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Abstract COACK-I

Selection of transformation-efficient barley genotypes based on TFA (transformation amenability) haplotype and higher resolution mapping of the TFA loci

Barley (Hordeum vulgare) cv. ‘Golden Promise’ is one of the most useful and well-studied cultivars for genetic manipulation. In a previous report, we identified several transformation amenability (TFA) loci responsible for Agrobacterium-mediated transformation using the F2 generation of immature embryos, derived from ‘Haruna Nijo’ × ‘Golden Promise,’ as explants. In this report, we describe higher density mapping of these TFA regions with additional SNP markers using the same transgenic plants. To demonstrate the robustness of transformability alleles at the TFA loci, we genotyped 202 doubled haploid progeny from the cross ‘Golden Promise’ × ‘Full Pint.’ Based on SNP genotype, we selected lines having ‘Golden Promise’ alleles at TFA loci and used them for transformation. Of the successfully transformed lines, DH120366 came the closest to achieving a level of transformation efficiency comparable to ‘Golden Promise.’ The results validate that the genetic substitution of TFA alleles from ‘Golden Promise’ can facilitate the development of transformation-efficient lines from recalcitrant barley cultivars

Inactivation of class II PI3K-C2 alpha induces leptin resistance, age-dependent insulin resistance and obesity in male mice

AIMS/HYPOTHESIS: While the class I phosphoinositide 3-kinases (PI3Ks) are well-documented positive regulators of metabolism, the involvement of class II PI3K isoforms (PI3K-C2α, -C2β and -C2γ) in metabolic regulation is just emerging. Organismal inactivation of PI3K-C2β increases insulin signalling and sensitivity, whereas PI3K-C2γ inactivation has a negative metabolic impact. In contrast, the role of PI3K-C2α in organismal metabolism remains unexplored. In this study, we investigated whether kinase inactivation of PI3K-C2α affects glucose metabolism in mice. METHODS: We have generated and characterised a mouse line with a constitutive inactivating knock-in (KI) mutation in the kinase domain of the gene encoding PI3K-C2α (Pik3c2a). RESULTS: While homozygosity for kinase-dead PI3K-C2α was embryonic lethal, heterozygous PI3K-C2α KI mice were viable and fertile, with no significant histopathological findings. However, male heterozygous mice showed early onset leptin resistance, with a defect in leptin signalling in the hypothalamus, correlating with a mild, age-dependent obesity, insulin resistance and glucose intolerance. Insulin signalling was unaffected in insulin target tissues of PI3K-C2α KI mice, in contrast to previous reports in which downregulation of PI3K-C2α in cell lines was shown to dampen insulin signalling. Interestingly, no metabolic phenotypes were detected in female PI3K-C2α KI mice at any age. CONCLUSIONS/INTERPRETATION: Our data uncover a sex-dependent role for PI3K-C2α in the modulation of hypothalamic leptin action and systemic glucose homeostasis. ACCESS TO RESEARCH MATERIALS: All reagents are available upon request

Phosphoinositide 3-Kinaseγ Controls the Intracellular Localization of CpG to Limit DNA-PKcs-Dependent IL-10 Production in Macrophages

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG) stimulate innate immune responses. Phosphoinositide 3-kinase (PI3K) has been implicated in CpG-induced immune activation; however, its precise role has not yet been clarified. CpG-induced production of IL-10 was dramatically increased in macrophages deficient in PI3Kγ (p110γ−/−). By contrast, LPS-induced production of IL-10 was unchanged in the cells. CpG-induced, but not LPS-induced, IL-10 production was almost completely abolished in SCID mice having mutations in DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Furthermore, wortmannin, an inhibitor of DNA-PKcs, completely inhibited CpG-induced IL-10 production, both in wild type and p110γ−/− cells. Microscopic analyses revealed that CpG preferentially localized with DNA-PKcs in p110γ−/− cells than in wild type cells. In addition, CpG was preferentially co-localized with the acidic lysosomal marker, LysoTracker, in p110γ−/− cells, and with an early endosome marker, EEA1, in wild type cells. Over-expression of p110γ in Cos7 cells resulted in decreased acidification of CpG containing endosome. A similar effect was reproduced using kinase-dead mutants, but not with a ras-binding site mutant, of p110γ. Thus, it is likely that p110γ, in a manner independent of its kinase activity, inhibits the acidification of CpG-containing endosomes. It is considered that increased acidification of CpG-containing endosomes in p110γ−/− cells enforces endosomal escape of CpG, which results in increased association of CpG with DNA-PKcs to up-regulate IL-10 production in macrophages

