Curcumin Antagonizes Glucose Fluctuation-Induced Renal Injury by Inhibiting Aerobic Glycolysis via the miR-489/LDHA Pathway

It has been considered that glucose fluctuation (GF) plays a role in renal injury and is related to diabetic nephropathy (DN) development. But the mechanism is still unclear. Aerobic glycolysis has become a topical issue in DN in recent years. There is an internal connection between GF, aerobic glycolysis, and DN. Curcumin (Cur) is a principal curcuminoid of turmeric and possesses specific protective properties in kidney functions. Cur also participates in the regulation of aerobic glycolysis switch. In this study, we first measured the levels of aerobic glycolysis and evaluated Cur’s inhibitory ability in a cell model of HEK-293 under the condition of oscillating high glucose. The results indicated that GF exacerbated inflammation injury, oxidative stress, and apoptosis in HEK-293 cell, while Cur alleviated this cytotoxicity induced by GF. We found that GF increased aerobic glycolysis in HEK-293 cells and Cur presented a dose-dependent weakening effect to this exacerbation. Next, we built a panel of 17 miRNAs and 8 lncRNAs that were previously reported to mediate the Warburg effect. Our RT-qPCR results indicated that GF reduced the miR-489 content in the HEK-293 cell model and Cur could prevent this downregulation. Then, we planned to explore the character of miR-489 in Cur-triggered attenuation of the Warburg effect under GF condition. Our findings presented that Cur prevented GF-triggered aerobic glycolysis by upregulating miR-489 in HEK-293 cells. Next, we choose the miR-489/LDHA axis for further investigation. We confirmed that Cur prevented GF-triggered aerobic glycolysis via the miR-489/LDHA axis in HEK-293 cells. In conclusion, this study presented that Cur prevented GF-triggered renal injury by restraining aerobic glycolysis via the miR-489/LDHA axis in the HEK-293 cell model.

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Functional diversity of PFKFB3 splice variants in glioblastomas

10.1101/2020.10.09.332817 ◽

2020 ◽

Author(s):

Ulli Heydasch ◽

Renate Kessler ◽

Jan-Peter Warnke ◽

Klaus Eschrich ◽

Nicole Scholz ◽

...

Keyword(s):

Splice Variant ◽

Splice Variants ◽

Aerobic Glycolysis ◽

Diagnostic Value ◽

Hek 293 ◽

Hek 293 Cells ◽

Specific Effects ◽

293 Cells ◽

Common Notion ◽

The Impact

AbstractTumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells.Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered.Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

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Functional diversity of PFKFB3 splice variants in glioblastomas

PLoS ONE ◽

10.1371/journal.pone.0241092 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0241092

Author(s):

Ulli Heydasch ◽

Renate Kessler ◽

Jan-Peter Warnke ◽

Klaus Eschrich ◽

Nicole Scholz ◽

...

Keyword(s):

Splice Variant ◽

Splice Variants ◽

Aerobic Glycolysis ◽

Diagnostic Value ◽

Hek 293 ◽

Hek 293 Cells ◽

Specific Effects ◽

293 Cells ◽

Common Notion ◽

The Impact

Tumor cells tend to metabolize glucose through aerobic glycolysis instead of oxidative phosphorylation in mitochondria. One of the rate limiting enzymes of glycolysis is 6-phosphofructo-1-kinase, which is allosterically activated by fructose 2,6-bisphosphate which in turn is produced by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2 or PFKFB). Mounting evidence suggests that cancerous tissues overexpress the PFKFB isoenzyme, PFKFB3, being causing enhanced proliferation of cancer cells. Initially, six PFKFB3 splice variants with different C-termini have been documented in humans. More recently, additional splice variants with varying N-termini were discovered the functions of which are to be uncovered. Glioblastoma is one of the deadliest forms of brain tumors. Up to now, the role of PFKFB3 splice variants in the progression and prognosis of glioblastomas is only partially understood. In this study, we first re-categorized the PFKFB3 splice variant repertoire to simplify the denomination. We investigated the impact of increased and decreased levels of PFKFB3-4 (former UBI2K4) and PFKFB3-5 (former variant 5) on the viability and proliferation rate of glioblastoma U87 and HEK-293 cells. The simultaneous knock-down of PFKFB3-4 and PFKFB3-5 led to a decrease in viability and proliferation of U87 and HEK-293 cells as well as a reduction in HEK-293 cell colony formation. Overexpression of PFKFB3-4 but not PFKFB3-5 resulted in increased cell viability and proliferation. This finding contrasts with the common notion that overexpression of PFKFB3 enhances tumor growth, but instead suggests splice variant-specific effects of PFKFB3, apparently with opposing effects on cell behaviour. Strikingly, in line with this result, we found that in human IDH-wildtype glioblastomas, the PFKFB3-4 to PFKFB3-5 ratio was significantly shifted towards PFKFB3-4 when compared to control brain samples. Our findings indicate that the expression level of distinct PFKFB3 splice variants impinges on tumorigenic properties of glioblastomas and that splice pattern may be of important diagnostic value for glioblastoma.

