High Cholesterol Diet Research Articles

Deficiency of germinal center kinase TRAF2 and NCK-interacting kinase (TNIK) in B cells does not affect atherosclerosis.

Atherosclerosis is the underlying cause of many cardiovascular diseases, such as myocardial infarction or stroke. B cells, and their production of pro- and anti-atherogenic antibodies, play an important role in atherosclerosis. In B cells, TRAF2 and NCK-interacting Kinase (TNIK), a germinal center kinase, was shown to bind to TNF-receptor associated factor 6 (TRAF6), and to be involved in JNK and NF-κB signaling in human B cells, a pathway associated with antibody production. We here investigate the role of TNIK-deficient B cells in atherosclerosis. ApoE-/-TNIKfl/fl (TNIKBWT) and ApoE-/-TNIKfl/flCD19-cre (TNIKBKO) mice received a high cholesterol diet for 10 weeks. Atherosclerotic plaque area did not differ between TNIKBKO and TNIKBWT mice, nor was there any difference in plaque necrotic core, macrophage, T cell, α-SMA and collagen content. B1 and B2 cell numbers did not change in TNIKBKO mice, and marginal zone, follicular or germinal center B cells were unaffected. Total IgM and IgG levels, as well as oxidation specific epitope (OSE) IgM and IgG levels, did not change in absence of B cell TNIK. In contrast, plasma IgA levels were decreased in TNIKBKO mice, whereas the number of IgA+ B cells in intestinal Peyer's patches increased. No effects could be detected on T cell or myeloid cell numbers or subsets. We here conclude that in hyperlipidemic ApoE-/- mice, B cell specific TNIK deficiency does not affect atherosclerosis.

Concomitant western diet and chronic-binge alcohol dysregulate hepatic metabolism.

There is significant overlap between non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) with regards to risk factors and disease progression. However, the mechanism by which fatty liver disease arises from concomitant obesity and overconsumption of alcohol (syndrome of metabolic and alcohol-associated fatty liver disease; SMAFLD), is not fully understood. Male C57BL6/J mice were fed chow diet (Chow) or high-fructose, high-fat, high-cholesterol diet (FFC) for 4 weeks, then administered either saline or ethanol (EtOH, 5% in drinking water) for another 12 weeks. The EtOH treatment also consisted of a weekly 2.5 g EtOH/kg body weight gavage. Markers for lipid regulation, oxidative stress, inflammation, and fibrosis were measured by RT-qPCR, RNA-seq, Western blot, and metabolomics. Combined FFC-EtOH induced more body weight gain, glucose intolerance, steatosis, and hepatomegaly compared to Chow, EtOH, or FFC. Glucose intolerance by FFC-EtOH was associated with decreased hepatic protein kinase B (AKT) protein expression and increased gluconeogenic gene expression. FFC-EtOH increased hepatic triglyceride and ceramide levels, plasma leptin levels, hepatic Perilipin 2 protein expression, and decreased lipolytic gene expression. FFC and FFC-EtOH also increased AMP-activated protein kinase (AMPK) activation. Finally, FFC-EtOH enriched the hepatic transcriptome for genes involved in immune response and lipid metabolism. In our model of early SMAFLD, we observed that the combination of an obesogenic diet and alcohol caused more weight gain, promoted glucose intolerance, and contributed to steatosis by dysregulating leptin/AMPK signaling. Our model demonstrates that the combination of an obesogenic diet with a chronic-binge pattern alcohol intake is worse than either insult alone.

Probiotics supplementation attenuates high cholesterol diet-induced hypercholesterolemia in rat model

