Insulin-dependent diabetes mellitus in mice does not alter liver heparan sulfate

Abstract

Diabetes -associated hyperlipidemia is generally attributed to reduced clearance of plasma lipoproteins, especially remnant lipoproteins enriched in cholesterol and triglycerides. Hepatic clearance of remnants occurs via low density lipoprotein receptors and the heparan sulfate proteoglycan, syndecan-1. Previous studies have suggested alterations in heparan sulfate proteoglycan metabolism in rat and mouse diabetic models, consistent with the idea that diabetic dyslipidemia might be caused by alterations in proteoglycan expression in the liver. In this study we analyzed the content and composition of liver heparan sulfate in streptozotocin-induced insulin-deficient diabetic mice that displayed fasting hypertriglyceridemia and delayed clearance of dietary triglyceride-rich lipoproteins. No differences between normal and diabetic littermates in liver heparan sulfate content, sulfation, syndecan-1 protein levels, or affinity for heparin-binding ligands, such as apolipoprotein E or fibroblast growth factor-2, were noted. Decreased incorporation of [(35)S]sulfate in insulin-deficient mice in vivo was observed, but the decrease was due to increased plasma inorganic sulfate, which reduced the efficiency of labeling of liver heparan sulfate. These results show that hyperlipidemia in insulin-deficient mice is not due to changes in hepatic heparan sulfate composition.

FIGURE 1.

Hypertriglyceridemia in IDDM mice. A, total fasting plasma triglycerides. STZ-treated diabetic (open circles) or untreated control (filled circles) C57BL/6 mice were fasted for 4 h in the morning, and blood was taken from the retroorbital sinus for triglyceride analysis. Average values ± S.D. were 50 ± 12 mg/dl in control mice versus 81 ± 19 mg/dl in STZ-treated animals, respectively (n = 12). B, chylomicron clearance measurement by retinyl ester excursion. Fasted control mice (closed circles, n = 4) and IDDM mice (open circles, n = 4) were given 200 μl of corn oil containing [³H]retinol by oral gavage. Blood samples were taken at the indicated times, and radioactivity remaining in plasma samples (10 μl) was determined by liquid scintillation counting. The values are expressed as mean ± S.D. (error bars) of three samples and are representative of at least three separate experiments.

FIGURE 2.

Analysis of liver heparan sulfate in IDDM mice. A, Western blot analysis of syndecan-1 expression in IDDM hepatocytes. Syndecan-1 was detected with mAb 281-2 in freshly prepared hepatocytes from untreated and STZ-treated hypertriglyceridemic diabetic mice (n = 4/strain). β-Actin was used as a loading control. B, analysis of endogenous liver [³⁵S]heparan sulfate. Untreated (n = 3) and STZ-treated hypertriglyceridemic diabetic (n = 3) wild-type mice and mutant mice bearing a hepatocyte specific deletion of Ndst1 (n = 3) were given [³⁵S]sulfate intraperitoneally to radiolabel newly made heparan sulfate chains in vivo. Two hours later [³⁵S]heparan sulfate was purified from the liver and quantified by liquid scintillation counting. Results were normalized/g of liver and are presented as a percentage of labeling in untreated control mice (1 ± 0.05 × 10⁵ cpm/g). The values are expressed as mean ± S.D. (error bars) and are representative of three separate studies (n = 9 mice). C, liver heparan sulfate measured chemically. Liver heparan sulfate was purified from untreated (n = 9) and STZ-treated hypertriglyceridemic diabetic (n = 9) wild-type mice and Ndst1 mutant mice (n = 4). The mass of heparan sulfate purified from each animal was determined by GRIL-LC/MS (see “Experimental Procedures”). Results were normalized to the amount of liver that had been analyzed (wet weight). D, sulfation of liver heparan sulfate. The disaccharide composition of liver heparan sulfate isolated in C was determined by GRIL-LC/MS. N-Sulfate and 6-O-sulfate groups in glucosamine moieties and 2-O-sulfate groups in uronic acids were calculated from the recovery of the individual disaccharides (see “Experimental Procedures”).

FIGURE 3.

Liver heparan sulfate binding to protein ligands. Liver [³⁵S]heparan sulfate from diabetic, Ndst1^f/fAlbCre⁺, and control mice (10⁴ cpm) was incubated with 10 μg of recombinant apoE (A) or fibroblast growth factor-2 (FGF-2; B), and [³⁵S]heparan sulfate bound to the proteins was collected by membrane filtration (see “Experimental Procedures”). Unbound [³⁵S]heparan sulfate does not bind to the membrane. Each experiment was done in triplicate, and the average values are shown. The values are expressed as mean ± S.D. (error bars) and are representative of two separate experiments.

FIGURE 4.

Plasma sulfate levels in IDDM mice. Sulfate levels were measured in untreated, diabetic, and Ndst1-mutant plasma samples (n = 6, respectively) by high performance liquid chromatography.