GPR146 Deficiency Protects against Hypercholesterolemia and Atherosclerosis

In the paper

“ERK1/2 signaling has been previously shown to regulate SREBP2 activity in HepG2 cells via direct phosphorylation (Arito et al., 2008; Kotzka et al., 2004; Kotzka et al., 2000)”

What the sources say

• Partly supported

  The Ser 455 results (page not available) link ERK activation to SREBP-2 phosphorylation and reduced sumoylation, while the HepG2 experiments show that inhibiting the ERK cascade increases SREBP-2 sumoylation. The phosphorylation experiments described in our copy were performed in COS-1 cells, however; they do not directly demonstrate ERK1/2 phosphorylation of SREBP-2 in HepG2 cells. (LLM, conf=0.84)

  cites Arito et al., 2008 sourceExperimental — accuracy not yet measured
• Supported

  The Results report that activating the Erk-MAPK pathway with MEK2D increased SREBP-2 trans-activity in HepG2 cells, while mutation of its phosphorylation sites greatly reduced or eliminated that response. The paper also identifies Ser-432 and Ser-455 as sites directly phosphorylated by Erk1/2. No page number is available in our copy. (LLM, conf=0.93)

  cites Kotzka et al., 2004 source
• Partly supported

  The Results report that MAP-kinase pathway activation increased SREBP-2 transcriptional activity in HepG2 cells, and that ERK1 or ERK2 directly phosphorylated recombinant SREBP-2 in vitro. The Abstract describes SREBP-2 as *possibly* a direct ERK1/2 substrate; it does not establish that direct phosphorylation mediates the activity change in HepG2 cells. Page numbers are not available in the supplied passages. (LLM, conf=0.88)

  cites Kotzka et al., 2000 sourceExperimental — accuracy not yet measured

In the paper

“Abundant evidence demonstrates that hepatic SREBP2 activity and expression of its target genes such as HMGCR and LDLR fall while fasting and rise substantially upon refeeding”

What the sources say

• Partly supported

  The Results and Discussion report that nuclear SREBP-2 and LDL receptor mRNA decline during fasting and return to approximately basal levels after refeeding. But the Results say HMG-CoA reductase (HMGCR) mRNA changes little during fasting, so the claim that this target gene falls while fasting is not supported. The paper also distinguishes the return of SREBP-2 and cholesterol-related genes to basal levels from the above-basal overshoot seen for SREBP-1 and lipogenic genes. (LLM, conf=0.88)

  cites Horton et al., 1998 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Liang et al., 2002 source
• Partly supported

  The Results (page not supplied) report that fasting reduced hepatic SREBP-2 mRNA and SREBP target-gene mRNAs in wild-type mice, and that refeeding raised several target-gene mRNAs above normal, notably HMG-CoA reductase. The passage does not establish that SREBP-2 activity rose substantially on refeeding or that LDL receptor expression had a similarly substantial rise. (LLM, conf=0.82)

  cites Matsuda et al., 2001 sourceExperimental — accuracy not yet measured

In the paper

“There are over 300 loci uncovered to be associated with plasma lipid levels in humans (Klarin et al., 2018; Liu et al., 2017; Teslovich et al., 2010; Willer et al., 2013).”

What the sources say

• Supported

  The Results section ('Lipid Genetic Association and Conditional Analyses') reports 118 novel and 268 previously identified loci associated with plasma lipid levels—a total of 386. The Discussion describes this as nearly 400 known independent genetic lipid loci, supporting the claim that there are over 300. (LLM, conf=0.94)

  cites Klarin et al., 2018 source
• Partly supported

  The paper reports loci associated with plasma lipid levels in humans, but its Abstract gives 250 loci—75 novel and 175 previously implicated—not over 300. It therefore supports the existence of lipid-associated loci but does not establish the cited sentence’s numerical threshold. (LLM, conf=0.87)

  cites Liu et al., 2017 sourceExperimental — accuracy not yet measured
• Partly supported

  The “Genome-Wide Association Mapping in >100,000 Individuals” section reports 95 lipid-associated loci, including 59 reported for the first time. This supports the existence of loci associated with human lipid levels, but this source does not establish the citing sentence’s total of over 300 loci. (LLM, conf=0.88)

  cites Teslovich et al., 2010 sourceExperimental — accuracy not yet measured

In the paper

“Substantial evidence supports the conclusion that hepatic SREBP2 activity and cholesterol biosynthesis rate fall while fasting and rise greatly upon refeeding, indicating the existence of feeding-responsive signaling pathways upregulating hepatic cholesterol synthesis in the presence of dietary nutrition (Horton et al., 1998; Liang et al., 2002; Matsuda et al., 2001; Slakey et al., 1972).”

