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Reducing glycosphingolipid content in adipose tissue of obese mice restores insulin sensitivity, adipogenesis and reduces inflammation.

van Eijk M, Aten J, Bijl N, Ottenhoff R, van Roomen CP, Dubbelhuis PF, Seeman I, Ghauharali-van der Vlugt K, Overkleeft HS, Arbeeny C, Groen AK, Aerts JM - PLoS ONE (2009)

Bottom Line: In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM.As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN).In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. m.c.vaneijk@amc.uva.nl

ABSTRACT
Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (Lep(Ob)) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of Lep(Ob) mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) gamma, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

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Adipose tissue analysis after lowering of glycosphingolipid content.Haematoxylin and eosin staining of adipose tissue of (A) lean mice, (B) LepOb mice and (C) LepOb mice following reduction of glycosphingolipid content. (D) Analysis of adipocyte cell size distribution in lean mice (black line), in LepOb mice (red line) and in AMP-DNM treated LepOb mice (green line). (E) EWAT weight. Data are depicted on the Y-axis as mean±S.E.M. (n = 5 per group). Actual p values are depicted in the graphs. Bars in the photographs represent 100 µm. The arrows in panel B indicate crown-like structures. For the distribution of adipocyte size at least 150 cells were analysed per animal (for details see methods).
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pone-0004723-g001: Adipose tissue analysis after lowering of glycosphingolipid content.Haematoxylin and eosin staining of adipose tissue of (A) lean mice, (B) LepOb mice and (C) LepOb mice following reduction of glycosphingolipid content. (D) Analysis of adipocyte cell size distribution in lean mice (black line), in LepOb mice (red line) and in AMP-DNM treated LepOb mice (green line). (E) EWAT weight. Data are depicted on the Y-axis as mean±S.E.M. (n = 5 per group). Actual p values are depicted in the graphs. Bars in the photographs represent 100 µm. The arrows in panel B indicate crown-like structures. For the distribution of adipocyte size at least 150 cells were analysed per animal (for details see methods).

Mentions: First, the effect of AMP-DNM treatment of LepOb animals on adipocyte function was investigated. For this purpose, tissue slides stained with haematoxylin and eosin were examined. Adipocytes appeared larger in LepOb mice when compared to those in lean mice (Fig. 1B versus 1A). When analyzing the average EWAT adipocyte size, calculated per animal, we observed a significant difference (p<0.0001) between lean mice (2923±455 µm2) and LepOb mice (7992±494 µm2). We however did not observe a significant difference between LepOb animals and AMP-DNM treated animals (Fig. 1A–C). Interestingly, when studying individual adipocytes we observed a change in distribution of size (Fig. 1D). As expected the adipocytes in lean mice (black curve) were predominantly smaller than adipocytes in LepOb mice (red curve). Following treatment with AMP-DNM a reduction of the median adipocyte size was observed in LepOb mice (green curve), mainly due to a shift to the left of the 2nd and 3th quartiles of the distribution, reflecting reduced numbers of the large adipocytes. Adipocyte size did not normalize to sizes observed in lean mice. The distribution frequencies were analysed in detail by using a non parametric 2-tailed Kolmogorov-Smirnov Z test. The distributions are significantly different (lean versus LepOb p≤0.0005; LepOb versus LepOb+AMP-DNM p = 0.002). We also studied adipocytes in the omental adipose tissue (OAT) depot. In general the adipocyte sizes appeared smaller in the OAT depot when compared to the EWAT depot. We observed a comparable shift to the left with AMP-DNM and the distributions again appeared significantly different using the Kolmogorov-Smirnov Z test (lean versus LepOb p≤0.0005; LepOb versus LepOb+AMP-DNM p<0.0005) (data not shown). The amount of EWAT in LepOb animals (both pads 3920±175.1 mg) tended to be lower upon AMP-DNM treatment (both pads 336±655 mg), but the reduction failed to reach significance (p = 0.10) (Fig. 1E). No significant reduction in EWAT weight was observed when normalized for body weight.


