SH3 domain binding kinase 1 (SBK1) is a novel enhancer of hepatic insulin signaling

Abstract

Enhanced hepatic gluconeogenesis is a predominant contributor to hyperglycemia in type 2 diabetes mellitus (T2DM). In healthy people, hepatic gluconeogenesis is tightly regulated by insulin via insulin receptor (IR), insulin receptor substrate 1 (IRS-1), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) pathway. In contrast, hepatocytes in T2DM patients are insensitive to insulin stimulation, resulting in excessive gluconeogenesis. Pharmacotherapeutic strategies aiming to restrain hepatic gluconeogenesis are unsatisfactory because of periodical therapeutic efficacy and side effects. Thus, identifying new regulatory signaling molecules/pathways of hepatic gluconeogenesis is necessary for developing better therapeutic methods against hyperglycemia. This project aims to delineate the role of a novel SH3 domain binding kinase 1 (SBK1) in glucose homeostasis under normal and disease status.

We postulated that SBK1 is a protective but unappreciated signaling molecule to compensate insulin resistance and relieve hyperglycemia in T2DM. To validate our hypothesis, we overexpressed SBK1 in a murine hepatoma cell line (Hepa1-6) cells, mouse primary hepatocytes, and chow diet-fed mice liver via adenovirus-mediated SBK1 overexpression. Hepatic insulin signaling including Akt and forkhead box protein O1 (FoxO1) phosphorylations was augmented in SBK1-overexpressed cells and mice. Moreover, pyruvate tolerance in SBK1-overexpressed mice were improved, and the fasted glucose level was reduced, which were associated with lower expression of gluconeogenic genes, including glucose 6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1). We further studied the effect of Sbk1 ablation on hepatic insulin sensitivity using liver-specific Sbk1 knockout (LKSO) mice. The fasted glucose level and gluconeogenesis in LSKO mice were higher than their littermate control because of the defective hepatic insulin signaling as demonstrated by the reduced Akt and FoxO1 phosphorylations in their liver and primary hepatocytes. On the other hand, liver-specific SBK1 overexpression alleviated high fructose diet (HFruD)- and HFD-induced the hepatic insulin resistance as revealed by the improved pyruvate intolerance, the reduced gluconeogenic gene (G6pc and Pck1) expression, lower blood glucose level, and the increased hepatic insulin signaling. To elucidate the molecular mechanism of how SBK1 affects insulin signaling, a BioID assay was applied to screen the potential SBK1 partners. IRS1, the crucial insulin signaling molecule, was specked. A series of biochemical assays including GST pull-down, co-immunoprecipitation assay, and in vitro kinase assay revealed that SBK1 interacts and phosphorylates IRS1 on T300, S324, and S336. Moreover, cycloheximide assay demonstrated that Sbk1 ablation reduced IRS1 stability in primary hepatocytes. To sum up, our studies demonstrated that SBK1 is a new member of insulin signaling in regulating hepatic gluconeogenesis, which may provide a new therapeutic target to ameliorate hyperglycemia and insulin resistance in T2DM.