The Sirt1 activator SRT3025 provides atheroprotection in Apoe−/− mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression

In the paper

“Thus, similar to previously described Sirt1 activators,15 SRT3025 acts by allosteric binding to Sirt1 at the lysine site 230, which is located at its catalytic core.”

What the sources say

• Disputed

  The paper supports allosteric activation of SIRT1 by previously described activators, but it identifies residue 230 as glutamate (Glu230), not lysine, and places it immediately N-terminal to the catalytic core, not within it (Abstract section; page unavailable). It reports that replacing Glu230 with lysine attenuated activation. Our copy does not mention SRT3025, so it cannot establish that compound’s binding site. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [15] Hubbard, 2013, Evidence for a common mechanism of SIRT1 regulation by allosteric activators, Science sourceExperimental — accuracy not yet measured

In the paper

“Ldlr was reported to be the main route of Pcsk9 clearance.22”

What the sources say

• Supported

  The Discussion explicitly concludes that LDLR is the primary route for serum PCSK9 clearance. It supports this with a markedly longer PCSK9 half-life in LDLR-deficient mice than in wild-type mice. (LLM, conf=0.95)

  cites [22] Tavori, 2013, Serum proprotein convertase subtilisin/kexin type 9 and cell surface low-density lipoprotein receptor: evidence for a reciprocal regulation, Circulation source

In the paper

“Sirt1 has been shown to be atheroprotective in apolipoprotein E-deficient (Apoe−/−) mice2–4 whereas genetic overexpression of Sirt1 in LDL-receptor-deficient (Ldlr−/−) mice enhanced atherosclerosis.5”

What the sources say

• Supported

  The Discussion reports that endothelium-specific SIRT1 overexpression reduced atherosclerosis in ApoE-deficient mice compared with controls. This supports the claim that SIRT1 has been shown to be atheroprotective in that mouse model. (LLM, conf=0.94)

  cites [2] Zhang, 2008, Endothelium-specific overexpression of class III deacetylase SIRT1 decreases atherosclerosis in apolipoprotein E-deficient mice, Cardiovasc Res source
• Supported

  In Results, under “SIRT1 protects against atherosclerosis,” ApoE−/− mice with two SIRT1 alleles had fewer plaques than those with one. This supports the claim that SIRT1 is atheroprotective in ApoE−/− mice. (LLM, conf=0.94)

  cites [3] Stein, 2010, SIRT1 decreases Lox-1-mediated foam cell formation in atherogenesis, Eur Heart J source
• ?Not checked — no full text of the sourcecites [4] Gorenne, 2013, Vascular smooth muscle cell sirtuin 1 protects against DNA damage and inhibits atherosclerosis, Circulation source

In the paper

“Conversely, administration of a Sirt1 activating drug to elderly volunteers and cigarette smokers decreased plasma levels of total and LDL-cholesterol.8,9”

What the sources say

• Partly supported

  The Results (Exploratory Pharmacodynamic Measures; page unavailable) report decreased total and LDL cholesterol after SRT2104 treatment in elderly volunteers. The Methods (Study Participants, Design and Treatment; page unavailable) explicitly exclude users of tobacco or nicotine products, so this paper does not support the claim about cigarette smokers. (LLM, conf=0.90)

  cites [8] Libri, 2012, A pilot randomized, placebo controlled, double blind phase I trial of the novel SIRT1 activator SRT2104 in elderly volunteers, PLoS One sourceExperimental — accuracy not yet measured
• Supported

  In the Discussion, this trial of otherwise healthy cigarette smokers reports mean reductions of 7% in total cholesterol and 11% in LDL cholesterol with SRT2104. This source supports the cigarette-smoker portion of the sentence’s two-citation claim. (LLM, conf=0.93)

  cites [9] Venkatasubramanian, 2013, Cardiovascular effects of a novel SIRT1 activator, SRT2104, in otherwise healthy cigarette smokers, J Am Heart Assoc source

In the paper

“Ldlr plays a critical role in the regulation of plasma LDL-cholesterol levels,10,11 a key determinant of atherogenesis.17”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Dietschy, 1984, Regulation of low density lipoprotein uptake and degradation in different animals species, Agents Actions Suppl source
• Supported

  The paper states that changes in LDLR activity profoundly alter plasma LDL-cholesterol concentration. Its reported 14-fold increase in plasma LDL-cholesterol after LDLR deletion directly supports the attributed claim (study summary; page not provided). The separate atherogenesis phrase is attached to citation 17, not this source. (LLM, conf=0.94)

  cites [11] Osono, 1995, Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse, J Clin Invest source

In the paper

“Hepatic deletion of Sirt1 in C57BL/6 mice fed a high-cholesterol diet induced mild hypercholesterolemia.7”

