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Increased thermogenesis by a noncanonical pathway in ANGPTL3/8-deficient mice

Banfi, Serena, Gusarova, Viktoria, Gromada, Jesper, Cohen, Jonathan C., Hobbs, Helen H.·2018·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1717420115PMC PMC5819435
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Abstract

Significance: White adipose tissue (WAT) serves as an energy reservoir during fasting and is replenished with fatty acids from circulating triglycerides upon refeeding. We showed previously that postprandial partitioning of fatty acids between oxidative and storage tissues is mediated by angiopoietin-like proteins 3 (A3) and 8 (A8). Here, we show that disruption of both Angptl3 and Angptl8 in mice causes striking alterations in energy metabolism: reduced fat mass, hyperthermia, increased metabolic rate, and beiging of subcutaneous WAT. The hypermetabolic features of the A3 −/− A8 −/− mice were most pronounced in fed animals, and attenuated with fasting and β3-adrenergic receptor blockade. These data indicate that A3 and A8 promote efficient energy utilization by tissues and limit the increase in energy expenditure associated with food intake.

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Fig. 1. Metabolic characterization of A3−/−, A8−/−, A3−/−A8−/−, and WT male mice. (A) Body weights of A3−/−, A8−/−, A3−/−A8−/−, and WT mice (n = 10–12 per group) were measured every other week starting at 6 wk of age (Right). Body weight (A) and composition (B) were measured in mice aged 8–10 wk (n = 6 per group). The experiment was repeated once, and the results were similar. (C) Food intake (Left) and total activity (Right) were assessed in mice housed individually in metabolic cages for 7 d. (D) Both O2 consumption and CO2 output were measured for 1 min at 30-min intervals for four consecutive days while the mice were housed in metabolic cages (n = 9; age 8–12 wk). Values are means ± SEM. Group means for VO2 consumption and VCO2 output were compared by ANCOVA with body weight as a covariate; similar results were obtained with an unpaired t test. Group means for other parameters were compared by using unpaired t tests. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 1. Metabolic characterization of A3−/−, A8−/−, A3−/−A8−/−, and WT male mice. (A) Body weights of A3−/−, A8−/−, A3−/−A8−/−, and WT mice (n = 10–12 per group) were measured every other week starting at 6 wk of age (Right). Body weight (A) and composition (B) were measured in mice aged 8–10 wk (n = 6 per group). The experiment was repeated once, and the results were similar. (C) Food intake (Left) and total activity (Right) were assessed in mice housed individually in metabolic cages for 7 d. (D) Both O2 consumption and CO2 output were measured for 1 min at 30-min intervals for four consecutive days while the mice were housed in metabolic cages (n = 9; age 8–12 wk). Values are means ± SEM. Group means for VO2 consumption and VCO2 output were compared by ANCOVA with body weight as a covariate; similar results were obtained with an unpaired t test. Group means for other parameters were compared by using unpaired t tests. *P < 0.05; **P < 0.01; ***P < 0.001.CC BY-NC-ND · PubMed Central
Fig. 2. Rectal temperatures of A3−/−, A8−/−, A3−/−A8−/−, and WT littermates at ambient temperature (21–23 °C) and after exposure to cold (6 °C). (A) Rectal temperatures were measured in male (Left) and female (Right) mice (n = 8–9 per group; age 7–9 wk) fed ad libitum. (B) Rectal temperatures (Upper) and indirect calorimetry measurements (Lower) were obtained in male mice (n = 11 per group, age 6–8 wk) that were fed ad libitum (Right Lower) and then after fasting for 24 h. Food was then provided, and the rectal temperature was remeasured at 24 h. VO2 consumption and CO2 output measurements were taken during the last 6 h of the fasting and refeeding periods. (C) Rectal temperatures were obtained every 2 h in male (Left) and female (Right) mice maintained for 6 h at 6 °C with free access to food (n = 5 or 6 per group, age 13–15 wk). Values are means ± SEM. Groups were compared by using unpaired t tests. *P < 0.05; **P < 0.01; ****P < 0.0001. The experiments were repeated twice, and the results were similar.