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Investigating the Effect of Kappa Opioid Receptor Agonists on Serotonin Transporter Function in HEK-293 Cells

10.26686/wgtn.17006281 ◽

2021 ◽

Author(s):

◽

Victoria Catherwood

Keyword(s):

Opioid Receptor ◽

High Throughput ◽

Cell Model ◽

Kappa Opioid Receptor ◽

Rotating Disc ◽

Kappa Opioid ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

High Throughput Assay

<p>The serotonin (5-HT) transporter (SERT) and the kappa opioid receptor (KOPr) are important brain proteins. Currently, it is known that KOPr modulates 5-HT concentrations but the biochemical mechanisms responsible remain enigmatic. The 5-HT and KOPr relationship is thought to involve SERT because both SERT and KOPr co-localise in the dorsal raphe nucleus (DRN), a brain region involved in drug addiction, affective disorders, and stress homeostasis. Thus, elucidating the KOPr and SERT relationship will clarify biomedical targets for these disorders. To investigate this relationship the effect of KOPr activation on SERT function was examined in HEK-293 cells co-expressing myc-rKOPr and eGFP-tagged hSERT. We found the KOPr agonists U50,488H, Salvinorin A (Sal A) and DS-3-240 had no acute (5 min) effect on SERT function as measured by rotating disc electrode voltammetry (RDEV) incubated with 1’-diethyl-2,2’-cyanine iodide (D-22; 1.5 mM) and by confocal microscopy. Interestingly, SERT function significantly decreased with chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) as measured with a novel high-throughput assay; this decrease was also attenuated with 5 min pre-treatment of the SERT inhibitor, fluoxitene (***p<0.001). Furthermore, this novel high-throughput assay also replicated our laboratories recent finding that chronic (30 min) exposure to U50,488H (**p<0.01) and Sal A (**p<0.01) significantly increase dopamine transporter (DAT) function in HEK-293 cells co-expressing myc-rKOPr and eYFP-tagged DAT. Collectively, these findings suggest in this cell model that chronic (30 min) KOPr activation by U50,488H and Sal A decreases SERT function.</p>

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Quercetin Antagonizes Glucose Fluctuation Induced Renal Injury by Inhibiting Aerobic Glycolysis via HIF-1α/miR-210/ISCU/FeS Pathway

Frontiers in Medicine ◽

10.3389/fmed.2021.656086 ◽

2021 ◽

Vol 8 ◽

Author(s):

Wei-long Xu ◽

Su Liu ◽

Nan Li ◽

Li-fang Ye ◽

Min Zha ◽

...

Keyword(s):

