasma HDL-C levels. We tested the hypothesis that genetic or pharmacological inhibition of CETP would preserve high-density lipoprotein levels and decrease mortality in clinical cohorts and animal models of sepsis. We examined the effect of a gain-of-function variant in CETP (rs1800777, p.Arg468Gln) and a genetic score for decreased CETP function on 28-day sepsis survival using Cox proportional hazard models adjusted for age and sex in the UK Biobank (n=5949), iSPAAR (Identification of SNPs Predisposing to Altered Acute Lung Injury Risk; n=882), Copenhagen General Population Study (n=2068), Copenhagen City Heart Study (n=493), Early Infection (n=200), St Paul's Intensive Care Unit 2 (n=203), and Vasopressin Versus Norepinephrine Infusion in Patients With Septic Shock studies (n=632). We then studied the effect of the CETP inhibitor, anacetrapib, in adult female APOE*3-Leiden mice with or without human CETP expression using the cecal-ligation and puncture model of sepsis. A fixed-effect meta-analysis of all 7 cohorts found that the CETP gain-of-function variant was significantly associated with increased risk of acute sepsis mortality (hazard ratio, 1.44 [95% CI, 1.22-1.70]; P<0.0001). In addition, a genetic score for decreased CETP function was associated with significantly decreased sepsis mortality in the UK Biobank (hazard ratio, 0.77 [95% CI, 0.59-1.00] per 1 mmol/L increase in HDL-C) and iSPAAR cohorts (hazard ratio, 0.60 [95% CI, 0.37-0.98] per 1 mmol/L increase in HDL-C). APOE*3-Leiden.CETP mice treated with anacetrapib had preserved levels of HDL-C and apolipoprotein-AI and increased survival relative to placebo treatment (70.6% versus 35.3%, Log-rank P=0.03), whereas there was no effect of anacetrapib on the survival of APOE*3-Leiden mice that did not express CETP (50.0% versus 42.9%, Log-rank P=0.87). Clinical genetics and humanized mouse models suggest that inhibiting CETP may preserve high-density lipoprotein levels and improve outcomes for individuals with sepsis.

Novel role of cholesteryl ester transfer protein (CETP): attenuation of adiposity by enhancing lipolysis and brown adipose tissue activity

ObjectiveThe systemic function of CETP has been well characterized. CETP plasma activity reduces HDL cholesterol and thus increases the risk of atherosclerosis. Here, we investigated whether CETP expression modulate adiposity. MethodsBody adiposity and energy metabolism related assays and gene/protein expression were compared in CETP transgenic and non-transgenic mice and in hamsters treated with CETP neutralizing antibody. ResultsWe found that transgenic mice expressing human CETP present less white adipose tissue mass and lower leptinemia than nontransgenic (NTg) littermates. No differences were found in physical activity, food intake, fat fecal excretion, lipogenesis or exogenous lipid accumulation in adipose depots. Nonetheless, adipose lipolysis rates and whole-body energy expenditure were elevated in CETP mice. In accordance, lipolysis-related gene expression and protein content were increased in visceral and brown adipose tissue (BAT). In addition, we verified increased BAT temperature and oxygen consumption. These results were confirmed in two other animal models: 1) hamsters treated with CETP neutralizing antibody and 2) an independent line of transgenic mice expressing simian CETP. ConclusionsThese findings reveal a novel anti-adipogenic role for CETP.

The lipid transfer properties of CETP define the concentration and composition of plasma lipoproteins

