Enhanced insulin signaling in density-enhanced phosphatase-1 (DEP-1) knockout mice

Abstract

Objective: Insulin resistance can be triggered by enhanced dephosphorylation of the insulin receptor or downstream components in the insulin signaling cascade through protein tyrosine phosphatases (PTPs). Downregulating density-enhanced phosphatase-1 (DEP-1) resulted in an improved metabolic status in previous analyses. This phenotype was primarily caused by hepatic DEP-1 reduction.

Methods: Here we further elucidated the role of DEP-1 in glucose homeostasis by employing a conventional knockout model to explore the specific contribution of DEP-1 in metabolic tissues. Ptprj (-/-) (DEP-1 deficient) and wild-type C57BL/6 mice were fed a low-fat or high-fat diet. Metabolic phenotyping was combined with analyses of phosphorylation patterns of insulin signaling components. Additionally, experiments with skeletal muscle cells and muscle tissue were performed to assess the role of DEP-1 for glucose uptake.

Results: High-fat diet fed-Ptprj (-/-) mice displayed enhanced insulin sensitivity and improved glucose tolerance. Furthermore, leptin levels and blood pressure were reduced in Ptprj (-/-) mice. DEP-1 deficiency resulted in increased phosphorylation of components of the insulin signaling cascade in liver, skeletal muscle and adipose tissue after insulin challenge. The beneficial effect on glucose homeostasis in vivo was corroborated by increased glucose uptake in skeletal muscle cells in which DEP-1 was downregulated, and in skeletal muscle of Ptprj (-/-) mice.

Conclusion: Together, these data establish DEP-1 as novel negative regulator of insulin signaling.

Keywords: DEP-1, density-enhanced phosphatase-1; Density-enhanced phosphatase-1; GTT, glucose tolerance test; Glucose homeostasis; HFD, high-fat diet; IL-6, interleukin 6; IR, insulin receptor; ITT, insulin tolerance test; Insulin resistance; Insulin signaling; KO, knockout; LFD, low-fat diet; MCP-1, monocyte chemotactic protein-1; PTP, protein tyrosine phosphatase; Phosphorylation; RER, respiratory exchange ratio; RTK, receptor tyrosine kinase; WT, wild-type.

Figure 1

DEP-1 expression and genotyping of wild-type and Ptprj^−/− mice. (A) DEP-1 expression based on activity measurements under reduced conditions (as outlined in the Materials and Methods section) in metabolic tissues derived from wild-type mice. (B) Wild-type and Ptprj^−/− mice were characterized by PCR. (C, D) Confirmation of DEP-1 knockout in liver tissue visualized by immunoblotting and DEP-1 activity measurements (n = 3–4 mice per genotype).

Figure 2

Metabolic phenotyping of wild-type and Ptprj^−/− mice. (A) Body weight of mice was determined twice weekly over 8 weeks. (B,C) ITT was performed after 4 h fasting and the AUC was calculated. (D,E) GTT was conducted after 12 h fasting and corresponding AUC was calculated (n = 8–10 mice per genotype). LFD WT vs. LFD Ptprj KO *p < 0.05; HFD WT vs. HFD Ptprj KO ^†p < 0.05. (F) Respiratory exchange ratio determined over 24 h and (G) mean of data recorded every 15 min (n = 6–10 mice per genotype). LFD WT vs. LFD Ptprj KO *p < 0.05, ***p < 0.001; HFD WT vs. HFD Ptprj KO ^†p < 0.05, ^†††p < 0.001.

Figure 3

Insulin signaling in the liver. (A) Tyrosine-phosphorylation levels of different insulin receptor (IR) and Akt phosphorylation sites were analyzed by immunoblotting. (B–E) Densitometric analyses of Akt phosphorylation at sites Ser⁴⁷³ and Thr³⁰⁸. Quantification was performed with all visualized mouse samples from all individual groups, with n = 3 without insulin challenge and n = 4 with insulin challenge. LFD WT vs. LFD Ptprj KO *p < 0.05; HFD WT vs. HFD Ptprj KO ^†p < 0.05.

