Journal: International Journal of Molecular Sciences

Article Title: Alogliptin Enhances Implant Osseointegration in Diabetes Through an Osteogenic–Angiogenic Immunomodulatory Procedure

doi: 10.3390/ijms27156800

Figure Lengend Snippet: Alogliptin promotes greater bone regeneration than metformin in type II diabetic rats. ( a ) Bone healing in the different groups was determined by micro-CT at 4 weeks after implantation. ( b ) Images of fluorescence labelling observed under an immunofluorescence microscope. ( c ) Osteogenesis volumes were measured by fluorescent labelling. ( d ) Images of Van Gieson (VG) staining under the microscope. ( e ) Differences in the bone area (BA; peri-implant bone tissue ratio) were quantified by VG staining. ( f ) Images of Toluidine Blue (TB) staining under the microscope. ( g ) TB staining quantified differences in the BIC (bone–implant longitudinal contact surface length/total longitudinal implant length). ( h ) HE, modified Masson staining, Safranin O/Fast Green decalcified alogliptin and metformin systemic samples were used. T: T2DM control group; A: Alogliptin group; M: Metformin group ( n = 7). * p < 0.05. Scale bar 1 cm:100 µm.

Article Snippet: The M-group rats received intragastric administration with 10 mM metformin (MedChemExpress, USA, the purity is 99.64%).

Techniques: Micro-CT, Fluorescence, Immunofluorescence, Microscopy, Staining, Modification, Control

Journal: International Journal of Molecular Sciences

Article Title: Alogliptin Enhances Implant Osseointegration in Diabetes Through an Osteogenic–Angiogenic Immunomodulatory Procedure

doi: 10.3390/ijms27156800

Figure Lengend Snippet: Greater osteo-inductivity of alogliptin is derived from activation of GLP1R/GSK-3β/β-catenin. ( a ) Immunofluorescent images of labelled GLP1R, β-catenin and GSK-3β around implants in the T2DM, alogliptin and metformin groups 4 weeks after the implant implantation in T2DM rats. ( b ) Statistical analysis of immunofluorescence. ( c ) Immunohistochemistry images labelled RUNX2, BMP2 and ALP around implants in the T2DM, alogliptin and metformin groups 4 weeks after the implant implantation in T2DM rats. ( d ) Statistical analysis of immunohistochemistry. T: T2DM control group; A: Alogliptin group; M: Metformin group ( n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars = 200 μm. The faint scale bar in the raw micrograph was retained to preserve the original imaging field of view; unified size calibration is defined in the caption.

Article Snippet: The M-group rats received intragastric administration with 10 mM metformin (MedChemExpress, USA, the purity is 99.64%).

Techniques: Derivative Assay, Activation Assay, Immunofluorescence, Immunohistochemistry, Control, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Alogliptin Enhances Implant Osseointegration in Diabetes Through an Osteogenic–Angiogenic Immunomodulatory Procedure

doi: 10.3390/ijms27156800

Figure Lengend Snippet: Cell identification and drug concentration. ( a ) Image of bone marrow mesenchymal stem cells (BMSCs) under microscope, scale bar = 200 μm. ( b ) Osteogenesis detected by ALP. ( c ) Adipogenesis detected by Oil Red. ( d ) Osteogenesis detected by Alizarin Red. ( e ) Identification of BMSCs through flow cytometry. ( f ) Identification of BMMs through flow cytometry. ( g ) Images of HUVECs under a microscope, scale bar = 200 μm. ( h ) Immunofluorescent staining of HUVECs stained with VE-cadherin, scale bar = 200 μm. ( i ) Drug concentration analysis of BMSC on alogliptin. ( j ) Drug concentration analysis of BMSC on metformin. ( n = 3). Scale bars = 200 μm. The faint scale bar in the raw micrograph was retained to preserve the original imaging field of view; unified size calibration is defined in the caption.

Article Snippet: The M-group rats received intragastric administration with 10 mM metformin (MedChemExpress, USA, the purity is 99.64%).

