Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway

Background@#Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. @*Methods@#Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco’s modified Eagle’s medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. @*Results@#SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. @*Conclusion@#These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

Role of autophagy in diabetes and endoplasmic reticulum stress of pancreatic beta-cells

Type 2 diabetes mellitus is characterized by insulin resistance and failure of pancreatic beta-cells producing insulin. Autophagy plays a crucial role in cellular homeostasis through degradation and recycling of organelles such as mitochondria or endoplasmic reticulum (ER). Here we discussed the role of beta-cell autophagy in development of diabetes, based on our own studies using mice with beta-cell-specific deletion of Atg7 (autophagy-related 7), an important autophagy gene, and studies by others. beta-cell-specific Atg7-null mice showed reduction in beta-cell mass and pancreatic insulin content. Insulin secretory function ex vivo was also impaired, which might be related to organelle dysfunction associated with autophagy deficiency. As a result, beta-cell-specific Atg7-null mice showed hypoinsulinemia and hyperglycemia. However, diabetes never developed in those mice. Obesity and/or lipid are physiological ER stresses that can precipitate beta-cell dysfunction. Our recent studies showed that beta-cell-specific Atg7-null mice, when bred with ob/ob mice, indeed become diabetic. Thus, autophagy deficiency in beta-cells could be a precipitating factor in the progression from obesity to diabetes due to inappropriate response to obesity-induced ER stress.

Determination of Genomospecies and Characterization of Antimicrobial Resistance of Multi-drug Resistant Acinetobacter spp. Isolates

Acinetobacter species are non-fermentative Gram-negative coccobacilli and they have emerged as important nosocomial pathogens which are associated with the significant multidrug resistance in recent years. Carbapenem-resistant A. baumannii (CRAB) and pandrug-resistant A. baumannii (PDRAB) were reported in 1991 and 1998, respectively. Fiftyeight isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005 were investigated for the existence of CRAB, PDRAB, extended-spectrum beta-lactamase (ESBL)-producing Acinetobacter and examined for their phenotypic and genotypic characteristics. Genomospecies of Acinetobacter species were determined by amplified rDNA restriction analysis (ARDRA) and antimicrobial susceptibility test was performed with 13 kinds of antimicrobial agents. Metallo-beta-lactamase (MBL) producers were screened by modified hodge test and confirmed by imipenem-EDTA disk synergy test. Detection of blaIMP-1, blaVIM-2, blaTEM, and blaPER-1 was performed by PCR. Genomic DNAs were analyzed by pulsed-field gel electrophoresis (PFGE). Among 58 isolates of Acinteobacter species, 40 isolates were identified as genospecies 2 (A. baumannii), 9 were 13TU, 5 were A. phenon 6/ct, and 4 were Acinetobacter genospecies 3 by ARDRA. Thirteen isolates were confirmed as MBL-producers and blaIMP-1 and blaVIM-2 were carried by 5 and 8 isolates of them, respectively. MBL-producers were mostly 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 and they were susceptible to ciprofloxacin and ampicillin-sulbactam. BlaPER-1 was carried by thirteen isolates and 12 isolates of them were PDRAB showing resistance to all antimicrobial agents tested, including ceftazidime, cefepime, aztreonam, ciprofloxacin, amikacin, gentamicin, ampicillin-sulbactam, and imipenem. In conclusion, most MBL-producers belonged to 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 which were susceptible to ciprofloxacin and ampicillin-sulbactam, whereas 12 of 13 PER-1-producers were PDRAB originated from the same clone.

Determination of Genomospecies and Characterization of Antimicrobial Resistance of Multi-drug Resistant Acinetobacter spp. Isolates

