Adipose tissue-derived PRXL2A suppresses hepatic lipogenesis in a study with male mice

Figures

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  Fig. 1. PRXL2A is a fasting-induced adipokine. A Silver-stained SDS-polyacrylamide gel displaying the profile of plasma proteins (left) and the identified PRXL2A fragments by LC-MS analysis (right) from 12-week-old male mice under ad libitum feeding (fed) or 6-hr fasting (fasted) conditions. Mouse plasma proteins were obtained using the ProteoMiner Protein Enrichment Kit after IgG removal. The red arrows indicate the differential protein bands between fed and fasted mouse plasma samples. Identified PRXL2A peptides are shown on the right. B Immunoblots and quantification showing the expression of PRXL2A in different tissues from 12-week-old male mice. eWAT, epididymal white adipose tissue. BAT, brown adipose tissue. n = 3. C Generation of Prxl2a adipose tissue-specific knockout (AKO) mice (top left), and immunoblots (bottom left) and quantification (right) showing AKO of Prxl2a. n = 3. D Effect of ad libitum feeding and fasting on plasma PRXL2A levels. Relative area under the curve (AUC) is shown on the right. At each indicated time point, plasma PRXL2A was measured by sandwich ELISA assay in 12-week-old male WT and Prxl2a AKO mice. Data are shown as means ± s.e.m. Statistical comparisons…

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  Fig. 2. Deficiency of Prxl2a increases hepatic DNL. A qPCR results display the relative mRNA levels of genes involved in lipogenesis from liver extracts of 12-week-old WT and Prxl2a AKO male mice under ad libitum feeding (fed) or 6 hr fasting (fasted) conditions. n = 8 mice. B Immunoblots and statistical data show the effect of Prxl2a deficiency on hepatic lipogenesis. Liver extracts were obtained from 12-week-old male mice under ad libitum feeding (fed) or 6 hr fasting (fasted) conditions. flSREBP-1, SREBP-1 precursor; nSREBP-1, nuclear-localized mature SREBP-1. n = 3. C–G Effect of Prxl2a deficiency on hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (C), and levels of hepatic non-esterified fatty acid (NEFA, D), hepatic triglyceride (TG, E), plasma NEFA (F) and plasma TG (G) in 12-week-old male mice after ad libitum feeding (fed) or 6 h fasting (fasted). H, I Results of glucose tolerance test (H) and insulin tolerance test (I) from 12-week-old WT and Prxl2a AKO male mice. Data are shown as means ± s.e.m. Comparison of different groups was carried out using the two-tailed unpaired Student’s ttest. n = 3 mice (C), n = 8 mice (D–I).

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  Fig. 3. Prxl2a AKO enhances hepatic DNL in diet-induced obese mice. A, B Effect of Prxl2a deficiency on body weight (A) and fat mass (B) in high-fat diet (HFD)-fed male mice. Ad libitum-fed mice were used. C qPCR results showing the relative mRNA levels of genes involved in lipogenesis from liver extracts of HFD-fed WT and Prxl2a AKO male mice. Ad libitum-fed mice were used. D Immunoblots and statistical data showing the effect of Prxl2a deficiency on hepatic lipogenesis from liver extracts of HFD-fed male mice. Ad libitum-fed mice were used. n = 3. E-N Effect of Prxl2a deficiency on hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (E), hepatic NEFA levels (F), hepatic TG levels (G), hepatic cholesterol levels (TC, H), liver morphology (I), plasma NEFA levels (J), plasma TG levels (K), plasma TC levels (L), glucose tolerance (M) and insulin tolerance (N) in HFD-fed male mice. Ad libitum-fed mice were used (E–L). Scale bars, 50 μm. Data are shown as means ± s.e.m. Comparison of different groups was carried out using the two-tailed unpaired Student’s ttest. n = 8 mice (A–C, F–N), n = 3 mice (E).

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  Fig. 4. Exogenous PRXL2A ameliorates hepatic lipid accumulation. A Plasma PRXL2A levels in regular diet (RD) or HFD-fed WT and Prxl2a AKO male mice. n = 6 mice. B Correlation of plasma PRXL2A levels with body mass index (BMI) in humans. The Pearson correlation coefficient (r) and P-value are shown. n = 210 humans. C qPCR results showing relative mRNA levels of genes involved in lipogenesis from liver extracts of HFD-fed male mice treated with or without PRXL2A. Mice were intraperitoneally injected with Trx-PRXL2A-6 × His (3 mg/kg). n = 8 mice. D Immunoblots and statistical data showing the effect of exogenous PRXL2A injection (3 mg/kg) on hepatic lipogenesis from liver extracts of HFD-fed male mice. n = 3. E–G Effect of exogenous PRXL2A injection (3 mg/kg) on hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (E), body weight (F), and fat mass (G) in HFD-fed male mice. n = 3 mice (E), n = 8 mice (F, G). H–P Effect of exogenous PRXL2A injection (3 mg/kg) on hepatic NEFA (H), hepatic TG (I), hepatic TC (J), hepatic lipid accumulation (K), plasma NEFA (L), plasma TG (M), plasma TC (N), glucose tolerance (O), and insulin tolerance (P) in HFD-fed male mice. n = 8 mice (H–P). Scale…