Techniques: Concentration Assay, Microscopy, Flow Cytometry, Staining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Alogliptin Enhances Implant Osseointegration in Diabetes Through an Osteogenic–Angiogenic Immunomodulatory Procedure

doi: 10.3390/ijms27156800

Figure Lengend Snippet: Alogliptin offers greater potential than metformin to establish the implant–bone connection by promoting BMSC viability, migration and adhesion. ( a ) Proliferation of BMSCs after 0 h, 24 h, 48 h, and 72 h was determined by CCK-8 assays. ( b ) Differences in the migration ability of BMSCs. ( c ) Differences in the adhesion ability of BMSCs. ( d ) Statistical analysis of the differences in the migratory capacity of the above cell type. ( e ) Statistical analysis of the differences in the adhesion capacity of the above cell type. Control: PBS intervention; Alogliptin: Alogliptin intervention. T: T2DM control group; A: Alogliptin group; M: Metformin group. ( n = 3). * p < 0.05, ** p < 0.01. Scale bars = 200 μm. The faint scale bar in the raw micrograph was retained to preserve the original imaging field of view; unified size calibration is defined in the caption.

Article Snippet: The M-group rats received intragastric administration with 10 mM metformin (MedChemExpress, USA, the purity is 99.64%).

Techniques: Migration, CCK-8 Assay, Control, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Alogliptin Enhances Implant Osseointegration in Diabetes Through an Osteogenic–Angiogenic Immunomodulatory Procedure

doi: 10.3390/ijms27156800

Figure Lengend Snippet: Effect of alogliptin on osteogenesis and osteogenic–vascular immune-coupling-related gene in BMSCs. ( a ) Alizarin Red assay was used to identify the difference in osteogenic differentiation ability of the above cell type. ( b ) Statistical analysis of the osteogenic differentiation ability of the above cell type. ( c ) ALP assay was used to identify the difference in osteogenic differentiation ability. ( d ) Statistical analysis of the osteogenic differentiation ability of the above cell type. ( e ) Analysis of qPCR of differences in expression of osteogenic-related genes. ( f ) Analysis of qPCR of differential expression of Wnt/β-catenin pathway. ( g ) Analysis of qPCR of angiogenic-related or inflammatory-related genes. ( h ). Analysis of ELISA of angiogenic-related or inflammatory-related proteins. T: T2DM control group; A: Alogliptin group; M: Metformin group. ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars = 200 μm. The faint scale bar in the raw micrograph was retained to preserve the original imaging field of view; unified size calibration is defined in the caption.

Article Snippet: The M-group rats received intragastric administration with 10 mM metformin (MedChemExpress, USA, the purity is 99.64%).

Techniques: ALP Assay, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Control, Imaging