What the sources say

• Partly supported

  The Results and Discussion report that hepatic nuclear SREBP-2 falls during fasting and recovers upon refeeding. But they specify that it returns only to the nonfasted level; the Abstract says cholesterol synthesis likewise returns only to its prefasted level. The paper therefore supports a fasting decline and refeeding recovery, but not an increase greatly beyond baseline. (LLM, conf=0.86)

  cites Horton et al., 1998 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Liang et al., 2002 source
• Partly supported

  The Results (page unavailable) report that fasting reduced hepatic SREBP-2 mRNA and cholesterol-synthesis target-gene mRNAs in mice; refeeding raised several target-gene mRNAs above their nonfasted levels. These findings support a feeding-responsive cholesterol-synthesis pathway, but the cited passage does not establish that SREBP-2 activity and the measured rate of cholesterol biosynthesis both fell during fasting and rose greatly upon refeeding. (LLM, conf=0.84)

  cites Matsuda et al., 2001 sourceExperimental — accuracy not yet measured

In the paper

“SREBP2 may maintain promoters of target genes in a receptive state for binding of those transcription factors (Gokey et al., 2011; Liang et al., 2002).”

What the sources say

• Supported

  The passage labeled Abstract describes a proposed model in which SREBP-2 maintains target promoters in a receptive state for binding by additional insulin-dependent transcription factors. The “Egr1 Regulates Cholesterol Synthesis” section repeats that permissive-factor model. The claim’s “may” preserves its tentative status; page numbers are not supplied in our copy. (LLM, conf=0.94)

  cites Gokey et al., 2011 source
• ?Not checked — no full text of the sourcecites Liang et al., 2002 source

In the paper

“In hepatocytes, many hormones and growth factors such as epidermal growth factors (EGF) and fibroblast growth factors (FGF) are able to stimulate ERK1/2 signaling cascade (Fremin et al., 2007; Guegan et al., 2012).”

What the sources say

• ?Not checked — no full text of the sourcecites Fremin et al., 2007 source
• Partly supported

  The Introduction and section 2 describe growth-factor activation of MEK–ERK signaling in hepatocytes and specifically discuss EGF, HGF, IGF-1, and growth hormone. They do not identify FGF as an ERK1/2 stimulus in hepatocytes, so that example is not supported by our copy. Page numbers are unavailable. (LLM, conf=0.86)

  cites Guegan et al., 2012 sourceExperimental — accuracy not yet measured

In the paper

“Previous studies in HepG2 cells showed that MEK1/ERK cascade directly phosphorylates SREBP2 at Ser-432 and Ser-455, which inhibits its sumoylation and subsequently promotes its transcription activity (Arito et al., 2008; Kotzka et al., 2004; Kotzka et al., 2000).”

What the sources say

• Partly supported

  In “Ser 455 of SREBP-2 Is Critical for SUMO-1 Conjugation” (page unavailable), the paper identifies Ser-432 and Ser-455 as phosphorylation sites, but reports that mutating Ser-432 had no effect on sumoylation; the inhibitory link is specific to Ser-455. The paper connects ERK-pathway activity, reduced sumoylation, and increased transcriptional activity, but its site-specific experiments were chiefly in COS-1 cells and do not directly demonstrate MEK1/ERK phosphorylation at both sites in HepG2 cells. (LLM, conf=0.88)

  cites Arito et al., 2008 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results identify Ser-432 and Ser-455 as Erk2 phosphorylation sites and show that activating the Erk pathway with MEK2D increases SREBP-2 transcriptional activity in HepG2 cells. Our copy does not establish that phosphorylation inhibits sumoylation, and the HepG2 experiment uses MEK2D rather than MEK1. (LLM, conf=0.89)

  cites Kotzka et al., 2004 sourceExperimental — accuracy not yet measured
• Partly supported

  The Results report that ERK1 and ERK2 phosphorylated SREBP-2 on serine residues in vitro, and that activating the MAP-kinase cascade increased SREBP-2 transcriptional activity in HepG2 cells. The paper does not identify Ser-432 or Ser-455, test sumoylation, or establish that phosphorylation at those sites causes the increased activity. (LLM, conf=0.91)

  cites Kotzka et al., 2000 sourceExperimental — accuracy not yet measured

In the paper

“Downregulation of cholesterol biosynthetic genes in the absence of GPR146 was observed under fasting-refeeding conditions, but not in mice after a prolonged 16-hour fasting”

In the paper

“Mice upon PD0325901 administration exhibited significantly reduced refed phosphor-ERK1/2 (pERK1/2) levels in liver, but not reduced levels of other MAPK families, such as phosphor-JNK and phosphor-P38”

In the paper

“there was a trend towards reduction in male Gpr146−/− mice and a significant decrease in female Gpr146−/− mice upon western diet feeding for two months, compared to control littermates”

In the paper

“It is worth noting that there remains a significant reduction of plasma TC levels in the absence of GPR146 upon a prolonged 16-hour fasting despite no changes of ERK1/2 and SREBP2 activities”

In the paper

“Metabolic profiling of mice fed chow showed no difference in either food/water intake or locomotor activity (Figure S2A–S2C) between Gpr146−/− and Gpr146+/+ littermates.”

