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. 2010 Dec;52(6):1957-67.
doi: 10.1002/hep.23916. Epub 2010 Nov 3.

Type 2 diabetes in mice induces hepatic overexpression of sulfatase 2, a novel factor that suppresses uptake of remnant lipoproteins

Keyang Chen  1 , Ming-Lin Liu, Lana Schaffer, Mingzhen Li, Guenther Boden, Xiangdong Wu, Kevin Jon Williams
Affiliations

Type 2 diabetes in mice induces hepatic overexpression of sulfatase 2, a novel factor that suppresses uptake of remnant lipoproteins

Keyang Chen et al. Hepatology. 2010 Dec.

Abstract

Type 2 diabetes mellitus (T2DM) impairs hepatic clearance of atherogenic postprandial remnant lipoproteins. Our work and that of others have identified syndecan-1 heparan sulfate proteoglycans (HSPGs) as remnant lipoprotein receptors. Nevertheless, defects in the T2DM liver have not been molecularly characterized, and neither has the correction that occurs upon caloric restriction. We used microarrays to compare expression of proteoglycan-related genes in livers from control db/m mice; obese, T2DM db/db littermates fed ad libitum (AL); and db/db mice pair-fed to match the intake of db/m mice. Surprisingly, the arrays identified only one gene whose dysregulation by T2DM would disrupt HSPG structure: the heparan sulfate glucosamine-6-O-endosulfatase-2 (Sulf2). SULF2 degrades HSPGs by removing 6-O sulfate groups, but had no previously known role in diabetes or lipoprotein biology. Follow-up quantitative polymerase chain reaction assays revealed a striking 11-fold induction of Sulf2 messenger RNA in the livers of AL T2DM mice compared with controls. Immunoblots demonstrated induction of SULF2 in AL livers, with restoration toward normal in livers from pair-fed db/db mice. Knockdown of SULF2 in cultured hepatocytes doubled HSPG-mediated catabolism of model remnant lipoproteins. Notably, co-immunoprecipitations revealed a persistent physical association of SULF2 with syndecan-1. To identify mechanisms of SULF2 dysregulation in T2DM, we found that advanced glycosylation end products provoked a 10-fold induction in SULF2 expression by cultured hepatocytes and an approximately 50% impairment in their catabolism of remnants and very low-density lipoprotein, an effect that was entirely reversed by SULF2 knockdown. Adiponectin and insulin each suppressed SULF2 protein in cultured liver cells and in murine livers in vivo, consistent with a role in energy flux. Likewise, both hormones enhanced remnant lipoprotein catabolism in vitro.

Conclusion: SULF2 is an unexpected suppressor of atherogenic lipoprotein clearance by hepatocytes and an attractive target for inhibition.