Hypercholesterolemia is characterized by elevated total serum cholesterol levels, reflecting a high LDL (low-density lipoprotein cholesterol) level, a high TG (triglyceride) level and a low HDL (high-density lipoprotein cholesterol) level. Hypercholesterolemia may cause cardiovascular diseases such as heart attack, stroke and coronary artery diseases. Nowadays, several drugs like statins that lower serum cholesterol are used to treat hypercholesterolemia. However, the cholesterol-lowering drugs have side effects including liver damage and kidney failure. For these reasons, alternatives like probiotics are expected to attenuate hypercholesterolemia instead. Recent studies have shown that probiotics protect against hypercholesterolemia and liver dysfunction via gut-liver axis. Probiotics such as lactic acid bacteria (LAB) may modulate gut microbiota, regulate lipid metabolism and lead to alleviate liver disease. This study aims to determine whether LABs have a protective effect on hypercholesterolemia. LAB strains were selected by acid tolerance, bile tolerance, and antibiotic activity through in vitro screening. 6-week SD rats were fed normal diet or high cholesterol diet (HCD) for 7 weeks. Treatment groups were fed HCD with statin and LABs respectively. LAB supplements effectively improved serum lipid profiles. LABs prevented HCD-induced intestinal and liver damage by enhancing tight junction, reducing inflammation. LABs suppressed lipid accumulation in the liver and enhanced the inhibition of the liver toxicity. Also, LAB supplements changed HCD-induced gut microbiota composition and influenced diversity of gut microbiome. The concentration of bile acid and short chain fatty acids in feces were increased by LAB supplements. Thus, results suggest that LABs are potential therapeutic agents for HCD-induced intestine and liver dysfunction. This work is supported by Cellbiotech Co., Ltd. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Toll-like receptor 4 activation contributes to systemic vascular dysfunction in pregnant rats fed a high cholesterol diet during pregnancy

Background: Hypercholesterolemia is a hallmark clinical feature of cardiovascular diseases. Vascular adaptations to pregnancy include withstanding pregnancy-induced hypercholesterolemia, a physiological process required for fetus development. However, excessive (pathological) hypercholesterolemia during pregnancy has been linked to an increased risk of complications (e.g., preeclampsia); still, the underlying mechanisms are unclear. Toll-like receptor 4 (TLR4) is an innate immune receptor modulated by high cholesterol levels (in non-pregnant animals), but whether TLR4 activation also plays a role in vascular dysfunction in pregnancies complicated with hypercholesterolemia is unknown. Hypothesis: Pathological hypercholesterolemia during pregnancy causes maternal vascular dysfunction via activation of TLR4. Methods: Sprague Dawley rats were fed a standard (CD) or high cholesterol diet (HCD; 2% cholesterol and 0.5% cholic acid) from gestational day (GD) 6 to GD20 (term=22 days; n=9-10). Blood pressure was assessed before pregnancy and on GD19, and the results are presented as delta change. On GD20, body weight and pregnancy outcomes were recorded, and mesenteric arteries (systemic resistance arteries) were assessed using wire myography to evaluate vasoconstriction capacity to phenylephrine and endothelial-dependent vasodilation responses to methacholine. Experiments were conducted in the presence or absence of L-NAME (a pan-NOS inhibitor, 100 mmol/l) and/or CLI-095 (a TLR4 inhibitor, 10 mmol/l). Functional data were summarized as Emax (maximum response) or pEC50 (concentration required to produce 50% of Emax). Statistics: Student’s t-test or two-way ANOVA with Sidak post hoc test (significance: p<0.05). Results: The HCD prevented the late gestation systolic (p=0.0004) and diastolic (p=0.0066) blood pressure reduction observed in the CD group, and impaired male (p=0.0062) and female (p=0.0352) fetoplacental efficiency. Total body weight gain during pregnancy was not different between CD and HCD dams (p>0.05). In mesenteric arteries, vasoconstriction responses to phenylephrine (Emax) were increased in HCD dams (p=0.0056), and TLR4 inhibition with CLI-095 reduced the sensitivity (pEC50) of phenylephrine in these vessels (p=0.0147). Additionally, HCD dams presented higher sensitivity (pEC50) to methacholine (p=0.0169), which was not modified by the TLR4 inhibitor (p>0.05). Incubation of arteries with L-NAME abolished the increased sensitivity to methacholine (p>0.05), which was not further altered by CLI-095 (p>0.05). Conclusion: Inhibition of TLR4 ( ex vivo) reduced the enhanced vasoconstriction observed in HCD dams but did not alter endothelial-dependent vasodilation responses. Thus, if inhibiting TLR4 could block the vasoconstrictor effects of excessive hypercholesterolemia during pregnancy, this may be a target for future therapy development. This work was supported by a foundation grant from CIHR (FS154313) and by the generosity of the Stollery Children's Hospital Foundation and the Alberta Women's Health Foundation through the Women and Children's Health Research Institute (WCHRI). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 592: Marfan Syndrome Mice Fed On High Fat Diet Have An Altered Metabolism