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: The timeline of experiments and the number of ovarian follicles in different stages in each group . ( A ) The timeline of experiments was shown. Metformin (MET), sirolimus (SIRO) and everolimus (EVE) were administered by gavage on Days 1 to 5 for 4 weeks. Cyclophosphamide (CP) was provided after gavage on Day 1 by intraperitoneal injection weekly for 3 weeks. Other experimental details were described in Materials and methods section. ( B – F ) The numbers of ovarian follicles in different stages during folliculogenesis in C57BL/6 mice are shown. The mice were treated with CP-alone, MET-alone, SIRO-alone, or CP in combination with MET, SIRO or EVE. After 4 weeks of treatment, the ovaries were processed into paraffin blocks, sectioned, mounted and hematoxylin and eosin (H&E) stained for follicular counting. The number of primordial and tertiary follicles, and corpus luteum decreased in the CP-alone group compared with the control group ( P = 0.0014, 0.00003, 0.073, respectively). The deleterious effects of CP on follicular counts were diminished when oral MET was given to mice (Primordial follicle: P = 0.0274). The other two specific mTOR inhibitors, SIRO and EVE, also exhibited significant protective effects against CP damage (primary follicles: CP-alone versus CP + SIRO: P = 0.024; tertiary follicles: CP-alone versus CP + EVE: P = 0.0046). (B) and (C) The Y axis represented the average follicular counts per high-power field (HPF). N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Each value represents the average of 2–3 HPF per animal. (D)–(F) The Y axis represented the total follicular counts per ovarian section. N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Data are expressed as the mean ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Injection, Staining, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: The serum hormone concentration and the number of offspring in each group. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone, or CP in combination with MET, SIRO or everolimus (EVE). After 4 weeks of treatment, the mice were sacrificed and serum was collected for hormonal determination. ( A ) The serum levels of anti-Müllerian hormone (AMH) were significantly decreased in the CP-alone group (compared with control group: P < 0.0001). Although the AMH levels increased in CP + MET, CP + SIRO and CP + EVE group, the data did not reach statistical significance probably due to limited case number. ( B ) The serum levels of estradiol were significantly decreased in the CP-alone group ( P < 0.0001), and tended to increase in CP + MET group, CP + SIRO group and CP + EVE group. ( C ) The serum levels of progesterone were significantly decreased in the CP-alone group ( P < 0.0001) and tended to increase in CP + MET group and CP + EVE group. ( D ) A breeding test was conducted 1 week after the 4-week treatment. Only one round of timed mating was conducted per female mouse and the outcome of the first pregnancy in each mouse was evaluated. The number of the offspring was significantly decreased in the CP-alone group ( P < 0.0001) and tended to increase in the CP + MET group. In (A)–(C), n = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. In (D), n = 6 mice per group. Data are expressed as the mean ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Concentration Assay, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Immunohistochemical (IHC) staining of the proteins involved in mTOR signaling pathway. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone or CP in combination with MET or SIRO. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the IHC detection of p-mTOR ( A , B ) and p-AMPK ( C , D ) proteins. The percentage of granulosa cells with positive staining was calculated by dividing the number of positive stained cells with the total number of granulosa cells in ovarian follicles under a microscopy at ×400 magnification. One largest tertiary follicle in each section was selected to calculate the number of positively stained cells. Five sections per mice were counted from a total of 5 mice in each group (n = 5 mice). Data are expressed as the mean (%) ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01. The scale bar is 50 μm. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, Microscopy, Standard Deviation, Control

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: IHC staining of the cellular apoptotic and proliferative markers. C57BL/6 mice were treated with CP-alone, metformin (MET)-alone, sirolimus (SIRO)-alone or CP in combination with MET or SIRO. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the TUNEL assay (A, B) and Ki67 staining (C, D) to separately evaluate the degree of cellular apoptosis and proliferation. The percentage of granulosa cells with positive staining was calculated by dividing the number of positive stained cells with the total number of granulosa cells in ovarian follicles under a microscopy at ×400 magnification. One largest tertiary follicle in each section was selected to calculate the number of positively stained cells. Five sections per mice were counted. N = 10 mice in the control and CP-alone group, while n = 5 mice in the other groups. Data are expressed as the mean (%) ± standard deviation. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The scale bar is 50 μm. Note: The double slash mark on the X axis separated the MET-alone and SIRO-alone group from other groups because these two control groups were run in a separate experiment.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Immunohistochemistry, TUNEL Assay, Staining, Microscopy, Control, Standard Deviation

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Cell cycle phase analysis by flow cytometry using cultured mouse granulosa cells. Mouse granulosa cells were treated with CP-alone (1ug/ml) or with CP + metformin (MET) (10 mM) or with MET-alone for 72 h. Fresh granulosa cells were collected from five mice, and then mixed and cultured together. ( A ) The cell cycles, including subG1 (apoptotic phase), G1, S and G2/M of the assayed cells were determined by quantifying their DNA contents with propidium iodide (PI) staining and flow cytometry. The M1, M2, M3 and M4 bars represent the subG1, G1, S and G2/M phases, respectively. ( B ) The quantitative results of the cell cycle phases in each group were shown. The percentage of cells in the subG1 phase was significantly increased in the CP group ( P = 0.0009) and tended to decrease when cells were cotreated with MET. There was no significant difference between control group and MET-alone group. All the experiments were repeated five times and therefore N = 5 per group. Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, *** P < 0.001.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Flow Cytometry, Cell Culture, Staining, Control

