Sun-Hyun Park

Glucose Deprivation Regulates KATP Channel Trafficking via AMP-Activated Protein Kinase in Pancreatic β-Cells

OBJECTIVE AMP-activated protein kinase (AMPK) and the ATP-sensitive K+ (KATP) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the KATP channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood. RESEARCH DESIGN AND METHODS Rat pancreatic β-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum KATP conductance (Gmax) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. KATP channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of KATP channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay. RESULTS Gmax and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in Gmax and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on KATP channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C. CONCLUSIONS KATP channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.

Development of sandwich enzyme-linked immunosorbent assay for the detection of Cronobacter muytjensii (formerly called Enterobacter sakazakii)

This study aimed to produce a polyclonal antibody against Cronobacter muytjensii (C. muytjensii, formerly called Enterobacter sakazakii) and to develop an immunoassay for its detection. The optimum production of rabbit anti‐C. muytjensii immunoglobulin G (IgG) and chicken anti‐C. muytjensii IgY was reached in weeks 8 and 9, respectively. Purification of rabbit anti‐C. muytjensii IgG from immunized rabbit sera was accomplished using the caprylic acid and ammonium sulfate precipitation method. As a result, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis produced two bands around 25 and 50 kDa, corresponding to a light and a heavy chain, respectively. The optimized conditions for sandwich enzyme‐linked immunosorbent assay were using rabbit anti‐C. muytjensii IgG (1 μg/mL) as a detection antibody and chicken anti‐C. muytjensii IgY (10 μg/mL) as a capture antibody. In this assay, no cross‐reactivity was observed with the other genera of pathogenic bacteria tested, which included Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. The developed assay did not show cross‐reactivity with other tested species of Cronobacter and Enterobacter genera such as C. turicensis, C. sakazakii, E. aerogenes, E. pulveris and E. helveticus. The detection limit of sandwich ELISA for C. muytjensii was found to be 2.0 × 104 colony forming units (CFU)/mL. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of sandwich ELISA for C. muytjensii, the detection limit being found to be 6.3 × 104 CFU/mL. These findings demonstrate that the developed method is able to detect all strains of C. muytjensii. Hence, this ELISA technique has potent application for the rapid and accurate detection of C. muytjensii in dietary foods.

ApoA-I mutants V156K and R173C promote anti-inflammatory function and antioxidant activities.

Background  Two mutants of apolipoprotein (apo) A‐I, V156K and A158E, showed markedly different structural and functional properties in lipid‐free and lipid‐bound states in the authors’ earlier report. The physiological activities of these mutants were compared with the wild‐type (WT) and R173C mutant using in vitro and in vivo experiments.

Immunoliposome-based immunomagnetic concentration and separation assay for rapid detection of Cronobacter sakazakii.

This study aimed to develop an immunoliposome-based immunomagnetic concentration and separation assay for the rapid detection of Cronobacter sakazakii (C. sakazakii), an acute opportunistic foodborne pathogenic bacterium, in both pure culture and infant formula. To develop the assay, magnetic nanoparticles (diameter 30 nm) were coated with immunoglobulin G (IgG), specifically anti-C. sakazakii IgG, and applied for the sensitive and efficient detection of C. sakazakii using immunoliposomes. The binding efficiency of anti-C. sakazakii IgG to the magnetic nanoparticles was 86.23 ± 0.59%. The assay developed in this study detected as few as 3.3 × 10(3) CFUmL(-1) of C. sakazakii in pure culture within 2h 30 min; in comparison, an indirect non-competitive enzyme-linked immunosorbent assay was able to detect 6.2 × 10(5) CFUmL(-1) of C. sakazakii in pure culture after 17 h. The developed assay did not show any cross-reactivity with other Cronobacter spp. or pathogens belonging to other genera. In addition, the method was able to detect 10(3) CFUmL(-1) of C. sakazakii in infant formula without any pre-incubation. These results confirm that the immunoliposome-based immunomagnetic concentration and separation assay may facilitate highly sensitive, efficient, and rapid detection of C. sakazakii.