Introduction: Marfan syndrome (MFS) is a genetic disorder, caused by different mutations in the fibrillin-1 gene ( FBN1 ) and affects the connective tissue. Patients suffer from a wide range of clinical symptoms at the cardiovascular, skeletal and ocular level. However, MFS patients are also often characterized as very slender. Body weight gain in these patients can be a challenge, and the reason for this is still not fully understood. Objective: To determine if individuals with MFS have an altered metabolism. Methods: 9-11 weeks old male Fbn1 C1041G/+ (MFS) mice and wild-type littermates were given either a chow, high cholesterol diet (HCD) or a high fat diet (HFD), for 12 weeks. These mice were then housed individually in a metabolic cage for 1 week before euthanasia was performed. Results: HFD resulted in higher body weights compared to chow diet, and the HFD groups became insulin resistant as measured by the intraperitoneal glucose tolerance test. However, body weight gain was not different in this MFS mouse model, as compared to WT mice, regardless of diet. The HFD groups had increased fat weight compared to chow groups. Interestingly, MFS mice had an enhanced heart weight compared to WT mice, independent of the diet. As expected, aortic root diameters were enlarged in MFS compared to WT mice. However, in the ascending aorta there was a significant increase in diameter in the WT HFD as compared to WT chow, indicating that enhanced lipid levels promote aortic growth. Metabolic cage data showed that the HFD groups had a lower respiratory exchange ratio (RER), indicating that HFD groups used fat as a predominant source of fuel and that chow groups used primarily carbohydrates as an energy source. MFS mice had overall lower RER compared to WT mice, revealing that MFS mice use more lipids under any condition, most likely to manage the significantly elevated activity in the MFS mice. Conclusion: Taken together, MFS mice seem metabolically different.

The effect of evolocumab alone and in combination with atorvastatin on atherosclerosis progression and TLRs expression.

Evolocumab, a PCSK-9 inhibitor, is known for its ability to reduce low-density lipoprotein cholesterol (LDL-C). This study aimed to investigate the effects of evolocumab, alone or in combination with atorvastatin, on the progression of atherosclerosis. Fifty male domestic rabbits were randomly assigned to five groups: control, high cholesterol diet, evolocumab vehicle (dimethyl sulfoxide, DMSO), evolocumab alone, and evolocumab plus atorvastatin. Serum levels of interleukin 10 (IL-10), IL-17, IL-1β, intracellular adhesion molecule (ICAM), and vascular adhesion molecule (VCAM) were measured. Toll-like receptor (TLR) expression on monocytes was evaluated using flow cytometry. Histopathological examination and measurement of intimal thickness (IT) were also conducted. The results revealed that the evolocumab produced a statistically significant (p<0.05) reduction in lipid profile at 5 weeks, with the peak effect occurring at 10 weeks. Furthermore, the inhibitor reduced TLRs at 10 weeks to 10.83±1.8 and intimal thickness to 160.66±9.45. IL-17, IL-1β, ICAM, and VCAM were significantly reduced by evolocumab treatment, with the improvement of the histopathological changes in the aortic wall. The combination of evolocumab and atorvastatin caused a more statistically significant reduction in TLRs at 10 weeks to 5.08±1.2 and intimal thickness to 121.79±5.3. IL-17, IL-1β, ICAM, and VCAM were significantly (p<0.05) reduced by the combination, and the histopathological changes in the aortic wall were significantly improved. In conclusion, evolocumab delays the progression of atherosclerosis by modulating inflammatory pathways.

