The PTP1B Inhibitor Trodusquemine (MSI-1436) Improves Glucose Uptake in Equine Metabolic Syndrome Affected Liver through Anti-Inflammatory and Antifibrotic Activity

Abstract

Background: Hyperactivation of protein tyrosine phosphatase (PTP1B) has been associated with several metabolic malfunctions ranging from insulin resistance, metaflammation, lipotoxicity, and hyperglycaemia. Liver metabolism failure has been proposed as a core element in underlying endocrine disorders through persistent inflammation and highly fibrotic phenotype.

Methods: In this study, the outcomes of PTP1B inhibition using trodusquemine (MSI-1436) on key equine metabolic syndrome (EMS)-related alterations including inflammation, fibrosis, and glucose uptake have been analyzed in liver explants collected from EMS-affected horses using various analytical techniques, namely, flow cytometry, RT-qPCR, and Western blot.

Results: PTP1B inhibition using trodusquemine resulted in decreased proinflammatory cytokines (IL-1β, TNF-α, and IL-6) release from liver and PBMC affected by EMS and regulated expression of major proinflammatory microRNAs such as miR-802 and miR-211. Moreover, MSI-1436 enhanced the anti-inflammatory profile of livers by elevating the expression of IL-10 and IL-4 and activating CD4⁺CD25⁺Foxp3⁺ regulatory T cells in treated PBMC. Similarly, the inhibitor attenuated fibrogenic pathways in the liver by downregulating TGF-β/NOX1/4 axis and associated MMP-2/9 overactivation. Interestingly, PTP1B inhibition ameliorated the expression of TIMP-1 and Smad7, both important antifibrotic mediators. Furthermore, application of MSI-1436 was found to augment the abundance of glycosylated Glut-2, which subsequently expanded the glucose absorption in the EMS liver, probably due to an enhanced Glut-2 stability and half-life onto the plasma cell membranes.

Conclusion: Taken together, the presented data suggest that the PTP1B inhibition strategy and the use of its specific inhibitor MSI-1436 represents a promising option for the improvement of liver tissue integrity and homeostasis in the course of EMS and adds more insights for ongoing clinical trials for human MetS management.

Figure 1

Inhibition of PTP1B with MSI-1436 alleviates inflammation in the liver and PBMC derived from EMS horses. (a) Relative gene expression of master mediators involved in inflammatory responses in liver tissue. (b) Relative gene expression of proinflammatory factors analyzed in equine PBMC. (c) Total levels of IL-1β and TNF-β proteins in the liver and PBMC lysates. (d) Relative protein expression of IL-6 and MCP-1 cytokines accompanied with their representative immunoblots for the liver and PBMC samples. Results were normalized to the expression of endogenous β-actin control. (e) Relative expression of key microRNA regulating proinflammatory responses determined in both liver and PBMC groups. Representative data from three independent experiments are shown ± SD (n = 3). An asterisk (^∗) indicates a comparison among healthy, EMS, and EMS-treated groups. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Eq Liver_HE: liver explants derived from healthy horses; Eq Liver_EMS: untreated liver explants derived from EMS horses. Eq Liver_EMS + 1 µM MSI-1436: liver explants derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor. Eq PBMCr_HE: PBMC derived from healthy horses; Eq PBMC_EMS: untreated PBMC derived from EMS horses. Eq PBMC_EMS + 1 µM MSI-1436: PBMC derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor.

Figure 2

MSI-1436 ameliorates anti-inflammatory signals in livers and PBMC affected by EMS. (a) Relative liver IL-4 and IL-10 protein expression. Results were normalized to the expression of endogenous β-actin control. (b) Levels of IL-4 and IL-10 transcripts in liver and PBMC samples. Relative expression of anti-inflammatory microRNAs in (c) liver and (d) PBMC groups. (e) Gating strategy for PBMCs flow cytometric analysis of CD4⁺, CD25⁺ and Foxp3⁺ T cells. Signal representative of stained markers: FITC (CD4), AF700 (CD25), and PE (FOXP3). The population of CD4/CD25/Foxp3 cells appears in Q2 quadrant. (f) Bar charts depicting the total percentage of CD4⁺, CD25⁺, Foxp3⁺, and triple positive CD4⁺CD25⁺Foxp3⁺ cells. Representative data from three independent experiments are shown ± SD (n = 3). An asterisk (^∗) indicates a comparison among healthy, EMS, and EMS-treated groups. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Eq Liver_HE: liver explants derived from healthy horses; Eq Liver_EMS: untreated liver explants derived from EMS horses. Eq Liver_EMS + 1 µM MSI-1436: liver explants derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor. Eq PBMCr_HE: PBMC derived from healthy horses; Eq PBMC_EMS: untreated PBMC derived from EMS horses. Eq PBMC_EMS + 1 µM MSI-1436: PBMC derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor.

Figure 3

PTP1B inhibition attenuates liver fibrogenesis pathways. (a) Relative gene expression of major profibrogenic mediators. (b) Protein levels of tissue VEGF. (c) Relative levels of liver fibrosis-associated marker transcripts. (d) Fold changes in gene expression of PTP1B-related profibrogenic effectors. Representative data from three independent experiments are shown ± SD (n = 3). An asterisk (^∗) indicates a comparison among healthy, EMS, and EMS-treated groups. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Eq Liver_HE: liver explants derived from healthy horses; Eq Liver_EMS: untreated liver explants derived from EMS horses. Eq Liver_EMS + 1 µM MSI-1436: liver explants derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor.

Figure 4

MSI-1436 regulates proteolytic ECM remodeling in EMS livers. (a) Relative gene expression of master metalloproteinases. (b) Relative protein expression of ECM-associated MMPs. Results were normalized to the expression of endogenous β-actin control. (c) Expression of metallopeptidase inhibitors 1 and 2. Representative data from three independent experiments are shown ± SD (n = 3). An asterisk (^∗) indicates a comparison among healthy, EMS, and EMS-treated groups. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Eq Liver_HE: liver explants derived from healthy horses; Eq Liver_EMS: untreated liver explants derived from EMS horses. Eq Liver_EMS + 1 µM MSI-1436: liver explants derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor.

Figure 5

MSI-1436 ameliorates liver glucose absorption and Glut-2 availability under EMS condition. (a) Western blot analysis of the liver Glut-2 protein level. Results were normalized to the expression of endogenous β-actin control. (b) Histograms showing D-glucose uptake by treated and untreated liver tissue. Representative data from three independent experiments are shown ± SD (n = 3). An asterisk (^∗) indicates a comparison among healthy, EMS, and EMS-treated groups. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. Eq Liver_HE: liver explants derived from healthy horses; Eq Liver_EMS: untreated liver explants derived from EMS horses. Eq Liver_EMS + 1 µM MSI-1436: liver explants derived from EMS horses and treated with 1 µM of MSI-1436 inhibitor.