Major facilitator superfamily domain-containing protein 2a (MFSD2A) has roles in body growth, motor function, and lipid metabolism

Abstract

The metabolic adaptations to fasting in the liver are largely controlled by the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARα), where PPARα upregulates genes encoding the biochemical pathway for β-oxidation of fatty acids and ketogenesis. As part of an effort to identify and characterize nutritionally regulated genes that play physiological roles in the adaptation to fasting, we identified Major facilitator superfamily domain-containing protein 2a (Mfsd2a) as a fasting-induced gene regulated by both PPARα and glucagon signaling in the liver. MFSD2A is a cell-surface protein homologous to bacterial sodium-melibiose transporters. Hepatic expression and turnover of MFSD2A is acutely regulated by fasting/refeeding, but expression in the brain is constitutive. Relative to wildtype mice, gene-targeted Mfsd2a knockout mice are smaller, leaner, and have decreased serum, liver and brown adipose triglycerides. Mfsd2a knockout mice have normal liver lipid metabolism but increased whole body energy expenditure, likely due to increased β-oxidation in brown adipose tissue and significantly increased voluntary movement, but surprisingly exhibited a form of ataxia. Together, these results indicate that MFSD2A is a nutritionally regulated gene that plays myriad roles in body growth and development, motor function, and lipid metabolism. Moreover, these data suggest that the ligand(s) that are transported by MFSD2A play important roles in these physiological processes and await future identification.

Figure 1. MFSD2A expression is nutritionally regulated and requires PPARα and glucagon signaling.

A, Representative Western blot of MFSD2A expression in the indicated tissues isolated from WT mice that were ad libitum fed, fasted for 24 h, or fasted for 24 h and ad libitum refed (n = 3 mice per condition). B, Western blot of liver samples from fed and fasted wildtype (WT) and PPARα knockout (KO) mice (n = 4 per genotype per condition). C, Western blot of livers from two glucagon receptor (Gcgr KO)-deficient mice and two WT fasted littermates probed for MFSD2A expression (n = 6–7). D, Western blot for MFSD2A from liver samples of 24 h fasted WT mice (0 h), and mice fasted, refed and sacrificed 1 to 9 h after refeeding. β-actin served as a loading control in all panels. Each lane represents a liver sample from an individual animal.

Figure 2. MFSD2A is rapidly turned over from the cell surface via the lysosome.

A, MFSD2A turnover as a function of time in cycloheximide-treated (CHX) HEK293 cells transfected with mouse Mfsd2a and primary hepatocytes from fasted mice. Time indicates duration of CHX treatment. Graph quantifies MFSD2A expression. B, Turnover of plasma membrane MFSD2A in Mfsd2a-transfected HEK293 cells. Cells were incubated with CHX for the indicated time period and then treated with membrane-impermeable maleimide-PEG₂-Biotin. Protein homogenates were immunoprecipitated with streptavidin beads, prior to Western blot analysis for plasma membrane localized MFSD2A. C and D, Mfsd2a-transfected HEK293 cells were treated with CHX as in panel A, with or without (C) chloroquine (CLQ) or (D) MG-132 (Western blot not shown). Protein extracts were analyzed by Western blot for MFSD2A. Graphs quantify MFSD2A expression. β-actin in A and C and Na-K-ATPase in B served as a loading control. Quantification represents experiments in triplicate, displayed as mean ± SD.

Figure 3. Generation of an Mfsd2a KO mouse.

A, Schema for conventional Mfsd2a knockout where exons 4–13 were replaced with a neomycin resistance cassette (NEO). B, PCR genotyping of WT, HET and KO mice with primers indicated in A. C, Western blots of MFSD2A expression in liver and brain from fasted WT, HET, and KO male mice. D, Western blot of MFSD2A in liver of WT and KO mice exposed to 4°C. E, Western blot analysis of MFSD2A expression in indicated tissues from fasted WT and KO mice. F, Western blot of MFSD2A expression in the indicated brain regions in WT fed, WT fasted, and KO fasted mice. β-actin is the loading control in panels C–F. Note: lack of β-actin in heart sample in D is expected based on actin isoform expression.

Figure 4. Mfsd2a KO mice are smaller and leaner than WT littermates, with reduced serum TG.

A, Number of genotype obtained for male and female day e18.5 embryos (n = 7 litters, 62 embryos) and post-natal day 10 pups (n = 156 litters, 1135 pups). Chi² analysis gives the probability that the observed ratios are Mendelian. B, Growth curves of weekly weights of offspring from heterozygous intercrosses for all three genotypes (average of n = 30 per genotype). Body weights between wildtype and KO mice are significantly different at all time points by week 2. C, Scatterplot of body lengths indicating average (from nose-to-anus) of WT, HET and KO littermates (n = 6–11). D, Absolute (left panel) and relative (right panel) adiposity of KO and WT littermates (n = 4–7) was determined by EchoMRI. E, Food intake per mouse, per 24 h (males, 14–16 weeks of age, n>5 per genotype). F, GTT of 14-week old chow-fed WT and KO males after overnight fast (n = 4). G, Metabolites and thyroid hormone from WT and KO male littermates, fed or post-18–24 h fast as indicated (n>4). Data are average ± SEM, * p<0.05, ** p<0.01, *** p<0.001, N.S. = not significant, N.D. = not determined.