Bifidobacterium supplementation attenuates hypercholesterolemia induced gut-brain-testis dysfunction in a rat model

Hypercholesterolemia is the state of high LDL blood cholesterol levels. This may lead to cholesterol accumulation in organs and cause diseases such as male infertility and NAFLD. Recent studies have shown strong correlations between Bifidobacterium strains and plasma cholesterol reduction. Bifidobacterium species are gram positive bacteria known to colonize the human gastrointestinal tract. Studies suggest that Bifidobacterium may reduce serum cholesterol levels by increasing bile salt hydrolase (BSH) activity. BSH increases fecal excretion of bile salt, and to compensate the loss of circulating bile acids, productions of bile acids using cholesterol are promoted. Currently, medications such as statins are commonly used. However, long term consumption could cause adverse effects on male reproductive organs. Bifidobacterium strains are known to regulate the hypothalamus-pituitary-gonadal (HPG) axis by modulating gut microbiota . Gut microbial bacteria such as Bifidobacterium contribute to the host metabolism by enhancing gut barrier integrity, reducing inflammation cytokine expressions, and increasing metabolite productions such as short chain fatty acids (SCFAs). Particularly, SCFAs like butyrate can cross the blood-brain barrier, and consequently affect the HPG axis. This suggests that Bifidobacterium strains may lower serum cholesterol while protecting reproductive organs. Therefore, this study aimed to evaluate and compare the serum cholesterol lowering and HPG axis protecting effects of Bifidobacterium strains with statin in the gut-brain-testis axis of a high cholesterol diet (HCD) induced hypercholesterolemia rat model. 6-week oral gavage of Bifidobacterium strains known to have BSH activity effectively reduced serum total cholesterol and LDL cholesterol levels in rats given HCD for the same period. Moreover, serum inflammatory cytokine IL-1β was reduced by Bifidobacterium treatment. Bifidobacterium also exerted protective effects against intestinal, testicular, and cerebral damage by attenuating disruption of histology and modulating transcript levels of inflammation markers. Notably, Bifidobacterium alleviated the HCD-induced reduction of male sex hormone synthesis markers in the testis, which statin had no effect. Fecal analysis revealed that Bifidobacterium treatment modulates gut microbiota compositions and metabolite levels such as SCFAs. Particularly, 16s rRNA genomic annotation showed that Bifidobacterium strains may have specific functions related to cholesterol reduction. Altogether, Bifidobacterium supplementation may reduce serum cholesterol levels by altering gut microbiota compositions, which affect gut metabolite compositions, inflammatory markers, and sex hormone synthesis markers. In conclusion, Bifidobacterium supplementation is a potential therapeutic approach for hypercholesterolemia-induced intestine, brain, and testis dysfunction. This work was supported by Cellbiotech Co., Ltd. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 107: CaMK4 is a Novel Regulator of Myeloid Phenotype and Function in Atherosclerosis

Background: Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent work by our group and others has demonstrated that advanced atherosclerosis is also characterized by the failure of an active repair process termed ‘inflammation resolution’. Strategies that boost resolution and break the cycle of chronic inflammation promotes plaque stability. Hypothesis: Our review of publicly available RNAseq data revealed an increase in expression of Calcium/calmodulin-dependent protein kinase 4 (CaMK4) in advanced/unstable regions of plaque within human carotid arteries as well as in myeloid cells derived from atherosclerotic murine aortas. Therefore, we hypothesized that CaMK4 promotes inflammation and impairs resolution. Results and Methods: Control and Camk4 -/- mice were injected with AAV-8 PCSK9 virus and fed a high-fat high-cholesterol Western diet for 12 weeks. Cross-sectional analysis of aortic roots showed that Camk4 -/- mice had significantly less plaque burden than Controls. Flow cytometric analysis revealed elevated monocyte numbers in Camk4 -/- mice compared to Control mice. Further analysis demonstrated increased skewing of Camk4 -/- monocyte populations toward a Ly6c low subset, indicating a more pro-reparative phenotype compared to Control mice. We considered whether CaMK4 may regulate a gene signature driving monocyte conversion and found that Camk4 -/- monocytes expressed higher levels of Nr4a1, Cebpb, and Klf2, which have been shown to promote conversion to Ly6c low monocytes. As Ly6c low monocytes give rise to pro-reparative macrophages, we generated bone marrow-derived macrophages and found that loss of CaMK4 in vitro led to higher levels of pro-reparative cytokines being produced in response to LPS stimulation as well as more efficient clearance of apoptotic cells when compared to Controls. Conclusions: Overall, these findings suggest that CaMK4 regulates myeloid phenotype in vitro and in vivo and that loss of CaMK4 promotes pro-resolving macrophage function. Therefore, targeting CaMK4 may offer a unique way to target atheroprogression.

