Katsuyoshi Kumagai

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr−/− mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-α signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-α signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

Overexpression of Monocyte Chemoattractant Protein-1 in Adipose Tissues Causes Macrophage Recruitment and Insulin Resistance *

Adipose tissue expression and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) correlate positively with adiposity. To ascertain the roles of MCP-1 overexpression in adipose, we generated transgenic mice by utilizing the adipocyte P2 (aP2) promoter (aP2-MCP-1 mice). These mice had higher plasma MCP-1 concentrations and increased macrophage accumulation in adipose tissues, as confirmed by immunochemical, flow cytometric, and gene expression analyses. Tumor necrosis factor-α and interleukin-6 mRNA levels in white adipose tissue and plasma non-esterified fatty acid levels were increased in transgenic mice. aP2-MCP-1 mice showed insulin resistance, suggesting that inflammatory changes in adipose tissues may be involved in the development of insulin resistance. Insulin resistance in aP2-MCP-1 mice was confirmed by hyperinsulinemic euglycemic clamp studies showing that transgenic mice had lower rates of glucose disappearance and higher endogenous glucose production than wild-type mice. Consistent with this, insulin-induced phosphorylations of Akt were significantly decreased in both skeletal muscles and livers of aP2-MCP-1 mice. MCP-1 pretreatment of isolated skeletal muscle blunted insulin-stimulated glucose uptake, which was partially restored by treatment with the MEK inhibitor U0126, suggesting that circulating MCP-1 may contribute to insulin resistance in aP2-MCP-1 mice. We concluded that both paracrine and endocrine effects of MCP-1 may contribute to the development of insulin resistance in aP2-MCP-1 mice.

Impaired Insulin Signaling in Endothelial Cells Reduces Insulin-Induced Glucose Uptake by Skeletal Muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.

Evi1 is essential for hematopoietic stem cell self-renewal, and its expression marks hematopoietic cells with long-term multilineage repopulating activity.

A new mouse in which an IRES-GFP cassette is knocked-in to the Evi1 locus reveals that HSC long-term multilineage repopulating activity specifically segregates with expression of the Evi1 transcription factor.

TCF7L2 in mouse pancreatic beta cells plays a crucial role in glucose homeostasis by regulating beta cell mass

Aims/hypothesisCommon genetic variations of the transcription factor 7-like 2 gene (encoded by TCF7L2), one of the T cell factor/lymphoid enhancer-binding factor transcription factors for the converging wingless-type MMTV integration site family (Wnt)/β-catenin signalling pathway, are known to be associated with type 2 diabetes. Individuals with at-risk alleles of TCF7L2 exhibit impaired insulin secretion. Although previous studies using animal models have revealed the existence of a relationship between the Wnt/β-catenin signalling pathway and glucose homeostasis, it remains unclear whether TCF7L2 in the pancreatic beta cells might be causally involved in insulin secretion in vivo. In this study, we investigated the role of TCF7L2 expressed in the pancreatic beta cells in glucose homeostasis.MethodsThree independent groups of genetically engineered mice (DN mice) were generated, in which expression of the dominant-negative form of Tcf7l2 was driven under a rat insulin promoter. Phenotypes of both adult and newborn mice were evaluated. The levels of genes and proteins expressed in isolated islets were determined by reverse transcription-quantitative PCR and western blot analysis, respectively.ResultsAdult DN mice showed impaired glucose tolerance and decreased insulin secretion in both oral and intraperitoneal glucose tolerance tests. Marked reduction of the beta cell area and whole-pancreas insulin content was observed in both the adult and newborn DN mice. Islets from the DN mice showed decreased gene expressions of Ccnd1, Ccnd2, Irs1, Irs2, Ins1, Ins2 and Mafa, consistent with the deleterious effects of the dominant-negative form of Tcf7l2 on beta cell proliferation and insulin production.Conclusions/interpretationTCF7L2 expressed in the pancreatic beta cells plays a crucial role in glucose metabolism through regulation of the beta cell mass.

Generation of Transgenic Mice on an NOD/SCID Background Using the Conventional Microinjection Technique

Humanized mice, which refers to immunodeficient mice repopulated with the human immune system, are powerful tools for study in the field of immunology. It has been difficult, however, to generate these transgenic (Tg) mice directly from such strains as the NOD/SCID mouse. In this study, we describe a method developed by us for the generation of Tg mice on an NOD/SCID background. First, we obtained fertilized eggs efficiently by means of in vitro fertilization (IVF); then, we attempted to generate CAG-EGFP Tg mice on an NOD/SCID background, finding that delayed timing of the microinjection after the IVF improved the time to development of the two-cell-stage embryos and the obtainment of newborns. We successfully generated Tg mice and confirmed the germ-line transmission in the offspring. In conclusion, we established a novel system for directly generating transgenic mice on an NOD/SCID background. This novel system is expected to allow improved efficiency of the generation of humanized mice.

