Effect of canagliflozin on renal threshold for glucose, glycemia, and body weight in normal and diabetic animal models

Background

Canagliflozin is a sodium glucose co-transporter (SGLT) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus (T2DM).

Methods

¹⁴C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human, rat, or mouse SGLT2 or SGLT1; ³H-2-deoxy-d-glucose uptake in L6 myoblasts; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed. Graded glucose infusions were performed to determine rate of urinary glucose excretion (UGE) at different blood glucose (BG) concentrations and the renal threshold for glucose excretion (RT_G) in vehicle or canagliflozin-treated Zucker diabetic fatty (ZDF) rats. This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity.

Results

Treatment with canagliflozin 1 mg/kg lowered RT_G from 415±12 mg/dl to 94±10 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RT_G. Canagliflozin dose-dependently decreased BG concentrations in db/db mice treated acutely. In ZDF rats treated for 4 weeks, canagliflozin decreased glycated hemoglobin (HbA1c) and improved measures of insulin secretion. In obese animal models, canagliflozin increased UGE and decreased BG, body weight gain, epididymal fat, liver weight, and the respiratory exchange ratio.

Conclusions

Canagliflozin lowered RT_G and increased UGE, improved glycemic control and beta-cell function in rodent models of T2DM, and reduced body weight gain in rodent models of obesity.     

Validation of bacterial replication termination models using simulation of genomic mutations

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point.     

Survival-promoting activity of pituitary adenylate cyclase-activating polypeptide in the presence of phosphodiesterase inhibitors on rat motoneurons in culture: cAMP-protein kinase A-mediated survival

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to be neurotrophic or neuroprotective in various neurons in culture. It is expressed in spinal motoneurons in vivo and its expression is increased markedly after axotomy, suggesting a neuroprotective role via an autocrine mechanism. However, neurotrophic activity of PACAP has not been reported for motoneurons. In the present study, we investigated the effects of PACAP on rat motoneurons in culture. In the presence of a phosphodiesterase inhibitor, PACAP showed significant neurotrophic activity at concentrations as low as 0.01 nM. Previously, we found that glutamate was excitotoxic to motoneurons even in the presence of brain-derived neurotrophic factor, which is neurotrophic for motoneurons. PACAP with a phosphodiesterase inhibitor protected motoneurons against this excitotoxicity. The activity of PACAP was inhibited by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide dihydrochloride, as was the case with the activity of forskolin, suggesting downstream involvement of a cAMP-protein kinase A signaling pathway. The present results may suggest a physiological role of PACAP in vivo, and implicate the PACAP-cAMP signaling pathway for the possible therapeutic target of amyotrophic lateral sclerosis as glutamate excitotoxicity was suggested in sporadic amyotrophic lateral sclerosis.     

Two distinct pathways for cyclooxygenase-2 protein degradation

Cyclooxygenases (COX-1 and COX-2) are N-glycosylated, endoplasmic reticulum-resident, integral membrane proteins that catalyze the committed step in prostanoid synthesis. COX-1 is constitutively expressed in many types of cells, whereas COX-2 is usually expressed inducibly and transiently. The control of COX-2 protein expression occurs at several levels, and overexpression of COX-2 is associated with pathologies such as colon cancer. Here we have investigated COX-2 protein degradation and demonstrate that it can occur through two independent pathways. One pathway is initiated by post-translational N-glycosylation at Asn-594. The N-glycosyl group is then processed, and the protein is translocated to the cytoplasm, where it undergoes proteasomal degradation. We provide evidence from site-directed mutagenesis that a 27-amino acid instability motif (27-IM) regulates posttranslational N-glycosylation of Asn-594. This motif begins with Glu-586 8 residues upstream of the N-glycosylation site and ends with Lys-612 near the C terminus at Leu-618. Key elements of the 27-IM include a helix involving residues Glu-586 to Ser-596 with Asn-594 near the end of this helix and residues Leu-610 and Leu-611, which are located in an apparently unstructured downstream region of the 27-IM. The last 16 residues of the 27-IM, including Leu-610 and Leu-611, appear to promote N-glycosylation of Asn-594 perhaps by causing this residue to become exposed to appropriate glycosyl transferases. A second pathway for COX-2 protein degradation is initiated by substrate-dependent suicide inactivation. Suicide-inactivated protein is then degraded. The biochemical steps have not been resolved, but substrate-dependent degradation is not inhibited by proteasome inhibitors or inhibitors of lysosomal proteases. The pathway involving the 27-IM occurs at a constant rate, whereas degradation through the substrate-dependent process is coupled to the rate of substrate turnover.     