Wheat Germ Supplementation Attenuates Obesity-Induced Neuroinflammation in the RVLM of IL-10 KO mice

Obesity is known to be a major factor in the increase of cardiovascular risk and inflammatory responses in the brain. It has been found that high-fat diet (HFD)-induced obesity increases sympathetic nerve activity (SNA) through a cascade of chronic metabolic processes such as an increase in oxidative stress and inflammation. The rostral ventrolateral medulla (RVLM) is a specific part of the brainstem that regulates SNA. Wheat germ (WG), a rich source of bioactive compounds including polyunsaturated fatty acid, has been shown to have an anti-inflammatory effect on the gut environment of HFD-fed mice. A healthy gut microbiota has been shown to have a regulatory effect on the gut-brain axis, especially under stress. In this study, we investigated the effects of WG supplementation on RVLM inflammation in high-fat high cholesterol diet (HFHC)-fed IL-10 KO mice. Six-week-old female C57BL/6 (WT) and IL-10 KO mice ( n = 4-5/group) were randomly assigned to treatment groups: WT and IL-10 KO fed a control diet (10% fat kcal), IL-10 KO fed HFHC (43.4% fat kcal, 1% cholesterol) diet with or without 10% WG for 12 weeks. At the end of 12 wks of treatment, animals were sacrificed and RVLM mRNA expression of proinflammatory cytokines and chemokine were analyzed using qPCR. HFHC increased the expression of IL1b, TNFa, and MCP-1 in the RVLM. WG supplementation was able to reverse the mRNA expression of IL1b (p&lt;0.05), TNFa (p&lt;0.05), and MCP1 (p&lt;0.05). These results indicate that HFHC diet-induced obesity increases inflammation in the RVLM and WG supplementation could reverse these effects. Future studies would focus on understanding the mechanisms by which WG reverses the inflammation in the brain. NIH R15HL148844, AHA AIREA959725, and Oklahoma Agriculture Experiment Station 2153907 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 683: Improved Myocardial Blood Flow And Arteriogenesis With Calpain Inhibition May Be Mediated By Vascular Endothelial Growth Factor And Fibroblast Growth Factor Angiogenic Pathways In A Large-animal Model Of Metabolic Syndrome And Chronic Myocardial Ischemia

Objective: Ischemic heart disease is the leading cause of death worldwide. Under pathological conditions like myocardial ischemia, endothelial calpain proteases are activated by hypoxia, leading to aberrant angiogenesis. Our group has shown calpain inhibition increases myocardial blood flow and vascular density, microvascular vasodilation, and expression of proangiogenic vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1, VEGF-R2) in a porcine model of chronic myocardial ischemia and diet-induced metabolic syndrome. We aim to assess the role of downstream factors in angiogenic pathways. Methods: Yorkshire swine were fed a high cholesterol diet for four weeks, and then underwent placement of an ameroid constrictor to the left circumflex artery. After three weeks, pigs received either daily calpain inhibitor MDL28170 (0.25mg/kg, oral) for five weeks (n = 8) or no drug (n = 7), followed by tissue harvest of ischemic myocardium. Results: Treatment was associated with a significant increase in expression of proangiogenic mediators, fibroblast growth factor (FGF) receptor 1 (FGFR1, p &lt;0.001) and matrix metalloprotease-2 (MMP-2, p &lt;0.001). Cathepsins D and L have been implicated in both pro- and antiangiogenic actions. Cathepsin D was significantly increased (p = 0.004), but Cathepsin L was unchanged (p &gt;0.99). Cathepsin L proenzyme, procathepsin L, was significantly decreased (p = 0.04), while there was a strong trend toward decreased procathepsin L:cathepsin L ratio (p = 0.09). Proangiogenic Tie-2 (p = 0.28), and antiangiogenic collagen 18 (p = 0.54) and angiostatin (p = 0.24) were unchanged. Conclusions: In metabolic syndrome, calpain inhibition led to increased proangiogenic signaling in chronically ischemic myocardium, suggesting angiogenic VEGF- and FGF-mediated pathways may work in concert with improved microvascular vasodilation to explain the observed increase in vascular density and collateral-dependent coronary perfusion.

