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Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity.

ABSTRACT: The mitochondrial splice variant of the sulfonylurea receptor (SUR2A-55) is associated with protection from myocardial ischemia-reperfusion (IR) injury, increased mitochondrial ATP sensitive K⁺ channel activity (mitoK_ATP) and altered glucose metabolism. While mitoK_ATP channels composed of CCDC51 and ABCB8 exist, the mitochondrial K⁺ pore regulated by SUR2A-55 is unknown. We explored whether SUR2A-55 regulates ROMK to form an alternate mitoK_ATP. We assessed glucose uptake in mice overexpressing SUR2A-55 (TG^SUR2A-55) compared with WT mice during IR injury. We then examined the expression level of ROMK and the effect of ROMK modulation on mitochondrial membrane potential (Δψm) in WT and TG^SUR2A-55 mice. TG^SUR2A-55 had increased glucose uptake compared to WT mice during IR injury. The expression of ROMK was similar in WT compared to TG^SUR2A-55 mice. ROMK inhibition hyperpolarized resting cardiomyocyte Δψm from TG^SUR2A-55 mice but not from WT mice. In addition, TG^SUR2A-55 and ROMK inhibitor treated WT isolated cardiomyocytes had enhanced mitochondrial uncoupling. ROMK inhibition blocked diazoxide induced Δψm depolarization and prevented preservation of Δψm from FCCP perfusion in WT and to a lesser degree TG^SUR2A-55 mice. In conclusion, cardio-protection from SUR2A-55 is associated with ROMK regulation, enhanced mitochondrial uncoupling and increased glucose uptake.

SUBMITTER: El-Meanawy SK

PROVIDER: S-EPMC10146620 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity.

El-Meanawy Sarah K SK Dooge Holly H Wexler Allison C AC Kosmach Anna C AC Serban Lara L Santos Elizabeth A EA Alvarado Francisco J FJ Hacker Timothy A TA Ramratnam Mohun M

Life (Basel, Switzerland) 20230414 4

The mitochondrial splice variant of the sulfonylurea receptor (SUR2A-55) is associated with protection from myocardial ischemia-reperfusion (IR) injury, increased mitochondrial ATP sensitive K+ channel activity (mitoKATP) and altered glucose metabolism. While mitoKATP channels composed of CCDC51 and ABCB8 exist, the mitochondrial K+ pore regulated by SUR2A-55 is unknown. We explored whether SUR2A-55 regulates ROMK to form an alternate mitoKATP</su ...[more]

PMID: 37109544

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Project description:Background: Studies suggest that indomethacin (Indo) exhibits detrimental changes in the small intestine (microvascular disorder, villus shortening, and epithelial disruption), mainly due to mitochondrial uncoupling. The effects of Indo on colon and liver tissue are unclear. The aim of this study was to determine the effects of Indo on mitochondrial respiration in colonic and hepatic tissue. Methods: Mitochondrial oxygen consumption was assessed in colon and liver homogenates from healthy rats. Homogenates were incubated without drug (control) or Indo (colon: 0.36, 1, 30, 179, 300, 1,000, 3,000 μM; liver: 0.36, 1, 3, 10, 30, 100, 179 μM; n = 6). State 2 (substrate-dependent) and state 3 (ADP-dependent respiration) were evaluated with respirometry. The respiratory control index (RCI) was derived and the ADP/O ratio was calculated. Statistics: Data presented as % of control, min/median/max, Kruskal-Wallis+Dunn's correction, * p < 0.05 vs. control. Results: Indo had no effect on RCI of colonic mitochondria. ADP/O ratio increased in complex I at concentrations of 1,000 and 3,000 μM (Indo 1,000 μM: 113.9/158.9/166.9%*; Indo 3,000 μM: 151.5/183.0/361.5%*) and in complex II at concentrations of 179 and 3,000 μM vs. control (179 μM: 111.3/73.1/74.9%*; 3,000 μM: 132.4/175.0/339.4%*). In hepatic mitochondria RCI decreased at 179 μM for both complexes vs. control (complex I: 25.6/40.7/62.9%*, complex II: 57.0/73.1/74.9%*). The ADP/O ratio was only altered in complex I at a concentration of 179 μM Indo vs. control (Indo 179 μM: 589.9/993.7/1195.0 %*). Conclusion: Indo affected parameters of mitochondrial function in an organ-specific and concentration-dependent manner. In colonic tissue, RCI remained unaltered whereas the ADP/O ratio increased. Indo at the highest concentration decreased the RCI for both complexes in hepatic mitochondria. The large increase in ADP/O ratio in complex I at the highest concentration likely reflects terminal uncoupling.

Project description:BackgroundThe production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes.ResultsA set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities.ConclusionsThe use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

Dataset Information

Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity.

Publications

Overexpression of a Short Sulfonylurea Splice Variant Increases Cardiac Glucose Uptake and Uncouples Mitochondria by Regulating ROMK Activity.

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