Fig. 2. Rectal temperatures of A3−/−, A8−/−, A3−/−A8−/−, and WT littermates at ambient temperature (21–23 °C) and after exposure to cold (6 °C). (A) Rectal temperatures were measured in male (Left) and female (Right) mice (n = 8–9 per group; age 7–9 wk) fed ad libitum. (B) Rectal temperatures (Upper) and indirect calorimetry measurements (Lower) were obtained in male mice (n = 11 per group, age 6–8 wk) that were fed ad libitum (Right Lower) and then after fasting for 24 h. Food was then provided, and the rectal temperature was remeasured at 24 h. VO2 consumption and CO2 output measurements were taken during the last 6 h of the fasting and refeeding periods. (C) Rectal temperatures were obtained every 2 h in male (Left) and female (Right) mice maintained for 6 h at 6 °C with free access to food (n = 5 or 6 per group, age 13–15 wk). Values are means ± SEM. Groups were compared by using unpaired t tests. *P < 0.05; **P < 0.01; ****P < 0.0001. The experiments were repeated twice, and the results were similar.CC BY-NC-ND · PubMed Central
Fig. 3. Adipose tissue morphology and mRNA levels in chow-fed A3−/−A8−/− and WT littermates. (A) Representative images of tissue chunks (Upper) and H&E staining of adipose tissue sections (Lower) (n = 4; age 18 wk; three fields per mouse). (Magnification: 20×.) The experiment was repeated twice, and the results were similar. (Scale bars: 50 μm.) (B) Fold changes in levels of selected mRNAs encoding proteins involved in fatty synthesis and oxidation (Upper) and glycolysis and mitochondrial function (Lower) in WAT-SQ of male WT and A3−/−A8−/− mice (n = 3 per group; age 18 wk) fasted overnight and then fed for 4 h. Transcript levels were determined by RNA-seq as described in Materials and Methods. Values are means ± SEM. Groups were compared using unpaired t tests. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was repeated once, and the results were similar.
Fig. 3. Adipose tissue morphology and mRNA levels in chow-fed A3−/−A8−/− and WT littermates. (A) Representative images of tissue chunks (Upper) and H&E staining of adipose tissue sections (Lower) (n = 4; age 18 wk; three fields per mouse). (Magnification: 20×.) The experiment was repeated twice, and the results were similar. (Scale bars: 50 μm.) (B) Fold changes in levels of selected mRNAs encoding proteins involved in fatty synthesis and oxidation (Upper) and glycolysis and mitochondrial function (Lower) in WAT-SQ of male WT and A3−/−A8−/− mice (n = 3 per group; age 18 wk) fasted overnight and then fed for 4 h. Transcript levels were determined by RNA-seq as described in Materials and Methods. Values are means ± SEM. Groups were compared using unpaired t tests. *P < 0.05; **P < 0.01; ***P < 0.001. The experiment was repeated once, and the results were similar.CC BY-NC-ND · PubMed Central
Fig. 4. Mitochondrial number, protein content, and size in WAT-SQ from A3−/−A8−/− and WT mice. (A) Ratio of mitochondrial to nuclear DNA (Left) and mitochondrial protein content (Right) of WAT-Epi, WAT-SQ, and BAT was determined by using mitochondria isolated by centrifugation. The amount of protein was normalized by tissue sample weight (n = 6 mice per group; age 11–13 wk). (B) Electron micrographs (Left) and mean (±SE) diameter (Right) of the mitochondria from WAT-SQ. (Scale bars: 0.5 μm.) (C and D) A Seahorse XF24 analyzer was used to measure extracellular OCRs in adipose tissue in mice fed a chow diet ad libitum (C) and after a 14-h fast (D) (n = 6; with five replicates per mouse). Values are means ± SEM. Groups were compared by using unpaired t tests. *P < 0.05; ****P < 0.00001.