Journal: Molecular Human Reproduction

Article Title: Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy

doi: 10.1093/molehr/gaaa084

Figure Lengend Snippet: Metformin exerted AMPK/p53/p21-mediated anti-apoptotic effect on cultured mouse granulosa cells. ( A ) Mouse granulosa cells were treated with p53 siRNA (25 nM) or control siRNA for 24 h prior CP (1ug/ml) or metformin (MET) (10 mM) treatment, after 72 h. Apoptotic cells were determined by flow cytometry with propidium iodide staining and quantified by the subG1 ratio. The experiments were repeated five times and therefore N = 5 per group. The apoptosis rate significantly increased in the CP-alone group ( P = 0.0029) and tended to decrease after cotreating the cells with MET. The anti-apoptosis effect of MET was diminished after blocking the p53 activity with siRNA. ( B ) The experimental conditions were the same as A, except that p21 siRNA (25 nM) was applied instead of p53 siRNA. ( C ) Mouse granulosa cells were treated with AMPK inhibitor BML275(10 μM) for 30 min prior MET (10 mM) treatment. At indicated time periods, the expression of p53 mRNA was determined by qRT-PCR. The experiments were repeated four times and therefore N = 4 per group. Sequential measurement of p53 mRNA expression in culture granulosa cells significantly increased 8 h after the addition of MET ( P = 0.0098). The expression of p53 mRNA was blocked with the addition of cell-permeable AMPK inhibitor BML. ( D ) The expression of p21 mRNA was determined by qRT-PCR and sequential measurement of p21 mRNA expression in cultured granulosa cells significantly increased 24 h after the addition of MET ( P = 0.0095). The experiments were repeated five times and therefore N = 5 per group. ( E ) C57BL/6 mice were treated with CP-alone or in combination with MET or MET-alone. After 4 weeks of treatment, the ovaries were processed into paraffin sections for the p53 and p21 staining. Both p53 and p21 activity were generally low in normal untreated ovarian tissue but was elevated after CP treatment (p53: P = 0.6529; p21: P = 0.0452) as determined with IHC staining under microscopic examination at ×400 magnification. The coadministration of MET with CP increased p53 and p21 protein expression in the ovarian tissue even higher ( P < 0.0004 in both). Statistical analyses were performed by nonparametric Kruskal–Wallis test with Dunn's post-hoc for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001. ( F ) Representative p53 and p21 IHC images of each group were shown. The scale bar is 50 μm.

Article Snippet: For in-vitro studies, metformin hydrochloride (dissolved in PBS) and CP (dissolved in DMSO) was purchased from Sigma (St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK), respectively.

Techniques: Cell Culture, Control, Flow Cytometry, Staining, Blocking Assay, Activity Assay, Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Inhibition of mitochondria NADH-Ubiquinone oxidoreductase (complex I) sensitizes the radioresistant glioma U87MG cells to radiation.

doi: 10.1016/j.biopha.2020.110460

Figure Lengend Snippet: Fig. 4. Inhibition of Complex I sensitized the radioresistant glioma U87MG_F cells to radiation. (A) MTT assay was used to test the effects of rotenone on the growth of U87MG and U87MG_F cells. (B) We used 200 nM rotenone to treat U87MG, U87MG_F and U251 cells. This dose of rotenone does not influence the cell growth. The survivals of cells after treating with rotenone and 2 Gy radiation were analyzed, which showed rotenone can sensitize U87MG_F cells to 2 Gy radiation. However, rote- none did not obviously increase the sensitization of U87MG cells to ra- diation. (C) We used 12 mM metformin treat U87MG, U87MG_F and U251 cells. The survivals of cells after treating with metformin and 2 Gy radiation were analyzed, which showed metformin sensitized U87MG_F cells to 2 Gy radiation. These results demonstrate mitochondria meta- bolism play vital roles in fractioned radiation-induced radioresistance of glioma cells. (D) The Complex I inhibitor, rotenone (200 nM), increases the intracellular ROS production in U87MG and U87MG_F cells, while metformin (12 mM) decrease the ROS production. The ROS in control cells was set 1, and fold changes in the drug-treated cells were shown up. ***p < 0.001.