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Figures

Figure 1
Figure 1
Type 2 diabetes robustly induces hepatic overexpression of the heparan sulfate glucosamine-6-O-endosulfatase-2 (SULF2) in association with hyperlipoproteinemia, and caloric restriction corrects hepatic levels of this enzyme towards normal. RNA (panel A) and protein (panel B) were extracted from livers of 14-week old phenotypically lean db/m mice (controls), their obese, ad lib-fed T2DM db/db littermates (AL), and db/db mice that we pair-fed to match the intake of the db/m controls (PF). In Panel A, the y-axis displays individual Sulf2 mRNA levels assayed by qPCR, normalized to Ppia mRNA levels (ΔCt), and then expressed relative to the median control value (2-ΔΔCt). Median values are indicated by the short horizontal black lines, and individual values for all mice in each group are shown by X-symbols, upright triangles, and inverted triangles, respectively. P<0.001 by ANOVA on ranks; ** (P<0.01) or n.s. (not significant) for pairwise comparisons by SNK. Panel B displays immunoblots in triplicate for SULF2 and, as a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each lane represents a sample from a different animal. Panel C: Plot of plasma triglyceride concentrations vs. hepatic Sulf2 mRNA levels for all control (X-symbols) and all T2DM AL (upright triangles) mice. Panel D: Plot of plasma LDL concentrations vs. hepatic Sulf2 mRNA levels from the same mice. Also displayed are calculated Spearman rank correlation coefficients (r) and their P-values.
Figure 1
Figure 1
Type 2 diabetes robustly induces hepatic overexpression of the heparan sulfate glucosamine-6-O-endosulfatase-2 (SULF2) in association with hyperlipoproteinemia, and caloric restriction corrects hepatic levels of this enzyme towards normal. RNA (panel A) and protein (panel B) were extracted from livers of 14-week old phenotypically lean db/m mice (controls), their obese, ad lib-fed T2DM db/db littermates (AL), and db/db mice that we pair-fed to match the intake of the db/m controls (PF). In Panel A, the y-axis displays individual Sulf2 mRNA levels assayed by qPCR, normalized to Ppia mRNA levels (ΔCt), and then expressed relative to the median control value (2-ΔΔCt). Median values are indicated by the short horizontal black lines, and individual values for all mice in each group are shown by X-symbols, upright triangles, and inverted triangles, respectively. P<0.001 by ANOVA on ranks; ** (P<0.01) or n.s. (not significant) for pairwise comparisons by SNK. Panel B displays immunoblots in triplicate for SULF2 and, as a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each lane represents a sample from a different animal. Panel C: Plot of plasma triglyceride concentrations vs. hepatic Sulf2 mRNA levels for all control (X-symbols) and all T2DM AL (upright triangles) mice. Panel D: Plot of plasma LDL concentrations vs. hepatic Sulf2 mRNA levels from the same mice. Also displayed are calculated Spearman rank correlation coefficients (r) and their P-values.
Figure 2
Figure 2
Endogenous SULF2 strongly inhibits the catabolism of model remnant lipoproteins by cultured liver cells. McArdle hepatoma cells were pre-incubated for 24h with 100nM non-target (Control) or Sulf2 siRNA, as indicated, followed by an additional 44h at 37°C. Cells were then incubated for 4h at 37°C with model remnant lipoproteins that we prepared by combining 125I-labeled methylated LDL with lipoprotein lipase (LpL), a molecule that bridges between lipoproteins and HSPGs (background values were assessed in the absence of LpL). Panel A: Immunoblots of cellular homogenates, using anti-SULF2 antibodies or, as a loading control, anti-ß-actin antibodies. Panel B: LpL-dependent cellular catabolism of model remnant lipoproteins, shown as surface binding (Surf), internalization (Inter), and degradation (Degr), normalized to control values from cells treated with the nontarget siRNA (means±SEMs, n=3; the non-normalized control values were 194±2.9, 605±20.6, and 112±4.3 ng/mg, respectively). **, P<0.01 by the two-tailed Student’s t-test. Displayed are data from a representative experiment from a total of four independent knock-down studies. Panel C: Coimmunoprecipitation of SULF2 with the syndecan-1 HSPG. McArdle hepatocytes were extracted into NP-40 and subjected to immunoprecipitation with nonimmune IgG (IP: Mock) or anti-syndecan-1 IgG (IP: anti-SDC1), followed by electrophoretic separation. Displayed are immunoblots that were performed to detect SULF2 (IB: anti-SULF2), and then the same blots were stripped and reprobed to detect syndecan-1 (IB: anti-SDC1).
Figure 3
Figure 3
