Expression of miR-33 from an SREBP2 Intron Inhibits Cholesterol Export and Fatty Acid Oxidation*

In the paper

“However, SREBPs not only activate cholesterol synthesis but also fatty acid and phospholipid synthesis (2).”

What the sources say

• Supported

  The Abstract states that SREBPs activate genes involved in the synthesis of cholesterol, fatty acids, and phospholipids. The “SREBPs: two genes, three proteins” section also identifies an SREBP-1c target gene encoding an enzyme involved in phospholipid synthesis. Page numbers are not supplied for these passages. (LLM, conf=0.94)

  cites [2] Horton, 2002, J. Clin. Invest. source

In the paper

“High levels of low density lipoprotein cholesterol are a major risk factor for the development of arteriosclerosis and death from cardiovascular causes (41).”

What the sources say

• Supported

  The section on elevated LDL cholesterol and coronary artery disease states that LDL is a causal agent in many forms of coronary heart disease. It also reports that people with markedly elevated LDL due to homozygous familial hypercholesterolemia develop severe coronary atherosclerosis and usually die from myocardial infarction. (LLM, conf=0.89)

  cites [41] Nabel, 2003, N. Engl. J. Med. source

In the paper

“Genes involved in its synthesis are coordinately up-regulated by the action of SREBP transcription factors (2).”

What the sources say

• Supported

  The “Function of individual SREBP isoforms in vivo” section reports that overexpressing SREBP-2 increases mRNAs encoding all cholesterol biosynthetic enzymes. The “SREBPs: two genes, three proteins” section also identifies SREBP-2 as preferentially activating cholesterol synthesis. (LLM, conf=0.93)

  cites [2] Horton, 2002, J. Clin. Invest. source

In the paper

“It has been previously shown that the homologs of SREBPs do not respond to changes in cholesterol, but rather changes in palmitate and/or phospholipids (39,40).”

What the sources say

• ?Not checked — no full text of the sourcecites [39] Dobrosotskaya, 2002, Science source
• Partly supported

  The Results report that palmitate reduced nuclear dSREBP in Drosophila S2 cells, whereas a cholesterol-containing sterol mixture failed to do so. The Discussion proposes that palmitic acid or a lipid derived from it regulates this pathway, but does not establish phospholipids as the signal. The claim also generalizes beyond the Drosophila homolog studied here. (LLM, conf=0.86)

  cites [40] Seegmiller, 2002, Dev. Cell sourceExperimental — accuracy not yet measured

In the paper

“It has been previously noted that cellular miR-33 levels are dependent on culture conditions and exogenous lipid supply (36).”

What the sources say

• Partly supported

  The Results (Screening of miRNAs related with differentiation; page unavailable) report that miR-33a increased in T84 cells between culture days 0 and 7, supporting a limited link between its level and culture conditions. The paper does not report that exogenous lipid supply affects miR-33 levels; its cell-culture methods describe serum-containing medium, not a lipid-supply comparison. (LLM, conf=0.87)

  cites [36] Tsuchiya, 2009, Nucleic Acids Res. sourceExperimental — accuracy not yet measured

In the paper

“This process seems to be essential for the formation of HDL particles because liver-and intestine-specific knockout mice for ABCA1 show strong reductions in HDL levels (45)(46)(47).”

What the sources say

• ?Not checked — no full text of the sourcecites [45] Brunham, 2009, Arterioscler. Thromb. Vasc. Biol. source
• Partly supported

  The Abstract reports an 80% reduction in plasma HDL cholesterol in liver-specific ABCA1 knockout mice, supporting the liver-specific part of the claim. The paper does not report an intestine-specific knockout or its HDL levels. Its Discussion also says low HDL reflects increased catabolism as well as decreased synthesis, so the reduction alone does not establish a defect solely in particle formation. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [46] Singaraja, 2006, Arterioscler. Thromb. Vasc. Biol. sourceExperimental — accuracy not yet measured
• Supported

  The Introduction says ABCA1 promotes cholesterol and phospholipid efflux to apoA-I in the rate-controlling step of HDL particle formation. The Discussion describes hepatic ABCA1 as essential to HDL biogenesis and reports that intestinal ABCA1 deletion significantly reduces plasma HDL; the Results quantify the intestinal-knockout reduction at approximately 30%. (LLM, conf=0.92)

  cites [47] Brunham, 2006, J. Clin. Invest. source

In the paper

“In a process termed reverse cholesterol transport, peripheral tissues export their excess cholesterol to HDL using the cholesterol export pumps ABCA1 and ABCG1 (44).”