Cleavage and functional loss of human apolipoprotein E by digestion of matrix metalloproteinase-14

By means of a degradomic approach applying proteomic techniques, we previously suggested that apolipoprotein E (apoE) is a substrate of matrix metalloproteinase‐14 (MMP‐14). Here we confirm that apoE is, in fact, a substrate of MMP‐14 and also of MMP‐7 and MMP‐2 to a lesser extent. The 34 kDa apoE protein was initially processed by MMP‐14 into fragments with molecular masses of 28, 23, 21, and 11 kDa. MMP‐14 cleavage sites within the apoE protein were determined by C‐terminal labeling of MMP‐14‐digested apoE fragments with isotope (18O/16O = 1:1) and identification of the doublet fragments or peptides showing 2 Da difference by MS, along with N‐terminal sequencing of the fragments. It was determined that the primary MMP‐14 cleavage sites were A176‐I177, P183‐L184, P202‐L203, and Q249‐I250. The MMP‐14‐mediated cleavage of apoE was consistent regardless of whether apoE existed in its lipid‐bound or lipid‐free form. Upon digestion with MMP‐14, apoE loses its ability to suppress the platelet‐derived growth factor‐induced migration of rat vascular smooth muscle cells. Considering the important role of apoE for lipid metabolism and atherosclerosis protection, our findings suggest that MMP‐14 plays an essential role for the development of hyperlipidemia and atherosclerosis as a result of degradation of apoE.

A point mutant of apolipoprotein A-I, V156K, exhibited potent anti-oxidant and anti-atherosclerotic activity in hypercholesterolemic C57BL/6 mice.

In our previous study, two point mutants of apolipoprotein A-I, designated V156K and A158E, revealed peculiar characteristics in their lipid-free and lipid-bound states. In order to determine the putative therapeutic potential of these mutants, several in vitro and in vivo evaluations were conducted. In the lipid-free state, V156K showed more profound antioxidant activity against LDL oxidation than did the wildtype (WT) or A158E variants in an in vitro assay. In the lipid-bound state, V156K-rHDL showed an enhanced cholesterol delivery activity to HepG2 cells in a time-dependent manner, as compared to WT-rHDL, A158E-rHDL, and R173C-rHDL. We assessed the physiological activities of the mutants in circulation, using hypercholesterolemic mice (C57BL6/J). Palmitoyloleoyl phosphatidylcholine (POPC)-rHDL preparations containing each of the apoA-I variants were injected into the mice at a dosage of 30 mg of apoA-I/kg of body weight. Forty eight hours after injection, the sera of the V156K-rHDL injected group showed the most potent antioxidant abilities in the ferric acid removal assay. The V156K-rHDL- or R173C-rHDL-injected mice showed no atherosclerotic lesions and manifested striking increases in their serum apo-E levels, as compared to the mice injected with WT-rHDL or A158E-rHDL. In conclusion, V156K-rHDL exhibited the most pronounced antioxidant activity and anti-atherosclerotic activity, both in vitro and in vivo. These results support the notion that HDL-therapy may prove beneficial due to its capacity to induce accelerated cholesterol excretion, as well as its enhanced antioxidant and anti-inflammatory effects and lesion regression effect.

Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

Cronobacter species (Cronobacter spp.) are hazardous foodborne pathogens associated with baby food, powdered infant formula (PIF). To develop a rapid and sensitive method for simultaneous detection of seven Cronobacter spp. in PIF, an indirect non-competitive enzyme-linked immunosorbent assay (INC-ELISA) was developed based on a novel immunoglobulin G (IgG), anti-Cronobacter IgG. The developed INC-ELISA was able to detect seven Cronobacter spp. at concentrations ranging from (5.6 ± 0.30) × 103 to (2.1 ± 0.01) × 105 colony forming unit (CFU)/mL in pure culture. Further, INC-ELISA employing anti-Cronobacter IgG was applicable for analysis of PIF samples contaminated with less than <10 cells of Cronobacter spp. per 25 g of PIF in 36 h. The developed antibody showed slight cross-reactivity with Franconibacter pulveris (LMG 24057) at high concentration (108 CFU/mL). The INC-ELISA method displayed excellent specificity without compromising cross-reactivity with other foodborne pathogens. The INC-ELISA assay method developed in this study using a novel anti-Cronobacter IgG facilitated highly sensitive, efficient, and rapid detection of Cronobacter spp. in baby food.

