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 5

Constitutive mTORC1 signaling inhibits CREBH activation.

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Constitutive mTORC1 signaling inhibits CREBH activation. (A) Immunoblot ...
(A) Immunoblot of phospho- and total S6 in liver extracts. Below, quantitation of phospho-S6 normalized to total and expressed in arbitrary units (AU); n = 4–5/group; 2-tailed Student’s t test. (B–E and G–K) Liver-specific TSC1–KO (LiTSC1-KO; TSC1fl/fl|Albumin-Cre+/–) mice and littermate controls (TSC1fl/fl|Albumin-Cre–/–) fed a complete (C) or protein-free (PF) diet for 1 week prior to analysis. (B) Immunoblot of CREBH full-length (FL) and cleaved fragment (CF) in liver extracts. Below, quantitation of CF normalized to FL (n = 3/group; 2-way ANOVA with Sidak post-hoc test between the indicated groups). (C) Hepatic Apoa5 mRNA expression (n = 7–8/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (D) Immunoblot of hepatic APOA5. Below, quantitation normalized to tubulin (n = 3/group; 2-way ANOVA with Sidak post-hoc test between indicated groups; interaction P = 0.01/30% variation; genotype P = 0.02/22% variation; diet P = 0.02/24% variation). (E) Apoa5 mRNA expression from livers of LiTSC1-KO mice injected once daily with rapamycin (1 mg/kg) or vehicle (Veh) for indicated days of treatment (Tx); n = 3–4/group; 1-way ANOVA with Dunnett post-hoc test compared with vehicle control. (F) Immunoblot of hepatic CREBH in mice with hepatocyte-specific Raptor deletion (LiRaptor-KO; Raptorfl/fl|Albumin-Cre+/–) vs. littermate controls (Raptorfl/fl|Albumin-Cre–/–). Below, quantitation of CREBH CF normalized to FL (n = 3/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (G) Serum TG (n = 13–14/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (H) VLDL-bound APOA5 levels expressed as the ratio of serum APOA5 to APOB-100 (n = 5–7/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (I) Correlation analysis between serum TG and circulating APOA5. Each symbol represents an individual mouse; r, Pearson’s coefficient. (J) TG content of VLDL particles purified from serum of WT and LiTSC1-KO mice expressed per unit APOB-100 indicative of VLDL particle lipidation (n = 3/group; 2-way ANOVA with Sidak post-hoc test between indicated groups). (K) Serum FGF21 levels (n = 4/group; 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.