What the sources say

• Partly supported

  The Animal experiments section describes liver-specific SIRT1 knockout mice on a C57BL/6 background, and the Results describe feeding them a western diet containing cholesterol. The Results and Discussion report increased cholesterol in the liver, but do not establish the claimed mild hypercholesterolemia (elevated blood cholesterol). No page numbers are available in our copy. (LLM, conf=0.84)

  cites [7] Purushotham, 2009, Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation, Cell Metab sourceExperimental — accuracy not yet measured

In the paper

“Hepatic Ldlr clears LDL-cholesterol from the blood stream.10,11”

What the sources say

• ?Not checked — no full text of the sourcecites [10] Dietschy, 1984, Regulation of low density lipoprotein uptake and degradation in different animals species, Agents Actions Suppl source
• Supported

  The paper’s summary (page not provided) reports that 79% of LDL-cholesterol degradation in normal mice occurred in the liver and that 91% of receptor-dependent transport was hepatic. It also states that most LDL-cholesterol returns directly to the liver, supporting the claim about hepatic LDLR-mediated clearance from blood. (LLM, conf=0.92)

  cites [11] Osono, 1995, Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse, J Clin Invest source

In the paper

“Transcription of Ldlr is controlled by the sterol-responsive element binding protein 2 (Srebp2),12 while its turnover depends on proprotein convertase subtilisin/kexin type 9 (Pcsk9), a serine protease.13”

What the sources say

• Supported

  The Abstract states that PCSK9 apparently enhances degradation of the LDLR protein. This supports the claim that PCSK9 regulates LDLR turnover; the paper leaves the mechanism unresolved. (LLM, conf=0.90)

  cites [13] Attie, 2005, Dual regulation of the LDL receptor—some clarity and new questions, Cell Metab source

In the paper

“Sirt1 has been shown to be atheroprotective in apolipoprotein E-deficient (Apoe−/−) mice2–4 whereas genetic overexpression of Sirt1 in LDL-receptor-deficient (Ldlr−/−) mice enhanced atherosclerosis.5”

What the sources say

• Supported

  The Results section, “Increased dyslipidemia and atherosclerosis in SirBACO:Ldlr−/− mice,” reports that SirT1-overexpressing Ldlr−/− mice fed a Western-type diet had a 28% larger aortic-root atherosclerotic lesion area than Ldlr−/− controls (P<0.05). This directly supports the claim in the studied dietary condition. (LLM, conf=0.95)

  cites [5] Qiang, 2011, Proatherogenic abnormalities of lipid metabolism in SirT1 transgenic mice are mediated through Creb deacetylation, Cell Metab source

In the paper

“Furthermore, constitutive Sirt1 overexpression in Ldlr−/− mice was associated with increased atherosclerosis.5”

What the sources say

• Supported

  The Results section (“Increased dyslipidemia and atherosclerosis in SirBACO:Ldlr−/− mice”) describes crossing SirT1-overexpressing mice with Ldlr−/− mice. After a Western-type diet, the double-mutant mice had a 28% greater aortic-root atherosclerotic lesion area than Ldlr−/− controls (P<0.05). (LLM, conf=0.94)

  cites [5] Qiang, 2011, Proatherogenic abnormalities of lipid metabolism in SirT1 transgenic mice are mediated through Creb deacetylation, Cell Metab source

In the paper

“These studies suggest that Sirt1 regulates plasma LDL-cholesterol.”

What the sources say

• Partly supported

  The Results section reports higher liver cholesterol in SIRT1-deficient mice, and the Discussion describes altered hepatic cholesterol metabolism. Those passages support a role for SIRT1 in cholesterol metabolism, but they do not establish regulation of plasma LDL-cholesterol; the Methods mention measuring serum cholesterol, not LDL-cholesterol specifically. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  cites [7] Purushotham, 2009, Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation, Cell Metab sourceExperimental — accuracy not yet measured
• Supported

  The Results (Exploratory Pharmacodynamic Measures, Serum Lipid Profile) report decreased LDL cholesterol after treatment with the SIRT1 activator SRT2104. The Discussion describes the lipid changes as consistent with target modulation, supporting the citing sentence’s cautious suggestion that SIRT1 regulates LDL cholesterol. (LLM, conf=0.88)

  cites [8] Libri, 2012, A pilot randomized, placebo controlled, double blind phase I trial of the novel SIRT1 activator SRT2104 in elderly volunteers, PLoS One source
• Supported