Figure 5. Mfsd2a KO mice have decreased liver TG with normal liver metabolism.

Oil red-O histology of fasted A, WT and B, KO liver illustrates neutral lipid stores. C, Quantification of TG extracted from fed and fasted WT and KO livers. D, Assay of β-oxidation in liver homogenates from fasted WT and KO littermates (n = 3–4 per genotype, data representative of three independent experiments). E, In vivo uptake of ¹⁴C-oleate in indicated tissues from WT and KO mice, normalized to heart uptake (n = 3 per genotype). F, TLC of lipid extract illustrating in vitro incorporation of ¹⁴C-glycerol and ¹⁴C-oleate by control and Mfsd2a-transfected HEK293 cells (n = 3). CE, cholesterol ester; M/D/TAG, mono-, di-, tri-acylglycerol; PL, phospholipids. G-H, In vivo VLDL production was assayed by measuring G, serum TG and H, cholesterol in fasted WT and KO mice (n>5 per genotype) following treatment with LPL-inhibitor F-127. I, Real-time PCR analysis of PPARα targets and fatty acid synthesis genes from fasted WT and KO liver (n = 3–4). J, Pyruvate tolerance test of 12–14-week old chow-fed WT and KO littermates after an overnight fast (n = 6). Data are displayed as mean ± SEM. * p<0.05, ** p<0.01.

Figure 6. Mfsd2a KO mice have decreased adiposity but normal lipolysis.

A, WT and B, KO WAT depots stained with H&E. C, NEFA measurements after a 4 h fast (baseline) and 15 min after an injection of adrenergic agonist CL316243 (stimulated). D, Lipolysis was assayed by measuring NEFA (left panel) and glycerol (right panel) production over two hours, with and without isoproterenol (ISO) stimulation, from WAT explants isolated from fasted WT and KO mice. Data are displayed as mean ± SEM. ** p<0.01, *** p<0.001.

Figure 7. Mfsd2a KO mice have increased energy expenditure.

A–D, Indirect calorimetric analysis of male WT (white bars) and KO (dark bars) mice (n = 4, 12–14 weeks of age) under ad libitum fed (left column) and fasted (right column) conditions for A, VO₂, B, VCO₂, C, energy expenditure (EE), and D, respiratory quotient (RER). § p<0.05, # p<0.001.

Figure 8. Mfsd2a KO mice have increased, ataxic movement.

A, X-Y movement and B, Z-axis movement under ad libitum fed (left column) and fasted (right column) conditions for WT and KO cohorts from Fig. 7. C, Rotarod analysis to quantitatively assess motor coordination in WT and KO littermates (n>7). Data are displayed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Figure 9. Mfsd2a KO mice develop glucose intolerance but are resistant to diet-induced obesity.

A, Growth curves for WT and KO male mice fed chow or high-fat diet (HFD) starting at 6 weeks of age (n = 4–8 per genotype). B and C, Average body weight (left), absolute adiposity (center), and relative adiposity (right) for WT (open bar) and KO (closed bar) cohorts fed B, 10 days and C, 10 weeks of HFD. D, WT and E, KO livers stained with ORO for neutral lipid following 10 weeks of HFD. F, GTT after overnight fast of WT and KO males fed HFD for 10 weeks (n = 3–4 mice per genotype, data representative of two independent experiments). Data are displayed as mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001, N.S. = not significant.

Figure 10. Mfsd2a KO mice have increased BAT β-oxidation.

H&E staining of BAT depots from fasted A, WT and B, KO mice. C, Quantification of TG from WT and KO BAT (n>7 per genotype). D, Assay of β-oxidation in BAT explants from fasted WT and KO littermates (n = 4). E, Real-time PCR analysis of PPARα and PPARα targets in BAT from fasted WT and KO mice. F, Quantification of mitochondrial genome numbers from the ddCT of real-time PCR signal for COX-II (mitochondrial) to UCP1 (nuclear) DNA from BAT (n>4). G, Western blots probed for BAT mitochondrial membrane proteins Tom20, COX-IV and UCP1 from fasted WT and KO mice (n = 3). H, Body temperature measurements to assess cold-induced (non-shivering) thermogenesis in cohorts of fasted WT, HET, and KO mice (n = 3–4 per genotype) exposed to 4°C. Data are displayed as mean ± SEM. * p<0.05, ** p<0.01.