Hepatocyte-Specific Phgdh-Deficient Mice Culminate in Mild Obesity, Insulin Resistance, and Enhanced Vulnerability to Protein Starvation

Abstract

l-Serine (Ser) is synthesized de novo from 3-phosphoglycerate via the phosphorylated pathway committed by phosphoglycerate dehydrogenase (Phgdh). A previous study reported that feeding a protein-free diet increased the enzymatic activity of Phgdh in the liver and enhanced Ser synthesis in the rat liver. However, the nutritional and physiological functions of Ser synthesis in the liver remain unclear. To clarify the physiological significance of de novo Ser synthesis in the liver, we generated liver hepatocyte-specific Phgdh KO (LKO) mice using an albumin-Cre driver. The LKO mice exhibited a significant gain in body weight compared to Floxed controls at 23 weeks of age and impaired systemic glucose metabolism, which was accompanied by diminished insulin/IGF signaling. Although LKO mice had no apparent defects in steatosis, the molecular signatures of inflammation and stress responses were evident in the liver of LKO mice. Moreover, LKO mice were more vulnerable to protein starvation than the Floxed mice. These observations demonstrate that Phgdh-dependent de novo Ser synthesis in liver hepatocytes contributes to the maintenance of systemic glucose tolerance, suppression of inflammatory response, and resistance to protein starvation.

Keywords: Phgdh; glucose tolerance; insulin signaling; l-serine deficiency; liver.

Figure 1

Targeted inactivation of Phgdh in liver. (A) Genotyping PCR from Phgdh(+) allele and Phgdh(−) allele in the liver of Floxed and LKO mice at 30 weeks (n = 6 each). (B) mRNA level of Phgdh in the liver of Floxed (n = 3) and LKO (n = 4) mice at 30 weeks. (C) Protein level of Phgdh in the liver of Floxed (n = 4) and LKO (n = 6) mice at 30 weeks. Comparable staining of β-actin was used to verify equivalent protein loading. Student’s t-test, * p < 0.05, ** p < 0.005.

Figure 2

Hepatocyte-specific deletion of Phgdh causes the weight gain. (A) Growth curve of Floxed (white node) and LKO (black node) mice from 4 to 30 weeks (n = 6 each). (B) The body weight and the organ weight of the liver, epididymal white adipose tissue (eWAT), parametrial white adipose tissue (pWAT), and mesenteric white adipose tissue (mWAT) were measured in Floxed and LKO mice at 30 weeks (n = 6 each). (C,D) Histological evaluation of the liver in Floxed (C) and LKO (D) mice at 28 weeks (n = 5 each). Representative images are shown. Scale bar, 500 μm. Student’s t-test, * p < 0.05, ** p < 0.005.

Figure 3

Measurement of serum biochemical markers. (A) The values of the serum biochemical test of lipoprotein (Lipopro), total cholesterol (Total-Cho), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), aspartate transaminase (AST), alanine transaminase (ALT), and non-esterified fatty acid (NEFA) (n = 6 each). (B) Blood glucose level in Floxed (black square node) and LKO (white triangle node) mice after glucose administration (Floxed: n = 4, LKO: n = 6). Left graph shows the concentration of blood glucose, and right graph shows the rate of increase of blood glucose. Student’s t-test, * p < 0.05. N.S.: not significant.

Figure 4

Phgdh deletion in hepatocytes induces inflammation and stress response in the liver. Protein levels of phosphorylated NK-κB in the liver of Floxed and LKO mice at 30 weeks (n= 6 each). Comparable staining of NK-κB was used to verify equivalent protein loading.

Figure 5

Identification of positively and negatively correlated gene sets in the liver of LKO mice in GO terms and KEGG pathways by gene set enrichment analysis (GSEA). The enrichment plots of GSEA showed positively correlated gene sets in fatty acid beta oxidation (A), electron transport chain (B), negatively correlated gene sets in regulation of cytoplasmic translation (C), and RNA phosphodiester bond hydrolysis (D) of GO terms. The enrichment plots of GSEA showed positively correlated gene sets in Alzheimer’s disease (E) and glutathione metabolism (F) and negatively correlated gene sets in RNA degradation (G) and basal transcription factors (H) of KEGG pathways. Nominal enrichment scores (NESs) and p-values are indicated in each enrichment plot.

Figure 6

Phgdh deletion in hepatocytes impairs insulin signaling in the liver. (A) Protein level of phosphorylated Akt in the liver of Floxed and LKO mice at 30 weeks. Comparable staining of Akt was used to verify equivalent protein loading. (B) Protein level of phosphorylated GSK3β in the liver of Floxed and LKO mice at 30 weeks. Comparable staining of GSK3β was used to verify equivalent protein loading. (C,D) Protein level of phosphorylated IRS-1 on Ser612 (C) and Ser636/639 (D) residue in the liver of Floxed and LKO mice at 30 weeks. Comparable staining of IRS-1 was used to verify equivalent protein loading. n = 6 each. Student’s t-test, ** p < 0.005, *** p < 0.0005.

Figure 7

Phgdh deletion in hepatocytes alters signaling pathway upstream of IRS1 in liver. (A) Protein level of phosphorylated Erk1/2 in the liver of Floxed and LKO mice at 30 weeks. Comparable staining of Erk1/2 was used to verify equivalent protein loading. (B) Protein level of phosphorylated SAPK/JNK in the liver of Floxed and LKO mice at 30 weeks. Comparable staining of SAPK/JNK was used to verify equivalent protein loading. (C,D) mRNA (C) and protein (D) level of Egr-1 in the liver of Floxed and LKO mice at 30 weeks. N = 6 each. Student’s t-test, * p < 0.05, ** p < 0.005.

Figure 8

Phgdh deletion in hepatocytes induces the reduction of glycolysis in the liver. (A,B) mRNA levels of Pfkfb3 (A) and Pdk4 (B) in the liver of Floxed and LKO mice at 30 weeks. (C,D) mRNA levels of Fasn in liver(C) and eWAT (D) in the liver of Floxed and LKO mice at 30 weeks. n = 6 each. Student’s t-test, * p < 0.05.

Figure 9

Effect of body weight and survival rate by feeding LKO mice a protein-free diet. (A) The transition of body weight in Floxed mice (white node) and LKO mice (black node) after feeding protein-free diet. (B) The transition of survival rate in Floxed and LKO mice during feeding protein-free diet. n = 5 each. Student’s t-test, * p < 0.05, ** p < 0.005.

Figure 10

A summary of the molecular mechanisms caused by Phgdh deletion in hepatocytes, as inferred by this study. Phgdh deletion in hepatocytes impairs insulin signaling via IRS-1 phosphorylation. Akt inactivation by impaired insulin signaling induced the suppression of gluconeogenesis. Enhanced glycolysis resulted in mild obesity and glucose intolerance.