What the sources say

• Partly supported

  The Introduction (page not provided) describes HDL-mediated cholesterol removal from peripheral tissues and identifies ABCA1 and ABCG1 as cholesterol-efflux transporters. It distinguishes their acceptors, however: ABCA1 exports to lipid-poor apoA-1, not mature HDL particles, whereas ABCG1 exports to HDL. Thus the claim is too broad in saying both pumps export cholesterol to HDL. (LLM, conf=0.86)

  cites [44] Yvan-Charvet, 2007, J. Clin. Invest. sourceExperimental — accuracy not yet measured

In the paper

“Previous studies have shown that reduction of one or the other component in heterozygote knock-out mice leads to a concomitant reduction of the other subunit (31)(32)(33).”

What the sources say

• Supported

  The section “Generation and molecular characterization of MTP-deficient mice” reports that heterozygous Mtpa knockout mice had intermediate expression of both α and β subunits. This supports the claim for reduction of the α component in heterozygous mice accompanied by reduction of the β component. (LLM, conf=0.90)

  cites [31] Ibdah, 2001, J. Clin. Invest. source
• Partly supported

  The Assessment of the Temporal Relationship section (page unavailable) reports lower hepatic MTP α-subunit expression in α-heterozygous mice than in wild-type mice. The Background & Aims section says α-null homozygotes lack both subunits, but the paper does not show a concomitant reduction of the β-subunit in heterozygotes or examine β-heterozygous knockouts. (LLM, conf=0.85)

  cites [32] Ibdah, 2005, Gastroenterology sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion and Western blot and enzyme activity assay section report that both MTP subunits were reduced in affected mice with a mutation in the β-subunit. But the Identification of the mutated gene section identifies the affected mice as homozygous for a missense mutation, not heterozygous knockout mice; the paper therefore does not establish the claim for that genotype. (LLM, conf=0.89)

  cites [33] Kao, 2006, Hum. Mol. Genet. sourceExperimental — accuracy not yet measured

In the paper

“The reciprocal relationship also appears to be true in that increased gene dosage of ABCA1 increases plasma HDL levels and protects from arteriosclerosis in animal models of hyperlipidemia (48).”

What the sources say

• Partly supported

  The Results report significantly higher HDL-C in ABCA1 BAC transgenic mice than in control littermates, including when fed an atherogenic diet. The paper does not report an arteriosclerosis outcome or establish protection from arteriosclerosis in hyperlipidemic animal models; feeding an atherogenic diet does not by itself establish that protection. (LLM, conf=0.88)

  cites [48] Singaraja, 2001, J. Biol. Chem. sourceExperimental — accuracy not yet measured

In the paper

“D. melanogaster is a cholesterol auxotroph and activation of SREBP is regulated by reduced levels of palmitate and/or phospholipids.”

What the sources say

• ?Not checked — no full text of the sourcecites [39] Dobrosotskaya, 2002, Science source
• Partly supported

  The paper says insects require dietary sterols, supporting the description of D. melanogaster as a cholesterol auxotroph. The Results and Discussion show that added palmitate inhibits dSREBP processing in cultured Drosophila cells, but the Discussion says it is not clear whether palmitate itself or a more complex lipid, possibly a phospholipid, is responsible. It does not establish that reduced phospholipid levels activate SREBP. (LLM, conf=0.85)

  cites [40] Seegmiller, 2002, Dev. Cell sourceExperimental — accuracy not yet measured

In the paper

“The mitochondrial trifunctional protein consists of an ␣ subunit (encoded by HADHA) and a ␤ subunit (encoded by HADHB) (31).”

What the sources say

• Supported

  The Introduction states that mitochondrial trifunctional protein is a hetero-octamer of α and β subunits and explicitly identifies the genes coding for them as HADHA and HADHB, respectively. (LLM, conf=0.95)

  cites [31] Ibdah, 2001, J. Clin. Invest. source

In the paper

“This was observed both in mice that carry a hypomorphic allele of the HADHB gene and, notably, in mice heterozygote for a null mutation of the HADHA gene (encoding the ␣-subunit) (32,33).”

What the sources say

• Supported

  The Background & Aims identifies the studied mice as heterozygous for an MTP α-subunit null allele, and Evaluation for Nonalcoholic Fatty Liver Disease reports hepatic steatosis in those mice. The finding was in aging mice; the paper reports no steatosis in young heterozygous mice. The separate HADHB example is not attributed to this source alone in the two-citation sentence. (LLM, conf=0.92)

  cites [32] Ibdah, 2005, Gastroenterology source
• Supported

  The Identification of the mutated gene section identifies a homozygous Hadhb M404K mutation in the affected mice, and the Abstract reports hepatic steatosis in those mice. The enzyme activity results show reduced, rather than absent, activity, consistent with a hypomorphic allele. The HADHA heterozygote clause is accompanied by a separate citation and is not attributed to this paper. (LLM, conf=0.91)

  cites [33] Kao, 2006, Hum. Mol. Genet. source

In the paper

“Localization and sequence of this miRNA family is highly conserved down to insects and chordates, but not in worms.”

