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, 98 (1), 54-66

Hyperglycemia Aggravates Acute Liver Injury by Promoting Liver-Resident Macrophage NLRP3 Inflammasome Activation via the Inhibition of AMPK/mTOR-mediated Autophagy Induction

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Hyperglycemia Aggravates Acute Liver Injury by Promoting Liver-Resident Macrophage NLRP3 Inflammasome Activation via the Inhibition of AMPK/mTOR-mediated Autophagy Induction

Qi Wang et al. Immunol Cell Biol.

Abstract

Although the detrimental effects of diabetes mellitus/hyperglycemia have been observed in many liver disease models, the function and mechanism of hyperglycemia regulating liver-resident macrophages, Kupffer cells (KCs), in thioacetamide (TAA)-induced liver injury remain largely unknown. In this study, we evaluated the role of hyperglycemia in regulating NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome activation by inhibiting autophagy induction in KCs in the TAA-induced liver injury model. Type I diabetes/hyperglycemia was induced by streptozotocin treatment. Compared with the control group, hyperglycemic mice exhibited a significant increase in intrahepatic inflammation and liver injury. Enhanced NLRP3 inflammasome activation was detected in KCs from hyperglycemic mice, as shown by increased gene induction and protein levels of NLRP3, cleaved caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain and interleukin-1β, compared with control mice. NLRP3 inhibition by its antagonist CY-09 effectively suppressed inflammasome activation in KCs and attenuated liver injury in hyperglycemic mice. Furthermore, inhibited autophagy activation was revealed by transmission electron microscope detection, decreased LC3B protein expression and p-62 protein degradation in KCs isolated from TAA-stressed hyperglycemic mice. Interestingly, inhibited 5' AMP-activated protein kinase (AMPK) but enhanced mammalian target of rapamycin (mTOR) activation was found in KCs from TAA-stressed hyperglycemic mice. AMPK activation by its agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or mTOR signaling knockdown by small interfering RNA restored autophagy activation, and subsequently, inhibited NLRP3 inflammasome activation in KCs, leading to ultimately reduced TAA-induced liver injury in the hyperglycemic mice. Our findings demonstrated that hyperglycemia aggravated TAA-induced acute liver injury by promoting liver-resident macrophage NLRP3 inflammasome activation via inhibiting AMPK/mTOR-mediated autophagy. This study provided a novel target for prevention of toxin-induced acute liver injury under hyperglycemia.

Keywords: AMPK; mTOR; Kupffer cell; NLRP3 inflammasome; autophagy; hyperglycemia; liver injury; thioacetamide.