Advanced glycosylation end-products (AGEs) augment expression of SULF2 by cultured liver cells, thereby inhibiting catabolism of model remnant lipoproteins and VLDL. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein induction by AGEs. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of AGEs, plus an amount of BSA to bring the total supplemented protein in each well to 400 μg/ml. As described in the Methods, cells in each time course of SULF2 regulation in vitro were harvested simultaneously. Here, 200 μg AGEs/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates, using anti-SULF2 or anti-ß-actin antibodies. Panel B: Time course of Sulf2 mRNA induction by AGEs (200 μg/ml). Displayed are Sulf2 mRNA levels assayed by qPCR, normalized to Ppia mRNA levels, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student- Newman-Keuls test. Panels C and D: Effects of AGEs on catabolism of remnant lipoproteins (C) or VLDL (D) by cultured liver cells, without and with SULF2 knock-down. McArdle hepatoma cells were incubated for three consecutive 24-h periods at 37°C. The first 24-h period was with 100nM non-target siRNA or Sulf2 siRNA, as indicated by minus (−) and plus (+) Sulf2 siRNA, respectively. Cells were rinsed to remove the siRNAs, then incubated in serum-containing medium for a second 24-h period. During the final 24h, 200μg of either BSA or AGEs per ml were added, as indicated by minus and plus AGEs, respectively. Lipoprotein catabolism was examined during the last 4h of this final 24-h period. The upper images in these two panels show immunoblots of cellular homogenates. In panel C, the column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells treated with the non-target siRNA and without AGEs (means±SEMs, n=3; the non-normalized control values were 242±3.4, 626±7.1, and 103±4.6ng/mg, respectively). In panel D, the column graph displays normalized values for surface binding, internalization, and degradation of labeled VLDL (means±SEMs, n=3; the non-normalized control values were 18.58±0.3, 78.86±0.9, and 59.89±0.35ng/mg, respectively). Within each of the three displayed groups of quantitative data, P<0.001 by ANOVA, and columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Displayed are data from representative experiments from a total of four independent dose-response, time-course, and lipoprotein catabolism studies.
Figure 3
Figure 3
Advanced glycosylation end-products (AGEs) augment expression of SULF2 by cultured liver cells, thereby inhibiting catabolism of model remnant lipoproteins and VLDL. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein induction by AGEs. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of AGEs, plus an amount of BSA to bring the total supplemented protein in each well to 400 μg/ml. As described in the Methods, cells in each time course of SULF2 regulation in vitro were harvested simultaneously. Here, 200 μg AGEs/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates, using anti-SULF2 or anti-ß-actin antibodies. Panel B: Time course of Sulf2 mRNA induction by AGEs (200 μg/ml). Displayed are Sulf2 mRNA levels assayed by qPCR, normalized to Ppia mRNA levels, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student- Newman-Keuls test. Panels C and D: Effects of AGEs on catabolism of remnant lipoproteins (C) or VLDL (D) by cultured liver cells, without and with SULF2 knock-down. McArdle hepatoma cells were incubated for three consecutive 24-h periods at 37°C. The first 24-h period was with 100nM non-target siRNA or Sulf2 siRNA, as indicated by minus (−) and plus (+) Sulf2 siRNA, respectively. Cells were rinsed to remove the siRNAs, then incubated in serum-containing medium for a second 24-h period. During the final 24h, 200μg of either BSA or AGEs per ml were added, as indicated by minus and plus AGEs, respectively. Lipoprotein catabolism was examined during the last 4h of this final 24-h period. The upper images in these two panels show immunoblots of cellular homogenates. In panel C, the column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells treated with the non-target siRNA and without AGEs (means±SEMs, n=3; the non-normalized control values were 242±3.4, 626±7.1, and 103±4.6ng/mg, respectively). In panel D, the column graph displays normalized values for surface binding, internalization, and degradation of labeled VLDL (means±SEMs, n=3; the non-normalized control values were 18.58±0.3, 78.86±0.9, and 59.89±0.35ng/mg, respectively). Within each of the three displayed groups of quantitative data, P<0.001 by ANOVA, and columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Displayed are data from representative experiments from a total of four independent dose-response, time-course, and lipoprotein catabolism studies.
Figure 3
Figure 3
Advanced glycosylation end-products (AGEs) augment expression of SULF2 by cultured liver cells, thereby inhibiting catabolism of model remnant lipoproteins and VLDL. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein induction by AGEs. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of AGEs, plus an amount of BSA to bring the total supplemented protein in each well to 400 μg/ml. As described in the Methods, cells in each time course of SULF2 regulation in vitro were harvested simultaneously. Here, 200 μg AGEs/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates, using anti-SULF2 or anti-ß-actin antibodies. Panel B: Time course of Sulf2 mRNA induction by AGEs (200 μg/ml). Displayed are Sulf2 mRNA levels assayed by qPCR, normalized to Ppia mRNA levels, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student- Newman-Keuls test. Panels C and D: Effects of AGEs on catabolism of remnant lipoproteins (C) or VLDL (D) by cultured liver cells, without and with SULF2 knock-down. McArdle hepatoma cells were incubated for three consecutive 24-h periods at 37°C. The first 24-h period was with 100nM non-target siRNA or Sulf2 siRNA, as indicated by minus (−) and plus (+) Sulf2 siRNA, respectively. Cells were rinsed to remove the siRNAs, then incubated in serum-containing medium for a second 24-h period. During the final 24h, 200μg of either BSA or AGEs per ml were added, as indicated by minus and plus AGEs, respectively. Lipoprotein catabolism was examined during the last 4h of