Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagenbinding integrin, a2b1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrinlinked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of b-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK lox/lox ) and muscle-specific ILK-deficient (ILK lox/lox HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK lox/lox and ILK lox/lox HSAcre mice. However, HF-fed ILK lox/lox HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK lox/lox mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK lox/lox HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries.Insulin resistance is a commonly associated risk factor for many pathophysiological conditions, including diabetes, cardiovascular diseases, neurological changes, liver diseases, and sleep apnea (1,2). A defect in glucose utilization in the skeletal muscle is a major component of insulin resistance. The pathogenesis of muscle insulin resistance is not fully understood, and pharmacological interventions that reduce insulin resistance are limited and often lose efficacy over time (e.g., biguanides) or have adverse side effects (e.g., thiazolidinediones) (3). Our recent studies have suggested a novel role for extramyocellular factors in regulating muscle insulin action. Expansion of extracellular matrix (ECM) components, such as the collagens and the expression of the collagen-binding integrin a2b1, are associated with muscle insulin resistance induced by a high-fat (HF) diet (4). The ECM is in direct contact with the muscle capillaries. Defects in recruitment of muscle capillaries contribute to the development of muscle insulin resistance (5). Transduction of ECM signals through integrins requires interaction of the integrin cytoplasmic domains with cellular proteins. To investigate the link between ECM-integrin signaling and muscle insulin resistance, we studied a highly conserved central downstream component of the ECM-integrin signaling, integrin-linked kinase (ILK), and its role in muscle insulin resistance.ILK, a pseudokinase with adaptor function, is a central component of the ILK-PINCH-parvin complex (IPP) (6). This complex binds to the cytoplasmic domain of b-integrin subunits. The IPP co...

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient ( cd44−/−) mice and wild-type littermates ( cd44+/+) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44−/− mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44−/− mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44+/+ mice but absent in cd44−/− mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44−/− compared with cd44+/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44−/− mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44−/− mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.

Objective Increased deposition of the extracellular matrix (ECM) in adipose tissue (AT) during obesity contributes to insulin resistance. The integrin receptors transmit changes in the extracellular environment causing corresponding intracellular adaptations. Integrin-linked kinase (ILK), an adaptor protein, is a central hub for intracellular signaling of integrins. This study determined the role of ILK in adipose function and insulin resistance. Methods The pathogenic role of ILK in obesity and insulin resistance was studied in human adipose tissue and adipocyte-specific ILK-deficient mice (ILK lox/lox AdCre ). ILK lox/lox AdCre mice together with wild-type littermates (ILK lox/lox ) were fed a chow diet or 60% high-fat (HF) diet for 16 weeks. In vivo insulin sensitivity was determined by hyperinsulinemic-euglycemic clamps. Results AT ILK expression was increased by HF diet feeding in mice and increased in visceral fat of morbidly obese humans. The HF-fed ILK lox/lox AdCre mice displayed reduced fat mass and improved glucose tolerance relative to the HF-fed ILK lox/lox mice. During a hyperinsulinemic-euglycemic clamp, the HF-fed ILK lox/lox AdCre mice exhibited partially improved insulin resistance in AT. Lipolysis was suppressed to a greater extent by insulin and glucose uptake in brown AT (BAT) increased. Increased inhibition of lipolysis may have been attributed to increased vascularization in white AT, while increased glucose uptake in BAT was associated with increased Akt phosphorylation and P38/JNK dephosphorylation. Notably, AT insulin sensitivity in lean mice was not affected by ILK deletion. Moreover, reduced fat mass in the HF-fed ILK lox/lox AdCre mice may have been attributed to decreased free fatty acid uptake into adipocytes via the downregulation of CD36 gene expression. Consistent with the results in the mice, knockdown and knockout of ILK in 3T3-L1 cells decreased lipid accumulation and CD36 gene expression during adipogenesis. Conclusions These data show that adipocyte ILK is important for regulating HF diet-mediated insulin resistance in AT in a manner consistent with AT function.

A suspension cultured cell line was established from the cultivar of Rosa hybrida 'Charleston' as a study model to understand the response of the anthocyanin biosynthesis pathway to environmental cues. The major identified anthocyanin in cell cultures was cyanidin 3-glucoside (chrysanthemin). The anthocyanin yield was enhanced by culturing cells in the EM medium with added sucrose at high concentration under additional UV-B radiation to white light. Three cDNA fragments were cloned with degenerate primers by RT-PCR and the obtained sequences shared high homology with putative key enzymes (DFR, ANS, and UF3GT) of other species. The expression levels of these three genes were promoted under optimum conditions for anthocyanin accumulation. These results suggest that expression levels of these genes were closely correlated with a temporal buildup of anthocyanins in response to environmental factors.Key words: Anthocyanin, chrysanthemin, cyanin, Rosa hybrida, suspension cell culture.Plant Biotechnology 23, 379-385 (2006) Original PaperThis article can be found at http://www.jspcmb.jp/ pelargonidin, cyanidin and peonidin type anthocyanins (Biolley et al. 1994). Recently, tissue culturing has become an important biotechnological technique to produce various secondary metabolites including anthocyanins on a mass scale for industrial and academic interest (Hirner et al. 2001;Gantet and Memelink 2002). Despite the fact that some plant species have been shown to produce anthocyanins in cell cultures, little information of the cell culture techniques that are capable of producing in vitro anthocyanins has been reported in rose.The biosynthesis of anthocyanins in plant cell culture is stimulated by not only genetic factors but also environmental factors such as light, temperature, growth regulators and nutrition (Sato et al. 1996;Zhang et al. 1997;Zhang et al. 2002). The identity of the environmental stimuli that lead to the accumulation of anthocyanins in rose is of considerable interest. To date, Sato et al. (1996) has examined the intensity of white light that induces anthocyanin production in strawberry cell culture lines; however, no information was obtained about the effect of light quality on the production of anthocyanins in cultural cells. Therefore, the originality of this research is to investigate anthocyanin biosynthesis in cultural cells exposed to UV-B (290-320 nm) radiation.In this study, we used Rosa hybrida cultivar 'Charleston', a climbing type, originated in Australia. The open flowers of 'Charleston' undergo a striking color change from yellow to red over 10-12 days under natural daylight. We established a suspension cultured cell line from this cultivar of rose. Anthocyanin content was quantified by HPLC to determine the most efficient conditions, such as light including UV-B radiation and media, for continuous production of the pigments in the rose cell suspension. The expression levels of three genes encoding putative key enzymes (DFR, ANS, and UF3GT) for anthocyanin biosynthesis were ana...

Irradiation of UV-B Induces Biosynthesis of Anthocyanins in Flower Petals of Rose, Rosa hybrida cv. •e Charleston' and•eEhigasa'