Impaired Adipocyte SLC7A10 Promotes Lipid Storage

Abstract and Introduction

Abstract

Context: The neutral amino acid transporter SLC7A10/ASC-1 is an adipocyte-expressed gene with reduced expression in insulin resistance and obesity. Inhibition of SLC7A10 in adipocytes was shown to increase lipid accumulation despite decreasing insulin-stimulated uptake of glucose, a key substrate for de novo lipogenesis. These data imply that alternative lipogenic substrates to glucose fuel continued lipid accumulation during insulin resistance in obesity.

Objective: We examined whether increased lipid accumulation during insulin resistance in adipocytes may involve alter flux of lipogenic amino acids dependent on SLC7A10 expression and activity, and whether this is reflected by extracellular and circulating concentrations of marker metabolites.

Methods: In adipocyte cultures with impaired SLC7A10, we performed RNA sequencing and relevant functional assays. By targeted metabolite analyses (GC-MS/MS), flux of all amino acids and selected metabolites were measured in human and mouse adipose cultures. Additionally, SLC7A10 mRNA levels in human subcutaneous adipose tissue (SAT) were correlated to candidate metabolites and adiposity phenotypes in 2 independent cohorts.

Results: SLC7A10 impairment altered expression of genes related to metabolic processes, including branched-chain amino acid (BCAA) catabolism, lipogenesis, and glyceroneogenesis. In 3T3-L1 adipocytes, SLC7A10 inhibition increased fatty acid uptake and cellular content of glycerol and cholesterol. SLC7A10 impairment in SAT cultures altered uptake of aspartate and glutamate, and increased net uptake of BCAAs, while increasing the net release of the valine catabolite 3- hydroxyisobutyrate (3-HIB). In human cohorts, SLC7A10 mRNA correlated inversely with total fat mass, circulating triacylglycerols, BCAAs, and 3-HIB.

Conclusion: Reduced SLC7A10 activity strongly affects flux of BCAAs in adipocytes, which may fuel continued lipogenesis during insulin resistance, and be reflected in increased circulating levels of the valine-derived catabolite 3-HIB.

Introduction

Altered adipose tissue function, in close association with systemic insulin resistance, adipocyte hypertrophy, local and systemic low-grade inflammation and ectopic lipid accumulation, contributes to chronic metabolic diseases such as type 2 diabetes and cardiovascular diseases.^[1–4] New insight into the cellular and molecular processes underlying lipid storage and insulin resistance may enable novel strategies for prevention and therapy. Mature white adipocytes store excess energy primarily as triacylglycerols (TAG),^[5] and fatty acids (FAs) required for TAG synthesis are sourced from exogenous FA uptake^[6] and de novo lipogenesis.^[7] While approximately 60% of glycerol for TAG synthesis in the liver comes from pyruvate,^[8] TAG synthesis in adipocytes largely depends on glyceroneogenesis, a process functionally similar to hepatic gluconeogenesis.^[9,10] Activity of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PCK1/PEPCK), and other glyceroneogenic enzymes expressed in adipocytes can drive glycerol formation from, for example, lactate, pyruvate, and other TCA cycle intermediates, which partly also depends on the availability and metabolism of specific amino acids.^[11–14]

Metabolomic analyses have revealed altered circulating concentrations of amino acids and metabolites related to insulin resistance,^[15] including the branched-chain amino acids (BCAAs; leucine, isoleucine, and valine). Genetic variants associated with insulin resistance modulate BCAA catabolic pathways, and activation of BCAA catabolism has been shown to improve insulin sensitivity and lipid metabolism in rats and mice.^[16] Moreover, variants associated with BCAA catabolic pathways are associated with an increased risk of type 2 diabetes.^[17,18] Elevated circulating BCAA levels partly depend on the loss of steps in BCAA catabolism and/or downregulation of enzymes responsible for BCAA oxidation in adipose tissue.^[15,19–22] Increased catabolism of BCAAs during adipogenesis is associated with lipid accumulation^[23,24] and suggests that these essential amino acids provide carbon for lipid and/or glycerol synthesis.^[25] Altered blood concentrations of BCAA-related metabolites have been found to reflect metabolic changes and mediate insulin resistance, and an increased extracellular concentration of 3-hydroxyisobutyrate (3-HIB),^[26] a valine catabolite, is shown to be strongly associated with insulin resistance and type 2 diabetes.^[27] Higher 3-HIB levels reflect adipocyte lipid accumulation, and also directly influence core metabolic processes in white as well as brown adipocytes.^[27]