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  Fig. 5. PRXL2A activates PTAFR-coupled calcium signaling. A Purified TRX-PRXL2A-6×His was cross-linked to Protein A/G Sepharose for pull-down assays in primary hepatocytes. A silver-stained SDS-PAGE (left) shows PRXL2A-interacting proteins in hepatocytes, confirmed by immunoblots (middle) and LC-MS (right) identifying plasma transmembrane proteins. The red arrow marks the differential band after TRX-PRXL2A-6×His pull-down. B Flow cytometry analysis of Trx-PRXL2A-6 × His binding on HEK293T cells with or without overexpression of the potential receptors (left) and immunoblots showing expression of the transfected receptors in HEK293T cells (right). C qPCR results showing relative mRNA levels of Ptafr in different tissues from 10-week-old male mice. n = 6 mice. D Binding assay on liver tissue from Ptafr+/+ and Ptafr–/– mice. Tissue slices were incubated with 50 nM TRX-6 × His or TRX-PRXL2A-6 × His. Scale bars, 10 μm. E Quantification of the binding affinity between PRXL2A and WT PTAFR or its mutant (W83A) by MST. ΔFnorm indicates the change in the normalized fluorescence. n = 3. F Effect of WT PTAFR or its mutant (W83A) on PRXL2A binding in hepatocytes. n = 3. G Schematic diagram…

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  Fig. 6. Ptafr liver-specific knockout promotes DNL. A qPCR results showing relative mRNA levels of lipogenesis genes in liver extracts from 10-week-old fl/fl and Ptafr liver-specific knockout (LKO) male mice under ad libitum feeding (fed) or 6 h fasting (fasted) conditions. n = 6 mice. B Immunoblots and statistical data showing the effect of Ptafr knockout on hepatic lipogenesis in liver extracts from 10-week-old male mice under ad libitum feeding (fed) or 6 h fasting (fasted) conditions. n = 3. C–G Effect of Ptafr knockout on hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (C), hepatic NEFA levels (D), hepatic TG levels (E), plasma NEFA levels (F), and plasma TG levels (G) in mice under ad libitum feeding (fed) or 6 h fasting (fasted) conditions. H, I Results of glucose tolerance test (H) and insulin tolerance test (I) from 10-week-old fl/fl and Ptafr LKO male mice. n = 3 mice (C), n = 6 mice (D–G), n = 12 mice for fl/fl and 11 mice for Ptafr LKO (H), n = 8 mice for fl/fl and 9 mice for Ptafr LKO (I). Data are presented as means ± s.e.m., and statistical analysis was conducted using the two-tailed unpaired Student’s ttest.

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  Fig. 7. PRXL2A suppresses hepatic DNL via PTAFR. A–FEffect of WT PTAFR or its mutant (W83A) on expression of lipogenic genes evaluated via qPCR (A), expression of lipogenic proteins evaluated by immunoblots and quantification (B), hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (C), hepatic NEFA levels (D), hepatic TG levels (E), plasma NEFA levels (F) and plasma TG levels (G). Mice received an intravenous (I.V.) tail vein injection of adenovirus expressing either WT PTAFR or its mutant (W83A), followed by PRXL2A protein injections (3 mg/kg) twice per week for two weeks. Data are presented as means ± s.e.m., and statistical analysis was conducted using a two-tailed unpaired Student’s ttest. n = 6 mice (A, D–G), n = 3 mice (C).

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  Fig. 8. AMPK mediates the suppressive effect of PRXL2A on hepatic DNL. A–F Expression of lipogenic genes evaluated by qPCR (A), expression of lipogenic proteins evaluated by immunoblots and statistical data (B), hepatic DNL measured by synthesis rate of hepatic 2H-palmitate (C), hepatic NEFA levels (D), hepatic TG levels (E), plasma NEFA levels (F), and plasma TG levels (Q) in ad libitum-fed WT (fl/fl) or AMPKα1&2 (Ampk) LKO mice. To create Ampk LKO mice, Ampk fl/fl mice received an intravenous (I.V.) tail vein injection of adenovirus expressing Cre recombinase, followed by PRXL2A protein injections (3 mg/kg) twice per week for three weeks. Data are presented as means ± s.e.m., and statistical analysis was conducted using the two-tailed unpaired Student’s ttest. n = 6 mice (A, D–G), n = 3 mice (C). H During fasting, PRXL2A is secreted from adipose tissues and binds PTAFR in the liver, thereby mobilizing calcium and subsequently activating AMPK to suppress SREBP1-controlled lipogenesis.