Deletion of pleiotrophin impairs glucose tolerance and liver metabolism in pregnant mice: Moonlighting role of glycerol kinase

Abstract

Pleiotrophin is a pleiotropic cytokine that has been demonstrated to have a critical role in regulating energy metabolism, lipid turnover and plasticity of adipose tissue. Here, we hypothesize that this cytokine can be involved in regulatory processes of glucose and lipid homeostasis in the liver during pregnancy. Using 18-days pregnant Ptn-deficient mice, we evaluated the biochemical profile (circulating variables), tissue mRNA expression (qPCR) and protein levels of key enzymes and transcription factors involved in main metabolic pathways. Ptn deletion was associated with a reduction in body weight gain, hyperglycemia and glucose intolerance. Moreover, we observed an impairment in glucose synthesis and degradation during late pregnancy in Ptn^-/- mice. Hepatic lipid content was significantly lower (73.6%) in Ptn^-/- mice and was associated with a clear reduction in fatty acid, triacylglycerides and cholesterol synthesis. Ptn deletion was accompanying with a diabetogenic state in the mother and a decreased expression of key proteins involved in glucose and lipid uptake and metabolism. Moreover, Ptn^-/- pregnant mice have a decreased expression of transcription factors, such as PPAR-α, regulating lipid uptake and glucose and lipid utilization. Furthermore, the augmented expression and nuclear translocation of glycerol kinase, and the decrease in NUR77 protein levels in the knock-out animals can further explain the alterations observed in hepatic glucose metabolism. Our results point out for the first time that pleiotrophin is an important player in maintaining hepatic metabolic homeostasis during late gestation, and further highlighted the moonlighting role of glycerol kinase in the regulation of maternal glucose homeostasis during pregnancy.

Keywords: NR4A1; glycerol kinase; liver; metabolism; pleiotrophin; pregnancy.

FIGURE 1

Ptn ^−/− mice show decreased body weight and less weight gain during pregnancy. (A) Body weight evolution, (B) increase of the body weight during pregnancy, (C) conceptus weight (fetus‐placenta structure), and (D) retroperitoneal adipose tissue in Ptn^+/+ (grey lines and bars) and Ptn^−/− (blue lines and bars) pregnant mice. Data are expressed as mean ± SEM (n = 15–19 animals/group) *p < .05, **p < .01, ***p < .001 for differences between Ptn^−/− and Ptn^+/+ mice

FIGURE 2

Altered fasting plasma biochemical parameters, hormones, plasma lipoproteins, and glucose tolerance in vivo in 18 days pregnant Ptn ^−/− mice. (A) Glucose; (B) cholesterol; (C) glycerol, (D) non‐esterified fatty acids (NEFA), (E) triacylglycerides, (F) VLDL/IDL, (G) LDL, (H) HDL, (I) insulin, (J) glucagon, (K) glucose‐induced insulinotropic peptide (GIP), (L) glucose tolerance curve (GTT), (M) Area under the curve (AUC) for glucose during the GTT, and (N) HOMA‐IR. Data are expressed as mean ± SEM (n = 4–9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn^−/− and Ptn^+/+ mice

FIGURE 3

Effect of pleiotrophin deletion on liver weight, total lipid content, and hepatic lipid fractions at day 18 of pregnancy. (A) liver weight, (B) total lipid content, (C) triacylglycerides, (D) phospholipids, (E) cholesterol, and (F) cholesteryl esters. Data are expressed as mean ± SEM (n = 5–6 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn ^−/− and Ptn ^+/+ mice

FIGURE 4

Deletion of pleiotrophin impairs hepatic glucose metabolism in 18 days pregnant mice. (A) Glut2 mRNA, (B) Glucokinase (Gck) mRNA, (C) Phosphofructokinase I (Pfk1) mRNA, (D) Pyruvate kinase (Pk) mRNA, (E) Lactate dehydrogenase (Ldh) mRNA, (F) Pyruvate dehydrogenase α1 (Pdha1) mRNA, (G) Pyruvate carboxylase (Pc) mRNA, (H) Phosphoenolpyruvate carboxykinase (Pepck) mRNA, (I) Fructose 1,6 bisphosphatase (Fbp1) mRNA, (J) Glucose 6‐phosphatase (G6pc) mRNA, and (K) Glucose 6‐phosphate dehydrogenase (G6pd) mRNA. Data are expressed as mean ± SEM (n = 9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn ^−/− and Ptn ^+/+ mice