A transgenic mutant mouse line accompanied by the complete deletion of interleukin-33 showed insulin and leptin resistances

Interleukin (IL) −33 has been identified as a member of the IL-1 family. Members of the IL-1 family have been reported to be involved in the regulation of energy homeostasis and glucose metabolism. Homozygous transgenic mutant mice of FLP14 line, that we previously generated, unexpectedly developed mature-onset obesity and diabetes. Through genetic investigations, we found that insertion of the transgenes had resulted in complete deletion of the Il33 gene. These obese male homozygous mutant mice exhibited hyperphagia with hyperleptinemia and insulin resistance caused by increased hepatic gluconeogenesis and decreased glucose uptake in skeletal muscle. As a result of examining preobese male homozygous mutant mice to investigate with the exclusion of the effect of obesity, hyperphagia with hyperleptinemina and insulin resistance caused by decreased glucose uptake in skeletal muscle were already observed, but the increased hepatic glucose production was not. To investigate whether the insulin resistance was caused by deletion of the Il33 gene, we treated these preobese homozygous mutant mice with recombinant IL-33 protein and noted a significant improvement in insulin resistance. Thus, insulin resistance in these homozygous mutant mice was caused, at least in part, by IL-33 deficiency, suggesting a favorable role of IL-33 for glucose metabolism in the skeletal muscle.

An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice

Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.

Abstract 3901: Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Chronic myeloid leukemia (CML) is a myeloproliferative disorder by BCR-ABL, which can transform hematopoietic stem cells (HSCs) into leukemic stem cells (LSCs) with a limitless capacity for self-renewal. Despite substantial prognostic improvement of CML by a specific debulking of tumor burden with a tyrosine kinase inhibitor (TKI) targeting ABL kinase, imatinib-treated CML patients in chronic phase can relapse and progress to blast phase due to the remnant CML stem cells. Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intra-individual EVI1-high cells. In this study, we hypothesized that EVI1 is a valuable marker of CML stem cells as well as HSCs and aimed to cover in depth the regulation of CML stem cells by Evi1. Introduction of CML into Evi1-IRES-GFP knock-in mice, a versatile HSC-reporter strain, enabled us to separate Evi1-high CML cells from the individual and revealed that Evi1 was predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity. Comparison of gene expression profiles between Evi1-high and -low CML cells revealed that Evi1-high CML cells had a more quiescent feature and a less differentiated feature than Evi1-low CML cells, suggesting that Evi1-high CML LSK cells could keep self-renewal capacity. Furthermore, Evi1-high CML cells exhibited apparent resistance to TKIs in vivo. Given that Evi1 heterozygosity ameliorated CML development in vivo and that the combination of Evi1 and BCR-ABL caused acute myeloid leukemia (AML) in mice resembling blastic transformation of CML, Evi1 could regulate CML development as a potent driver. In accordance to our data of Evi1-trafficking CML mouse, our single-cell analysis of primitive or differentiated subsets from primary CML-CP samples show that EVI1 was highly expressed in stem cell-enriched CD34+CD38-CD90+ cells, which implied that EVI1 could mark CML stem cells as well as normal HSCs. This point can be translated to human CML cases like that high EVI1 means the increasing number of CML stem cells. As opposed to Evi1-reporter CML models, in AML model by MLL-ENL oncogene, Evi1-high MLL-ENL leukemic cells showed no advantage in leukemia initiation compared to Evi1-low cells. Other Evi1-reporter AML models by MOZ-TIF2 and TEL-PDGFR+AML1-ETO never generated Evi1-high fraction, suggesting the high affinity of Evi1 for stem cell disease such as CML. In conclusion, high Evi1 can define the population of CML stem cells which are resistant to nilotinib. Combinatorial analyses of Evi1-IRES-GFP allele CML animals and single cells from primary CML-CP patients also covered in depth the critical regulation of CML stem cells by Evi1. Citation Format: Tomohiko Sato, Susumu Goyama, Keisuke Kataoka, Ryo Nasu, Takako Tsuruta-Kishino, Yuki Kagoya, Arika Nukina, Katsuyoshi Kumagai, Naoto Kubota, Masahiro Nakagawa, Shunya Arai, Akihide Yoshimi, Hiroaki Honda, Takashi Kadowaki, Mineo Kurokawa. Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3901. doi:10.1158/1538-7445.AM2014-3901