In the paper

“Cotransfection of these constructs with preMIR-33 significantly reduced activation of constructs containing the wild type 3Ј UTR when compared with transfection with a preMIR control (Fig. 5, B, D, and F).”

In the paper

“With regard to their sequence, miR-33a and miR-33b differ only by two nucleotides outside the seed sequence (Fig. 1B).”

In the paper

“Most species (including the mouse), however, only possess miR-33a in the SREBP2 gene and have lost the sequence of miR-33b within the SREBP1 gene (Fig. 1A and supplemental Fig. S3).”

In the paper

“overexpression of competitive antagonists of miR-33 action (i.e.miRNA sponges) in HepG2 cells led to an up-regulation of ABCA1 protein levels, which was accompanied by a significant increase in apoAI-dependent cellular cholesterol export (Fig. 3, C and D).”

In the paper

“Several explanations seem possible to explain why we did not observe an increase in ␤-oxidation and HADHB levels upon inhibition of miR-33.”

In the paper

“The discovery that miR-33 inhibits ABCA1 expression and consequentially cellular cholesterol export suggests that one genetic locus increases synthesis and limits export of cholesterol through two distinct mechanisms (Fig. 8A).”

• Also stated in: § miR-33 in Cholesterol and Fatty Acid Metabolism · plain

  “miR-33 reduces endogenous ABCA1 protein levels and modulates cellular cholesterol export in HepG2 cells.”

• Also stated in: § miR-33 in Cholesterol and Fatty Acid Metabolism · plain

  “miR-33 reduces endogenous ABCA1 protein levels and modulates cellular cholesterol export in THP1 macrophages.”

In the paper

“Inhibition of miR-33 with miRNA sponges or antisense oligonucleotides did not cause a reproducible increase in fatty acid ␤-oxidation or alteration of HADHB and CPT1A protein levels in HepG2 cells (data not shown).”

In the paper

“Site-directed mutagenesis of one or two of the predicted seed binding sites completely abolished this response (mutated nucleotides indicated by asterisks).”

In the paper

“Culturing cells at high density and/or in lipid-free medium leads to an increase of miR-33 (supplemental Fig. S12A).”

In the paper

“Notably, these two changes lead to a perfect complementarity of miR-33b with the CROT 3Ј UTR over a total of 18 nucleotides, which could allow miR-33b to induce Ago2-dependent cleavage of the CROT transcript (supplemental Fig. S8, B versus C).”

In the paper

“It should be noted that several prediction algorithms do not recognize CPT1A as a miR-33 target gene.This is due to the fact that these algorithms utilize a short 3Ј UTR generated from an alternative exon (supplemen- tal Fig. S9A).”

In the paper

“When analyzing cellular extracts by TLC we observed a significant increase in cellular free fatty acid levels and triacylglycerides (Fig. 6D and supplemental Fig. S11).”

In the paper

“miR-33 reduces endogenous ABCA1 protein levels and modulates cellular cholesterol export in HepG2 cells.”

In the paper

“miR-33 reduces endogenous ABCA1 protein levels and modulates cellular cholesterol export in THP1 macrophages.”

In the paper

“miR-33a localization within SREBP2 and sequences are evolutionary conserved.”

In the paper

“In fact, there was a small, but not significant, trend for reduced miR-33 levels, as one might intuitively expect in a situation of hyperalimentation.”

In the paper

“miR-33 reduces ␤-oxidation and alters cellular fatty acid metabolism.”

In the paper

“CPT1A is the liver-specific isoform of the enzyme that converts acyl-CoA to acylcarnitine and thereby allows fatty acids to enter the mitochondrion where fatty acid ␤-oxidation takes place (27).”

What the sources say

• ?Not checked — no full text of the sourcecites [27] Bonnefont, 2004, Mol. Aspects Med. source

In the paper

“ABCA1 acts as a cellular cholesterol export pump.Although there are a number of cholesterol efflux pumps, ABCA1 has the unique capability to export cholesterol to lipid-free apolipoprotein AI (apoAI) and is therefore involved in the initial step of high-density lipoprotein particle formation in liver, as well as reverse cholesterol transport (26).”

What the sources say

• ?Not checked — no full text of the sourcecites [26] Tall, 2008, Cell Metab. source

In the paper

“The genetic locus encoding SREBP2 and miR-33 therefore contains a protein that increases lipid synthesis and a miRNA that prevents export and degradation of newly synthesized lipids.”

In the paper

“CROT, sometimes abbreviated COT, is a peroxisomal enzyme that couples short chain fatty acids to carnitine and thereby allows their entry in the mitochondrial matrix.”