FIGURE 5

Impaired expression of enzymes involved in fatty acid oxidation in Ptn ^−/− 18 days pregnant mice. (A) Carnitine palmitoyl transferase 1α (Cpt1α) mRNA, (B) Acyl‐CoA dehydrogenase long chain (Acadl) mRNA, (C) Acyl‐CoA dehydrogenase very long chain (Acavdl) mRNA, (D) 3‐Hydroxyacyl‐Coa dehydrogenase α (Hadha) mRNA, and (E) Acyl‐CoA oxidase 1 (Acox1) mRNA. Data are expressed as mean ± SEM (n = 9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn ^−/− and Ptn ^+/+ mice

FIGURE 6

At late pregnancy deletion of pleiotrophin is associated with changes in key enzymes involved in fatty acids, triacylglycerides and cholesterol synthesis. (A) ATP citrate lyase (Acly) mRNA, (B) Acetyl‐CoA carboxylase (Acc) mRNA, (C) Fatty acid synthase (Fas) mRNA, (D) Stearoyl‐CoA desaturase‐1 (Scd1) mRNA, (E) Lipin 2 (Lpin2) mRNA, (F) Glycerol phosphate acyltransferase (Gpat) mRNA, (G) Diacyglycerol acyltransferase‐1 (Dgat1) mRNA, (H) Diacyglycerol acyltransferase‐2 (Dgat2) mRNA, (I) Acetyl‐CoA acetyltransferase‐2 (Acat2) mRNA, (J) Hydroxymethylglutaryl‐CoA synthase (Hmgs) mRNA, (K) Hydroxymethylglutaryl‐CoA reductase (Hmgr) mRNA, (L) Sterol O‐acyltransferase (Soat) mRNA, (N) Acetyl‐CoA carboxylase total protein/HSP90, (M) ratio of phospho‐acetyl‐CoA carboxylase/acetyl‐CoA carboxylase protein, and (O) representative blots of phospho‐acetyl‐CoA carboxylase, acetyl‐CoA carboxylase, and HSP90. Data are expressed as mean ± SEM (n = 8–9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn ^−/− and Ptn ^+/+ mice

FIGURE 7

Pleiotrophin deletion alters transporters and enzymes of lipoprotein metabolism in mice on day 18 of pregnancy. (A) Lipoprotein lipase (Lpl) mRNA, (B) Hepatic lipase (Lipc) mRNA, (C) Aquaporin 9 (Aqp9) mRNA, (D) Fatty acid transporter protein 4 (Fatp4) mRNA, (E) Fatty acid transporter protein 5 (Fatp5) mRNA, (F) Perilipin 2 (Plin2) mRNA, (G) Apo C‐II (ApoC2) mRNA, and (H) Apo B‐100 (ApoB100) mRNA. Data are expressed as mean ± SEM (n = 9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn ^−/− and Ptn ^+/+ mice

FIGURE 8

Deletion of pleiotrophin is associated with differential expression of genes involved in the regulation of glucose and lipid metabolism in the liver of 18 days pregnant mice. (A) Ppar‐α mRNA, (B) Ppar‐γ1 mRNA, (C) Ppar‐βδ mRNA; (D) Pgc1‐α mRNA, (E) Rxr‐α mRNA, (F) Srebp1 mRNA, (G) Fgf21 mRNA, (H) Cebp‐α mRNA, (I) Nur77 mRNA, (J) NUR77 protein/β‐actin, (K) Glycerol kinase (Gyk) mRNA, (L) Glycerol kinase protein/HSP90, (M) Correlation analysis of glycerol kinase (Gyk) mRNA and Nur77 mRNA, (N) Glycerol kinase nuclear protein/TBP protein. Data are expressed as mean ± SEM (n = 3–9 animals/group) *p < .05, **p < .01, and ***p < .001 for differences between Ptn^−/− and Ptn^+/+ mice

FIGURE 9

Summary of the molecular mechanism by which Ptn depletion alters liver metabolism during pregnancy, and the involvement of glycerol kinase as a regulatory factor. Pregnancy involves a series of metabolic changes that in turn can be affected by the deletion of the Ptn gene. In our study, Ptn ^−/− pregnant mice exhibit diminished glycerol and fatty acid uptake caused by a decrease in the expression of the transporters Aqp9, Fatp4, and Fatp5. Moreover, in the condition of lacking Ptn several transcription factors (PPARα, RXRα, SREBP‐1c) and key proteins involved in lipid metabolic pathways are downregulated, contributing to the reduction in lipid depots and circulating levels of triacylglycerides (TG) and NEFA. On the other hand, these factors are regulated by glycerol kinase, which links to carbohydrate metabolism. Gyk, which is upregulated in Ptn ^−/− pregnant mice, inhibits the transcriptional function of Nur77, a key regulator of glucose metabolism, including impaired gluconeogenesis. Both altered lipid and carbohydrate metabolism in the liver may favor the development of diabetes in pregnancy