In the paper

“Mating between Gpr146+/− mice produced offspring in the expected Mendelian 1:2:1 ratio.”

In the paper

“it is well established that many GPCRs activate ERK1/2 signaling upon ligand binding (van Biesen et al., 1996)”

What the sources say

• ?Not checked — no full text of the sourcecites van Biesen et al., 1996 source

In the paper

“Gpr146−/− mice were viable, fertile and normal in appearance.”

In the paper

“elevated plasma LDL-C and TG levels are major risk factors (Defesche et al., 2017; Do et al., 2013; Hegele, 2009; Hokanson and Austin, 1996; Rader, 2016)”

What the sources say

• ?Not checked — no full text of the sourcecites Defesche et al., 2017 source
• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Do et al., 2013
• ?Not checked — no full text of the sourcecites Hegele, 2009 source

In the paper

“over 95% of HoFH patients have loss-of-function mutations in both LDLR alleles (Defesche et al., 2017)”

What the sources say

• ?Not checked — no full text of the sourcecites Defesche et al., 2017 source

In the paper

“thereby increasing SREBP2 activities and VLDL secretion rate”

In the paper

“Melting curve analysis was carried out to confirm the RT-PCR products.”

In the paper

“In summary, we provide several lines of evidence that the GPR146/ERK/SREBP2 axis regulates plasma cholesterol levels and depletion of GPR146 protects against hypercholesterolemia and atherosclerosis in LDLR-deficient mice.”

In the paper

“No statistical methods were used to predetermine sample size.”

In the paper

“RNA quality was verified using the Agilent Bioanalyzer and the 6000 nano kit.”

In the paper

“We further put forward a signaling pathway in which GPR146 promotes ERK1/2 signaling cascade in hepatocytes upon feeding, which in turn increases SREBP2 activities and VLDL secretion rate”

In the paper

“Defects in this regulation lead to dramatically elevated plasma LDL-C levels in HoFH patients due to increased hepatic cholesterol synthesis as well as decreased LDL-C clearance (Brown and Goldstein, 1975, 1986; Goldstein and Brown, 1973).”

What the sources say

• ?Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  cites Brown and Goldstein, 1975, 1986
• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Goldstein and Brown, 1973 source

In the paper

“Both male and female 6- to 36-week-old littermate mice were used in this study to match age and sex.”

In the paper

“Moreover, there is longstanding evidence showing that, besides SREBP2, additional feeding-responsive transcription factors are likely to promote expression of cholesterol biosynthetic genes.”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Gokey et al., 2011 source
• ?Not checked — no full text of the sourcecites Liang et al., 2002 source

In the paper

“GPR146 upon ligand binding might directly activate ERK1/2 through activated Gα and Gβγ subunits and downstream signaling cascade; or GPR146 signaling might cross-talk with other growth factor/hormone signaling pathways to modulate ERK1/2 activities.”

In the paper

“GPR146 upon ligand binding might directly activate ERK1/2 through activated Gα and Gβγ subunits and downstream signaling cascade; or GPR146 signaling might cross-talk with other growth factor/hormone signaling pathways to modulate ERK1/2 activities.”

In the paper

“Our results show that depletion of GPR146 reduces circulating cholesterol and TG levels substantially and confers great protection to LDLR-deficient mice against atherosclerosis.”

In the paper

“It is well documented that many GPCRs upon ligand binding activate ERK signaling and further regulate cell growth, division and differentiation (van Biesen et al., 1996).”

What the sources say

• ?Not checked — no full text of the sourcecites van Biesen et al., 1996 source

In the paper

“Feedback suppression of SREBP2 pathway by LDLR plays a pivotal role in maintaining the balance of cholesterol levels between liver and blood (Brown and Goldstein, 1986).”

What the sources say

• ?Not checked — no full text of the sourcecites Brown and Goldstein, 1986 source

In the paper

“All image capture and quantitation were done in a blinded fashion.”

In the paper

“The early growth response gene family (EGR1, EGR2 and EGR3), which are ERK1/2 target genes, have previously been indicated as additional transcription factors to upregulate cholesterol biosynthetic gene expression upon feeding (Gokey et al., 2011).”

What the sources say

• Not checked yet

  Checked before — its earlier verdict is held back until a new check runs.

  cites Gokey et al., 2011 source

In the paper

“To reduce the frequency of homologous recombination of long terminal repeat (LTRs), E. coli stbl3 strain cultured in LB broth was used to clone all of the lentiviral vectors.”

In the paper

“All animal care and experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee at Harvard University and Beth Israel Deaconess Medical Center.”

In the paper

“Genome-edited F1 Gpr146−/− and Gpr146+/fl mice were bred with C57BL/6 mice for two rounds to dilute the off-target effects.”