Cell Viability ◽

Renal Injury ◽

Cell Model ◽

Aerobic Glycolysis ◽

Dependent Manner ◽

Glomerular Mesangial Cells ◽

Mitochondrial Energy Metabolism ◽

Glucose Fluctuation ◽

Repressive Effect ◽

Quercetin Treatment

Background and Objective: Glucose fluctuation (GF) has been reported to induce renal injury and diabetic nephropathy (DN). However, the mechanism still remains ambiguous. Mitochondrial energy metabolism, especially aerobic glycolysis, has been a hotspot of DN research for decades. The activation of HIF-1α/miR210/ISCU/FeS axis has provided a new explanation for aerobic glycolysis. Our previous studies indicated quercetin as a potential therapeutic drug for DN. This study aims to evaluate levels of aerobic glycolysis and repressive effect of quercetin via HIF-1α/miR210/ISCU/FeS axis in a cell model of GF.Methods: The mouse glomerular mesangial cells (MCs) were exposed in high or oscillating glucose with or without quercetin treatment. Cell viability was measured by CCK8 assay. Aerobic glycolysis flux was evaluated by lactate acid, pH activity of PFK. Apoptosis level was confirmed by Annexin V-APC/7-AAD double staining and activity of caspase-3. TNF-α and IL-1β were used to evaluate inflammation levels.Results: GF deteriorated inflammation damage and apoptosis injury in MCs, while quercetin could alleviate this GF-triggered cytotoxicity. GF intensified aerobic glycolysis in MCs and quercetin could inhibit this intensification in a dose-dependent manner. Quercetin prevented activities of two FeS-dependent metabolic enzymes, aconitase, and complex I, under GF injury in MCs. The mRNA expression and protein contents of HIF-1α were increased after GF exposure, and these could be alleviated by quercetin treatment. Knockdown of ISCU by siRNA and Up-regulating of miR-210 by mimic could weaken the effects of quercetin that maintained protein levels of ISCU1/2, improved cell viability, relieved inflammation injury, decreased apoptosis, and reduced aerobic glycolysis switch in MCs.Conclusion: Quercetin antagonizes GF-induced renal injury by suppressing aerobic glycolysis via HIF-1α/miR-210/ISCU/FeS pathway in MCs cell model. Our findings contribute to a new insight into understanding the mechanism of GF-induced renal injury and protective effects of quercetin.

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Investigating the Effect of Kappa Opioid Receptor Agonists on Serotonin Transporter Function in HEK-293 Cells

10.26686/wgtn.17006281.v1 ◽

2021 ◽

Author(s):

◽

Victoria Catherwood

Keyword(s):

Opioid Receptor ◽

High Throughput ◽

Cell Model ◽

Kappa Opioid Receptor ◽

Rotating Disc ◽

Kappa Opioid ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

High Throughput Assay

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Characteristics and requirements of basal autophagy in HEK 293 cells

Autophagy ◽

10.4161/auto.25455 ◽

2013 ◽

Vol 9 (9) ◽

pp. 1407-1417 ◽

Cited By ~ 41

Author(s):

Patience Musiwaro ◽

Matthew Smith ◽

Maria Manifava ◽

Simon A. Walker ◽

Nicholas T. Ktistakis

Keyword(s):

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

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Halothane Inhibition of Recombinant Cardiac L-type Ca2+ Channels Expressed in HEK-293 Cells

Anesthesiology ◽

10.1097/00000542-200512000-00009 ◽

2005 ◽

Vol 103 (6) ◽

pp. 1156-1166 ◽

Cited By ~ 7

Author(s):

Kevin J. Gingrich ◽

Son Tran ◽

Igor M. Nikonorov ◽

Thomas J. Blanck

Keyword(s):

Charge Transfer ◽

Calcium Channels ◽

Dependent Manner ◽

Current Time ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Dependent Inactivation ◽

Voltage Dependent

Background Volatile anesthetics depress cardiac contractility, which involves inhibition of cardiac L-type calcium channels. To explore the role of voltage-dependent inactivation, the authors analyzed halothane effects on recombinant cardiac L-type calcium channels (alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1), which differ by the alpha2/delta1 subunit and consequently voltage-dependent inactivation. Methods HEK-293 cells were transiently cotransfected with complementary DNAs encoding alpha1C tagged with green fluorescent protein and beta2a, with and without alpha2/delta1. Halothane effects on macroscopic barium currents were recorded using patch clamp methodology from cells expressing alpha1Cbeta2a and alpha1Cbeta2aalpha2/delta1 as identified by fluorescence microscopy. Results Halothane inhibited peak current (I(peak)) and enhanced apparent inactivation (reported by end pulse current amplitude of 300-ms depolarizations [I300]) in a concentration-dependent manner in both channel types. alpha2/delta1 coexpression shifted relations leftward as reported by the 50% inhibitory concentration of I(peak) and I300/I(peak)for alpha1Cbeta2a (1.8 and 14.5 mm, respectively) and alpha1Cbeta2aalpha2/delta1 (0.74 and 1.36 mm, respectively). Halothane reduced transmembrane charge transfer primarily through I(peak) depression and not by enhancement of macroscopic inactivation for both channels. Conclusions The results indicate that phenotypic features arising from alpha2/delta1 coexpression play a key role in halothane inhibition of cardiac L-type calcium channels. These features included marked effects on I(peak) inhibition, which is the principal determinant of charge transfer reductions. I(peak) depression arises primarily from transitions to nonactivatable states at resting membrane potentials. The findings point to the importance of halothane interactions with states present at resting membrane potential and discount the role of inactivation apparent in current time courses in determining transmembrane charge transfer.

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