  The Results (Lipid Profile) show lower LDL cholesterol after treatment with SRT2104 than with placebo. The Discussion reports an 11% mean reduction in serum LDL cholesterol and says the findings suggest a beneficial role for SIRT1 activation, while noting that the mechanism is unclear. This supports the citing sentence’s hedged conclusion. (LLM, conf=0.88)

  cites [9] Venkatasubramanian, 2013, Cardiovascular effects of a novel SIRT1 activator, SRT2104, in otherwise healthy cigarette smokers, J Am Heart Assoc source

In the paper

“Of note, statin therapy not only increases hepatic Ldlr, but also plasma Pcsk9 activity, thus limiting its effect on LDL-cholesterol.23”

What the sources say

• Partly supported

  The Discussion says statins increase LDLR and PCSK9 gene expression, and reports increased circulating PCSK9 protein after atorvastatin treatment. It proposes that increased PCSK9 may explain the limited additional LDL-C lowering from higher statin doses, but explicitly calls that explanation unproven. The study measures PCSK9 protein levels, not plasma PCSK9 activity. (LLM, conf=0.88)

  cites [23] Welder, 2010, High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol, J Lipid Res sourceExperimental — accuracy not yet measured

In the paper

“By binding to the EGF-A domain of Ldlr, Pcsk9 targets this receptor for lysosomal degradation rather than for recycling to the cell surface.20,21”

What the sources say

• Supported

  The Introduction states that PCSK9 binds LDLR and targets it for lysosomal degradation. The Fig. 4 caption describes PCSK9 binding as preventing the conformational changes associated with receptor recycling, ultimately leading to lysosomal LDLR degradation. (LLM, conf=0.92)

  cites [20] Lo Surdo, 2011, Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH, EMBO Rep source
• Partly supported

  The Figure 2 and Figure 3 passages report PCSK9-mediated LDLR degradation and reduced cell-surface LDLR in hepatocytes. They do not establish that the degradation is lysosomal rather than recycling to the surface; the paper also shows binding to the LDLR extracellular domain, not specifically the EGF-A domain. Page numbers are unavailable in our copy. (LLM, conf=0.84)

  cites [21] Lagace, 2006, Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice, J Clin Invest sourceExperimental — accuracy not yet measured

In the paper

“Co-administration of Pcsk9 [3 ng ml−1, based upon concentrations measured in the supernatants of untreated AML12 (Figure 4D)] and SRT3025 to AML12 hepatocytes attenuated the drug-dependent increase in Ldlr protein expression (Figure 5A).”

In the paper

“Incubation of AML12 cells with 10 μM SRT3025 was associated with a time-dependent decrease in Pcsk9 secretion into the supernatant (Figure 4D).”

In the paper

“Pharmacological Sirt1 activation increased liver expression of Abca1, but did not affect Abcg5, Abcg8, Ldlr, Pcsk9, Pparα, Pparγ, Srebp1, and Srebp2 (Figure 3A).”

In the paper

“In addition, we observed reduced plasma levels of Mcp-1 and Il-6 (Figure 1G) and lower hepatic mRNA expression of these cytokines (Figure 3A).”

In the paper

“Increased deacetylation of known Sirt1 targets upon SRT3025 treatment, hepatic p65 and forkhead transcription factor family O1 (Foxo1) in skeletal muscle (Figure 2B and C) indicate successful Sirt1 activation in vivo.”

In the paper

“In agreement with this, we found reduced plasma Pcsk9 protein levels following pharmacological Sirt1 activation in Apoe−/− mice compared with placebo-treated controls (Figure 3C).”

In the paper

“Interestingly, plasma levels of total-, LDL- and VLDL-cholesterol were significantly lower after SRT3025 treatment compared with placebo (Figure 1E and F).”

In the paper

“Moreover, co-immunoprecipitation of endogenous Pcsk9 and Ldlr after 24 h incubation with 10 μM SRT3025 revealed that Pcsk9 binding to Ldlr was impaired after SRT3025 treatment compared with vehicle control (Figure 4E).”

In the paper

“Fluorescence analysis revealed an increase in labelled LDL uptake upon 10 μM SRT3025 treatment compared with vehicle controls (Figure 5B), suggesting a functional relevance of the SRT3025-dependent increase in Ldlr expression.”

In the paper

“Plasma lipids showed an increase in total and VLDL-cholesterol in SRT3025-treated Ldlr−/− mice compared with placebo-treated controls (Figure 7E and F), whereas plasma LDL-cholesterol, HDL-cholesterol, and triglycerides were unaffected by drug treatment (Figure 7F and Supplementary material online, Figure S2A).”

In the paper

“Conversely, genetic overexpression of Sirt1 increased Ldlr protein expression in AML12 cells compared with control transfection (Figure 6B).”

