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Soluble CLEC2 Extracellular Domain Improves Glucose and Lipid Homeostasis by Regulating Liver Kupffer Cell Polarization.

Wu X, Zhang J, Ge H, Gupte J, Baribault H, Lee KJ, Lemon B, Coberly S, Gong Y, Pan Z, Rulifson IC, Gardner J, Richards WG, Li Y - EBioMedicine (2015)

Bottom Line: Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models.Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance.These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.

View Article: PubMed Central - PubMed

Affiliation: Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.

ABSTRACT
The polarization of tissue resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to positively impact obesity and insulin resistance. Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models. Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance. Furthermore, Fc-CLEC2(ECD) treatment improves cytokine profiles and increases both the M2 macrophage population and the genes involved in the oxidation of lipid metabolism in the liver. These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.

No MeSH data available.


Related in: MedlinePlus

High expression of Clec2 in the non-parenchymal fraction of liver cells. (A) RT-qPCR of different human tissues shows CLEC2 enrichment in the testis, liver and PBL. (B) RT-qPCR of different mouse tissues. (C) Clec2 is highly expressed in non-parenchymal cells of liver compared to hepatocytes. Results are expressed as the mean ± SEM of triplicates and are representative of two independent experiments.
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f0005: High expression of Clec2 in the non-parenchymal fraction of liver cells. (A) RT-qPCR of different human tissues shows CLEC2 enrichment in the testis, liver and PBL. (B) RT-qPCR of different mouse tissues. (C) Clec2 is highly expressed in non-parenchymal cells of liver compared to hepatocytes. Results are expressed as the mean ± SEM of triplicates and are representative of two independent experiments.

Mentions: During a quest to identify potential novel regulators of metabolism, we noted the relatively high expression levels of CLEC2 in human liver samples among a panel of tissues examined (Fig. 1A). Clec2 is also expressed in the mouse liver tissue although the relative level to other tissues is not as high as in humans (Fig. 1B). Fractionation of cells recovered from mouse liver revealed that Clec2 was highly expressed specifically in non-parenchymal cells compared to hepatocytes (Fig. 1C), consistent with the detection of CLEC2 on Kupffer cells (Tang et al., 2010). As liver plays major roles in the metabolic control of lipid, glucose, and energy metabolism, we wondered if CLEC2 could affect metabolism. Since no soluble ligands have been identified for CLEC2, we took the approach to overexpress a soluble form of CLEC2, which could potentially act as a decoy antagonistic receptor as a way to study CLEC2 function. To achieve a stable and high level expression of CLEC2, the extracellular domain of murine CLEC2 (residues 51–229) was fused with human Fc to generate Fc-CLEC2(ECD) (Fig. 2A). Purified recombinant Fc-CLEC2(ECD) can bind to the known ligand, rhodocytin, and block rhodocytin-induced platelet aggregation in vitro (Fig. 2B and C), demonstrating that this form of CLEC2 is functional. A DNA expression construct carrying Fc-CLEC2(ECD) was delivered in vivo through HTV injection in a diet induced obesity (DIO) mouse model. 12-week-old male B6D2F1/J DIO mice were randomized into two groups based on body weight and baseline glucose levels. The mice in the treatment group were injected with the HTV Fc-CLEC2(ECD) DNA construct and mice in the control group were injected with a DNA construct carrying Fc alone. The expression of full length Fc-CLEC2(ECD) protein was confirmed by Western blot using an anti-human Fc antibody (data not shown). Serum exposure was quantitated by ELISA and revealed that high levels of protein exposure were achieved after HTV delivery (Fig. 2D). Fc-CLEC2(ECD) did not induce significant changes to body weight during the experiment period (Fig. 2E), while a significant reduction in fasting blood glucose levels was observed 13 days after Fc-CLEC2(ECD) treatment (Fig. 2F). At day 13, an oGTT demonstrated a significant improvement in the animals expressing Fc-CLEC2(ECD) compared to the mice in the control group (Fig. 2G). Furthermore, mice in the treatment group also had significantly lowered serum insulin levels (Fig. 2H), suggesting an improvement in insulin sensitivity. Because hepatic steatosis is commonly associated with obesity and diabetes, livers were collected from the mice at day 18 for triglyceride (TG) measurement. Liver TG levels were significantly reduced in the treatment group compared to the control mice (Fig. 2I). No signs of fibrosis were found in livers from either group as indicated by the negative immunohistochemical staining for α smooth muscle actin (αSMA) (Supplementary Fig. 1A). Histopathological analysis of multiple organs, including the liver, lymph node, pancreas, skeletal muscle, spleen, and white fat, also did not reveal any gross abnormalities in mice from either group (data not shown).


