Dietary protein restriction reduces circulating VLDL triglyceride levels via CREBH-APOA5–dependent and –independent mechanisms
J. Humberto Treviño-Villarreal, Justin S. Reynolds, Alexander Bartelt, P. Kent Langston, Michael R. MacArthur, Alessandro Arduini, Valeria Tosti, Nicola Veronese, Beatrice Bertozzi, Lear E. Brace, Pedro Mejia, Kaspar Trocha, Gustavo S. Kajitani, Alban Longchamp, Eylul Harputlugil, Rose Gathungu, Susan S. Bird, Arnold D. Bullock, Robert S. Figenshau, Gerald L. Andriole, Andrew Thompson, Jöerg Heeren, C. Keith Ozaki, Bruce S. Kristal, Luigi Fontana, James R. Mitchell
J. Humberto Treviño-Villarreal, Justin S. Reynolds, Alexander Bartelt, P. Kent Langston, Michael R. MacArthur, Alessandro Arduini, Valeria Tosti, Nicola Veronese, Beatrice Bertozzi, Lear E. Brace, Pedro Mejia, Kaspar Trocha, Gustavo S. Kajitani, Alban Longchamp, Eylul Harputlugil, Rose Gathungu, Susan S. Bird, Arnold D. Bullock, Robert S. Figenshau, Gerald L. Andriole, Andrew Thompson, Jöerg Heeren, C. Keith Ozaki, Bruce S. Kristal, Luigi Fontana, James R. Mitchell
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Research Article Metabolism

Dietary protein restriction reduces circulating VLDL triglyceride levels via CREBH-APOA5–dependent and –independent mechanisms

Abstract

Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Dietary interventions based on protein restriction (PR) reduce circulating triglycerides (TGs), but underlying mechanisms and clinical relevance remain unclear. Here, we show that 1 week of a protein-free diet without enforced calorie restriction significantly lowered circulating TGs in both lean and diet-induced obese mice. Mechanistically, the TG-lowering effect of PR was due, in part, to changes in very low–density lipoprotein (VLDL) metabolism both in liver and peripheral tissues. In the periphery, PR stimulated VLDL-TG consumption by increasing VLDL-bound APOA5 expression and promoting VLDL-TG hydrolysis and clearance from circulation. The PR-mediated increase in Apoa5 expression was controlled by the transcription factor CREBH, which coordinately regulated hepatic expression of fatty acid oxidation–related genes, including Fgf21 and Ppara. The CREBH-APOA5 axis activation upon PR was intact in mice lacking the GCN2-dependent amino acid–sensing arm of the integrated stress response. However, constitutive hepatic activation of the amino acid–responsive kinase mTORC1 compromised CREBH activation, leading to blunted APOA5 expression and PR-recalcitrant hypertriglyceridemia. PR also contributed to hypotriglyceridemia by reducing the rate of VLDL-TG secretion, independently of activation of the CREBH-APOA5 axis. Finally, a randomized controlled clinical trial revealed that 4–6 weeks of reduced protein intake (7%–9% of calories) decreased VLDL particle number, increased VLDL-bound APOA5 expression, and lowered plasma TGs, consistent with mechanistic conservation of PR-mediated hypotriglyceridemia in humans with translational potential as a nutraceutical intervention for dyslipidemia.

Authors

J. Humberto Treviño-Villarreal, Justin S. Reynolds, Alexander Bartelt, P. Kent Langston, Michael R. MacArthur, Alessandro Arduini, Valeria Tosti, Nicola Veronese, Beatrice Bertozzi, Lear E. Brace, Pedro Mejia, Kaspar Trocha, Gustavo S. Kajitani, Alban Longchamp, Eylul Harputlugil, Rose Gathungu, Susan S. Bird, Arnold D. Bullock, Robert S. Figenshau, Gerald L. Andriole, Andrew Thompson, Jöerg Heeren, C. Keith Ozaki, Bruce S. Kristal, Luigi Fontana, James R. Mitchell

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Figure 3

CREBH regulates Apoa5 expression upon protein restriction.

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CREBH regulates Apoa5 expression upon protein restriction. (A–D) B6D2F1 ...
(A–D) B6D2F1 mice were fed complete (C) or protein free (PF) diet for 1 week prior to analysis. (A) Immunoblot of CREBH full-length (FL) and cleaved fragment (CF) in liver extracts; 2 left lanes, CREBH-KO antibody specificity controls. Right, quantitation of CREBH CF normalized to FL and expressed in arbitrary units (AU; n = 5/group; 2-tailed Student’s t test). (B) CREBH CF immunoblot in liver nuclear extracts. Right, quantitation normalized to lamin A/C (n = 5/group; 2-tailed Student’s t test). (C) Representative confocal microscopic images (20× objective) of livers stained for CREBH amino-terminus (fire-red look-up intensity table visualization), DAPI-stained nuclei (DNA, blue) super-imposed onto the corresponding differential interference contract (DIC) image, and composite. Scale bars: 50 µm. Right micrograph, 10× digital magnification of indicated area. (D) Hepatic mRNA expression of CREBH targets Apoa4, Cyp4a10, Pepck, and Fgf21 (n = 5–10/group; 2-tailed Student’s t test). (E) Representative confocal microscopic images (63× objective) of HepG-2 cells transfected with hemagglutinin-tagged (HA-tagged) human CREBH CF (hCREBH [CF]-HA) vs. empty vector (Mock) and labeled with anti-HA antibody or DAPI (DNA) as indicated. Scale bars: 10 µm. (F) mRNA expression of human Apoa5 from cells transfected as in E; data in triplicate from one representative experiment of 3; 2-tailed Student’s t test. (G) ChIP of CREBH from livers of mice on the indicated diet, followed by qPCR analysis of the region flanking the predicted CREBH binding motif in the Apoa5 promoter (n = 3/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (H–N) CREBH-KO or WT control littermates were fed the indicated diet for 1 week prior to analysis. (H–J) Hepatic mRNA levels of Apoa5 (H), Fgf21 (I), and the Pparα targets Acadvl, Ctp1a, and Pparα itself (J) (n = 4/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (K) Immunoblot of hepatic APOA5; below, quantitation normalized to tubulin (n = 3/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (L–M) VLDL-bound APOA5 levels expressed as the ratio of serum APOA5 to APOB-100 (L) and serum FGF21 levels (M) (n = 4/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (N) Fasted serum TG concentrations pooled from 3 independent experiments including male and female WT and CREBH-KO mice (2-way ANOVA with Sidak post-hoc test between indicated groups). Data expressed as mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.