Reducing glycosphingolipid content in adipose tissue of obese mice restores insulin sensitivity, adipogenesis and reduces inflammation.

van Eijk M, Aten J, Bijl N, Ottenhoff R, van Roomen CP, Dubbelhuis PF, Seeman I, Ghauharali-van der Vlugt K, Overkleeft HS, Arbeeny C, Groen AK, Aerts JM - PLoS ONE (2009)

Adipose tissue analysis after lowering of glycosphingolipid content.Haematoxylin and eosin staining of adipose tissue of (A) lean mice, (B) LepOb mice and (C) LepOb mice following reduction of glycosphingolipid content. (D) Analysis of adipocyte cell size distribution in lean mice (black line), in LepOb mice (red line) and in AMP-DNM treated LepOb mice (green line). (E) EWAT weight. Data are depicted on the Y-axis as mean±S.E.M. (n = 5 per group). Actual p values are depicted in the graphs. Bars in the photographs represent 100 µm. The arrows in panel B indicate crown-like structures. For the distribution of adipocyte size at least 150 cells were analysed per animal (for details see methods).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654925&req=5

pone-0004723-g001: Adipose tissue analysis after lowering of glycosphingolipid content.Haematoxylin and eosin staining of adipose tissue of (A) lean mice, (B) LepOb mice and (C) LepOb mice following reduction of glycosphingolipid content. (D) Analysis of adipocyte cell size distribution in lean mice (black line), in LepOb mice (red line) and in AMP-DNM treated LepOb mice (green line). (E) EWAT weight. Data are depicted on the Y-axis as mean±S.E.M. (n = 5 per group). Actual p values are depicted in the graphs. Bars in the photographs represent 100 µm. The arrows in panel B indicate crown-like structures. For the distribution of adipocyte size at least 150 cells were analysed per animal (for details see methods).
Mentions: First, the effect of AMP-DNM treatment of LepOb animals on adipocyte function was investigated. For this purpose, tissue slides stained with haematoxylin and eosin were examined. Adipocytes appeared larger in LepOb mice when compared to those in lean mice (Fig. 1B versus 1A). When analyzing the average EWAT adipocyte size, calculated per animal, we observed a significant difference (p<0.0001) between lean mice (2923±455 µm2) and LepOb mice (7992±494 µm2). We however did not observe a significant difference between LepOb animals and AMP-DNM treated animals (Fig. 1A–C). Interestingly, when studying individual adipocytes we observed a change in distribution of size (Fig. 1D). As expected the adipocytes in lean mice (black curve) were predominantly smaller than adipocytes in LepOb mice (red curve). Following treatment with AMP-DNM a reduction of the median adipocyte size was observed in LepOb mice (green curve), mainly due to a shift to the left of the 2nd and 3th quartiles of the distribution, reflecting reduced numbers of the large adipocytes. Adipocyte size did not normalize to sizes observed in lean mice. The distribution frequencies were analysed in detail by using a non parametric 2-tailed Kolmogorov-Smirnov Z test. The distributions are significantly different (lean versus LepOb p≤0.0005; LepOb versus LepOb+AMP-DNM p = 0.002). We also studied adipocytes in the omental adipose tissue (OAT) depot. In general the adipocyte sizes appeared smaller in the OAT depot when compared to the EWAT depot. We observed a comparable shift to the left with AMP-DNM and the distributions again appeared significantly different using the Kolmogorov-Smirnov Z test (lean versus LepOb p≤0.0005; LepOb versus LepOb+AMP-DNM p<0.0005) (data not shown). The amount of EWAT in LepOb animals (both pads 3920±175.1 mg) tended to be lower upon AMP-DNM treatment (both pads 336±655 mg), but the reduction failed to reach significance (p = 0.10) (Fig. 1E). No significant reduction in EWAT weight was observed when normalized for body weight.

Bottom Line: In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM.As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN).In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. m.c.vaneijk@amc.uva.nl

ABSTRACT
Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (Lep(Ob)) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of Lep(Ob) mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) gamma, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals.

Show MeSH
Related in: MedlinePlus