Article Snippet: Rotenone and metformin (Selleckchem, USA) was incubated with the cells for 24 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was incubated with the cells for 4 h. Culture medium was removed and 150 μL DMSO (Sangon Inc, China) was added.

Techniques: Inhibition, MTT Assay, Control

Journal: PLoS ONE

Article Title: AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes

doi: 10.1371/journal.pone.0081870

Figure Lengend Snippet: A : Increase in AMP/ATP and ADP/ATP ratios in HepG2 cells. The cells were treated with 0.05% DMSO (open bar) or 200 nM R419 (solid bar) for 6 hours. Nucleotide levels were measured by HPLC. The data are presented as mean (bar) ± SEM (line) of triplicate cultures. Unpaired two-tailed t-tests were performed between DMSO control and R419-treated group. Asterisks ** and *** represent p < 0.01 and p < 0.001. B : Dose-dependent reduction of oxygen consumption rate (OCR) in HepG2 cells. OCR in the presence of DMSO or R419 was measured using an XF24 Seahorse instrument. Time point of R419 or DMSO injection is indicated by arrow. The data are presented as mean (symbol) ± SEM (line) of triplicate cultures. Repeated measures two-way ANOVA followed by the Dunnett ad-hoc test was performed and the multiple comparisons were done against the corresponding DMSO control at each timepoint. Asterisks *, **, *** and # represent p < 0.05, p<0.01, p<0.001 and p < 0.0001. C : Intact succinate-driven respiration in the presence of R419 in purified mouse liver mitochondria. The assay was conducted using an XF24 Seahorse instrument according to manufacturer’s protocols. 5 µg of purified mitochondria per well were used. Rotenone (Ro), succinate (Suc), antimycin A (Ant) and ascorbate/N,N,N',N'-tetramethyl-p-phenylene diamine (As/T) mixture were introduced at timepoints indicated by arrows. DMSO (n=6) or R419 (n=4) was introduced immediately prior to the assay. The data are presented as mean (symbol) ± SEM (line). Repeated measures two-way ANOVA followed by the Sidak ad-hoc test was performed and the multiple comparison test was done against the corresponding DMSO control at each timepoint. Except for time 0, significant differences in OCR between the two groups were not observed. Asterisk * represents p < 0.01. D : Inhibition of complex I-mediated NADH oxidation by R419 in purified mouse liver mitochondria. R419 or metformin was added to a mitochondrial lysate preparation (330 µg/ml) containing 2 mM NADH and incubated for 20 minutes. NADH to NAD + conversion was measured by monitoring the absorbance at 340 nm. Difference between the initial absorbance and the absorbance after 20-minute incubation was presented as ΔA340 a.u. (absorbance units). The data are presented as mean (symbol) ± range between two measures (line) of duplicate cultures. Statistical analyses were not performed for this Data Set. E : Reduction of NAD + /NADH ratio by R419. HepG2 cells were treated with 0.05% DMSO or R419 for 2 hours. NAD + and NADH levels in cell lysates were measured using a commercially available kit. The data are presented as mean (bar) ± range between two measures (line) of duplicate cultures. Ordinary one-way ANOVA with the Dunnett ad-hoc test was performed. The multiple comparison test was done against the DMSO control. Asterisk ** represents p < 0.01.

Article Snippet: AICAR and metformin were from Toronto Research Chemicals Inc. Rotenone, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), antimycin, and oligomycin were purchased from Seahorse Biosciences.