this final 24-h period. The upper images in these two panels show immunoblots of cellular homogenates. In panel C, the column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells treated with the non-target siRNA and without AGEs (means±SEMs, n=3; the non-normalized control values were 242±3.4, 626±7.1, and 103±4.6ng/mg, respectively). In panel D, the column graph displays normalized values for surface binding, internalization, and degradation of labeled VLDL (means±SEMs, n=3; the non-normalized control values were 18.58±0.3, 78.86±0.9, and 59.89±0.35ng/mg, respectively). Within each of the three displayed groups of quantitative data, P<0.001 by ANOVA, and columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Displayed are data from representative experiments from a total of four independent dose-response, time-course, and lipoprotein catabolism studies.
Figure 4
Figure 4
Adiponectin suppresses SULF2 expression by cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by adiponectin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of adiponectin, where zero indicates adiponectin-free medium. In the time course, 6 μg adiponectin/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA suppression by a physiologic concentration of adiponectin (6 μg/ml). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student-Newman-Keuls test. Panel C: Regulation of hepatic SULF2 protein levels by adiponectin in vivo. Protein was extracted from livers of wild-type C57BL/6J mice (WT, n=3) and adiponectinknockout mice that had been injected with either saline (KO+Saline, n=4) or fulllength adiponectin (KO+fAd, n=3, 25μg/mouse, three times/day for four days). Plasma adiponectin concentrations in these mice 4h after the last injection were 20.86±1.23, 0.41±0.013, and 6.16±1.08 μg/ml, respectively. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Regulation of hepatic Sulf2 mRNA levels by adiponectin in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA level were assessed by qPCR, normalized to Ppia, and then expressed relative to wild-type (means±SEM, n=3-4), P<0.001 by ANOVA. Columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Panel E: Effects of adiponectin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h in adiponectin-free medium (minus symbols and open columns) or medium supplemented with 6 μg adiponectin/ml (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without adiponectin (means±SEMs, n=3; the non-normalized control values were 179±3.5, 618±43.4, and 37±1.1ng/mg, respectively). **, P<0.01 by the two-tailed Student’s t-test.
Figure 4
Figure 4
Adiponectin suppresses SULF2 expression by cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by adiponectin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of adiponectin, where zero indicates adiponectin-free medium. In the time course, 6 μg adiponectin/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA suppression by a physiologic concentration of adiponectin (6 μg/ml). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student-Newman-Keuls test. Panel C: Regulation of hepatic SULF2 protein levels by adiponectin in vivo. Protein was extracted from livers of wild-type C57BL/6J mice (WT, n=3) and adiponectinknockout mice that had been injected with either saline (KO+Saline, n=4) or fulllength adiponectin (KO+fAd, n=3, 25μg/mouse, three times/day for four days). Plasma adiponectin concentrations in these mice 4h after the last injection were 20.86±1.23, 0.41±0.013, and 6.16±1.08 μg/ml, respectively. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Regulation of hepatic Sulf2 mRNA levels by adiponectin in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA level were assessed by qPCR, normalized to Ppia, and then expressed relative to wild-type (means±SEM, n=3-4), P<0.001 by ANOVA. Columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Panel E: Effects of adiponectin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h in adiponectin-free medium (minus symbols and open columns) or medium supplemented with 6 μg adiponectin/ml (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without adiponectin (means±SEMs, n=3; the non-normalized control values were 179±3.5, 618±43.4, and 37±1.1ng/mg, respectively). **, P<0.01 by the two-tailed Student’s t-test.
Figure 4
Figure 4
Adiponectin suppresses SULF2 expression by cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by adiponectin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of adiponectin, where zero indicates adiponectin-free medium. In the time course, 6 μg adiponectin/ml was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA suppression by a physiologic concentration of adiponectin (6 μg/ml). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3). P<0.001 by ANOVA; **, P<0.01 compared to the unexposed control by the Student-Newman-Keuls test. Panel C: Regulation of hepatic SULF2 protein levels by adiponectin in vivo. Protein was extracted from livers of wild-type C57BL/6J mice (WT, n=3) and adiponectinknockout mice that had been injected with either saline (KO+Saline, n=4) or fulllength adiponectin (KO+fAd, n=3, 25μg/mouse, three times/day for four days). Plasma adiponectin concentrations in these mice 4h after the last injection were 20.86±1.23, 0.41±0.013, and 6.16±1.08 μg/ml, respectively. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Regulation of hepatic Sulf2 mRNA levels by adiponectin in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA level were assessed by qPCR, normalized to Ppia, and then expressed relative to wild-type (means±SEM, n=3-4), P<0.001 by ANOVA. Columns labeled with different lowercase letters are statistically different by the Student-Newman-Keuls test (P<0.01). Panel E: Effects of adiponectin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h in adiponectin-free medium (minus symbols and open columns) or medium supplemented with 6 μg adiponectin/ml (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without adiponectin (means±SEMs, n=3; the non-normalized control values were 179±3.5, 618±43.4, and 37±1.1ng/mg, respectively). **, P<0.01 by the two-tailed Student’s t-test.