Acinetobacter species are non-fermentative Gram-negative coccobacilli and they have emerged as important nosocomial pathogens which are associated with the significant multidrug resistance in recent years. Carbapenem-resistant A. baumannii (CRAB) and pandrug-resistant A. baumannii (PDRAB) were reported in 1991 and 1998, respectively. Fiftyeight isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005 were investigated for the existence of CRAB, PDRAB, extended-spectrum beta-lactamase (ESBL)-producing Acinetobacter and examined for their phenotypic and genotypic characteristics. Genomospecies of Acinetobacter species were determined by amplified rDNA restriction analysis (ARDRA) and antimicrobial susceptibility test was performed with 13 kinds of antimicrobial agents. Metallo-beta-lactamase (MBL) producers were screened by modified hodge test and confirmed by imipenem-EDTA disk synergy test. Detection of blaIMP-1, blaVIM-2, blaTEM, and blaPER-1 was performed by PCR. Genomic DNAs were analyzed by pulsed-field gel electrophoresis (PFGE). Among 58 isolates of Acinteobacter species, 40 isolates were identified as genospecies 2 (A. baumannii), 9 were 13TU, 5 were A. phenon 6/ct, and 4 were Acinetobacter genospecies 3 by ARDRA. Thirteen isolates were confirmed as MBL-producers and blaIMP-1 and blaVIM-2 were carried by 5 and 8 isolates of them, respectively. MBL-producers were mostly 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 and they were susceptible to ciprofloxacin and ampicillin-sulbactam. BlaPER-1 was carried by thirteen isolates and 12 isolates of them were PDRAB showing resistance to all antimicrobial agents tested, including ceftazidime, cefepime, aztreonam, ciprofloxacin, amikacin, gentamicin, ampicillin-sulbactam, and imipenem. In conclusion, most MBL-producers belonged to 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 which were susceptible to ciprofloxacin and ampicillin-sulbactam, whereas 12 of 13 PER-1-producers were PDRAB originated from the same clone.

Prevalence of CTX-M Extended-spectrum Beta-lactamases in Clinical Isolates of Enterobacteriaceae in Korea

The evolution and dissemination of extended-spectrum beta-lactamases (ESBL) have compromised the clinical use of third-generation cephalosporins worldwide. Although most ESBLs belong to the TEM and SHV beta-lactamase families, the members of CTX-M, a novel ESBL family, are increasing worldwide in Gram-negative bacteria. We examined the prevalence of CTX-M ESBL in clinical isolates of the family Enterobacteriaceae collected from three university hospitals located in three different cities in Korea. Among a total of 603 isolates collected, 163 isolates (27.0%) revealed > or =2 microgram/ ml of MIC against cefotaxime, and 93 isolates (15.4%) produced ESBL confirmed by the double disk synergy test. Among 93 ESBL-producing isolates, blaCTX-M genes were detected in 41 isolates by PCR method and they included 1 isolate of C. freundii, 3 of E. aerogenes, 2 of E. cloacae, 17 of E. coli, 9 of K. pneumoniae, and 9 of S. marcescens. Thus, the overall prevalence of CTX-M ESBL-producing isolates among the family Enterobacteriaceae was 6.8% (41 of 603 isolates) and the proportion of CTX-M-producers among the ESBL-producing isolates was 44.1% (41 of 93 isolates). Further determination of the blaCTX-M subtype by nucleotide sequencing revealed blaCTX-M-3 in 17, blaCTX-M-15 in 11, blaCTX-M-14 in 9, and blaCTX-M-9 in 4 isolates. To our knowledge, this is the first report on the dissemination of CTX-M ESBL among clinical isolates of the family Enterobacteriaceae in Korea.

Prevalence of CTX-M Extended-spectrum Beta-lactamases in Clinical Isolates of Enterobacteriaceae in Korea

The evolution and dissemination of extended-spectrum beta-lactamases (ESBL) have compromised the clinical use of third-generation cephalosporins worldwide. Although most ESBLs belong to the TEM and SHV beta-lactamase families, the members of CTX-M, a novel ESBL family, are increasing worldwide in Gram-negative bacteria. We examined the prevalence of CTX-M ESBL in clinical isolates of the family Enterobacteriaceae collected from three university hospitals located in three different cities in Korea. Among a total of 603 isolates collected, 163 isolates (27.0%) revealed > or =2 microgram/ ml of MIC against cefotaxime, and 93 isolates (15.4%) produced ESBL confirmed by the double disk synergy test. Among 93 ESBL-producing isolates, blaCTX-M genes were detected in 41 isolates by PCR method and they included 1 isolate of C. freundii, 3 of E. aerogenes, 2 of E. cloacae, 17 of E. coli, 9 of K. pneumoniae, and 9 of S. marcescens. Thus, the overall prevalence of CTX-M ESBL-producing isolates among the family Enterobacteriaceae was 6.8% (41 of 603 isolates) and the proportion of CTX-M-producers among the ESBL-producing isolates was 44.1% (41 of 93 isolates). Further determination of the blaCTX-M subtype by nucleotide sequencing revealed blaCTX-M-3 in 17, blaCTX-M-15 in 11, blaCTX-M-14 in 9, and blaCTX-M-9 in 4 isolates. To our knowledge, this is the first report on the dissemination of CTX-M ESBL among clinical isolates of the family Enterobacteriaceae in Korea.