In the paper

“Compared with Apoe−/− mice, baseline plasma Pcsk9 levels in Ldlr−/− mice were about 20-fold higher (Figure 7G).”

In the paper

“Histomorphometry of thoraco-abdominal aortae en face and cross-sections of aortic roots revealed a significant reduction in plaque size in SRT3025-treated Apoe−/− mice compared with placebo-treated controls (Figure 1A and B).”

In the paper

“As in Apoe−/− mice, SRT3025 prevented an increase in weight gain and epididymal white adipose tissue without affecting food intake (Supplementary material online, Figure S2B–D), and lowered plasma levels of pro-inflammatory cytokines Mcp-1 and Il-6 (Figure 7G).”

In the paper

“SRT3025 induced a minimal but significant increase in plasma Pcsk9 levels in Ldlr−/− mice (Figure 7G).”

In the paper

“As observed in vivo, mRNA levels of Ldlr and Pcsk9 were not altered by SRT3025 (Figure 4C), indicating that post-translational effects cause the changes in protein expression.”

In the paper

“Pharmacokinetic analysis of the drug in Apoe−/− mice showed that the drug indeed reached target tissues (Supplementary material online, Table S1).”

In the paper

“We observed a concentration- and time-dependent increase in Ldlr and Pcsk9 protein expression upon SRT3025 administration in cell lysates (Figure 4A and B).”

In the paper

“In addition, we observed reduced plasma levels of Mcp-1 and Il-6 (Figure 1G) and lower hepatic mRNA expression of these cytokines (Figure 3A).”

In the paper

“Plasma lipids showed an increase in total and VLDL-cholesterol in SRT3025-treated Ldlr−/− mice compared with placebo-treated controls (Figure 7E and F), whereas plasma LDL-cholesterol, HDL-cholesterol, and triglycerides were unaffected by drug treatment (Figure 7F and Supplementary material online, Figure S2A).”

In the paper

“Pharmacological Sirt1 activation increased liver expression of Abca1, but did not affect Abcg5, Abcg8, Ldlr, Pcsk9, Pparα, Pparγ, Srebp1, and Srebp2 (Figure 3A).”

In the paper

“In contrast to control scramble siRNA-treated cells, cells transfected with Sirt1 siRNA did not show an increase in Ldlr expression upon SRT3025 addition (Figure 6A).”

In the paper

“Plaque analyses of thoraco-abdominal aortae en face and cross-sections of aortic roots revealed no difference in the extent of atherosclerosis between drug- and placebo-treated mice (Figure 7A and B).”

In the paper

“Moreover, co-administration of exogenous active Pcsk9 also attenuated the drug-induced increase in LDL uptake (Figure 5B), consistent with the observed attenuation of Ldlr protein expression (Figure 5A).”

In the paper

“This intracellular accumulation of Pcsk9 protein with no change in mRNA suggests a disturbed transport and/or secretion of the protein.”

In the paper

“These data indicate that the internalization and degradation process of Ldlr is not defective and that limited extracellular availability of Pcsk9 contributes to the increase in Ldlr protein expression.”

In the paper

“These data indicate that SRT3025 decreases hepatic Pcsk9 release, impairs Pcsk9 binding to hepatic Ldlr, and thereby prevents hepatic Ldlr degradation.”

In the paper

“We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice.”

In the paper

“Therefore, our data suggest that not the reduction in inflammation, which still occurs in the absence of Ldlr, but the decrease in plasma LDL-cholesterol accounted for reducing atherosclerosis in Apoe−/− mice treated with SRT3025.”

In the paper

“These data indicate that SRT3025 decreases hepatic Pcsk9 release, impairs Pcsk9 binding to hepatic Ldlr, and thereby prevents hepatic Ldlr degradation.”

In the paper

“Moreover, since we observed a reduction in weight gain in both Apoe−/− and Ldlr−/− mice despite unaltered food intake, the atherosclerotic phenotype is also independent of changes in body weight.”

In the paper

“These data indicate that SRT3025 decreases hepatic Pcsk9 release, impairs Pcsk9 binding to hepatic Ldlr, and thereby prevents hepatic Ldlr degradation.”

In the paper

“We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice.”

In the paper

“These data indicate that the SRT3025-associated changes in Ldlr expression are Sirt1-dependent.”

In the paper

“These data indicate that the internalization and degradation process of Ldlr is not defective and that limited extracellular availability of Pcsk9 contributes to the increase in Ldlr protein expression.”

In the paper

“Thus, Sirt1 is required for SRT3025-induced increase in Ldlr protein expression.”

In the paper

“Taken together, we demonstrate that the increased expression of hepatic Ldlr is essential for the cholesterol-lowering effects of SRT3025.”