Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism

Metformin is widely used as a hypoglycemic agent for the treatment of type 2 diabetes. Both metformin and rotenone, an inhibitor of respiratory chain complex I, suppressed glucose-6-phosphatase (G6pc), a rate limiting enzyme of liver glucose production, mRNA expression in a rat hepatoma cell line accompanied by a reduction of intracellular ATP concentration and an activation of AMP-activated protein kinase (AMPK). When yeast NADH-quinone oxidoreductase 1 (NDI1) gene was introduced into the cells, neither inhibition of ATP synthesis nor activation of AMPK was induced by these agents. Interestingly, in contrast to rotenone treatment, G6pc mRNA down-regulation was observed in the NDI1 expressing cells after metformin treatment. Since NDI1 can functionally complement the complex I under the presence of metformin or rotenone, our results indicate that metformin induces down-regulation of G6pc expression through an inhibition of complex I and an activation of AMPK-independent mechanism.     

A Novel Biosynthetic Pathway of Archaetidyl-myo-inositol via Archaetidyl-myo-inositol Phosphate from CDP-archaeol and d-Glucose 6-Phosphate in Methanoarchaeon Methanothermobacter thermautotrophicus Cells

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[¹⁴C]inositol 1-phosphate, which was enzymatically prepared from [¹⁴C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.     

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in <Emphasis Type="Italic">Cyanophora paradoxa</Emphasis>

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177–187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3–3.4 μm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.     

Novel effects of diosgenin on skin aging

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor α, estrogen receptor β or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor α, estrogen receptor β or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17β-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17β-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly²²-Asn³⁴, in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C₁₅, C₁₆ and C₁₇ chain lengths and C₁₅ chain length, respectively, and it was considered that these fatty acids are bound to glycerol–Cys¹⁸. Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys¹⁸ → Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.     

Activation regimens for full‐term development of rabbit oocytes injected with round spermatids

The present study was designed to investigate the effect of activation regimens on full‐term development of rabbit oocytes after round spermatid injection (ROSI). In the first series, rabbit oocytes were treated with 5 µM ionomycin before ROSI, after ROSI, or before and after ROSI. In addition, non‐treated oocytes were subjected to intracytoplasmic sperm injection (ICSI) using ejaculated spermatozoa. Cleavage rate of ROSI oocytes activated before and after ROSI (55%) was comparable with that of ICSI oocytes (60%), and significantly higher than those of ROSI oocytes activated either before or after ROSI (29–39%; P < 0.05). No offspring were produced by transfer of the cleaving ROSI oocytes, while 8% of the cleaving ICSI oocytes transferred gave birth to offspring. In the second series, oocytes were exposed to 5, 10, or 20 µM ionomycin, followed by ROSI, 5 µM ionomycin treatment, and incubation with 5 µg/ml cycloheximide (CHX) + 2 mM 6‐dimethylaminopurine (DMAP). Significantly higher cleavage rates were derived from oocytes activated with 10 and 20 µM ionomycin before ROSI (91% and 82%, respectively; P < 0.05) compared to those activated with 5 µM ionomycin before ROSI (53%). Live offspring were obtained when the cleaving ROSI oocytes with the initial ionomycin treatment at 5 and 10 µM were transferred (offspring rate 2% and 4%, respectively). These activation regimens, however, were not valid for the ROSI using cryopreserved round spermatids. In conclusion, rabbit ROSI oocytes were capable of developing into full‐term when the oocytes were activated with a combined treatment of ionomycin and CHX/DMAP. Mol. Reprod. Dev. 76: 573–579, 2009. © 2008 Wiley‐Liss, Inc.     

Dihydro-alpha-lipoic acid has more potent cytotoxicity than alpha-lipoic acid

Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.