Angiotensin receptor blocker ameliorates hypertension, improves glucose intolerance, and reduces visceral fat in OLETF rats fed a high-cholesterol diet

Metabolic Syndrome (MetS) affects more than 30% of the US population and unmanaged can lead to cardiovascular disease and type II diabetes mellitus. MetS is a cluster of conditions, which include elevated arterial pressure, poor glucose tolerance with or without insulin resistance, and abdominal obesity. Existing literature has demonstrated high cholesterol diet’s (HCD) potential to develop MetS cluster factor conditions like dyslipidemia. Our lab has previously shown that angiotensin receptor blockers (ARBs), a common therapy to treat hypertension by disrupting the renin-angiotensin-aldosterone system (RAAS), attenuate cluster factor conditions of MetS. However, the efficacy of ARBs during the consumption of a HCD remains unclear. Otsuka Long Evan Tokushima Fatty (OLETF) rats (a MetS model) were fed an HCD and at presentation of elevated arterial pressure, they were treated with ARB (10 mg olmesartan/kg/d x 3 wks) by oral gavage. Blood pressure was monitored in real-time with surgically implanted radio telemeters. ARB reduced systolic blood pressure by 15±1mmHg (11%; p&lt;0.05). ARB also reduced BM by 3% (p&lt;0.05) in chow-fed and 4% (p&lt;0.05) in HCD-fed OLETF compared to OLETF. However, there was no difference in BM between OLETF+ARB and OLETF+HCD+ARB. Neither, HCD nor ARB alone reduced abdominal fat in OLETF rats. Although, ARB in OLETF+HCD induced visceral fat loss (37%; p&lt;0.05) compared to OLETF. Additionally, ARB reduced AUCglucose by 28% (P&lt;0.05) and tended to reduce fasting blood glucose by 19% (p=0.06). These findings demonstrate that chronic treatment of ARB can effectively ameliorate MetS-associated hypertension, hyperglycemia, and abdominal obesity despite the additional insult of a HCD. This work was supported by the National Institutes of Health NCMHD 9T37MD001480 and National Institutes of Health, National Center on Minority Health and Health Disparities. E.E.J was supported by NIH GRISE BIO-STeP. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Human Genetics and Dyslipidemia: From Non-synonymous Variants to Non-coding Variants

Dyslipidemia has strong heritability, with over 50% of dyslipidemia is caused by genetics in general population. Previously studies identify many non-synonymous variants in genes of LDLR, PCSK9, ANGPTL3, ABCG5/8, ARH et.al, which lay the fundation for durg development. However, these known non-synonymous variants can only explain about 10-20% of genetic risks. Genome-wide association studies (GWAS) have identified hundreds of genetic variants associated with dyslipidemia in large population. However, about 95% of these variants are located in genome noncoding regions and cluster in different loci. The disease-causing variant for each locus and underline mechanism remain largely unknown. We systematically analyzed these noncoding variants and found that rs1997243 is the disease-causing variant in locus 7p22, which is strongly associated with hypercholesterolemia. The rs1997243 risk allele is associated with increased expression of GPR146 in human and specifically up regulates GPR146 expression in cultured hepatocytes. GPR146 is an orphan G-protein coupled receptor that is located on plasma membrane and responses to stimulation of heat-inactivated serum. Disrupting gpr146 specifically in the liver decreases the blood cholesterol level and prevents high-fat or high-fat high-cholesterol diets induced hypercholesterolemia in mice. Mechanistic study suggests that GPR146 regulates blood cholesterol level mainly through regulating the catabolism of cholesterol rich lipoprotein particles. Thus we uncovered a novel G-protein coupled receptor that regulates blood cholesterol levels in both humans and mice. Our results also suggest that antagonizing GPR146 function will be an effective strategy to treat hypercholesterolemia. National Key Research and Development Program of China (2018YFA0800700), the National Natural Science Foundation of China (92057207, 91754101, 32021003, 31771304) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of immune system humanization on atherosclerosis in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO/LDL-R KO mice

Aim: Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing. Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background. Methods: LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse TKO-LDLR KO recipient of human hematopoietic stem cells (hCD34+). Results: TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both males and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next, we tested the impact of immune system humanization. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. Conclusions: We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mouse. This mouse model presents human B and T cells and could represent an important tool to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients.A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Galanin ameliorates liver inflammation and fibrosis in mice by activating AMPK/ACC signaling and modifying macrophage inflammatory phenotype.

