Hepatic overexpression of glycerol-sn-3-phosphate acyltransferase 1 in rats causes insulin resistance

Fatty liver is commonly associated with insulin resistance and type 2 diabetes, but it is unclear whether triacylglycerol accumulation or an excess flux of lipid intermediates in the pathway of triacyglycerol synthesis are sufficient to cause insulin resistance in the absence of genetic or diet-induced obesity. To determine whether increased glycerolipid flux can, by itself, cause hepatic insulin resistance, we used an adenoviral construct to overexpress glycerol-sn-3-phosphate acyltransferase-1 (Ad-GPAT1), the committed step in de novo triacylglycerol synthesis. After 5-7 days, food intake, body weight, and fat pad weight did not differ between Ad-GPAT1 and Ad-enhanced green fluorescent protein control rats, but the chow-fed Ad-GPAT1 rats developed fatty liver, hyperlipidemia, and insulin resistance. Liver was the predominant site of insulin resistance; Ad-GPAT1 rats had 2.5-fold higher hepatic glucose output than controls during a hyperinsulinemic-euglycemic clamp. Hepatic diacylglycerol and lysophosphatidate were elevated in Ad-GPAT1 rats, suggesting a role for these lipid metabolites in the development of hepatic insulin resistance, and hepatic protein kinase Cepsilon was activated, providing a potential mechanism for insulin resistance. Ad-GPAT1-treated rats had 50% lower hepatic NF-kappaB activity and no difference in expression of tumor necrosis factor-alpha and interleukin-beta, consistent with hepatic insulin resistance in the absence of increased hepatic inflammation. Glycogen synthesis and uptake of 2-deoxyglucose were reduced in skeletal muscle, suggesting mild peripheral insulin resistance associated with a higher content of skeletal muscle triacylglycerol. These results indicate that increased flux through the pathway of hepatic de novo triacylglycerol synthesis can cause hepatic and systemic insulin resistance in the absence of obesity or a lipogenic diet.     

Electric Stimulus Opens Intercellular Spaces in Skin

Iontophoresis is a technology for transdermal delivery of ionic small medicines by faint electricity. Since iontophoresis can noninvasively deliver charged molecules into the skin, this technology could be a useful administration method that may enhance patient comfort. Previously, we succeeded in the transdermal penetration of positively charged liposomes (diameters: 200–400 nm) encapsulating insulin by iontophoresis (Kajimoto, K., Yamamoto, M., Watanabe, M., Kigasawa, K., Kanamura, K., Harashima, H., and Kogure, K. (2011) Int. J. Pharm. 403, 57–65). However, the mechanism by which these liposomes penetrated the skin was difficult to define based on general knowledge of principles such as electro-repulsion and electro-osmosis. In the present study, we confirmed that rigid nanoparticles could penetrate into the epidermis by iontophoresis. We further found that levels of the gap junction protein connexin 43 protein significantly decreased after faint electric stimulus (ES) treatment, although occludin, CLD-4, and ZO-1 levels were unchanged. Moreover, connexin 43 phosphorylation and filamentous actin depolymerization in vivo and in vitro were observed when permeation of charged liposomes through intercellular spaces was induced by ES. Ca²⁺ inflow into cells was promoted by ES with charged liposomes, while a protein kinase C inhibitor prevented ES-induced permeation of macromolecules. Consequently, we demonstrate that ES treatment with charged liposomes induced dissociation of intercellular junctions via cell signaling pathways. These findings suggest that ES could be used to regulate skin physiology.     

Stimulation of tetrahydrobiopterin synthesis by cyclosporin A in mouse brain microvascular endothelial cells

We examined the effect of the immunosuppressant, cyclosporin A (CsA) on the synthesis of tetrahydrobiopterin (BH4), a cofactor for nitric oxide (NO) synthase and a scavenger of reactive oxygen species (ROS), in mouse brain microvascular endothelial cells. Treatment with CsA increased the BH4 content and the expression of mRNA level of GTP cyclohydrolase I, the rate-limiting enzyme of BH4 synthesis. 2,4-Diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, strongly reduced the CsA-induced increase in BH4 content. Cycloheximide (CHX), a protein synthesis inhibitor, also reduced CsA-induced BH4 synthesis. These findings suggest that CsA stimulates BH4 synthesis via a de novo pathway with the induction of GTP cyclohydrolase I. Moreover, CsA-induced the mRNA level of the inducible type of NO synthase, and stimulated the L-citrulline formation from L-arginine, which is a marker for NO synthesis. The CsA-stimulated L-citrulline formation was attenuated by the co-treatment with GTP cyclohydrolase I inhibitor. The expression of the endothelial type of NO synthase was low under basal condition, and was not affected by the treatment with CsA. These findings suggest that increase in BH4 content induced by CsA is coupled with NO production by inducible type of NO synthase.     

Surf4 modulates STIM1-dependent calcium entry

Store-operated Ca(2+) entry (SOCE) is crucial for various physiological responses in immune cells. Although it is known that STIM1 relocates into discrete puncta juxtaposed to the plasma membrane to initiate SOCE, the machinery modulating the function of STIM1 remains unclear. We explored to find its modulators using affinity purification for STIM1-binding proteins and identified surfeit locus protein 4 (Surf4). Surf4 associated with STIM1 in the endoplasmic reticulum. Deletion of Surf4 in DT40 B cells resulted in marked increase of SOCE and facilitation of STIM1 clustering upon store-depletion. These findings suggest the modulatory function of Surf4 for STIM1-mediated SOCE.     

Protein phosphatase 4 catalytic subunit regulates Cdk1 activity and microtubule organization via NDEL1 dephosphorylation

Protein phosphatase 4 catalytic subunit (PP4c) is a PP2A-related protein serine/threonine phosphatase with important functions in a variety of cellular processes, including microtubule (MT) growth/organization, apoptosis, and tumor necrosis factor signaling. In this study, we report that NDEL1 is a substrate of PP4c, and PP4c selectively dephosphorylates NDEL1 at Cdk1 sites. We also demonstrate that PP4c negatively regulates Cdk1 activity at the centrosome. Targeted disruption of PP4c reveals disorganization of MTs and disorganized MT array. Loss of PP4c leads to an unscheduled activation of Cdk1 in interphase, which results in the abnormal phosphorylation of NDEL1. In addition, abnormal NDEL1 phosphorylation facilitates excessive recruitment of katanin p60 to the centrosome, suggesting that MT defects may be attributed to katanin p60 in excess. Inhibition of Cdk1, NDEL1, or katanin p60 rescues the defective MT organization caused by PP4 inhibition. Our work uncovers a unique regulatory mechanism of MT organization by PP4c through its targets Cdk1 and NDEL1 via regulation of katanin p60 distribution.     

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.     

Development and application of a T-RFLP data analysis method using correlation coefficient matrices

Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s.     

Antiapoptotic activity of herpes simplex virus type 2: the role of US3 protein kinase gene

In order to determine the ability of herpes simplex virus type 2 (HSV-2) to suppress apoptosis, we examined the effect of HSV-2 infection on apoptosis induced in HEp-2 cells by treatment with 1 M sorbitol. Although a wild-type strain of HSV-2 induced apoptosis in a significant fraction of the infected cells, HSV-2 could suppress sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that HSV-2, like HSV-1, has an antiapoptosis gene. Characterization of the cells infected with a US3-deletion mutant of HSV-2 revealed the necessity of a US3 gene in the antiapoptotic activity of this virus.