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Cell lysis-free quantum dot multicolor cellular imaging-based mechanism study for TNF-α-induced insulin resistance.

Kim MJ, Rangasamy S, Shim Y, Song JM - J Nanobiotechnology (2015)

Bottom Line: Through the measurement of the glycogen level in HepG2 cell treated with TNF-α, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid.The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs.The results showed that amygdalin and cinnamic acid inhibit serine phosphorylation of IRS-1 through targeting JNK serine kinase and enhance insulin sensitivity.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, South Korea. jmsong@snu.ac.kr.

ABSTRACT

Background: TNF-α is an inflammatory cytokine that plays an important role in insulin resistance observed in obesity and chronic inflammation. Many cellular components involved in insulin signaling cascade are known to be inhibited by TNF-α. Insulin receptor substrate (IRS)-1 is one of the major targets in TNF-α-induced insulin resistance. The serine phosphorylation of IRS-1 enables the inhibition of insulin signaling. Until now, many studies have been conducted to investigate the mechanism of TNF-α-induced insulin resistance based on Western blot. Intracellular protein kinase crosstalk is commonly encountered in inflammation-associated insulin resistance. The crosstalk among the signaling molecules obscures the precise role of kinases in insulin resistance. We have developed a cell lysis-free quantum dots (QDots) multicolor cellular imaging to identify the biochemical role of multiple kinases (p38, JNK, IKKβ, IRS1ser, IRS1tyr, GSK3β, and FOXO1) in inflammation-associated insulin resistance pathway with a single assay in one run. QDot-antibody conjugates were used as nanoprobes to simultaneously monitor the activation/deactivation of the above seven intracellular kinases in HepG2 cells. The effect of the test compounds on the suppression of TNF-α-induced insulin resistance was validated through kinase monitoring. Aspirin, indomethacin, cinnamic acid, and amygdalin were tested.

Results: Through the measurement of the glycogen level in HepG2 cell treated with TNF-α, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid. The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. QDot multicolor cellular imaging demonstrated that amygdalin and cinnamic acid selectively acted via the JNK1-dependent pathway to suppress the inflammation-induced insulin resistance and improve insulin sensitivity.

Conclusion: The regulatory function of multiple kinases could be monitored concurrently at the cellular level. The developed cellular imaging assay provides a unique platform for the understanding of inflammation and insulin resistance signaling pathways in type II diabetes mellitus and how they regulate each other. The results showed that amygdalin and cinnamic acid inhibit serine phosphorylation of IRS-1 through targeting JNK serine kinase and enhance insulin sensitivity.

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Related in: MedlinePlus

Effect of four different chemicals on inflammation-associated insulin signaling pathway in HepG2 cells. The QDot HCS assay was developed for various key kinases involved in inflammation-associated IR that are indicated with a red arrow and validated by specific inhibitors and anti-inflammatory drugs as shown. The p38α, JNK1, and IKKβ involved in the inflammatory pathway and IRS1ser307, IRS1tyr, FOXO1, and GSK3β associated with the insulin signaling pathway were simultaneously monitored by QDot multicolor cellular imaging.
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Related In: Results  -  Collection

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Fig1: Effect of four different chemicals on inflammation-associated insulin signaling pathway in HepG2 cells. The QDot HCS assay was developed for various key kinases involved in inflammation-associated IR that are indicated with a red arrow and validated by specific inhibitors and anti-inflammatory drugs as shown. The p38α, JNK1, and IKKβ involved in the inflammatory pathway and IRS1ser307, IRS1tyr, FOXO1, and GSK3β associated with the insulin signaling pathway were simultaneously monitored by QDot multicolor cellular imaging.

Mentions: Figure 1 shows a schematic model for TNF-α-induced insulin resistance. TNF-α is the most extensively studied cytokine from the point of view of insulin resistance. TNF-α acts by direct and indirect mechanisms to converge at multiple signaling nodes in the way of insulin action. Direct mechanisms include the stimulation of the serine phosphorylation of IRS-1 and decreasing the expression of IRS-1 and GLUT-4 [12]. Indirectly, TNF-α also interacts with muscle cells, liver cells, and adipose tissue to evoke insulin resistance. These stimuli may activate overlapping signal pathways via common upstream kinases in the insulin pathway, such as IRS1-4 proteins, PI3K and AKT/protein kinase B (PKB), and the activation of several kinases, such as p38α, JNK1, and IKKβ in inflammatory signaling. The crosstalk between inflammation and insulin signaling is a major hurdle for understanding the molecular link between the two pathways. The present approaches to studying cellular signaling mechanisms suffer from fluctuation in the activities of intracellular kinases and reproducible results. Therefore, we developed a novel QDot-based quantitative multicolor cellular imaging for simultaneous analysis and monitoring of the activity of various kinases in both disease mechanisms. As shown in Figure 2(a), the phospho-p38α, phospho-JNK1, phospho-IKKβ, phospho-IRS1ser307, phospho-IRS1tyr, phospho-GSK3β, and phospho-FOXO1 antibodies were conjugated with 565, 565, 565, 605, 705, 525, and 655 QDots, respectively, using an antibody conjugation kit for deciphering the insulin resistance pathway. The conjugate between QDot and antibody was verified through a dynamic light scattering and zeta potential measurement. Figure 2(b) and (c) show particle sizes of QDot605 and QDot605-antibody conjugate measured with dynamic light scattering. As a result of conjugation, the size of QDot605-antibody conjugate increased compared to QDot605. Table 1 summarizes particle sizes and zeta potential values of QDots and QDot-antibody conjugates used in this study. Antibodies conjugated to QDots have different structures and charges. As a result, the size of QDot-antibody conjugate was not proportional linearly to that of QDot. Zeta potential values of QDot-antibody conjugates were larger than those of QDots due to the conjugation between QDot and antibody. Zeta potential values of QDot-antibody conjugates were hardly changed three weeks after the conjugation. Emission spectra of five different QDots are very narrow (Figure 2[d]), which is appropriate for the elimination of crosstalk in high-content cell-based assay. In addition, acousto-optic tunable filter (AOTF) can contribute greatly to the spectral overlap-free high-content monitoring. AOTF allows cellular imaging at particular single wavelengths devoid of spectral overlap among QDot-antibody conjugates. The crosstalk-free high-content monitoring of inflammation and insulin signal transduction was attempted using QDot-antibody conjugates and AOTF in this work. We investigated the effect of various inflammatory kinase inhibitors acting on insulin signaling and evaluated the compound efficacies for the functional ability to inhibit gluconeogenesis and enhance glycogen content, with the goal of identifying potential kinase for suppressing inflammation-induced insulin resistance. Our experiment also addresses the suitability of multicolor cellular imaging for studying kinase crosstalk operating in complex diseases, such as inflammation-associated insulin resistance, at a single shot in a single cell. Surprisingly, amygdalin and cinnamic acid acted selectively via the JNK1-dependent pathway to improve insulin sensitivity. The HepG2 cell line was chosen for this study as it is a perpetual, epithelial cell line derived from well-differentiated hepatocellular carcinoma. These cells solely depend upon exogenous growth factors for survival and respond to stimuli, such as TNF-α, adiponectin, leptin, etc. To study the causal role of elevated cytokines levels and MAP kinase inhibitors in hepatic insulin resistance, HepG2 cells were treated with inhibitors 30 min prior to stimulation with 10 ng/ml TNF-α for 5 h. The cells were then treated with 100 nM insulin for 10 min. Past studies using the obese TNF-α+/+ mice model have shown disturbances in insulin signaling in response to TNF-α [13]. Therapies that have successfully targeted TNF-α, including genetic silencing of the TNF-α or TNF receptor in mice, have demonstrated significant efficacy in the treatment of obesity-related insulin resistance. Therefore, we established the insulin resistance model in vitro using TNF-α stimulation of HepG2 cells. The glycogen amount decreased in the TNF-α treated group, suggesting compromised insulin sensitivity. The MAP kinase inhibitors suppressed the effect of TNF-α on the insulin transduction pathway and consequently resulted in elevated levels of glycogen content (Figure 3a). The inflammatory signaling inhibitors, such as p38α kinase inhibitors (SB203580), JNK1 inhibitors (SP600125), IKKβ inhibitors, aspirin, and indomethacin increased glycogen levels by almost two-fold in comparison to amygdalin and cinnamic acid. Glycogen content in HepG2 cells treated with p38, JNK1, IKKβ inhibitors, aspirin, and indomethacin were four to five times higher than that of the control; whereas, glycogen content in amygdalin or cinnamic acid-treated HepG2 cells were two to three times higher than that of the control [14]. As shown by a glucose production assay, exposure of HepG2 cells to inhibitors reduced gluconeogenesis in comparison to the control group (Figure 3b). Cinnamic acid was much more potent in suppressing glucose production than MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. The rate of hepatic glucose production by amygdalin was similar to that of aspirin.Figure 1


Cell lysis-free quantum dot multicolor cellular imaging-based mechanism study for TNF-α-induced insulin resistance.

Kim MJ, Rangasamy S, Shim Y, Song JM - J Nanobiotechnology (2015)

Effect of four different chemicals on inflammation-associated insulin signaling pathway in HepG2 cells. The QDot HCS assay was developed for various key kinases involved in inflammation-associated IR that are indicated with a red arrow and validated by specific inhibitors and anti-inflammatory drugs as shown. The p38α, JNK1, and IKKβ involved in the inflammatory pathway and IRS1ser307, IRS1tyr, FOXO1, and GSK3β associated with the insulin signaling pathway were simultaneously monitored by QDot multicolor cellular imaging.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4310030&req=5

Fig1: Effect of four different chemicals on inflammation-associated insulin signaling pathway in HepG2 cells. The QDot HCS assay was developed for various key kinases involved in inflammation-associated IR that are indicated with a red arrow and validated by specific inhibitors and anti-inflammatory drugs as shown. The p38α, JNK1, and IKKβ involved in the inflammatory pathway and IRS1ser307, IRS1tyr, FOXO1, and GSK3β associated with the insulin signaling pathway were simultaneously monitored by QDot multicolor cellular imaging.
Mentions: Figure 1 shows a schematic model for TNF-α-induced insulin resistance. TNF-α is the most extensively studied cytokine from the point of view of insulin resistance. TNF-α acts by direct and indirect mechanisms to converge at multiple signaling nodes in the way of insulin action. Direct mechanisms include the stimulation of the serine phosphorylation of IRS-1 and decreasing the expression of IRS-1 and GLUT-4 [12]. Indirectly, TNF-α also interacts with muscle cells, liver cells, and adipose tissue to evoke insulin resistance. These stimuli may activate overlapping signal pathways via common upstream kinases in the insulin pathway, such as IRS1-4 proteins, PI3K and AKT/protein kinase B (PKB), and the activation of several kinases, such as p38α, JNK1, and IKKβ in inflammatory signaling. The crosstalk between inflammation and insulin signaling is a major hurdle for understanding the molecular link between the two pathways. The present approaches to studying cellular signaling mechanisms suffer from fluctuation in the activities of intracellular kinases and reproducible results. Therefore, we developed a novel QDot-based quantitative multicolor cellular imaging for simultaneous analysis and monitoring of the activity of various kinases in both disease mechanisms. As shown in Figure 2(a), the phospho-p38α, phospho-JNK1, phospho-IKKβ, phospho-IRS1ser307, phospho-IRS1tyr, phospho-GSK3β, and phospho-FOXO1 antibodies were conjugated with 565, 565, 565, 605, 705, 525, and 655 QDots, respectively, using an antibody conjugation kit for deciphering the insulin resistance pathway. The conjugate between QDot and antibody was verified through a dynamic light scattering and zeta potential measurement. Figure 2(b) and (c) show particle sizes of QDot605 and QDot605-antibody conjugate measured with dynamic light scattering. As a result of conjugation, the size of QDot605-antibody conjugate increased compared to QDot605. Table 1 summarizes particle sizes and zeta potential values of QDots and QDot-antibody conjugates used in this study. Antibodies conjugated to QDots have different structures and charges. As a result, the size of QDot-antibody conjugate was not proportional linearly to that of QDot. Zeta potential values of QDot-antibody conjugates were larger than those of QDots due to the conjugation between QDot and antibody. Zeta potential values of QDot-antibody conjugates were hardly changed three weeks after the conjugation. Emission spectra of five different QDots are very narrow (Figure 2[d]), which is appropriate for the elimination of crosstalk in high-content cell-based assay. In addition, acousto-optic tunable filter (AOTF) can contribute greatly to the spectral overlap-free high-content monitoring. AOTF allows cellular imaging at particular single wavelengths devoid of spectral overlap among QDot-antibody conjugates. The crosstalk-free high-content monitoring of inflammation and insulin signal transduction was attempted using QDot-antibody conjugates and AOTF in this work. We investigated the effect of various inflammatory kinase inhibitors acting on insulin signaling and evaluated the compound efficacies for the functional ability to inhibit gluconeogenesis and enhance glycogen content, with the goal of identifying potential kinase for suppressing inflammation-induced insulin resistance. Our experiment also addresses the suitability of multicolor cellular imaging for studying kinase crosstalk operating in complex diseases, such as inflammation-associated insulin resistance, at a single shot in a single cell. Surprisingly, amygdalin and cinnamic acid acted selectively via the JNK1-dependent pathway to improve insulin sensitivity. The HepG2 cell line was chosen for this study as it is a perpetual, epithelial cell line derived from well-differentiated hepatocellular carcinoma. These cells solely depend upon exogenous growth factors for survival and respond to stimuli, such as TNF-α, adiponectin, leptin, etc. To study the causal role of elevated cytokines levels and MAP kinase inhibitors in hepatic insulin resistance, HepG2 cells were treated with inhibitors 30 min prior to stimulation with 10 ng/ml TNF-α for 5 h. The cells were then treated with 100 nM insulin for 10 min. Past studies using the obese TNF-α+/+ mice model have shown disturbances in insulin signaling in response to TNF-α [13]. Therapies that have successfully targeted TNF-α, including genetic silencing of the TNF-α or TNF receptor in mice, have demonstrated significant efficacy in the treatment of obesity-related insulin resistance. Therefore, we established the insulin resistance model in vitro using TNF-α stimulation of HepG2 cells. The glycogen amount decreased in the TNF-α treated group, suggesting compromised insulin sensitivity. The MAP kinase inhibitors suppressed the effect of TNF-α on the insulin transduction pathway and consequently resulted in elevated levels of glycogen content (Figure 3a). The inflammatory signaling inhibitors, such as p38α kinase inhibitors (SB203580), JNK1 inhibitors (SP600125), IKKβ inhibitors, aspirin, and indomethacin increased glycogen levels by almost two-fold in comparison to amygdalin and cinnamic acid. Glycogen content in HepG2 cells treated with p38, JNK1, IKKβ inhibitors, aspirin, and indomethacin were four to five times higher than that of the control; whereas, glycogen content in amygdalin or cinnamic acid-treated HepG2 cells were two to three times higher than that of the control [14]. As shown by a glucose production assay, exposure of HepG2 cells to inhibitors reduced gluconeogenesis in comparison to the control group (Figure 3b). Cinnamic acid was much more potent in suppressing glucose production than MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. The rate of hepatic glucose production by amygdalin was similar to that of aspirin.Figure 1

Bottom Line: Through the measurement of the glycogen level in HepG2 cell treated with TNF-α, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid.The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs.The results showed that amygdalin and cinnamic acid inhibit serine phosphorylation of IRS-1 through targeting JNK serine kinase and enhance insulin sensitivity.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, South Korea. jmsong@snu.ac.kr.

ABSTRACT

Background: TNF-α is an inflammatory cytokine that plays an important role in insulin resistance observed in obesity and chronic inflammation. Many cellular components involved in insulin signaling cascade are known to be inhibited by TNF-α. Insulin receptor substrate (IRS)-1 is one of the major targets in TNF-α-induced insulin resistance. The serine phosphorylation of IRS-1 enables the inhibition of insulin signaling. Until now, many studies have been conducted to investigate the mechanism of TNF-α-induced insulin resistance based on Western blot. Intracellular protein kinase crosstalk is commonly encountered in inflammation-associated insulin resistance. The crosstalk among the signaling molecules obscures the precise role of kinases in insulin resistance. We have developed a cell lysis-free quantum dots (QDots) multicolor cellular imaging to identify the biochemical role of multiple kinases (p38, JNK, IKKβ, IRS1ser, IRS1tyr, GSK3β, and FOXO1) in inflammation-associated insulin resistance pathway with a single assay in one run. QDot-antibody conjugates were used as nanoprobes to simultaneously monitor the activation/deactivation of the above seven intracellular kinases in HepG2 cells. The effect of the test compounds on the suppression of TNF-α-induced insulin resistance was validated through kinase monitoring. Aspirin, indomethacin, cinnamic acid, and amygdalin were tested.

Results: Through the measurement of the glycogen level in HepG2 cell treated with TNF-α, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid. The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. QDot multicolor cellular imaging demonstrated that amygdalin and cinnamic acid selectively acted via the JNK1-dependent pathway to suppress the inflammation-induced insulin resistance and improve insulin sensitivity.

Conclusion: The regulatory function of multiple kinases could be monitored concurrently at the cellular level. The developed cellular imaging assay provides a unique platform for the understanding of inflammation and insulin resistance signaling pathways in type II diabetes mellitus and how they regulate each other. The results showed that amygdalin and cinnamic acid inhibit serine phosphorylation of IRS-1 through targeting JNK serine kinase and enhance insulin sensitivity.

Show MeSH
Related in: MedlinePlus