Galanin is a naturally occurring peptide that plays a critical role in regulating inflammation and energy metabolism, with expression in the liver. The exact involvement of galanin in non-alcoholic fatty liver disease and related fibrosis remains controversial. The effects of subcutaneously administered galanin were studied in mice with non-alcoholic steatohepatitis (NASH) induced by a high-fat and high-cholesterol diet for 8 weeks, and in mice with liver fibrosis induced by CCl4 for 7 weeks. The underlying mechanism was also studied in vitro on murine macrophage cells (J774A.1 and RAW264.7). Galanin reduced inflammation, CD68-positive cell count, MCP-1 level, and mRNA levels of inflammation-related genes in the liver of NASH mice. It also mitigated liver injury and fibrosis caused by CCl4. In vitro, galanin had anti-inflammatory effects on murine macrophages, including reduced phagocytosis and intracellular reactive oxygen species (ROS). Galanin also activated AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling. Galanin ameliorates liver inflammation and fibrosis in mice, potentially by modifying macrophage inflammatory phenotype and activating AMPK/ACC signaling.

The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested.To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr−/−)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment.Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr−/− mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

Effects of Dietary Cholesterol Regulation on Spermatogenesis of Gobiocypris rarus Rare Minnow.

Cholesterol is an important component of cell membranes, and also a precursor for the synthesis of sex hormones, playing an important role in reproduction. However, few studies have focused on cholesterol and reproductive health. To investigate the toxic effects of different cholesterol levels on the spermatogenesis of rare minnows, we regulate the cholesterol content in fish by feeding them a high-cholesterol diet and cholesterol inhibitor pravastatin, and cholesterol levels, sex hormone (T and 11KT) levels, testis histology, sperm morphology and function, and the expression of genes related to sex hormone synthesis were investigated. The research findings indicate that increasing cholesterol levels significantly increases the liver weight and hepatic-somatic index, as well as the total cholesterol and free cholesterol levels in the testis, liver, and plasma of rare minnow, while inhibiting cholesterol has the opposite effect (p < 0.05). However, both increasing and decreasing cholesterol levels can suppress rare minnow testicular development, as evidenced by a decrease in testis weight, lowered gonadosomatic index, suppressed sex hormone levels, and reduced mature sperm count. Further exploration revealed that the expression of sex hormone synthesis-related genes, including star, cyp19a1a, and hsd11b2, was significantly affected (p < 0.05), which may be an important reason for the decrease in sex hormone synthesis and consequent inhibition of testicular development. At the same time, the fertilization ability of mature sperm in both treatment groups significantly decreased. Scanning electron microscopy and fluorescence polarization tests showed that reducing cholesterol levels significantly increased the rate of sperm head cell membrane damage, while both increasing and decreasing cholesterol levels led to a reduction in sperm cell membrane fluidity, which may be the main reason for the decrease in sperm fertilization ability. This study demonstrates that both increasing and decreasing the levels of cholesterol are detrimental to the fish spermatogenesis, providing fundamental information for the study of fish reproduction and also a reference for the causes of male reproductive dysfunction.

Ameliorating effect of probiotic on nonalcoholic fatty liver disease and lipolytic gene expression in rabbits

