Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84

Abstract

Fatty acid receptors have been recognized as important players in glycaemic control. This study is the first to describe a role for the medium-chain fatty acid (MCFA) receptor G-protein-coupled receptor (Gpr) 84 in skeletal muscle mitochondrial function and insulin secretion. We are able to show that Gpr84 is highly expressed in skeletal muscle and adipose tissue. Mice with global deletion of Gpr84 [Gpr84 knockout (KO)] exhibit a mild impairment in glucose tolerance when fed a MCFA-enriched diet. Studies in mice and pancreatic islets suggest that glucose intolerance is accompanied by a defect in insulin secretion. MCFA-fed KO mice also exhibit a significant impairment in the intrinsic respiratory capacity of their skeletal muscle mitochondria, but at the same time also exhibit a substantial increase in mitochondrial content. Changes in canonical pathways of mitochondrial biogenesis and turnover are unable to explain these mitochondrial differences. Our results show that Gpr84 plays a crucial role in regulating mitochondrial function and quality control.-Montgomery, M. K., Osborne, B., Brandon, A. E., O'Reilly, L., Fiveash, C. E., Brown, S. H. J., Wilkins, B. P., Samsudeen, A., Yu, J., Devanapalli, B., Hertzog, A., Tolun, A. A., Kavanagh, T., Cooper, A. A., Mitchell, T. W., Biden, T. J., Smith, N. J., Cooney, G. J., Turner, N. Regulation of mitochondrial metabolism in murine skeletal muscle by the medium-chain fatty acid receptor Gpr84.

Keywords: insulin resistance; insulin secretion; mitochondrial function.

Figure 1

A, B) Tissue characterization of Gpr84 in mice. Relative (A) and absolute (B) quantification of Gpr84 mRNA expression. C) Tissue distribution of Gpr84 at the protein level. D) Specificity of the Gpr84 antibody was determined through overexpression (OE) or knockdown (KD) of Gpr84 in tibialis anterior muscle using in vivo electrotransfer. Shown are means ± sem, n =4 mice/group and assessment.

Figure 2

Phenotypic characterization of Gpr84 KO mice and their WT littermates, fed either chow diet (control) or an MCFA‐enriched high‐fat diet for 8 wk. Gpr84 mRNA (A) and protein levels (B) in quadriceps muscle of WT and KO mice, body weight (C), fat mass as determined by EchoMRI (D), heat production as a measure of whole‐body energy expenditure (E), and RER (F). Shown are means ± sem, n = 6‐12/group. Statistical differences were determined by 2‐way ANOVA followed by Bonferroni post hoc test.

Figure 3

A‐D) Glucose metabolism in Gpr84 KO mice and their WT littermates, fed either a chow diet (control) or an MCFA‐enriched high‐fat diet for 8 wk. Glucose excursion during an ip.GTT (A), insulin levels during the GTT (B), GSIS (C), and insulin content of islets isolated from Gpr84 KO and WT mice, and incubated in either control or lauric acid‐enriched medium (D). E, F) Relative islet insulin content based on insulin staining and representative pancreatic images from chow and MCFA‐fed WT and Gpr84 KO mice. G‐L) Hyperinsulinemic euglycemic clamp data: GIR (G), glucose disposal rate (RD) (H), suppression of hepatic glucose output (I), and peripheral glucose uptake in quadriceps muscle (J), gastrocnemius muscle (K), and heart (L). Shown are means ± sem, n = 6‐12/group. Statistical differences were determined by 2‐way ANOVA followed by Bonferroni post hoc test. A) Asterisks show statistical difference between MCFA KO and CHOW WT as well as CHOW KO at respective time points. B) Asterisks show statistical difference between MCFA WT and CHOW WT.

Figure 4

A— C) Lipidomics analysis of quadriceps muscle of Gpr84 KO mice and their WT littermates, fed either a chow diet (control) or an MCFA‐enriched high‐fat diet for 8 wk. Total TAG content (B) as well as the respective TAG species in control (A) and MCFA‐fed mice (C). D‐F) Total DAG content (D) as well as the respective DAG species in control (E) and MCFA‐fed mice (F). G—I) Total ceramide content (G) as well as the respective ceramide species in control (H) and MCFA‐fed mice (I). Shown are means ± sem, with n = 5/group. Statistical differences were determined by 2‐way ANOVA followed by Bonferroni post hoc test (for total lipid content), or by Student's t test comparing specific lipid species between WT and KO mice.

Figure 5

Skeletal muscle metabolic characterization of Gpr84 KO mice and their WT littermates, fed either a chow diet (control) or an MCFA‐enriched high‐fat diet for 8 wk. A‐D) Succinate‐driven state 2, 3, and 4 respiration in isolated mitochondria (A), hydrogen peroxide generation in isolated mitochondria in the presence of succinate or succinate and rotenone (B), as well as determination of TBARS (C) and LOOH content in whole muscle homogenates (D). E) Respiration of intact muscle fibers in the presence of glutamate, glutamate and ADP, and succinate and ADP. F) Immunoblotting analysis of various markers of mitochondrial capacity. Representative immunoblots show n = 2/group; however, indicated percentage changes and statistical significance refer to n = 8/group. G—I) Mitochondrial DNA/nuclear DNA ratio as an indicator of mitochondrial content (G), SDH (H), and β‐HAD activity (I) in muscle lysates. J) Immunoblotting analysis of autophagy regulators/markers in whole quad lysates. Shown are means ± sem, n = 7‐10 mice/assessment/group. Statistical differences were determined by 2‐way ANOVA followed by Bonferroni post hoc test; or by Student's t test for the immunoblotting analysis.

Figure 6

RNA‐Seq transcriptomic analysis of quadriceps muscle (n = 4/group). Shown is hierarchical clustering of genes encoding a broad suite of autophagy/mitophagy regulators (A), ETC proteins (B), respiratory complex assembly factors (C), and mitochondrial import/export proteins (D) for Gpr84 KO mice and their WT littermates, fed either a chow diet (con) or an MCFA‐enriched high‐fat diet for 8 wk.