Our recent studies revealed the sodium independent amino acid transporter SLC7A10 (also known as ASC-1), carrying small neutral amino acids such as serine, glycine, and alanine, as a novel player regulating adipocyte lipid accumulation and metabolism in obesity and insulin resistance.^[28,29] SLC7A10 mRNA expression in adipose tissue is highly heritable and correlates inversely with risk alleles in the KLF14 type 2 diabetes risk locus; it is strongly associated with insulin resistance and adipocyte hypertrophy.^[30] In human primary and mouse 3T3-L1 adipose cultures, SLC7A10 impairment reduced serine uptake, production of the antioxidant glutathione, mitochondrial respiration, and insulin-stimulated glucose uptake, and increased reactive oxygen species generation and lipid accumulation.^[28] Another study, inhibiting the amino acid carrier in cultured human deep neck white adipocytes, confirmed reduction in serine uptake, but also found significantly reduced uptake of cysteine, glycine, and alanine compared to controls.^[31] SLC7A10 has recently also been implicated in thermogenesis and regulation of metabolism in different adipocyte subtypes.^[31–34] Therefore, understanding the effects of perturbed SLC7A10 activity in adipocytes in more detail may elucidate cellular and molecular pathways underlying the development of unhealthy adipose tissue expansion and insulin resistance. In particular, studying the means by which SLC7A10 inhibition promotes lipid accumulation while suppressing insulin-stimulated glucose uptake can identify alternate carbon sources for lipogenesis in adipocytes. Additionally, identifying circulating biomarkers that reflect these cellular changes may allow earlier detection of adipose-dependent development of insulin resistance.

In the present study, we examine changes in amino acid metabolism that may drive lipid accumulation in conditions of reduced insulin-stimulated glucose uptake, by profiling changes in the flux of amino acids and related metabolites in human adipocytes with and without pharmacologic inhibition of SLC7A10 activity. The clinical relevance of SLC7A10-dependent changes in the metabolism of BCAAs and other amino acids is supported by clinical cohorts showing strong correlations between adipose SLC7A10 mRNA, circulating amino acid and 3-HIB concentrations, adiposity traits, and systemic insulin resistance.

Authors and Disclosures

Regine Å. Jersin^1,2,*, Divya Sri Priyanka Tallapragada^1,2,*, Linn Skartveit^1,2, Mona S. Bjune^1,2, Maheswary Muniandy³, Sindre Lee-Ødegård⁴, Sini Heinonen³, Marcus Alvarez⁵, Kåre Inge Birkeland⁴, Christian André Drevon⁶, Päivi Pajukanta^5,7,8, Adrian McCann⁹, Kirsi H. Pietiläinen^3,10, Melina Claussnitzer^11,12, Gunnar Mellgren^1,2 and Simon N. Dankel^1,2

¹Mohn Nutrition Research Laboratory, Department of Clinical Science, University of Bergen, N-5021 Bergen, Norway
²Hormone Laboratory, Department of Medical Biochemistry and Pharmacology, Haukeland University Hospital, N-5021 Bergen, Norway
³Obesity Research Unit, Research Program for Clinical and Molecular Metabolism, Faculty of Medicine, University of Helsinki, FIN-00014 Helsinki, Finland
⁴Department of Transplantation Medicine, The University of Oslo, Institute of Clinical Medicine, and Oslo University Hospital, N-0372 Oslo, Norway
⁵Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
⁶Department of Nutrition, The University of Oslo, Institute of Basic Medical Sciences, N-0372 Oslo, Norway
⁷Bioinformatics Interdepartmental Program, UCLA, Los Angeles, CA 90095, USA
⁸Institute for Precision Health, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
⁹Bevital A/S, Laboratoriebygget, Haukeland University Hospital, N-5021 Bergen, Norway
¹⁰Obesity Center, Endocrinology, Abdominal Center, Helsinki University Hospital and University of Helsinki, FIN-00014 Helsinki, Finland
¹¹Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
¹²Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA

Correspondence
Simon N. Dankel, PhD, Department of Clinical Science, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. Email: simon.dankel@uib.no.

*These authors contributed equally.

Author Contributions
R.Å.J., D.S.P.T., L.S., and S.N.D. designed the study, carried out the experiments, analyzed and interpreted results, and wrote the manuscript. M.S.B. assisted with experiments. S.L-Ø., K.I.B., and C.D. conducted the MyoGlu study, and S.L-Ø. analyzed the data. S.H. and K.H.P. contributed to sample/data collection in the twin cohorts; M.A. and P.P. to generation and analysis of RNA-seq data; and M.M. and K.H.P. to the design, transcriptomics and metabolite data analysis, and interpretation for these cohorts. A.M. performed metabolite analyses. S.N.D. and G.M. facilitated the laboratory work. All authors reviewed and edited the manuscript. S.N.D. is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of all the analyses.

Disclosure
The authors declare no competing interests.