Fig. 4. Mitochondrial number, protein content, and size in WAT-SQ from A3−/−A8−/− and WT mice. (A) Ratio of mitochondrial to nuclear DNA (Left) and mitochondrial protein content (Right) of WAT-Epi, WAT-SQ, and BAT was determined by using mitochondria isolated by centrifugation. The amount of protein was normalized by tissue sample weight (n = 6 mice per group; age 11–13 wk). (B) Electron micrographs (Left) and mean (±SE) diameter (Right) of the mitochondria from WAT-SQ. (Scale bars: 0.5 μm.) (C and D) A Seahorse XF24 analyzer was used to measure extracellular OCRs in adipose tissue in mice fed a chow diet ad libitum (C) and after a 14-h fast (D) (n = 6; with five replicates per mouse). Values are means ± SEM. Groups were compared by using unpaired t tests. *P < 0.05; ****P < 0.00001.CC BY-NC-ND · PubMed Central
Fig. 5. Effect of thermoneutrality and β-AR blockade on rectal temperature in A3−/−A8−/− and WT chow-fed mice. (A) Six-week-old male A3−/−A8−/− and WT mice (n = 8 per group) were maintained at thermoneutrality for 5 wk. Rectal temperatures, body weights, VO2 consumption, and VCO2 output were measured as described in the Materials and Methods. (B) Rectal temperatures were obtained in mice fed ad libitum (first 4 bars) or after a 14-h fast (second 4 bars) (n = 6; age 9–12 wk). The mice were then treated with a nonselective β-adrenergic blocker (propranolol; 5 mg/kg) and provided food. Temperatures were monitored as described in Materials and Methods. (C) Male mice (n = 5 per group) were treated chronically with propranolol (0.5 mg/mL in the drinking water) starting at 6 wk of age for 5 wk. Heart rates were measured as a control for propranolol activity. (D) Mice (n = 5 per group; age 7–9 wk) were treated with l-748,337 compound, a β3-receptor blocker (5 mg/kg). (E) Male mice were maintained at thermoneutrality for 5 wk and then injected twice with CL 316,243 (1 mg/kg, 18 h apart) (n = 4; age 16–17 wk). Values are means ± SEM. Group means were compared by using unpaired t tests. Experiments were repeated once, and the results were similar. (F) cAMP levels were measured in tissue lysates from WAT-SQ, BAT, and WAT-Epi of WT and A3−/−A8−/− mice fed ad libitum (n = 5; age 8–11 wk). Levels were measured by using an ELISA, as described in Materials and Methods. Values are means ± SEM. Groups were compared by using unpaired t tests. Experiments were repeated once, and the results were similar. CL, CL 316,243; L, l-748,337; P, propranolol. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 5. Effect of thermoneutrality and β-AR blockade on rectal temperature in A3−/−A8−/− and WT chow-fed mice. (A) Six-week-old male A3−/−A8−/− and WT mice (n = 8 per group) were maintained at thermoneutrality for 5 wk. Rectal temperatures, body weights, VO2 consumption, and VCO2 output were measured as described in the Materials and Methods. (B) Rectal temperatures were obtained in mice fed ad libitum (first 4 bars) or after a 14-h fast (second 4 bars) (n = 6; age 9–12 wk). The mice were then treated with a nonselective β-adrenergic blocker (propranolol; 5 mg/kg) and provided food. Temperatures were monitored as described in Materials and Methods. (C) Male mice (n = 5 per group) were treated chronically with propranolol (0.5 mg/mL in the drinking water) starting at 6 wk of age for 5 wk. Heart rates were measured as a control for propranolol activity. (D) Mice (n = 5 per group; age 7–9 wk) were treated with l-748,337 compound, a β3-receptor blocker (5 mg/kg). (E) Male mice were maintained at thermoneutrality for 5 wk and then injected twice with CL 316,243 (1 mg/kg, 18 h apart) (n = 4; age 16–17 wk). Values are means ± SEM. Group means were compared by using unpaired t tests. Experiments were repeated once, and the results were similar. (F) cAMP levels were measured in tissue lysates from WAT-SQ, BAT, and WAT-Epi of WT and A3−/−A8−/− mice fed ad libitum (n = 5; age 8–11 wk). Levels were measured by using an ELISA, as described in Materials and Methods. Values are means ± SEM. Groups were compared by using unpaired t tests. Experiments were repeated once, and the results were similar. CL, CL 316,243; L, l-748,337; P, propranolol. *P < 0.05; **P < 0.01; ***P < 0.001.CC BY-NC-ND · PubMed Central