Rac-mediated actin remodeling and myosin II are involved in KATP channel trafficking in pancreatic β-cells.

AMP-activated protein kinase (AMPK) is a metabolic sensor activated during metabolic stress and it regulates various enzymes and cellular processes to maintain metabolic homeostasis. We previously reported that activation of AMPK by glucose deprivation (GD) and leptin increases KATP currents by increasing the surface levels of KATP channel proteins in pancreatic β-cells. Here, we show that the signaling mechanisms that mediate actin cytoskeleton remodeling are closely associated with AMPK-induced KATP channel trafficking. Using F-actin staining with Alexa 633-conjugated phalloidin, we observed that dense cortical actin filaments present in INS-1 cells cultured in 11 mM glucose were disrupted by GD or leptin treatment. These changes were blocked by inhibiting AMPK using compound C or siAMPK and mimicked by activating AMPK using AICAR, indicating that cytoskeletal remodeling induced by GD or leptin was mediated by AMPK signaling. AMPK activation led to the activation of Rac GTPase and the phosphorylation of myosin regulatory light chain (MRLC). AMPK-dependent actin remodeling induced by GD or leptin was abolished by the inhibition of Rac with a Rac inhibitor (NSC23766), siRac1 or siRac2, and by inhibition of myosin II with a myosin ATPase inhibitor (blebbistatin). Immunocytochemistry, surface biotinylation and electrophysiological analyses of KATP channel activity and membrane potentials revealed that AMPK-dependent KATP channel trafficking to the plasma membrane was also inhibited by NSC23766 or blebbistatin. Taken together, these results indicate that AMPK/Rac-dependent cytoskeletal remodeling associated with myosin II motor function promotes the translocation of KATP channels to the plasma membrane in pancreatic β-cells.

A Caucasian male with very low blood cholesterol and low apoA-II without evidence of atherosclerosis

Background  It is well known that a high level of apolipoprotein (apo) A‐II can be associated with familial combined hyperlipidaemia, and that high apolipoprotein profiles can contribute to the development of atherosclerosis. The serum lipoprotein/apolipoprotein profile of a Caucasian patient who had unusually low serum total cholesterol (83 mg dL−1) and triglyceride (28 mg dL−1) levels despite a high body mass index (33·5 kg m−2), is the subject of this report.

Glucose Deprivation Regulates KATP Channel Trafficking via AMPK in Pancreatic Beta-Cells

AMP-activated protein kinase (AMPK) and ATP-sensitive K+ channel (KATP channel) are metabolic sensors that are activated during metabolic stress. The importance of AMPK has been appreciated by its role as a regulator of metabolism, whereas KATP channel is known as a regulator of cellular excitability. Cross-talks between two systems are not well understood. In pancreatic β-cells or INS-1 cells, we measured KATP currents by the patch clamp technique and examined distributions of KATP channel proteins (Kir6.2 and SUR1) using immunofluorescence imaging and surface biotinylation studies. When KATP channels were activated by washout of intracellular ATP using a ATP- and Mg2+-free internal solution, the increase in whole cell conductance was surprisingly small in cells incubated in 11.1 mM glucose medium, but the increase was significantly higher in cells pre-incubated in glucose-free medium for 2 hrs. We confirmed that KATP channel proteins were mostly internalized in 11.1 mM glucose, but recruited to the plasma membrane by glucose deprivation without changes in total levels. The effects of glucose deprivation on KATP channels were abolished by an AMPK inhibitor or a knockdown of AMPK using siRNA, but mimicked by an AMPK activator. These results suggest that regulation of KATP channel trafficking by AMPK is a prerequisite for KATP channel activation in pancreatic β-cells in response to glucose deprivation. The interplay between AMPK and KATP channels may play a key role in inhibiting cellular excitability and insulin secretion under low energy status.