The transcription factor cyclic AMP-responsive element-binding protein H regulates triglyceride metabolism

Abstract

Here we report that the transcription factor cyclic AMP-responsive element-binding protein H (CREB-H, encoded by CREB3L3) is required for the maintenance of normal plasma triglyceride concentrations. CREB-H-deficient mice showed hypertriglyceridemia secondary to inefficient triglyceride clearance catalyzed by lipoprotein lipase (Lpl), partly due to defective expression of the Lpl coactivators Apoc2, Apoa4 and Apoa5 (encoding apolipoproteins C2, A4 and A5, respectively) and concurrent augmentation of the Lpl inhibitor Apoc3. We identified multiple nonsynonymous mutations in CREB3L3 that produced hypomorphic or nonfunctional CREB-H protein in humans with extreme hypertriglyceridemia, implying a crucial role for CREB-H in human triglyceride metabolism.

Figure 1. Creb3l3^−/− mice display HTG secondary to inefficient TG clearance catalyzed by Lpl

(a)Plasma TG, FFA, and cholesterol levels measured after a 16 h fast. Each dot represents an individual mouse. (b) We separated lipoproteins by density gradient ultracentrifugation of pooled plasma (n = 3 per each) after a 24 h fast. TG levels in and density of fractions (1 ml each) sequentially collected from top to bottom. (c) Plasma TG levels in WT (n = 9) and Creb3l3^−/− (n = 7) male mice deprived of food, starting at 8 AM. (d) Hepatic TG levels measured at fed state or after a 24h fast. n = 5 per group. (e) After concentration, fractions from (b) were separated on a 4–20% gradient SDS-polyacrylamide gels. The gel was stained by Coomassie Brilliant Blue G-250. Arrowhead indicates Apoc3 identified by mass spectrometry. VLDL, d < 1.006 g ml⁻¹; IDL d = 1.006–1.019 g ml⁻¹; LDL, d = 1.019–1.063 g ml⁻¹; HDL, d = 1.063–1.21 g ml⁻¹. (f) Apoc3 western blot of VLDL fractions. (g) Plasma TG concentrations measured after a 4h fasting followed by i.v. injection with tyloxapol (500 mg kg⁻¹). n = 4 per group. (h) Plasma TG concentrations measure after a 16 h fasting followed by oral gavage of olive oil (10 ml kg⁻¹). n = 6 per group. (i) Post-heparin Lpl activity (n = 6 per group) measured in the presence of heat inactivated serum pooled from WT or Creb3l3^−/− mice as Apoc sources. (j) Recombinant LPL was incubated with triolein substrate in the presence of plasma collected from WT or Creb3l3^−/− mice. Values represent FFA concentration released from triolein. n = 8 per group. *P < 0.05, ***P < 0.0001, compared to WT mice.

Figure 2. CREB-H controls genes involved in TG metabolism and mutations of CREB3L3 are associated with human HTG

(a)Hepatic mRNA levels determined by qRT-PCR. Mice were fasted for 24h before sacrifice. n = 3 mice per group. Error bars indicate standard deviation. (b) Apoc2, Apoa4, Apoa5 and Fgf21 mRNA levels at fed state or after a 24 h fast in WT and Creb3l3^−/− mice as determined by qRT-PCR. n = 4 mice per group. (c) Locations of the nonsynonymous mutations found in individuals with HTG, and predicted amino acid changes. bZIP: basic leucine zipper; TM: transmembrane domain. (d) Hepa1.6 cells were co-transfected with Apoa4, Apoc2, or Fgf21 promoterdriven luciferase reporters and the indicated CREB-H(N) constructs. Values represent fold induction of luciferase activities compared to the reporter only transfection. *P < 0.05, **P < 0.01, ***P < 0.0001.