Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2

Abstract

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

Keywords: ceramide; hepatocyte; insulin resistance; sphingolipids; type 2 diabetes.

Fig. 1.

Physiological characteristics of Sphk2-LKO mice. (A) Body weight (BW) gain of hepatocyte-specific Sphk2 knockout (Sphk2-LKO) and floxed control mice on a CD or HFD was monitored every other week for 20 wk. After 20 wk of feeding, mice were fasted for 16 h before sacrifice; n = 7. The liver weight (B) and fat weight (C) were normalized to body weight; n = 7. (D–G) Levels of NEFA (D), TG (E), TC (F), and ALT (G) in plasma; n = 7. (H) Levels of S1P, sphingosine (Sph), and ceramide mass in the liver; n = 6. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

Hepatocyte-specific knockout of Sphk2 results in glucose intolerance and insulin resistance. Hepatocyte-specific Sphk2 knockout (Sphk2-LKO) and floxed control mice were fed on a CD or HFD for 20 wk. Levels of fasting blood glucose (A) and plasma insulin (B) were examined. (C) HOMA-IR score was calculated as fasting insulin (ng/mL) × fasting blood glucose (mM)/22.5. Oral glucose tolerance test (D) and i.p. insulin tolerance test (F) were performed and quantified as area under curve in (E) and (G), respectively. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, Sphk2-LKO CD vs. Control CD, if not specified; ^#P < 0.05, ^##P < 0.01, Sphk2-LKO HFD vs. Control HFD; n = 7.

Fig. 3.

Knockout of Sphk2 impairs insulin-mediated suppression of hepatic gluconeogenesis. (A and B) i.p. PTT was performed in hepatocyte-specific Sphk2 knockout (Sphk2-LKO, n = 9) and floxed control (n = 5) mice on a normal chow diet (A), and quantified as area under curve (B). (C) mRNA expression of Pck1, G6pc, Gck, and Pklr were examined in liver tissues using RT-qPCR; n = 5. (D–G) Primary hepatocytes were isolated from WT and global Sphk2 knockout (KO) mice. Western blot analyses were performed in cells stimulated with insulin at indicated concentrations for 15 min (D) or at 10 nM for indicated times (F). Primary hepatocytes were stimulated with 100 nM insulin for 6 h, then HGP (E) was examined in the culture medium, and mRNA expression levels of Pck1 and G6pc (G) were quantified using RT-qPCR; n = 6. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Knockdown of SphK2 impairs hepatic insulin signaling. SphK2 was knocked down in Huh7 hepatic cell line using lentiviral-based shRNA. Huh7 cells were treated with insulin at indicated concentrations for 15 min (A) or at 10 nM for indicated times (C). (B and D) Levels of indicated phosphorylated protein vs. total protein were quantified. Data are expressed as mean ± SD; **P < 0.01, ***P < 0.001, n = 3. (E) Huh7 parental cells were treated with vehicle (Veh; dimethyl sulfoxide), K145, or ABC294640 (ABC) for the indicated concentrations and times, prior to 15 min treatment with 10 nM insulin. (F) FLAG-tagged SphK1 or SphK2 were stably overexpressed in shSphK2 Huh7 cells. Cells were stimulated with 10 nM insulin for 15 min. Western blot analyses were performed.

Fig. 5.

Effect of SphK2 on insulin-induced PI3K activation. SphK2 was knocked down in Huh7 cells using lentiviral-based shRNA. (A) PIP₃ was visualized by the transfection of GFP-tagged Akt-PH. Bar, 10 μm. (B) PIP₃ level was quantified using ELISA; n = 5. (C) Coimmunoprecipitation assay detecting the physical interaction between IRS1 and p85 subunit of PI3K. (D and E) Phosphorylation of the indicated proteins in insulin-signaling pathway was examined in cells treated with 10 nM insulin for 15 min by Western blot analyses (D) and normalized to total protein (E); n ≥ 3. (F) Level of phospho-S6 was examined in cells treated with 1 mM leucine for the indicated times. (G) Knockdown of SphK2 inhibited 740 Y-P–induced Akt phosphorylation. Cells were treated with 10 nM insulin or 25 μM 740 Y-P for 15 min. Data are expressed as mean ± SD; **P < 0.01, ***P < 0.001.

Fig. 6.

SphK2 regulates hepatic insulin signaling primarily via sphingosine. SphK2 was knocked down in Huh7 cells using lentiviral-based shRNA. (A–F and H) Western blot analyses were performed in cells treated with 10 nM insulin for 15 min following the indicated cotreatment. (A) FLAG-tagged WT-SphK2 or its DN mutant were stably overexpressed in shSphK2 Huh7 cells. (B–E) Cells were treated with 1 μM S1P for the indicated times (B), myriocin (C), fumonisin b1 (Fb1, D), or ARN14974 (ARN, E) at the indicated concentrations for 24 h. (F) Cells were transfected with negative control siRNA or siRNA against ASAH1 (siASAH1) for 48 h prior to insulin treatment. (H) Cells were treated with l- and D-form of sphingosine (So) or sphinganine (Sa) at 250 nM for 1 h. (G) Levels of ceramides (Cer), Sph, and S1P were quantified using lipidomics in indicated cells, untreated or treated with ARN14974 at 10 μM for 24 h; n = 6. (I) PI3K activity was examined following the treatment with 20 nM insulin for 15 min, in control cells, SphK2 knockdown (shSphK2) cells, and control cells pretreated with 250 nM sphingosine for 1 h; n = 3. Data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001.