Effect of arm swing direction on forward and backward jump performance

This study quantified and compared how the directional differences in arm swing affected mechanical and physiological parameters during forward and backward jumping. Seven subjects maximally performed three types of forward and backward squat jumps-no arm swing (FJ, BJ), forward arm swing (FJF, BJF), and backward arm swing (FJB, BJB) from a force platform. All performances were captured with a 3-D motion capture system. Electromyograms (EMGs) of the lower extremity muscles were obtained. Variables were calculated by combining kinematic and kinetic data. The jump displacement and center of mass velocity at take-off were significantly larger in FJF than in FJ or FJB and larger in BJB than in BJ or BJF, suggesting that the best performance was obtained by employing the same arm swing direction as a given jump direction. The total work by three lower and two upper extremity joints was significantly larger in FJF than in FJ or FJB and larger in BJB than in BJ or BJF. For the lower extremity joints, hip work was the greatest in FJF and BJB. The integrated EMG of the biceps femoris when the hip power was produced was significantly larger in FJF and BJB than under other conditions. These results suggest that if the arm swing direction is the same as a given jump direction, the activation level of the hip extensor is greater to counter large loads which make the hip joint flex during the push-off phase, which result in increased hip extension torque, power, and work.     

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near −40 °C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

Intracellular changes of metal elements by fucoidan extracted from brown seaweed (Cladosiphon okamuranus)

This study was undertaken to elucidate the intracellular changes of metal elements after the administration of fucoidan extracted from Cladosiphon okamuranus. TRL1215 cells (normal rat liver cell line) were treated with 0, 0.1, or 1.0 mg/ml fucoidan and incubated in 5% CO2 at 37 degrees C. The cellular levels of Mg, Al, Fe, and Zn were significantly increased in the 1.0 mg/ml fucoidan-treated cells compared to those of the 0.1 mg/ml fucoidan-treated cells and the control. Next, TRL1215 cells were cultured on Mylar film overnight. At 24 h after 5-bromo-2'-deoxyuridine dosing, 0, 0.1, or 1.0 mg/ml fucoidan was treated for 9 h. The cellular distribution of elements was analyzed using in-air micro-micro-particle induced X-ray emission. The X-ray spectra showed that yields of Al, Mg, and Zn were high in order of the 1.0 mg/ml fucoidan-treated sample, the 0.1 mg/ml fucoidan-treated sample, and the control. Fe yield was mildly increased by fucoidan administration. In fucoidan-treated cells, the focal accumulation of Br was correlated spatially with phosphorous-rich region, suggesting that Br was localized within the nucleus. Al distribution provided a spatial association with Br map. These data suggest that fucoidan increases the accumulations of Al, Mg, Fe, and Zn in normal rat hepatocytes, and fucoidan-binding Al is postulated to be transferred into the nucleus.     

Structure-based analysis reveals hydration changes induced by arginine hydrochloride

Arginine hydrochloride has been used to suppress protein aggregation during refolding and in various other applications. We investigated the structure of hen egg-white lysozyme (HEL) and solvent molecules in arginine hydrochloride solution by X-ray crystallography. Neither the backbone nor side-chain structure of HEL was altered by the presence of arginine hydrochloride. In addition, no stably bound arginine molecules were observed. The number of hydration water molecules, however, changed with the arginine hydrochloride concentration. We suggest that arginine hydrochloride suppresses protein aggregation by altering the hydration structure and the transient binding of arginine molecules that could not be observed.     

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.     

ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase.

Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma-imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.