What the sources say

• ?Not checked — no full text of the sourcecites [29] Wanders, 2010, Biochim. Biophys. Acta source

In the paper

“Although not strictly an enzyme of the ␤-oxidation, the activity of CPT1A is rate-limiting for ␤oxidation, and is the most highly regulated element in this metabolic pathway (28).”

What the sources say

• ?Not checked — no full text of the sourcecites [28] Saggerson, 2008, Annu. Rev. Nutr. source

In the paper

“Taken together, our data suggest that endogenous miR-33 targets and represses translation of ABCA1, and reduces cellular cholesterol export.”

In the paper

“It has recently been noted that cancer cell lines and proliferative cells often express transcripts with shorter 3Ј UTRs (51).”

What the sources say

• ?Not checked — no full text of the sourcecites [51] Sandberg, 2008, Science source

In the paper

“Loss-of-function alleles of ABCA1 are associated with a decrease in HDL levels and might be associated with an increase in cardiovascular mortality (42,49).”

What the sources say

• ?Not checked — no full text of the sourcecites [42] Brunham, 2008, JAMA source
• ?Not checked — no full text of the sourcecites [49] Frikke-Schmidt, 2008, JAMA source

In the paper

“Given its preventive effect in mice, increasing ABCA1 protein levels by inhibiting miR-33 might therefore prove useful in the prevention of cardiovascular diseases.”

In the paper

“Inactivating mutations in ABCA1 lead to the development of Tangier disease, which is characterized by the absence of HDL and the accumulation of cholesterol in peripheral tissues (42).”

What the sources say

• ?Not checked — no full text of the sourcecites [42] Brunham, 2008, JAMA source

In the paper

“Although CROT is localized to the peroxisomes, it serves to transfer remnants from peroxisomal ␤-oxidation to the mitochondria and therefore fuels mitochondria with fatty acids independently of CPT1A (29).”

What the sources say

• ?Not checked — no full text of the sourcecites [29] Wanders, 2010, Biochim. Biophys. Acta source

In the paper

“When decreases in cellular cholesterol levels trigger the activation of SREBP transcription factors, the presence of miR-33 prevents cholesterol from leaving the cell by suppressing expression of ABCA1, thus allowing cellular cholesterol levels to be restored (Fig. 8A).”

In the paper

“On the other hand, elevated levels of high density lipoprotein cholesterol reduces the risk of arteriosclerosis (42,43).”

What the sources say

• ?Not checked — no full text of the sourcecites [42] Brunham, 2008, JAMA source
• ?Not checked — no full text of the sourcecites [43] Kannel, 1983, Am. J. Cardiol. source

In the paper

“The gene HADHB encodes for the ␤-subunit of the mitochondrial trifunctional protein that carries out the last three steps of ␤-oxidation (30).”

What the sources say

• ?Not checked — no full text of the sourcecites [30] Eaton, 2000, Biochem. Soc. Trans. source

In the paper

“Peroxisomal ␤-oxidation is limited to the degradation of long-and medium-chain fatty acids as it is not able to oxidize short chain fatty acids (29).”

What the sources say

• ?Not checked — no full text of the sourcecites [29] Wanders, 2010, Biochim. Biophys. Acta source

In the paper

“Agents that alter HDL cholesterol levels therefore carry the potential to reduce cardiovascular diseases.”

In the paper

“Presently, non-alcoholic fatty liver disease (NAFLD) is by far the most common liver ailment (53).”

What the sources say

• ?Not checked — no full text of the sourcecites [53] Angulo, 2002, N. Engl. J. Med. source

In the paper

“Likewise, altering the levels of HADHB might change the potential maximum flux through ␤-oxidation, but might have little influence under most conditions where fatty acid supply in the mitochondria is rate-limiting for ␤-oxidation.”

In the paper

“Protein amounts of SREBPs therefore represent the maximum potential to respond to changes in lipid levels.”

In the paper

“Interfering with miR-33 function in vivo therefore not only holds promise in increasing HDL cholesterol levels but may also prove beneficial for treatment of NAFLD and NASH.”

In the paper

“With the increasing prevalence of obesity, it affects 10 -25% of the general population and up to 75% of obese type II diabetic patients (54).”

What the sources say

• ?Not checked — no full text of the sourcecites [54] Marchesini, 2008, J. Clin. Endocrinol. Metab. source

In the paper

“A recent study of Cabellero and co-workers (55) suggests that SREBP2 transcript levels in NAFLD and NASH are elevated 3-and 7-fold, respectively.”

What the sources say

• ?Not checked — no full text of the sourcecites [55] Caballero, 2009, J. Hepatol. source

In the paper

“The relevance of targeting HADHB is not quite as apparent, although reduced HADHB concentrations might help maintain adequate intermediary product levels in a situation where the enzyme that supplies the substrates for ␤-oxidation (CPT1A and CROT) is inhibited.”