Fig. 6. Effect of inactivating circulating A3, A8, and A3 plus A8 on rectal temperature. (A) Anti-human A8 (REGN3776; 10 mg/kg) and anti-mouse A3 (REGN1500; 10 mg/kg) or control monoclonal antibodies (CTRL IgG; RG1945) were given i.v. for 3 d to ANGPTL8hum/hum mice (n = 6 per group; age 9 wk). Values are means ± SEM. Groups were compared by using unpaired t tests. **P < 0.01. The experiment was repeated, and the results were similar. (B) Overview of postprandial TG metabolism in WAT-SQ from WT (Upper) and A3−/−A8−/− (Lower) mice. In fed WT mice, circulating dietary TGs are hydrolyzed by LPL, and FAs are taken up into adipocytes, where they are esterified and stored as TG in lipid droplets. Norepinephrine (NE) activates β3-AR, resulting in activation of ADCY6, which generates cAMP and subsequent phosphorylation and activation of protein kinase A (PKA-P). PKA-P activates both TG hydrolysis and oxidative phosphorylation; the rise in intracellular FA inhibits ADCY6, thus inhibiting these downstream pathways. In WAT-SQ from A3−/−A8−/− mice, LPL in oxidative tissues fails to be suppressed, resulting in diminished delivery of circulating TG to WAT (9). Glucose uptake and its conversion to FAs is increased in these animals. The reduced intracellular concentration of FA may fail to suppress ADCY6, accounting for the increased in cAMP levels. We hypothesize that this results in activation of PKA, causing an increase in both lipolysis and oxidative phosphorylation.
Fig. 6. Effect of inactivating circulating A3, A8, and A3 plus A8 on rectal temperature. (A) Anti-human A8 (REGN3776; 10 mg/kg) and anti-mouse A3 (REGN1500; 10 mg/kg) or control monoclonal antibodies (CTRL IgG; RG1945) were given i.v. for 3 d to ANGPTL8hum/hum mice (n = 6 per group; age 9 wk). Values are means ± SEM. Groups were compared by using unpaired t tests. **P < 0.01. The experiment was repeated, and the results were similar. (B) Overview of postprandial TG metabolism in WAT-SQ from WT (Upper) and A3−/−A8−/− (Lower) mice. In fed WT mice, circulating dietary TGs are hydrolyzed by LPL, and FAs are taken up into adipocytes, where they are esterified and stored as TG in lipid droplets. Norepinephrine (NE) activates β3-AR, resulting in activation of ADCY6, which generates cAMP and subsequent phosphorylation and activation of protein kinase A (PKA-P). PKA-P activates both TG hydrolysis and oxidative phosphorylation; the rise in intracellular FA inhibits ADCY6, thus inhibiting these downstream pathways. In WAT-SQ from A3−/−A8−/− mice, LPL in oxidative tissues fails to be suppressed, resulting in diminished delivery of circulating TG to WAT (9). Glucose uptake and its conversion to FAs is increased in these animals. The reduced intracellular concentration of FA may fail to suppress ADCY6, accounting for the increased in cAMP levels. We hypothesize that this results in activation of PKA, causing an increase in both lipolysis and oxidative phosphorylation.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Banfi, S., Gusarova, V., Gromada, J., Cohen, J. C., & Hobbs, H. H.
Affiliations
  • Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390;
  • Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591;
  • Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2018-01-22
Volume
115
Issue
6
ISSN
  • 0027-8424
  • 1091-6490
DOI
10.1073/pnas.1717420115
PMC
PMC5819435
Cited by
56
References
5
Language
en
Licence
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)
Funders
HHS | National Institutes of Health (5 PO1 HL20948.)

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