Fig 2: Mup1 levels in the liver and serum of placental mTORKOpl and TSC2KOpl adult offspring. (A) Comparison of reads for various Mup family genes detected in RNA sequencing analysis of TSC2KOpl female liver versus littermate controls. All Mup genes had fold-change > 2 and FDR < 0.05. Hepatic Mup1 mRNA expression of 90-day-old adult (B) TSC2KOpl females compared to littermate controls (n = 3), (C) mTORKOpl females compared to littermate controls (n = 4–7), (D) TSC2KOpl males compared to littermate controls (n = 5–6), (E) mTORKOpl males compared to littermate controls (n = 7–8). (F) Circulating Mup1 levels in placental mTORKOpl (n = 4) and TSC2KOpl (n = 3–5) female offspring on normal chow diet and high-fat diet and respective littermate controls. (G) Circulating Mup1 levels in placental mTORKOpl (n = 4) and TSC2KOpl (n = 3–4) male offspring on normal chow diet and high-fat diet and respective littermate controls. Statistical analyses were conducted using an unpaired two-tailed t-test and repeated measures two-way ANOVA, with significance *P < 0.05, #P < 0.1.

Fig 3: Acute rapamycin treatment in vivo reduces hepatic mTOR signaling with no impact on circulating Mup1. (A) Timeline of 2 mg/kg body weight intraperitoneal administration of rapamycin or saline as vehicle followed by the collection of tissue and serum. Posttreatment (B) body weight, (C) liver weight relative to body weight, and (D) blood glucose measurements. (E) Representative Western blot of phosphorylated S6 at serine 240/244, total S6, and vinculin from livers of vehicle- or rapamycin-treated females. (F) Quantification of pS6/total S6 represented as fold-change relative to vehicle (n = 3). (G) Circulating MUP1 levels in females at the end of 10-day treatment with the vehicle of rapamycin (n = 3). Statistical analyses were conducted using an unpaired two-tailed t-test, with significance *P < 0.05, **P < 0.01, and # P < 0.1.

Fig 4: MUP1 is regulated by high-fat diet (HFD) and exercise training (ET). Heatmaps (A) of proteins altered (q < 0.05 and fold-change +/−50%) by ET on Chow (top), HFD (middle), or ET on HFD (bottom heatmap) with corresponding proteins from the other groups. Proteins depicted in (B) were either altered in the same direction by ET and ET on HFD, or they were oppositely altered by HFD and ET on Chow or ET on HFD (‘+’ denotes increased, ‘−’ decreased abundance; a diagonal strikethrough means that the protein was not significantly regulated). Quantification and representative images of western blot performed for the indicated proteins (C–G, n = 5–10). The effects of ET and HFD on Mup1 gene expression in muscle (H, n = 7–10), as well as the effect of ET effect on Mup1 gene expression in AMPK KD mice (I, n = 8). Data are means +/− SEM. ***p < 0.001 main effect of diet (HFD vs Chow). ###p < 0.001 and ##p < 0.01 main effects for activity (ET vs sedentary (SED)). ¤¤p < 0.01 difference between indicated groups.

Fig 5: Mup1 mRNA is not altered by inhibited mTOR-S6 signaling in rapamycin-treated AML12 hepatocyte cells. (A) Western blots of phosphorylated S6 at serine 240/244, total S6, phosphorylated Akt at serine 473, total Akt, and vinculin from AML12 hepatocyte cells treated with 15 nM or 30 nM rapamycin for 24 h. (B) Quantification of pS6/total S6 represented as fold-change relative to control for 24-h rapamycin-treated cells (n = 3). (C) Quantification of pAkt/total Akt represented as fold-change relative to control for 24-h rapamycin-treated cells (n = 3). (D) Quantification of total S6/vinculin represented as fold-change relative to control for 24-h rapamycin-treated cells (n = 3). (E) Quantification of total Akt/vinculin represented as fold-change relative to control for 24-h rapamycin-treated cells (n = 3). (F) Mup1 mRNA expression of AML12 hepatocyte cells treated for 24 h with 15 nM or 30 nM rapamycin. (G) Mup1 mRNA expression of AML12 hepatocyte cells treated for 12 h with 30 nM rapamycin. Quantification of Western blots for (H) phosphorylated S6 and (I) phosphorylated Akt represented as fold-change relative to control (n = 3) for 30 nM rapamycin treatment for 12 h. Statistical analyses were conducted using an unpaired two-tailed t-test or a one-way ANOVA with Tukey’s post hoc test, with significance *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.