Figure 5
Figure 5
Insulin suppresses SULF2 protein, but not mRNA, in cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by insulin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of insulin. In the time course, 3 nM insulin was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA levels during exposure to a physiologic concentration of insulin (3 nM). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3; N.S. by ANOVA). Panel C: Regulation of hepatic SULF2 protein levels by insulin in vivo. Sprague-Dawley rats were subjected to euglycemic-hyperinsulinemic clamps (4.8 mU insulin/kg/min) for four hours (Insulin injected); control rats received saline/glycerol. Protein was extracted from frozen liver samples. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Hepatic Sulf2 mRNA levels after insulin injections in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA were measured by qPCR, normalized to Ppia, and then expressed relative to control (means±SEM, n=3-4; N.S. by ANOVA). Panel E: Effects of insulin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h without (minus symbols and open columns) or with 10 nM insulin (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without insulin (means±SEMs, n=3; this experiment was performed simultaneously with the one displayed in Figure 4E). **, P<0.01 by the two-tailed Student’s t-test.
Figure 5
Figure 5
Insulin suppresses SULF2 protein, but not mRNA, in cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by insulin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of insulin. In the time course, 3 nM insulin was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA levels during exposure to a physiologic concentration of insulin (3 nM). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3; N.S. by ANOVA). Panel C: Regulation of hepatic SULF2 protein levels by insulin in vivo. Sprague-Dawley rats were subjected to euglycemic-hyperinsulinemic clamps (4.8 mU insulin/kg/min) for four hours (Insulin injected); control rats received saline/glycerol. Protein was extracted from frozen liver samples. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Hepatic Sulf2 mRNA levels after insulin injections in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA were measured by qPCR, normalized to Ppia, and then expressed relative to control (means±SEM, n=3-4; N.S. by ANOVA). Panel E: Effects of insulin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h without (minus symbols and open columns) or with 10 nM insulin (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without insulin (means±SEMs, n=3; this experiment was performed simultaneously with the one displayed in Figure 4E). **, P<0.01 by the two-tailed Student’s t-test.
Figure 5
Figure 5
Insulin suppresses SULF2 protein, but not mRNA, in cultured liver cells and in murine livers in vivo, and enhances remnant lipoprotein catabolism in vitro. Panel A: Dose-response (upper images) and time course (lower images) of SULF2 protein suppression by insulin. The dose-response involved a 24-h incubation of McArdle hepatoma cells with the indicated concentrations of insulin. In the time course, 3 nM insulin was added at the indicated times before harvest. Displayed are immunoblots of cellular homogenates. Panel B: Time course of Sulf2 mRNA levels during exposure to a physiologic concentration of insulin (3 nM). Displayed are Sulf2 mRNA levels, normalized to Ppia, and then expressed relative to the unexposed control at 0 h (means±SEM, n=3; N.S. by ANOVA). Panel C: Regulation of hepatic SULF2 protein levels by insulin in vivo. Sprague-Dawley rats were subjected to euglycemic-hyperinsulinemic clamps (4.8 mU insulin/kg/min) for four hours (Insulin injected); control rats received saline/glycerol. Protein was extracted from frozen liver samples. Displayed are immunoblots for SULF2 and GAPDH. Panel D: Hepatic Sulf2 mRNA levels after insulin injections in vivo. RNA was extracted from the same livers as in Panel C. Levels of Sulf2 mRNA were measured by qPCR, normalized to Ppia, and then expressed relative to control (means±SEM, n=3-4; N.S. by ANOVA). Panel E: Effects of insulin on remnant lipoprotein catabolism by cultured liver cells. McArdle hepatoma cells were incubated for 24h without (minus symbols and open columns) or with 10 nM insulin (plus symbols and black columns). The upper images show immunoblots of cellular homogenates. The column graph displays LpL-dependent surface binding, internalization, and degradation of model remnant lipoproteins, normalized to control values from cells incubated without insulin (means±SEMs, n=3; this experiment was performed simultaneously with the one displayed in Figure 4E). **, P<0.01 by the two-tailed Student’s t-test.
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