Cholesteryl ester transfer protein (CETP) facilitates the net transfer of cholesteryl esters (CEs) and TGs between lipoproteins, impacting the metabolic fate of these lipoproteins. Previous studies have shown that a CETP antibody can alter CETP's preference for CE versus TG as transfer substrate, suggesting that CETP substrate preference can be manipulated in vivo. Hamster and human CETPs have very different preferences for CE and TG. To assess the effect of altering CETP's substrate preference on lipoproteins in vivo, here, we expressed human CETP in hamsters. Chow-fed hamsters received adenoviruses expressing no CETP, hamster CETP, or human CETP. Plasma CETP mass increased 2-fold in both the hamster and human CETP groups. Although the animals expressing human CETP still had low levels of hamster CETP, the CE versus TG preference of their plasma CETP was similar to that of the human ortholog. Hamster CETP overexpression had little impact on lipoproteins. However, expression of human CETP reduced HDL up to 50% and increased VLDL cholesterol 2.5-fold. LDL contained 20% more CE, whereas HDL CE was reduced 40%, and TG increased 6-fold. The HDL3:HDL2 ratio increased from 0.32 to 0.60. Hepatic expression of three cholesterol-related genes (LDLR, SCARB1, and CYP7A1) was reduced up to 40%. However, HDL-associated CE excretion into feces was unchanged. We conclude that expression of human CETP in hamsters humanizes their lipoprotein profile with respect to the relative concentrations of VLDL, LDL, HDL, and the HDL3:HDL2 ratio. Altering the lipid substrate preference of CETP provides a novel approach for modifying plasma lipoproteins.

Direct Delivery of Metformin to Subcutaneous White Adipose Tissue and Brown Adipose Tissue for Combating Obesity

Abstract Objectives Obesity and its comorbidities are major public health problems worldwide. The transformation of white adipose tissue (WAT) to brown adipose tissue (BAT); browning of WAT, may serve as a promising strategy for combating obesity. Metformin is not only the first line of drug for type 2 diabetes but also has an anti-obesity potential. Emerging evidence suggests that metformin can reduce body weight and enhance energy expenditure via activating BAT or browning of WAT. However, metformin delivery to adipose tissue is limited due to the lack of adipocyte-specific surface markers. Thus, the direct injection might be an alternative. Methods ApoE3-Leiden.human cholesteryl ester transfer protein (E3L.CETP) mice (5 mice/group) were fed a high-fat diet (HFD) for 15 weeks. From week 10 to 15, mice were randomly divided into 3 groups as 1. Metformin inguinal WAT (IgWAT) injection, 2. Metformin delivery to interscapular BAT (IBAT) and 3. Saline IgWAT injection (HFD control). Mice received injections twice per week (40 mg/kg/week). Bodyweight (BW), body composition, food intake, energy expenditure and glucose tolerance test (GTT) were measured. Gene expression of beige or brown makers was analyzed using real time-PCR. Results Compared to HFD control mice, IgWAT- and IBAT-treated mice lost 2.16% and 1.9% more of their body fat, respectively (P-value &amp;lt; 0.001). IgWAT- and IBAT-treated mice had 1.09- and 1.24-fold lower area under the curve calculated from the GTT time course than HFD control mice, respectively, but the differences were not statistically significant. The metabolic cage data indicated that both IgWAT- and IBAT-treated mice compared to HFD control mice had significantly decreased respiration exchange ratio (RER) (P &amp;lt; 0.0001). IgWAT-treated mice had significantly lower IgWAT weight than the HFD control mice (P &amp;lt; 0.05). IgWAT-treated compared to HFD control mice had 1.5-, 2-, 2.7- and 3-fold higher expression of UCP1, PRDM16, TMEM26 and Elovl3 in IgWAT, respectively. Conclusions This study demonstrated that local delivery of metformin to IgWAT and IBAT decreased BW and fat mass, which were associated with reduced RER and improved glucose homeostasis. Direct delivery of metformin to IgWAT and IBAT might be an efficient approach for combating obesity via inducing IgWAT browning and enhancing IBAT activity. Funding Sources NIH 1R15AT010395 and AHA 19AIREA34480011.

Alterations of lipid metabolism, blood pressure and fatty liver in spontaneously hypertensive rats transgenic for human cholesteryl ester transfer protein.