Techniques: Two Tailed Test, Control, Injection, Purification, Comparison, Inhibition, Incubation

Journal: PLoS ONE

Article Title: AMPK Activation through Mitochondrial Regulation Results in Increased Substrate Oxidation and Improved Metabolic Parameters in Models of Diabetes

doi: 10.1371/journal.pone.0081870

Figure Lengend Snippet: A : AMPK-dependent glucose uptake by R419 in primary mouse muscle cells. The cells were exposed to R419 or metformin for indicated times. Glucose uptake was assayed by incorporation of 2-deoxy-D-[ 3 H]glucose (1%Ci/ml, 26.2 Ci/mmol) into the cell lysate in 10 min. The data are presented as mean (bar) ± SEM (line) of 2~8 individual experiments performed in triplicate (line) (n=8 (DMSO), n=3 (0.5 hour), n=4 (1 hour), n=7 (2 hours), n=2 (6 hours) and n=3 (24 hours)). Ordinary two-way ANOVA followed by the Dunnett ad-hoc test was performed and the multiple comparison test within the same genotype was done against each DMSO control. Asterisks *, ** and *** represent p<0.05, p<0.01 and p < 0.001, respectively. B : AMPK-dependent ACC and ULK1 phosphorylation by R419. Primary muscle cells from AMPK wild type mice and AMPK α1/α2 KO mice were treated with A-769662 (300 µM), metformin (5 mM), AICAR (2 mM), and R419 (0.01, 0.1 and 1 µM) for one hour. Lysates were blotted using the indicated antibodies. C : AMPK-independent suppression of glucose production by R419. Primary hepatocytes from WT mice and AMPK α1/α2 KO mice were stimulated with Bt2-cAMP in the presence or absence of R419 or metformin for eight hours. The amount of glucose released into the media was normalized to protein content. Data are normalized to DMSO control and presented as mean (bar) ± SEM (line) of triplicate cultures. Ordinary two-way ANOVA followed by the Dunnett ad-hoc test was performed and the multiple comparisons within the same genotype was done against each DMSO control. Significance against each DMSO control is indicated on top of the bars. Asterisks *** and # represent p<0.001 and p < 0.0001, respectively. Genotype (WT vs KO) is not a significant source of variation by ordinary two-way ANOVA. D: AMPK-dependent ACC and AMPK phosphorylation in hepatocytes by R419. Primary hepatocytes from wild type mice (WT) and AMPK α1/α2 KO mice (AMPK KO) were stimulated with 100 µM Bt2-cAMP in the presence or absence of R419 (0.1, 0.2, 0.5 and 1 µM), A-769662 (30 µM) or metformin (0.5 mM) for eight hours. Lysates were blotted using the indicated antibodies. Quantities of transferred protein on the membrane were examined with Ponceau S staining solution (Ponceau). The images of western blots and the Ponceau S-stained membrane are trimmed and different parts of the same blots are grouped.

Article Snippet: AICAR and metformin were from Toronto Research Chemicals Inc. Rotenone, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), antimycin, and oligomycin were purchased from Seahorse Biosciences.

Techniques: Comparison, Control, Phospho-proteomics, Membrane, Staining, Western Blot

Journal: International Journal of Applied Pharmaceutics

Article Title: PHARMACOKINETIC PROFILE OF METFORMIN HYDROCHLORIDE IN DRIED BLOOD SPOT OF HEALTHY SUBJECTS USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY–PHOTODIODE ARRAY

doi: 10.22159/ijap.2018.v10s1.78

Figure Lengend Snippet: Fig. 1: Mean pharmacokinetic profile versus time of metformin

Article Snippet: The materials used included metformin hydrochloride and calcium atorvastatin were purchased from British Pharmacopoeia, HPLC grade acetonitrile (Merck), HPLC grade methanol (Merck), sodium dihydrogen phosphate monohydrate (Merck), sodium dodecyl sulfate (SDS) (SDS; Merck), orthophosphoric acid (Merck), sodium hydroxide (NaOH) (NaOH; Merck), aquabidest (Ikapharmindo), DBS paper (Perkin Elmer), human blood (Indonesian Red Cross), and 850-mg immediate release metformin hydrochloride tablets (Merck).

Techniques: