A novel quantitative assay for analysis of GLUT4 translocation using high content screening

Komakula, SaiSantosh Babu; Tiwari, Ashok Kumar; Singh, Shashi

doi:10.1016/j.biopha.2020.111032

Cited by 6 publications

(7 citation statements)

References 42 publications

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“…These observations showed that BCE possessed phytochemicals that could induce glucose uptake into the myotubes independent of insulin stimulation as well as properties that would further augment glucose uptake under insulin stimulation. Such observations have also been reported earlier with natural products (Komakula et al, 2021).…”

Section: Effect Of Bce On Insulin-dependent and Non-insulin-dependent...supporting

confidence: 90%

“…This process is facilitated by family of glucose transporters. GLUT4 is the main facilitator of insulin‐regulated uptake in muscle cells (Komakula et al., 2021). However, in insulin resistance state, the translocation of GLUT4 to plasma membrane is compromised (Maegawa et al., 1991).…”

Section: Resultsmentioning

confidence: 99%

“…In fact, our plant‐based food materials not only serve the purpose of supplying nutrition through macro‐ and micronutrients but also deliver a number of non‐nutrient phytochemicals possessing medicinal properties. Phytochemicals present in vegetables have been observed to promote cellular glucose uptake by multiple mechanisms (Komakula et al., 2021) and exhibit antihyperglycemic activities (Anand et al., 2021; Tiwari et al., 2011).…”

Section: Introductionmentioning

confidence: 99%

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Bombax ceiba calyx displays antihyperglycemic activity via improving insulin secretion and sensitivity: Identification of bioactive phytometabolomes by UPLC‐QTof‐MS/MS

Komati

Anand

Nagendla

et al. 2022

Journal of Food Science

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Vegetables are considered good food for the management of hyperglycemia. Bombax ceiba L. (family: Bombacaceae) calyces are part of traditional vegetables. This study evaluated its usefulness on various parameters responsible for the development of hyperglycemia and conducted phytometabolomic analysis to identify phytochemicals responsible for the observed activities. It was found that the aqueous methanol extract of its calyces (B. ceiba calyx extract, BCE) reduced (12.4%) significantly (p < 0.05) the development of sucrose‐induced postprandial hyperglycemic load in rats. In‐vitro studies revealed that BCE improved glucose‐stimulated insulin secretory activity in MIN6 cells plausibly by decreasing ADP/ATP ratio. BCE also augmented concentration‐dependent (5 µg, 10 µg, and 20 µg) increase in glucose uptake in hyperglycemic L6 myotubes both by non‐insulin‐dependent manner (35%, 68%, and 132%, respectively) and insulin‐dependent manner (42%, 59%, and 172%, respectively). The insulin‐stimulated GLUT4 translocation was compromised (34%) significantly (p < 0.05) under hyperglycemic condition; however, it was improved by 23% and 72% (p < 0.001) when L6 myotubes were primed with 10 and 20 µg of BCE, respectively. Hyperglycemia aggravated reactive oxygen species (ROS) generation in L6 myotubes. The ROS generation was significantly (p < 0.001) reduced by priming myotubes with BCE before challenging myotubes to hyperglycemic environment, possibly by preserving cellular antioxidant enzymes catalase, glutathione peroxidase, and reduced glutathione levels. Phytometabolomic analysis disclosed a number of phytochemicals present in B. ceiba calyces known to display these activities. This is the first study reporting antihyperglycemic activity in B. ceiba calyces, its mechanisms of action, and phytometabolomic profile applying UPLC‐QTof‐MS/MS technique. Practical Application B. ceiba calyces are part of traditional vegetables. Our study finds that B. ceiba calyces contain phytochemicals possessing antihyperglycemic, insulin secretory, insulin sensitization properties, and potentials for preserving hyperglycemia‐induced vitiations in cellular antioxidant defense. These observations provide foundation for exploring further possibilities of B. ceiba calyces to become valuable dietary inclusion in the diet of people suffering from metabolic disorders.

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Section: Effect Of Bce On Insulin-dependent and Non-insulin-dependent...supporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bombax ceiba calyx displays antihyperglycemic activity via improving insulin secretion and sensitivity: Identification of bioactive phytometabolomes by UPLC‐QTof‐MS/MS

Komati

Anand

Nagendla

et al. 2022

Journal of Food Science

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“…Recently, Komakula et al [77] introduced a highcontent screen for real-time readout of GLUT4 translocation. The nuclei of CHO cells expressing myc-GLUT4-eGFP were stained with Hoechst 33342 to identify the perinuclear region.…”

Section: Confocal Microscopymentioning

confidence: 99%

Fluorescence microscopy-based quantitation of GLUT4 translocation

Heckmann

Klanert²,

Sandner

et al. 2022

Methods Appl. Fluoresc.

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Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

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“…It was shown that these cells traffic recombinant Glut4 to the cell membrane in insulin dependent manner and also respond to other stimuli, similar to differentiated 3T3-L1 adipocytes. Since these cells express Glut4 with Myc tag at the N-terminal region (which is exposed extracellularly) and GFP tag at the C-terminal end (intracellular), the Glut4 translocation can be analysed in these cells by FACS and microscopic methods, and have been used to understand the signalling components involved in Glut4 translocation in presence of Insulin and amino acids ( 121314 ) or used for drug screening 15 . We have previously shown that prolonged exposure of insulin without glucose stimulus to these CHO-K1 cells expressing human insulin receptor and Glut4 transporter, leads to IR 16 .…”

Section: Introductionmentioning

confidence: 99%

Prolonged exposure to insulin causes epigenetic alteration leading to insulin resistance

Bano

Mongad

et al. 2022

Preprint

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Glucose homeostasis is maintained by insulin. It has been observed that hyperinsulinemia precedes insulin resistance and Type 2 diabetes. Insulin resistance is caused by multiple factors including genetic and diet. The molecular mechanism underlying insulin resistance (IR) is not completely understood. Using Glut4 and insulin receptor-expressing CHO cells we had previously shown that prolonged exposure of these cells to insulin in the absence of high levels of glucose led to insulin resistance in the cells. In the present study, we have shown that the underlying cause for the impaired GLUT4 trafficking is the defective PI3K/AKT pathway. This insulin resistance is likely due to epigenetic alterations as it is stable and can be maintained for several generations even when insulin is not provided, and epigenetic modifiers can reverse the insulin resistance. We extended these studies to liver cell line (BRL-3A) and show that these cells also develop impaired insulin signaling upon exposure to insulin in the absence of high levels of glucose. Transcriptomic analysis of the insulin-sensitive and -resistance cells uncover altered signaling networks involved in chromatin remodelling, Rho GTPases, and ubiquitination. Pathway analysis reveals the role of demethylase Kdm5b and lysine methyltransferase (Kmt2a and Kmt2e) in the development of insulin resistance. It is also observed that trimethylation of histone H3 at lysine 4 (H3K4me3) is increased in insulin resistance cellular models. We further showed that mice injected with low doses of insulin when fasting develop insulin resistance with impaired glucose tolerance and increased HOMA-IR index. Altogether, these findings suggest dysregulated synthesis of insulin in the absence of glucose stimulus could lead to epigenetic alterations that may lead to insulin resistance.Summary StatementInsulin stimulation in the absence of glucose leads to insulin resistance. We have developed a cell and mouse model of insulin resistance in this study to characterise the molecular signalling involved in insulin resistance and early onset of type 2 diabetes. The transcriptomic analysis provides new insights on epi-transcriptomic regulation in insulin resistance.

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A novel quantitative assay for analysis of GLUT4 translocation using high content screening

Cited by 6 publications

References 42 publications

Bombax ceiba calyx displays antihyperglycemic activity via improving insulin secretion and sensitivity: Identification of bioactive phytometabolomes by UPLC‐QTof‐MS/MS

Bombax ceiba calyx displays antihyperglycemic activity via improving insulin secretion and sensitivity: Identification of bioactive phytometabolomes by UPLC‐QTof‐MS/MS

Fluorescence microscopy-based quantitation of GLUT4 translocation

Prolonged exposure to insulin causes epigenetic alteration leading to insulin resistance

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