Cholesteryl ester transfer protein (CETP) mediates a step in reverse cholesterol transport, which channels cholesterol from peripheral tissues back to the liver. Mice and rats are CETP-deficient species, which assumedly contribute to rodent atherosclerosis resistance. Both pro- and anti-atherogenic effects have been shown in studies of CETP-transgenic rodent models thus far. As the results of pharmacological studies of CETP modification are largely controversial in humans, further knowledge about the impact of CETP on atherogenic phenotypes is required to evaluate its clinical utility for the prevention of cardiovascular and other organ damage associated with metabolic syndrome. Therefore, we newly generated a human CETP-transgenic (Tg[hCETP]) strain on the genetic background of spontaneously hypertensive rats (SHRs), which are characterized by the spontaneous occurrence of hypertension and insulin resistance. This allowed us to assess the in vivo role of CETP on cardiometabolic phenotypes in combination with hypertension. In Tg[hCETP] SHRs fed normal rat chow, systolic blood pressure was markedly elevated by 20-37 mmHg throughout the study period, and the development of fatty liver was accelerated with appreciable changes in the plasma lipid profile (HDL cholesterol reduction and triglyceride elevation). These phenotypic changes are in accordance with the assumption of proatherogenic effects inducible by the overexpression of CETP. However, with plasma LDL cholesterol levels concomitantly reduced, no apparent progression of atherosclerosis was detected in either the aorta or coronary arteries of Tg[hCETP] SHRs fed a high-fat, high-cholesterol diet. Our data provide new insight into the multifaceted regulation of cardiometabolic phenotypes via the modification of CETP.

Small HDL, diabetes, and proinflammatory effects in macrophages

Atherosclerosis, an inflammatory disease of the arterial wall, is the commonest cause of cardiovascular disease (CVD) events. Elevated levels of high‐density lipoprotein (HDL) cholesterol are generally believed to protect against CVD and to reduce inflammatory activation of macrophages, a key cell type in lesions of atherosclerosis. However, HDL can also exert proinflammatory effects in macrophages by inducing cholesterol depletion. Because small HDL (7–8.5 nm diameter) is most effective in removing cholesterol from cells by the ABCA1 pathway, we hypothesized that small HDL is the most proinflammatory HDL population. Small‐, medium‐ and large HDL populations were isolated from a human apolipoprotein A‐I transgenic LDL receptor‐deficient mouse model (APOA1Tg;Ldlr−/−). Depletion of cholesterol by HDL increased the ability of toll‐like receptor (TLR) ligands to induce proinflammatory mediators in macrophages. This effect was prevented by cholesterol loading of the macrophages. Small HDL had a significantly stronger effect on Tnfa (3‐fold over LPS alone) and Il1b (2.5‐fold over LPS alone) gene expression than medium or large HDL particles. Inhibition or deletion of the sheddase ADAM17 in macrophages prevented the proinflammatory effects of HDL. Patients with type 1 diabetes mellitus (T1DM) exhibit an increased risk of CVD despite having normal or high HDL‐cholesterol. To investigate the effect of diabetes on HDL subpopulations, we crossed a virally‐induced mouse model of T1DM (Ldlr−/−;GpTg) with the human APOA1Tg;Ldlr−/− mouse. The mice (APOA1Tg;Ldlr−/−;GpTg, n=8–10) were injected with a liver‐specific adeno‐associated virus driving expression of human cholesteryl ester transfer protein (hCETP) to mimic human physiology. Diabetic mice had an increased HDL particle concentration (~2‐fold) associated with a significant increase in serum HDL cholesterol efflux capacity through both ABCA1 and ABCG1, compared with non‐diabetic mice. The largest increase was in the small HDL particle concentration (15 μM vs 6 μM in non‐diabetic mice). Moreover, resident peritoneal macrophages from diabetic mice exhibited increased cytokine gene expression, as compared with non‐diabetic littermates. Our findings demonstrate that higher levels of small HDL, as observed in a mouse model of diabetes, can exacerbate inflammatory activation in non‐lipid loaded macrophages in an ADAM17‐dependent manner. We have found similar changes in HDL concentration and function in humans with T1DM, suggesting that our mouse model might provide novel insights into CVD risk in T1DM patients.Support or Funding Informationfunded in part by the National Institutes of Health, grants DP3DK108209, P01HL092969, P30 DK017047, and P01HL128203This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of Increased LDL (Low-Density Lipoprotein) and Decreased Triglycerides With SGLT2 (Sodium-Glucose Cotransporter 2) Inhibition