Shikonin Increases Glucose Uptake in Skeletal Muscle Cells and Improves Plasma Glucose Levels in Diabetic Goto-Kakizaki Rats

Glucose is the most common substrate for energy metabolism. Despite the varying demands for glucose, the body needs to regulate its internal environment and maintain a constant and stable condition. Glucose homeostasis requires harmonized interaction between several tissues, achieving equilibrium between glucose output and uptake. In this thesis we aimed to investigate factors modulating glucose homeostasis in a rat model of type 2 diabetes, the Goto-Kakizaki (GK) rat. In addition, we investigated sex differences in hepatic carbohydrate and lipid metabolism in healthy rats. In Paper I, three-week but not three-day treatment with a Southeast Asian herb, Gynostemma pentaphyllum (GP), significantly reduced plasma glucose (PG) levels in GK rats. An intra-peritoneal glucose tolerance test (IPGTT) was significantly improved in GP-treated compared to placebo-treated group. In the GP treated rats, the glucose response in an intra-peritoneal pyruvate tolerance test was significantly lower, indicating decreased gluconeogenesis, and hepatic glucose output (HGO) was reduced. GP-treatment significantly reduced hepatic glycogen content, but not glycogen synthase activity. The study provides evidence that the GP extract exerted anti-diabetic effect in GK rats, reducing PG levels and HGO, suggesting that GP improves the hepatic insulin sensitivity by suppressing gluconeogenesis. In Paper II, shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increased glucose uptake in L6 myotubes, but did not phosphorylate Akt. Furthermore we found no evidence for the involvement of AMP activated protein kinase (AMPK) in shikonin induced glucose uptake. Shikonin increased the intracellular levels of calcium in these cells and stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myotubes. In GK rats treated with shikonin once daily for 4 days, PG levels were significantly decreased. In an insulin sensitivity test, the absolute PG levels were significantly lower in the shikonin-treated rats. These findings suggest that shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium. In Paper III, GK and control Wistar rats were injected daily for up to 4 weeks with either a non-hematopoietic erythropoietin analog ARA290 or with placebo. PG levels in GK but not Wistar rats were significantly lower in ARA290-treated compared to placebo. After 2 and 4 weeks, the IPGTT was significantly improved in ARA290 treated GK rats. In insulin and pyruvate tolerance tests, glucose responses were similar in ARA290 and placebo groups. In isolated GK rat islets, glucose-stimulated insulin release was two-fold higher and islet intracellular calcium concentrations in response to several secretagogues were significantly higher in ARA290-treated than in placebo-treated GK rats. These findings indicate that treatment with ARA290 significantly improved glucose tolerance in diabetic GK rats, most likely due to improvement of insulin release. In Paper IV, sex differences in hepatic carbohydrate and lipid metabolism were characterized in healthy rats. No sex-differences were observed regarding hepatic triglyceride content, fatty acid oxidation rates or insulin sensitivity. Male rats had higher ratios of insulin to glucagon levels, increased hepatic glycogen content, a lower degree of AMPK phosphorylation, a higher rate of glucose production and higher expression levels of gluconeogenic genes, as compared to female rats. A sex-dependent response to mild starvation was observed with males being more sensitive. In conclusion, sex-differences reflect a higher capacity of the healthy male rat liver to respond to increased energy demands. Key words: glucose homeostasis, type 2 diabetes, GK rats, L6 myotubes, hepatic glucose output, insulin sensitivity, sex differences