Fig 2: Expression of important factors in gluconeogenesis in primary hepatocytes and spexin gene knockout activated the gluconeogenesis signalling pathway. (A) Spexin inhibited the expression of FoxO1, PGC-1α, PEPCK, G-6-Pase mRNA in primary hepatocytes. (B) Spexin intervention increased the p-FoxO1 Ser256/FoxO1 ratio in primary hepatocytes. (C) Spexin inhibited the protein expression of FoxO1, PGC-1α, PEPCK, and G-6-Pase in primary hepatocytes. (D) The glucose concentration in the culture media of spexin gene knockout HepG2 cells was significantly increased. (E) The mRNA expression of FoxO1, PGC-1α, PEPCK, and G-6-Pase was significantly increased in spexin gene knockdown HepG2 cells. (F) Spexin gene knockdown decreased the Ser256/FoxO1 p-FoxO1 ratio in HepG2 cells. (G) The protein expression of FoxO1, PGC-1α, PEPCK, and G-6-Pase in spexin gene knockdown HepG2 cells was increased. * P < 0.05.

Fig 3: Autophagy is required for the osteogenic differentiation of hASCs. (A, B): Depletion of ATG7 decreased ALP staining (A) and activity (B) on the seventh day after induction of osteogenic differentiation of hASCs. (C, D): Extracellular matrix mineralization (C) and ARS quantification (D) were decreased in ATG7 knockdown cells after 14 days of osteogenic induction. (E): Protein levels of RUNX2, p62, and LC3B are indicated as shown. GAPDH was used as internal loading control. (F–M): Treatment with 5 mM 3‐MA decreased the osteogenic capacity of PCK2‐expressing cells. Relative mRNA expression of RUNX2 (F) and ALP (G) on the seventh day of osteogenic induction are shown. Relative mRNA expression of RUNX2 (H) and OCN (I) after 14 days of osteogenic induction is shown. ALP staining (J) and activity (K) on the seventh day of osteogenic induction are shown. Extracellular matrix mineralization (L) and ARS quantification (M) on the 14th day of osteogenic induction are presented. (N): Protein expression patterns of PCK2, RUNX2, p62, and LC3B are shown. The control vector cells and PCK2‐expressing cells were cultured in PM, PM containing 5 mM 3‐MA, OM, or OM containing 5 mM 3‐MA for 5 days. GAPDH was used as internal loading control. Data are represented as mean ± SD. **, p ≤ .01; *, p ≤ .05, Student's t test. Abbreviations: 3‐MA, 3‐methyladenine; ALP, alkaline phosphatase; ARS, alizarin red S; ATG7, autophagy‐related‐gene‐7; LC3B, microtubule associated protein 1 light chain 3 β; OCN, osteocalcin; NC, negative control for sh‐ATG7; NS, not significant; OM, osteogenic media; p62, p62/SQSTM1; PCK2, mitochondrial phosphoenolpyruvate carboxykinase; PM, proliferation media; RUNX2, runt‐related transcription factor 2.

Fig 4: PCK2 promotes anchorage independent growth and glutamine utilization. (A) Colorectal cancer cell lines were transduced with lentivirus producing shRNAs targeting PCK2 and subjected to immunoblot after 72 h to confirm knockdown. (B) Representative photomicrographs of colonies in soft agar two weeks after shRNA mediated depletion of PCK2 in four CRC cell lines (C) Quantification of colonies from panel B. (D) Intracellular metabolites were measured by LC-MS/MS 72 h after siRNA-mediated depletion of PCK2. (E) Oxygen consumption rates were measured using a Seahorse XFe96 analyzer in colorectal cancer cell lines 72 h after shRNA-mediated knockdown of PCK2 using only L-glutamine as a substrate. * = p-value less than 0.05; ** = p-value less than 0.01, *** = p-value less than 0.001. # = p-value less than 0.001 when compared to the non-targeting control using one way ANOVA with multiple comparisons.

Fig 5: Expression of Akt, CREB, FoxO1, PGC-1, PEPCK, and G-6-Pase in the liver tissues of rats. (A, B, C, D) The mRNA expression of FoxO1, PGC-1α, PEPCK, G-6-Paseα in the HFD + spexin group was significantly decreased as compared with that in the HFD group. (E) The spexin treatment increased the p-FoxO1 Ser256/FoxO1 ratio. (F) The spexin treatment increased the p-Akt /Akt ratio. (G) The intervention of spexin decreased the p-CREB /CREB ratio. (H) The protein expression of p-Akt, Akt, p-CREB, CREB, FoxO1, PGC-1, PEPCK, and G-6-Pase. * P < 0.05, # P > 0.05.