Nonalcoholic fatty liver disease (NAFLD) is a condition that affects about 24% of people worldwide. Increased liver fat, inflammation, and, in the most severe cases, cell death are all characteristics of NAFLD. However, NAFLD pathogenesis and therapy are still not clear enough. Thus, this study aimed to determine the effect of a high-cholesterol diet (HCD) inducing NAFLD on lipolytic gene expression, liver function, lipid profile, and antioxidant enzymes in rabbits and the modulatory effects of probiotic Lactobacillus acidophilus (L. acidophilus) on it. A total of 45 male New Zealand white rabbits, eight weeks old, were randomly divided into three groups of three replicates (5 rabbits/replicate). Rabbits in group I were given a basal diet; rabbits in group II were given a high-cholesterol diet that caused NAFLD; and rabbits in group III were given a high-cholesterol diet as well as probiotics in water for 8 weeks. The results showed that a high-cholesterol diet caused hepatic vacuolation and upregulated the genes for lipoprotein lipase (LPL), hepatic lipase (HL), and cholesteryl ester transfer protein (CETP). Downregulated low-density lipoprotein receptor (LDLr) gene, increased liver enzymes [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH)], cholesterol, triglycerides (TG), low-density lipoprotein (LDL), glucose, and total bilirubin. On the other hand, it decreased high-density lipoprotein (HDL), total protein, albumin, and liver antioxidants [glutathione peroxidase (GPx), catalase (CAT), reduced glutathione (GSH), and superoxide dismutase (SOD)]. Supplementing with probiotics helped to return all parameters to normal levels. In conclusion, probiotic supplementation, especially L. acidophilus, protected against NAFLD, and restored lipolytic gene expression, liver functions, and antioxidants to normal levels.

Excessive exogenous cholesterol activating intestinal LXRα-ABCA1/G5/G8 signaling pathway can not reverse atherosclerosis in ApoE−/− mice

BackgroundThe long-term excessive intake of exogenous cholesterol can lead to abnormally elevated blood lipid levels and induce cardiovascular and cerebrovascular diseases. However, the influence and relevance of exogenous cholesterol on plasma cholesterol components were still unclear, and the influence on intestinal lipid metabolism targets needs to be further explored.MethodsIn vivo, the C57BL/6 + NF group and ApoE−/− + NF group mice were fed a normal specific pathogen-free (SPF) diet; the ApoE−/− + HF group mice were fed a high-cholesterol SPF diet. The plasma and jejunum tissue homogenate were obtained for non-targeted lipid metabolomics. The lipid droplets in tissues were observed by transmission electron microscope and oil red O staining. Jejunum tissue morphology was observed by HE staining. The kits were used to detect lipid content in plasma, tissues, intestinal contents, and cells. Western blot, RT-PCR, immunohistochemistry (IHC), and immunofluorescence (IF) were used to observe the key target of lipid metabolism. In vitro, the final concentration of cholesterol was 100 μmol/L in Caco-cells. Oil red O staining, western blot, RT-PCR and immunofluorescence (IF) were used to observe the changes of lipid metabolism. Finally, the influence of liver X receptor alpha (LXRα) on intestinal cholesterol metabolism was clarified by applying the LXRα inhibitor GSK2033 and siRNA targeting LXRα.ResultsThe aortic arch and intestinal villi of the two groups of ApoE−/− mice showed apparent lesions and lipid accumulation, and there were significant changes in a variety of lipids in the plasma and jejunum. Additionally, jejunum LXRα was markedly activated. High cholesterol can significantly activate LXRα in Caco-2 cells. After LXRα was inhibited, the protein level of ATP-binding cassette transporter A1/G5/G8 (ABCA1/G5/G8) decreased, and the quantity and volume of intracellular lipids soared.ConclusionIn a high-cholesterol environment, the intestine promotes the excretion of cholesterol from the cell through the LXRα-ABCA1/G5/G8 pathway, reduces the intestinal intake of a variety of exogenous cholesterol, and reduces the risk of AS.Graphical

Hake heads proteins hydrolysates as a source of bioactive peptides with prevention against hypercholesterolemia in mice: The involvement of oxidative dysfunction in hepatic tissues

Food proteins are a source of nutraceutical and bioactive peptides that promote health and prevent diseases. The aim of this study was to evaluate the potential of Hake heads proteins hydrolysates (HHPH) in the prevention of hypercholesterolemia, oxidative stress and obesity in mice fed a high cholesterol diet. After six week of treatment serum lipid profiles (total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol), the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the thiobarbituric acid-reactive substances (TBARS) level, the advanced oxidation protein product (AOPP) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione (GSH) were tested. The high cholesterol diet led to dyslipidemia, oxidative stress and liver structure damage, compared with the control group. It is interesting to note that the addition of HHPH to the HCD attenuated these anomalies in a dose-dependent manner. The overall findings revealed that the hypocholesterolemic effect of HHPH could be due to their capacity to diminish serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels as well as to their antioxidant activities by improving the liver architecture.