Targeted Disruption of ROCK1 Causes Insulin Resistance in Vivo

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16–18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced ∼40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.The ability of insulin to acutely stimulate glucose uptake and metabolism in peripheral tissues such as skeletal muscle and adipose tissue is critical for the regulation of normal glucose homeostasis (1). Impairments in insulin secretion and in the response of peripheral tissues to insulin (i.e. insulin resistance) are major pathogenic features of type 2 diabetes and contribute to the morbidity of obesity (1, 2). Insulin action involves a series of signaling cascades initiated by insulin binding to its receptor, eliciting receptor autophosphorylation and activation of the receptor tyrosine kinase, resulting in tyrosine phosphorylation of insulin receptor substrates (IRSs)⁴ (3). Phosphorylation of IRSs leads to activation of phosphatidylinositol 3-kinase (PI3K) and subsequently to activation of Akt and its downstream mediator AS160, all of which are important steps for the stimulation of glucose transport induced by insulin (4–6). Although the mechanism(s) underlying insulin resistance are not completely understood in peripheral tissues such as skeletal muscle, they are thought to result, at least in part, from impaired insulin-stimulated signal transduction (7).Rho-kinase (ROCK) is a Ser/Thr protein kinase identified as a GTP-Rho-binding protein (8). There are two isoforms of Rhokinase, ROCK1 (also known as ROCKβ) (9, 10) and ROCK2 (also known as ROCKα) (9, 11). ROCK activity is enhanced by binding with RhoA GTP through a Rho-binding domain (8). Insulin activates geranylgeranyltranferase and increases the cellular amounts of geranlygeranylated RhoA, leading to increased RhoA activity (12). ROCK plays important roles in many cellular processes, including signal transduction, vesicle trafficking, and cytoskeletal organization (13, 14), key processes involved in insulin-stimulated glucose transport in myocytes and adipocytes (15–17). Previous studies have indicated that ROCK chemical inhibition is beneficial for reversing certain disease abnormalities in hypertension and diabetic nephropathy (18–20). Studies of the effects of ROCK inhibitors on glucose homeostasis in animals have yielded conflicting results, however. In obese Zucker rats, chronic treatment with the ROCK inhibitor fasudil decreases blood pressure and improves glucose tolerance (21). However, very recently, chronic treatment of obese db/db mice with the inhibitor fasudil was reported to have no effect on blood glucose levels (20). In contrast, in normal mice, we found that acute treatment with ROCK inhibitor Y-27632 causes insulin resistance in vivo by reducing insulin-mediated glucose uptake in skeletal muscle (22). In support of this, our previous work demonstrated that overexpression of dominant negative ROCK decreases insulin-stimulated glucose transport in L6 muscle cells, isolated soleus muscle ex vivo, and 3T3-L1 adipocytes via impairing PI3K activity (22). However, the use of different inhibitors, doses, treatment times, and animal models in these in vivo animal studies limits understanding of the roles of ROCK in regulating glucose homeostasis and insulin sensitivity in vivo. The fact that ROCK inhibitors target both ROCK isoforms and that their specificities may not be absolute further complicates interpretation of these studies (23).In this study, we examined the physiological role of ROCK1 in the regulation of glucose homeostasis, whole body insulin sensitivity, and insulin action in mice with particular emphasis on the molecular basis of insulin resistance. Here we provide the evidence that global ROCK1 deficiency in mice causes insulin resistance in vivo in part via serine 632/635 phosphorylation of IRS-1. These data identify ROCK1 as a novel regulator of whole body glucose homeostasis and insulin signaling in vivo.     

Human follicular dendritic cells promote the APC capability of B cells by enhancing CD86 expression levels

Follicular dendritic cells (FDCs) are an essential cellular component of the germinal center (GC) and are believed to exert regulatory effects on the various stages of GC reactions. According to our previous reports, human FDCs express prostacyclin synthase, and prostacyclin analogues augment adhesion and co-stimulatory molecules on the surface of activated B cells. These findings prompted us to investigate whether FDCs would contribute to the antigen-presenting capability of B cells by using the well-established FDC-like cells, HK cells, and tonsillar B cells. Our results show that HK cells significantly enhance the expression levels of CD54, CD80, and CD86 on the surface of activated B cells. The enhancing effect of HK cells on CD86 is impeded by indomethacin and an EP4 antagonist, implying that a certain prostaglandin is mediating the up-regulation. Prostacyclin indeed recapitulates the enhancing effect on CD86, which is inhibited by EP4 as well as IP antagonists. B cells co-cultured with HK cells exhibit an augmented APC activity, which is inhibited by CD86 neutralization. These results reveal another unrecognized function of human FDC.     

