Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

Introduction

Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy.

Materials and methods

Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks.

Results

PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney.

Conclusions

These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.     

A Dimer Is the Minimal Proton-Conducting Unit of the Influenza A Virus M2 Channel

When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function.     

Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.     

Isolation and Characterization of Two Squalene Epoxidase Genes from Botryococcus braunii,Race B

The B race of the green microalga Botryococcus braunii produces triterpene hydrocarbons, botryococcenes and methylsqualenes that can be processed into jet fuels with high heating values. In this alga, squalene is also converted into membrane sterols after 2,3-epoxidation. In the present study, cDNA clones of two distinct squalene epoxidases (BbSQE-I and -II) were isolated. Predicted amino acid sequences encoded on these genes are 45% identical with each other. Introduction of BbSQE-I or -II into Saccharomyces cerevisie erg1 mutants resulted in the complementation of ergosterol auxotrophy. The relative expression level of SQE-II increased 3.5-fold from the early stage to the middle phase of a culture period of 42 days, while that of SQE-I was almost constant throughout the culture period. Southern blot analyses suggested that these genes are single-copied genes. This is the first report on the isolation of functional SQEs that are encoded in duplicated loci in the algal genome.     

Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.     

Excitability Changes in Intracortical Neural Circuits Induced by Differentially Controlled Walking Patterns

Our previous single-pulse transcranial magnetic stimulation (TMS) study revealed that excitability in the motor cortex can be altered by conscious control of walking relative to less conscious normal walking. However, substantial elements and underlying mechanisms for inducing walking-related cortical plasticity are still unknown. Hence, in this study we aimed to examine the characteristics of electromyographic (EMG) recordings obtained during different walking conditions, namely, symmetrical walking (SW), asymmetrical walking 1 (AW1), and asymmetrical walking 2 (AW2), with left to right stance duration ratios of 1:1, 1:2, and 2:1, respectively. Furthermore, we investigated the influence of three types of walking control on subsequent changes in the intracortical neural circuits. Prior to each type of 7-min walking task, EMG analyses of the left tibialis anterior (TA) and soleus (SOL) muscles during walking were performed following approximately 3 min of preparative walking. Paired-pulse TMS was used to measure short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) in the left TA and SOL at baseline, immediately after the 7-min walking task, and 30 min post-task. EMG activity in the TA was significantly increased during AW1 and AW2 compared to during SW, whereas a significant difference in EMG activity of the SOL was observed only between AW1 and AW2. As for intracortical excitability, there was a significant alteration in SICI in the TA between SW and AW1, but not between SW and AW2. For the same amount of walking exercise, we found that the different methods used to control walking patterns induced different excitability changes in SICI. Our research shows that activation patterns associated with controlled leg muscles can alter post-exercise excitability in intracortical circuits. Therefore, how leg muscles are activated in a clinical setting could influence the outcome of walking in patients with stroke.     

Reduced CSF Water Influx in Alzheimer’s Disease Supporting the β-Amyloid Clearance Hypothesis

Objective

To investigate whether water influx into cerebrospinal fluid (CSF) space is reduced in Alzheimer’s patients as previously shown in the transgenic mouse model for Alzheimer’s disease.

Methods

Ten normal young volunteers (young control, 21-30 years old), ten normal senior volunteers (senior control, 60-78 years old, MMSE ≥ 29), and ten Alzheimer’s disease (AD) patients (study group, 59-84 years old, MMSE: 13-19) participated in this study. All AD patients were diagnosed by neurologists specializing in dementia based on DSM-IV criteria. CSF dynamics were analyzed using positron emission tomography (PET) following an intravenous injection of 1,000 MBq [¹⁵O]H₂O synthesized on-line.

Results

Water influx into CSF space in AD patients, expressed as influx ratio, (0.755 ± 0.089) was significantly reduced compared to young controls (1.357 ± 0.185; p < 0.001) and also compared to normal senior controls (0.981 ± 0.253, p < 0.05). Influx ratio in normal senior controls was significantly reduced compared to young controls (p < 0.01).

Conclusion

Water influx into the CSF is significantly reduced in AD patients. β-amyloid clearance has been shown to be dependent on interstitial flow and CSF production. The current study indicates that reduction in water influx into the CSF may disturb the clearance rate of β-amyloid, and therefore be linked to the pathogenesis of AD.

Trial Registration

UMIN Clinical Trials Registry UMIN000011939     

Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.