Objective- SGLT2 (sodium-glucose cotransporter 2) inhibition in humans leads to increased levels of LDL (low-density lipoprotein) cholesterol and decreased levels of plasma triglyceride. Recent studies, however, have shown this therapy to lower cardiovascular mortality. In this study, we aimed to determine how SGLT2 inhibition alters circulating lipoproteins. Approach and Results- We used a mouse model expressing human CETP (cholesteryl ester transfer protein) and human ApoB100 (apolipoprotein B100) to determine how SGLT2 inhibition alters plasma lipoprotein metabolism. The mice were fed a high-fat diet and then were made partially insulin deficient using streptozotocin. SGLT2 was inhibited using a specific antisense oligonucleotide or canagliflozin, a clinically available oral SGLT2 inhibitor. Inhibition of SGLT2 increased circulating levels of LDL cholesterol and reduced plasma triglyceride levels. SGLT2 inhibition was associated with increased LpL (lipoprotein lipase) activity in the postheparin plasma, decreased postprandial lipemia, and faster clearance of radiolabeled VLDL (very-LDL) from circulation. Additionally, SGLT2 inhibition delayed turnover of labeled LDL from circulation. Conclusions- Our studies in diabetic CETP-ApoB100 transgenic mice recapitulate many of the changes in circulating lipids found with SGLT2 inhibition therapy in humans and suggest that the increased LDL cholesterol found with this therapy is because of reduced clearance of LDL from the circulation and greater lipolysis of triglyceride-rich lipoproteins. Most prominent effects of SGLT2 inhibition in the current mouse model were seen with antisense oligonucleotides-mediated knockdown of SGLT2.

Abstract 167: Mechanism of Increased Low Density Lipoprotein Cholesterol and Decreased Triglycerides with Sodium-glucose Co-transporter 2 Inhibition

Objective: Although Sodium glucose cotransporter 2 (SGLT2) inhibition in humans sometimes leads to increased levels of low density lipoprotein (LDL) cholesterol, this therapy is associated with marked reduction in cardiovascular disease. In this study, we aimed to determine how SGLT2 inhibition alters circulating lipoproteins. Approach and Results: We used a mouse model in which plasma lipoprotein profiles were humanized by expression of human cholesteryl ester transfer protein and human apolipoprotein B100 to determine how SGLT2 inhibition alters lipoprotein profiles. The mice were fed a high fat diet and then were made partially insulin deficient using streptozotocin. SGLT2 was inhibited using a specific anti-sense oligonucleotide or canagliflozin, a clinically available oral SGLT2 inhibitor. Inhibition of SGLT2 increased circulating levels of LDL cholesterol and reduced plasma triglyceride levels. SGLT2 inhibition was associated with increased lipoprotein lipase activity in the post heparin plasma and decreased postprandial lipemia. The changes in lipoprotein metabolism were more significant in mice treated with SGLT2 ASO compared to canagliflozin. Conclusions: Our studies in mice recapitulate many of the changes in circulating lipids found with SGLT2 inhibition therapy in humans and suggest that the increased LDL cholesterol found with this therapy is due to greater lipolysis of triglyceride-rich lipoproteins. This change in lipoprotein physiology is likely, in part, to explain the reduced cardiovascular disease found with SGLT2 inhibition.

Abstract 390: A New Humanized Mouse Model to Study Lipid Abnormalities and HDL Function in Poorly Controlled Type 1 Diabetes Mellitus