Soluble CLEC2 Extracellular Domain Improves Glucose and Lipid Homeostasis by Regulating Liver Kupffer Cell Polarization.

Wu X, Zhang J, Ge H, Gupte J, Baribault H, Lee KJ, Lemon B, Coberly S, Gong Y, Pan Z, Rulifson IC, Gardner J, Richards WG, Li Y - EBioMedicine (2015)

High expression of Clec2 in the non-parenchymal fraction of liver cells. (A) RT-qPCR of different human tissues shows CLEC2 enrichment in the testis, liver and PBL. (B) RT-qPCR of different mouse tissues. (C) Clec2 is highly expressed in non-parenchymal cells of liver compared to hepatocytes. Results are expressed as the mean ± SEM of triplicates and are representative of two independent experiments.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489977&req=5

f0005: High expression of Clec2 in the non-parenchymal fraction of liver cells. (A) RT-qPCR of different human tissues shows CLEC2 enrichment in the testis, liver and PBL. (B) RT-qPCR of different mouse tissues. (C) Clec2 is highly expressed in non-parenchymal cells of liver compared to hepatocytes. Results are expressed as the mean ± SEM of triplicates and are representative of two independent experiments.
Mentions: During a quest to identify potential novel regulators of metabolism, we noted the relatively high expression levels of CLEC2 in human liver samples among a panel of tissues examined (Fig. 1A). Clec2 is also expressed in the mouse liver tissue although the relative level to other tissues is not as high as in humans (Fig. 1B). Fractionation of cells recovered from mouse liver revealed that Clec2 was highly expressed specifically in non-parenchymal cells compared to hepatocytes (Fig. 1C), consistent with the detection of CLEC2 on Kupffer cells (Tang et al., 2010). As liver plays major roles in the metabolic control of lipid, glucose, and energy metabolism, we wondered if CLEC2 could affect metabolism. Since no soluble ligands have been identified for CLEC2, we took the approach to overexpress a soluble form of CLEC2, which could potentially act as a decoy antagonistic receptor as a way to study CLEC2 function. To achieve a stable and high level expression of CLEC2, the extracellular domain of murine CLEC2 (residues 51–229) was fused with human Fc to generate Fc-CLEC2(ECD) (Fig. 2A). Purified recombinant Fc-CLEC2(ECD) can bind to the known ligand, rhodocytin, and block rhodocytin-induced platelet aggregation in vitro (Fig. 2B and C), demonstrating that this form of CLEC2 is functional. A DNA expression construct carrying Fc-CLEC2(ECD) was delivered in vivo through HTV injection in a diet induced obesity (DIO) mouse model. 12-week-old male B6D2F1/J DIO mice were randomized into two groups based on body weight and baseline glucose levels. The mice in the treatment group were injected with the HTV Fc-CLEC2(ECD) DNA construct and mice in the control group were injected with a DNA construct carrying Fc alone. The expression of full length Fc-CLEC2(ECD) protein was confirmed by Western blot using an anti-human Fc antibody (data not shown). Serum exposure was quantitated by ELISA and revealed that high levels of protein exposure were achieved after HTV delivery (Fig. 2D). Fc-CLEC2(ECD) did not induce significant changes to body weight during the experiment period (Fig. 2E), while a significant reduction in fasting blood glucose levels was observed 13 days after Fc-CLEC2(ECD) treatment (Fig. 2F). At day 13, an oGTT demonstrated a significant improvement in the animals expressing Fc-CLEC2(ECD) compared to the mice in the control group (Fig. 2G). Furthermore, mice in the treatment group also had significantly lowered serum insulin levels (Fig. 2H), suggesting an improvement in insulin sensitivity. Because hepatic steatosis is commonly associated with obesity and diabetes, livers were collected from the mice at day 18 for triglyceride (TG) measurement. Liver TG levels were significantly reduced in the treatment group compared to the control mice (Fig. 2I). No signs of fibrosis were found in livers from either group as indicated by the negative immunohistochemical staining for α smooth muscle actin (αSMA) (Supplementary Fig. 1A). Histopathological analysis of multiple organs, including the liver, lymph node, pancreas, skeletal muscle, spleen, and white fat, also did not reveal any gross abnormalities in mice from either group (data not shown).

Bottom Line: Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models.Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance.These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.

View Article: PubMed Central - PubMed

Affiliation: Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States.

ABSTRACT
The polarization of tissue resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to positively impact obesity and insulin resistance. Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models. Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance. Furthermore, Fc-CLEC2(ECD) treatment improves cytokine profiles and increases both the M2 macrophage population and the genes involved in the oxidation of lipid metabolism in the liver. These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease.

No MeSH data available.


Related in: MedlinePlus