Structural basis for electron and methyl-group transfer in a methyltransferase system operating in the reductive acetyl-CoA pathway

Several anaerobic acetogenic, methanogenic, hydrogenogenic, and sulfate-reducing microorganisms are able to use the reductive acetyl-CoA (Wood-Ljungdahl) pathway to convert CO₂ into biomass. The reductive acetyl-CoA pathway consists of two branches connected by the Co/Fe-containing corrinoid iron-sulfur protein (CoFeSP), which transfers a methyl group from a methyltransferase (MeTr)/methyltetrahydrofolate (CH₃-H₄ folate) complex to the reduced Ni-Ni-[4Fe-4S] cluster (cluster A) of acetyl-CoA synthase. We investigated the CoFeSP and MeTr couple of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans and show that the two proteins are able to catalyze the methyl-group transfer reaction from CH₃-H₄ folate to the Co(I) center of CoFeSP. We determined the crystal structures of both proteins. The structure of CoFeSP includes the previously unresolved N-terminal domain of the large subunit of CoFeSP, revealing a unique four-helix-bundle-like architecture in which a [4Fe-4S] cluster is shielded by hydrophobic amino acids. It further reveals that the corrinoid and the [4Fe-4S] cluster binding domains are mobile, which is mandatory for the postulated electron transfer between them. Furthermore, we solved the crystal structures of apo-MeTr, CH₃-H₄-folate-bound MeTr, and H₄-folate-bound MeTr, revealing a substrate-induced closure of the CH₃-H₄ folate binding cavity of MeTr. We observed three different conformations of Asn200 depending on the substrate bound in the active site, demonstrating its conformational modulation by hydrogen-bonding interactions with the substrate. The observed flexibility could be essential to stabilize the transition state during methyl-group transfer. The conformational space and role of Asn200 are likely conserved in homologous cobalamin-dependent MeTrs such as methionine synthase.     

PDH activation during in vitro muscle contractions in PDH kinase 2 knockout mice: effect of PDH kinase 1 compensation

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate oxidation in skeletal muscle. PDH is deactivated by a set of PDH kinases (PDK1, PDK2, PDK3, PDK4), with PDK2 and PDK4 being the most predominant isoforms in skeletal muscle. Although PDK2 is the most abundant isoform, few studies have examined its physiological role. The role of PDK2 on PDH activation (PDHa) at rest and during muscle stimulation at 10 and 40 Hz (eliciting low- and moderate-intensity muscle contractions, respectively) in isolated extensor digitorum longus muscles was studied in PDK2 knockout (PDK2KO) and wild-type (WT) mice (n = 5 per group). PDHa activity was unexpectedly 35 and 77% lower in PDK2KO than WT muscle (P = 0.043), while total PDK activity was nearly fourfold lower in PDK2KO muscle (P = 0.006). During 40-Hz contractions, initial force was lower in PDK2KO than WT muscle (P < 0.001) but fatigued similarly to ～75% of initial force by 3 min. There were no differences in initial force or rate of fatigue during 10-Hz contractions. PDK1 compensated for the lack of PDK2 and was 1.8-fold higher in PDK2KO than WT muscle (P = 0.019). This likely contributed to ensuring that resting PDHa activity was similar between the groups and accounts for the lower PDH activation during muscle contraction, as PDK1 is a very potent inhibitor of the PDH complex. Increased PDK1 expression appears to be regulated by hypoxia inducible factor-1α, which was 3.5-fold higher in PDK2KO muscle. It is clear that PDK2 activity is essential, even at rest, in regulation of carbohydrate oxidation and production of reducing equivalents for the electron transport chain. In addition, these results underscore the importance of the overall kinetics of the PDK isoform population, rather than total PDK activity, in determining transformation of the PDH complex and PDHa activity during muscle contraction.     

Five-Year Incidence of Primary Open-Angle Glaucoma and Rate of Progression in Health Center-Based Korean Population: The Gangnam Eye Study

Objective

To investigate the 5-year incidence and progression rate of primary open-angle glaucoma (POAG) in a health-center-based Korean population.

Methods

The study population comprised 5,021 subjects who participated in standardized health screening (including non-contact tonometry and fundus photography) at the Gangnam Healthcare Center during the period from January 2005 to December 2006 and again from January 2010 to December 2011. Among these subjects, 948 (18.9%) with findings suggestive of glaucoma were subjected to a comprehensive glaucoma evaluation, which included applanation tonometry and standard automated perimetry. Based on the results, the subjects were diagnosed as POAG suspect or definite POAG.

