Fig 1: (A) Breeding strategy to establish ß-cell specific knockout mice (Arfrp1ß-cell-/-). (B) Relative mRNA expression of Arfrp1 in different tissues from 2 mice per genotype. Data is shown as mean ± SEM. (C) Western blot analysis of indicated tissues from Arfrp1flox/flox and Arfrp1ß-cell-/- mice probed with indicated antibodies. (D) Relative expression levels of Arfrp1 measured in islets of Arfrp1flox/flox (n = 3) and Arfrp1ß-cell-/- (n = 6) female mice. Data is shown as mean ± SEM. ** = p < 0.01 (unpaired Student's t-test, Welch's correction). (E) Relative expression levels of Ins2 measured in islets of Arfrp1flox/flox (n = 3) and Arfrp1ß-cell-/- (n = 6) female mice. Data is shown as mean ± SEM. (F) Body weight measurement of male Arfrp1flox/flox and Arfrp1ß-cell-/- mice. (G) Fat mass of male Arfrp1flox/flox and Arfrp1ß-cell-/- mice. (H) Lean mass of male Arfrp1flox/flox and Arfrp1ß-cell-/- mice. Data in (F)–(H) are presented as mean ± SEM for Arfrp1flox/flox (n = 12) and Arfrp1ß-cell-/- mice (n = 14). * = p < 0.05 (2-way ANOVA). (I) Total pancreatic pro-insulin and (J) C-peptide from ad libitum fed male Arfrp1flox/flox (n = 5) and Arfrp1ß-cell-/- mice (n = 5). Data is represented as mean ± SEM. (K) Immunohistochemical staining of insulin (green) and glucagon (red) in pancreatic sections of female Arfrp1flox/flox and Arfrp1ß-cell-/- mice. Scale bar represents 40 µm. (L) Quantification of islet number per tissue section. (M) Quantification of islet size from pancreatic tissue sections. Data in (L) + (M) is represented as mean ± SEM from three pancreatic sections of 3 mice per genotype. * = p < 0.05 (unpaired Student's t-test, Welch's correction). (N) Immunohistochemical staining of GLUT2 (yellow) in pancreatic sections of female Arfrp1flox/flox and Arfrp1ß-cell-/- mice. Scale bar represents 40 µm.
Fig 2: Disruption of ARFRP1 dependent scaffold leads to degradation of SNAP25. (A) Immunofluorescent pictures of dispersed islet cells from Arfrp1flox/flox and Arfrp1ß-cell-/- mice stained for SNAP25. Nuclei are visualized by DAPI, scale bar represents 40 µm. (B) Western blot analysis of islets isolated from Arfrp1flox/flox and Arfrp1ß-cell-/- mice probed with indicated antibodies. (C) Quantification of Western blots shown in (B) of 8 mice per genotype. Relative SNAP25 levels normalized to loading control is shown as mean ± SEM. * = p < 0.05 (unpaired students t-test, Welch's correction). (D) Western blot analysis of whole cell lysates from Min6 cells treated with indicated adenoviruses at a MOI of 100 and harvested at indicated time points post-infection. Knockdown efficiency for Arfrp1 is 50% after 48 h, and 60–70% for Gopc after 48 h. Membranes were probed with indicated antibodies. (E) Immunofluorescent pictures of Min6 cells infected with indicated adenoviruses. Nuclei were visualized with DAPI. Scale bar represents 20 µm. (F) Quantification of Western blots from whole cell lysates of Min6 cells treated with indicated adenoviruses and cycloheximide (CHX) for indicated time points in 3 independent experiments. Membranes were probed with indicated antibodies. (G) Western blot analysis of whole cell lysates from Min6 cells treated with indicated adenoviruses and chloroquine (CQ) or solvent control (Co = MiliQ) and harvested 36 h post-infection. Membranes were probed with indicated antibodies.
Fig 3: Loss of adipocyte-specific Arfrp1 results in deteriorated liver insulin sensitivity, increased hepatic glucose production, and elevated fasting blood glucose levels. (A, B) Western blot analysis of phosphorylated (pAKT) and total AKT (tAKT) levels in liver (A) and quadriceps (B) of 10-week-old Arfrp1iAT-/- (white symbols) and control mice (Arfrp1flox/flox, black symbols) following injection with insulin (Ins, 1 IU/kg body weight, n = 3–5 mice per genotype) or vehicle (NaCl, n = 2 mice per genotype). (C) Band intensities (insulin treatment) were quantified by densitometric analysis, and the ratio of pAKT to tAKT was expressed as fold of control set to 1. (D, E) Expression of indicated proteins was determined in liver lysates of 10-week-old animals via immunoblotting (D). Signal intensities were quantified by densitometric analysis and expressed as fold of control set to 1 (n = 3–5 mice per genotype) (E). (F) Ten-week-old mice were subjected to pyruvate tolerance tests (PTT), and blood glucose concentration was determined at indicated time points (n = 17–20 animals per genotype). (G) The area under the curve (AUC) is depicted for blood glucose. (H) Transcript levels of gluconeogenic genes (G6pc, Pck1) were determined by qRT-PCR in livers of 10-week-old animals (n = 4–18 mice per genotype) after 6 h of fasting. (I) Blood glucose concentration was measured in 10-week-old mice at indicated time points (n = 10–17 mice per genotype). All data represent mean ± SEM, *P = 0.05, **P = 0.01, ***P = 0.001 by unpaired Student's t-test (C, E, G-I) or by two-way ANOVA with Bonferroni's post-test for multiple comparison (F).
Fig 4: Ablation of Arfrp1 in adipocytes selectively decreases adiponectin and adipsin secretion. (A) Circulating levels of indicated adipokines detected in plasma of 7-week-old Arfrp1iAT-/- (white bars) and control (Arfrpflox/flox, black bars) mice (n = 5–19 mice per genotype). (B) Adiponectin isoforms measured in plasma of 7-week-old animals. HMW: high molecular weight adiponectin. (C) Adiponectin transcript levels (Adipoq) determined in gonadal and subcutaneous white adipose tissue (gonWAT, scWAT) and in brown adipose tissue (BAT) of 7-week-old mice (n = 3–6 mice per genotype). (D, E) Released levels of adiponectin (D) and leptin (E) from gonWAT and scWAT explants of Arfrp1iAT-/- and control mice (n = 4–6 mice per genotype). All data are presented as mean ± SEM, **P = 0.01, ***P = 0.001 by unpaired Student's t-test.
Fig 5: Changes in protein and gene expression relating to lipolysis with a single bout of endurance exercise in human skeletal muscle.A) Representative blots of PLIN3, ATGL, ARF1, ARFRP1 and loading control GAPDH. B) Quantitative bar graph of skeletal muscle PLIN3 protein after an acute exercise bout (n = 19). C) Quantitative bar graph of skeletal muscle ATGL protein after an acute exercise bout (n = 19). D) mRNA levels of lipid droplet coatomer genes (n = 19), and (E) mRNA levels of oxidative genes, in skeletal muscle of healthy subjects in response to an acute exercise bout (n = 14–19). *p<0.05.
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