Elevated plasma triglycerides (TG) and VLDL are often present in poorly controlled type 1 diabetes mellitus (T1DM). Furthermore, T1DM patients exhibit an increased risk of cardiovascular disease (CVD) despite normal or high HDL-cholesterol. There is a need for new mouse models to study T1DM lipid abnormalities because mice do not express cholesteryl ester transfer protein (CETP), which transfers cholesteryl ester (CE) from HDL to VLDL and other lipoproteins in exchange for TG, and because mice do not exhibit human-like HDL populations. To develop a physiologically relevant model, we crossed a virally-induced mouse model of T1DM ( Ldlr -/- ;Gp Tg ) deficient in the LDL receptor with human apolipoprotein A-I ( APOA1 ) transgenic mice. These mice (n=8-10) were injected with a liver-specific adeno-associated virus driving expression of human CETP, mimicking the human CETP activity/apoA-I ratio, and the 3 major human HDL subpopulations. Using this model ( APOA1 Tg ;Ldlr -/- ;Gp Tg ), we examined the impact of diabetes on dyslipidemia, HDL particle concentration (HDL-P), and serum HDL function. Induction of diabetes increased plasma TG and cholesterol, primarily due to an increase in VLDL, as compared with non-diabetic (ND) littermates. The dyslipidemia was associated with increases in hepatic TG content and hepatic TG production rate (2-fold over ND). Moreover, resident peritoneal macrophages from diabetic mice exhibited a 5-fold increase in CE loading, increased lipid raft content (20%), and increased Tnfa (3-fold) and Il1b mRNA (4-fold), as compared with ND littermates. In addition, T1DM mice had increased small, medium and large HDL-P (2-fold) concentrations compared with ND controls. Serum HDL from T1DM mice showed a significant increase in its cholesterol efflux capacity through both ABCA1 and ABCG1. Thus, in this humanized mouse model, poorly controlled T1DM leads to elevated plasma TG and VLDL due in part to increased hepatic TG production. This dyslipidemia is associated with higher HDL-P and an increase in serum HDL’s cholesterol efflux capacity, likely due to the increased plasma HDL-P. We have found similar changes in HDL concentration and function in humans with T1DM, suggesting that our mouse model might provide insights into CVD risk in T1DM patients.

Corrigendum to "Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits".

[This corrects the article DOI: 10.1155/2017/3824276.].

A novel swine model for evaluation of dyslipidemia and atherosclerosis induced by human CETP overexpression

BackgroundThe mechanism of cholesteryl ester transfer protein (CETP) in lipid metabolism is still unclear. Furthermore, the relationship of CETP and atherosclerosis (AS) has been controversial. As pigs are a good model for both lipid and AS research, we investigated the lipid metabolism of human CETP (hCETP) transgenic pigs and explored the mechanism of CETP in lipid modulation.MethodsPlasmids expressing the hCETP gene were designed, successfully constructed, and transfected into porcine fetal fibroblasts by liposomes. Using somatic cell nuclear transfer technology and embryonic transfer, hCETP transgenic pigs were generated. After the DNA, RNA, and protein levels were identified, positive hCETP transgenic pigs were selected. Blood samples were collected at different ages to evaluate the phenotypes of biochemical markers, and the metabolomes of plasma samples were analyzed by liquid mass spectrometry.ResultsEight positive hCETP transgenic pigs and five negative cloned pigs were generated by transgenic technology. Finally, five hCETP transgenic and five cloned pigs were grown healthily. After feeding with a normal diet, hCETP transgenic pigs compared with unmodified pigs had no significant differences in body weight, liver function, kidney function, or plasma ions, while total cholesterol and low-density lipoprotein were higher than in unmodified pigs, and high-density lipoprotein was significantly decreased. Metabolomics analysis showed that there were differences in metabolic components between hCETP transgenic pigs, cloned pigs, and unmodified pigs.ConclusionsIn this study, we created hCETP transgenic pigs that could serve as an excellent model for lipid disorders and atherosclerosis.

Discovery of pentacyclic triterpene 3β-ester derivatives as a new class of cholesterol ester transfer protein inhibitors

A series of pentacyclic triterpene 3β-ester derivatives were designed, synthesized and evaluated as a new class of cholesteryl ester transfer protein (CETP) inhibitors for the treatment of dyslipidemia. In vitro screening assay showed that 5 out of 30 compounds displayed moderate inhibiting human CETP activity with IC50s less than 10 μM. Among them, compound 20 (IC50 = 2.3 μM) had the most potent biological activity, and effectively ameliorated plasma lipid levels of human adipose tissue specific CETP transgenic (ap2-CETPTg) mice and guinea pigs. Additional safety evaluation (no blood pressure elevation in guinea pigs) and pharmacokinetics studies indicated that the potential druggability for compound 20 which is a promising lead for development of a new class of CETP inhibitors for the treatment of dyslipidemia.

Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits.

High levels of plasma high-density lipoprotein-cholesterol (HDL-C) are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP) is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg) rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.