Results

The 5-year incidences of POAG suspect and definite POAG were 0.84% (42 subjects) and 0.72% (36 subjects), respectively. The rate of progression from POAG suspect to definite POAG was 4.75% per year. In subjects with a baseline intraocular pressure (IOP) >21 mmHg, the incidence of POAG suspect or definite POAG was significantly higher than in those with a baseline IOP≤21 mmHg (32% vs. 1.05%; P<0.001). A multivariate analysis showed that the progression from POAG suspect to definite POAG was significantly associated with older age (odds ratio [OR], 1.07; 95% confidence interval [CI], 1.03–1.10), higher baseline IOP (OR, 1.10; 95% CI, 1.01–1.24), higher body mass index (BMI) (OR, 1.15; 95% CI, 1.03–1.31), higher education level (OR, 1.57; 95% CI, 1.05–2.17), and higher hematocrit level (OR, 1.22; 95% CI, 1.08–1.43).

Conclusions

In the health-center-based Korean population, the 5-year incidence of POAG was 0.72%, and the rate of progression from POAG suspect to definite POAG was 4.75% per year. This study identified old age, high baseline IOP, high BMI, high level of education, and high hematocrit level as significant risk factors for incident POAG.     

Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFβ receptor interaction via CD44-PKCδ

Hyaluronic acid (HA) has been shown to promote angiogenesis. However, the mechanism behind this effect remains largely unknown. Therefore, in this study, the mechanism of HA-induced angiogenesis was examined. CD44 and PKCδ were shown to be necessary for induction of the receptor for HA-mediated cell motility (RHAMM), a HA-binding protein. RHAMM was necessary for HA-promoted cellular invasion and endothelial cell tube formation. Cytokine arrays showed that HA induced the expression of plasminogen activator-inhibitor-1 (PAI), a downstream target of TGFβ receptor signaling. The induction of PAI-1 was dependent on CD44 and PKCδ. HA also induced an interaction between RHAMM and TGFβ receptor I, and induction of PAI-1 was dependent on RHAMM and TGFβ receptor I. Histone deacetylase 3 (HDAC3), which is decreased by HA via rac1, reduced induction of plasminogen activator inhibitor-1 (PAI-1) by HA. ERK, which interacts with RHAMM, was necessary for induction of PAI-1 by HA. Snail, a downstream target of TGFβ signaling, was also necessary for induction of PAI-1. The down regulation of PAI-1 prevented HA from enhancing endothelial cell tube formation and from inducing expression of angiogenic factors, such as ICAM-1, VCAM-1 and MMP-2. HDAC3 also exerted reduced expression of MMP-2. In this study, we provide a novel mechanism of HA-promoted angiogenesis, which involved RHAMM-TGFβRI signaling necessary for induction of PAI-1.     

Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells

Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four active compounds (-)-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), kakuol (3), and '3,4,5-trimethoxytoluene' (=1,2,3-trimethoxy-5-methylbenzene; 4), in terms of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compounds 1-4 showed potent inhibition of NO production, with IC(50) values in the low nanomolar-to-micromolar range. Also isolated were the known compounds methylkakuol (5), '3,5-dimethoxytoluene', safrole, asaricin, methyleugenol, and (-)-asarinin, which were found to be inactive in the above assay. Among the ten known isolates, compounds 1, 2, and 5 were found for the first time in this plant.     

Baculovirus expression and bioactivity of a soluble 140 kDa extracellular cleavage fragment of L1 neural cell adhesion molecule

L1 neural cell adhesion molecule is the founding member of the L1 subfamily of the immunoglobulin superfamily and plays an important role in the overall development of both the central and peripheral nervous systems, making it an attractive candidate for promoting neural regeneration following injury. Currently, L1 used for experimental studies is primarily mammalian-derived; however, the insect cell expression system described here provides an alternative source of recombinant L1 with equivalent bioactivity. A 140 kDa L1 fragment based on a physiological plasmin cleavage site in the extracellular domain was cloned and expressed with a C-terminal 6x histidine tag. Recombinant insect cell-derived L1 was analyzed by Western blot using an antibody to human L1 to confirm immunogenicity and to optimize infection conditions for recombinant L1 production. The recombinant protein was secreted by insect cells, efficiently purified under non-denaturing conditions using dialysis followed by metal affinity chromatography, and analyzed by SDS-PAGE to produce a single band of the expected approximate 140 kDa size. The bioactivity of insect cell-derived L1 was compared to mammalian-derived L1-Fc and poly-L-lysine (PLL) using chick embryonic forebrain neurons. The results show comparable, robust neurite outgrowth at 24h on insect cell-derived L1 and mammalian-derived L1-Fc, with significantly longer neurites than those observed on PLL. Future studies will examine the immobilization of L1 to biomaterial surfaces in physiologically appropriate orientation via the C-terminal 6x histidine tag and will investigate their application in promoting axonal regeneration in the injured nervous system.