Figure 4

Insulin signaling in the skeletal muscle. (A) Tyrosine-phosphorylation levels of different IR and Akt phosphorylation sites were analyzed by immunoblotting. (B–E) Densitometric analyses of Akt phosphorylation at sites Ser⁴⁷³ and Thr³⁰⁸. Quantification was performed with all visualized mouse samples from all individual groups, with n = 3 without insulin challenge and n = 4 with insulin challenge. LFD WT vs. LFD Ptprj KO *p < 0.05, **p < 0.01; HFD WT vs. HFD Ptprj KO ^†p < 0.05.

Figure 5

Insulin signaling in adipose tissue. (A) Tyrosine-phosphorylation levels of different IR and Akt phosphorylation sites were analyzed by immunoblotting. Arrows indicate the phosphorylated IR. (B–E) Densitometric analyses of Akt phosphorylation at sites Ser⁴⁷³ and Thr³⁰⁸. Quantification was performed with all visualized mouse samples from all individual groups, with n = 3 without insulin challenge and n = 4 with insulin challenge. HFD WT vs. HFD Ptprj KO ^†p < 0.05, ^†††p < 0.001.

Figure 6

Glucose uptake in muscle. (A) Transcript analysis by quantitative real-time PCR of transfected myotubes with non-targeting siRNA and siRNA against DEP-1. The data are represented as means ± SEM of three independent experiments. (B) Glucose uptake was performed in C2C12 cells with or without DEP-1 downregulation. Data are expressed as means ± SEM, and based on unstimulated conditions. (C) Glucose uptake in isolated soleus muscle from WT and Ptprj KO mice subjected to insulin (n = 5–7 per genotype). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Serum parameters, blood pressure and morphometric pancreatic beta cell- and apoptosis analyses. (A–E) Serum parameters of insulin (n = 7–9 mice per genotype), leptin (n = 8–9 mice per genotype), resistin (n = 8–9 mice per genotype), MCP-1 (n = 7–9 mice per genotype) and IL-6 (n = 3–6 mice per genotype) were determined by Milliplex ELISA. (F) Systolic blood pressure was measured non-invasively (n = 8–10 per genotype). LFD WT vs. LFD Ptprj KO *p < 0.05; HFD WT vs. HFD Ptprj KO ^†p < 0.05. (G) Representative images of immunostained pancreatic sections showing insulin-positive cells. Scale bars represent 100 μm. (H) Pancreas beta cell area was analyzed morphometrically from LFD- and HFD WT, and LFD- and HFD Ptprj KO mice (n = 8–10 per group). (I) The ratio of Bax/Bcl2 in the pancreas was determined by quantitative real-time PCR analysis in all animal groups (n = 7–9 mice per genotype), and was normalized to the expression of Rn18s. HFD WT vs. HFD Ptprj KO ^†p < 0.05.

Figure 8

Schematic depiction of the role of DEP-1 in insulin signaling. DEP-1, a receptor-like protein tyrosine phosphatase, impacts on insulin signaling. DEP-1 comprises an eight fibronectin (FN)-like repeats-containing extracellular domain, a single transmembrane segment, and an intracellular catalytic domain with pure tyrosine affinity. Previously we demonstrated that DEP-1 is closely recruited to the insulin receptor in situ upon insulin challenge . DEP-1 targets the insulin receptor, depicted as an inhibitory arrow, resulting in lower tyrosine phosphorylation at the intracellular domain of the receptor (shown as lower brightness). Applying a conventional knockout model, here we show that mice with genetic DEP-1 disruption (lower brightness and dotted inhibitory arrow) are characterized by improved insulin signaling, in particular evident by enhanced phosphorylation of the downstream signaling molecule Akt at sites Ser⁴⁷³ and Thr³⁰⁸. This ultimately leads to facilitated glucose uptake through glucose transporters (for mechanistic illustration two transporters are shown on the right hand side), suggesting DEP-1 as potential novel drug target in insulin resistance.