Conflict of interest statement

All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Hyperglycemia exacerbates thioacetamide (TAA)‐induced acute liver injury. Diabetic streptozotocin (STZ) and control (CON) mice were prepared as described in the “Methods” section. TAA‐induced acute liver injury or a sham procedure was performed. (a) Blood glucose levels were measured in different groups (n = 6 mice/group). (bd) Serum alanine aminotransferase (sALT) and aspartate aminotransferase (sAST) levels (n = 6 mice/group) and liver histopathology (representative of six experiments) were used to evaluate the liver injury at 24 h after TAA administration. (e) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of the liver sections (200× magnification, representative of six mice/group). (f) The relative ratio of TUNEL‐positive cells in different groups (n = 6 mice/group). (g) The levels of Bcl‐2, Bcl‐xL and β‐actin proteins were measured by western blot. Representative of three experiments. *P < 0.05. DAPI, 4′,6‐diamidino‐2‐phenylindole.
Figure 2
Figure 2
Hyperglycemia induces the expression of the NLRP3 inflammasome in Kupffer cells (KCs) in thioacetamide (TAA)‐induced acute liver injury mice. Mice were subjected to streptozotocin (STZ) pretreatment and TAA administration as described in the “Methods” section. KCs were isolated from different experimental groups. (a) The expression of proinflammatory genes in KCs was measured by quantitative real‐timePCR (n = 6 mice/group). (b) Isolated KCs from different experimental groups were cultured for 6 h. Interleukin‐1 β (IL‐1β) and IL‐18 protein levels were measured in the culture supernatant by ELISA (n = 6 mice/group). (c) The levels of intracellular NLRP3, cleaved caspase‐1, procaspase‐1, ASC, IL‐1β, pro‐IL‐1β and β‐actin proteins were measured by western blot (representative of three experiments). (d) Immunofluorescence staining of NLRP3 in KCs (200× magnification, representative of three experiments). Diabetic and control mice were treated with the NLRP3 inhibitor CY‐09 (20 mg kg−1, intraperitoneally) once a day for 7 days prior to TAA administration. (e) Isolated KCs from different experimental groups were cultured for 6 h. IL‐1β and IL‐18 protein levels were measured in the culture supernatant by ELISA (n = 6 mice/group). (f) The levels of intracellular NLRP3, cleaved caspase‐1, procaspase‐1, ASC, IL‐1β, pro‐IL‐1β and β‐actin proteins were detected by western blot (representative of three experiments). (g) Immunofluorescence staining of NLRP3 in KCs (200× magnification, representative of three experiments). *P < 0.05. CON, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; mRNA, messenger RNA; NLRP3, NOD‐like receptor family pyrin domain‐containing 3 protein.
Figure 3
Figure 3
NLRP3 inhibitor treatment alleviates thioacetamide (TAA)‐induced acute liver injury in hyperglycemic mice. (a–c) Serum alanine aminotransferase (sALT), aspartate aminotransferase (sAST) levels (n = 6 mice/group) and liver histopathology (representative of six experiments) were used to evaluate liver injury in diabetic mice and controls after treatment with CY‐09 and TAA. (d) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of liver sections (200× magnification, representative of six experiments). (e) The ratio of TUNEL‐positive cells in different experimental groups (n = 6 mice/group). (f) The levels of Bcl‐2, Bcl‐xL and β‐actin proteins were measured by western blot (representative of three experiments). *P < 0.05. CON, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; HPF, high‐power field; NLRP3, NOD‐like receptor family pyrin domain‐containing 3 protein; STZ, streptozotocin.
Figure 4
Figure 4
Hyperglycemia inhibits mammalian target of rapamycin (mTOR)‐mediated autophagy in Kupffer cells (KCs) post‐ thioacetamide (TAA) treatment. (a) After 24 h of TAA treatment, KCs were isolated, and intracellular p‐mTOR, mTOR, LC3B, p62 and β‐actin protein levels were detected by western blot (representative of three experiments). (b) The expression of LC3B in KCs was measured by immunofluorescence staining (200× magnification, representative of three experiments). Diabetic and control mice were injected with mannose‐conjugated mTOR‐siRNA (2 mg kg−1) via the tail vein at 4 h prior to TAA treatment. (c) The knockdown efficiency of mannose‐conjugated mTOR‐siRNA was measured by PCR (n = 6 mice/group). (d) Immunofluorescence staining of LC3B in KCs from different experimental groups (200× magnification, representative of three experiments). (e) Autophagic microstructures in cells were detected by transmission electron microscopy ( images are representative of those from three experiments). The areas enclosed within black squares were further amplified (1200× and 5000× magnification; scale bars, 5 and 2 μm). (f) The levels of intracellular p‐mTOR, mTOR, LC3B, p62, NLRP3, cleaved caspase‐1, procaspase‐1, ASC, interleukin‐1β (IL‐1β), pro‐IL‐1β and β‐actin proteins were detected by western blot (representative of three experiments). (g) The expression of proinflammatory genes in KCs was measured by quantitative real‐timePCR (n = 6 mice/group). (h) Isolated KCs from different experimental groups were cultured for 6 h. IL‐1β and IL‐18 protein levels were measured in the culture supernatant by ELISA (n = 6 mice/group). *P < 0.05. CON, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; mRNA, messenger RNA; siRNA, small interfering RNA; STZ, streptozotocin.
Figure 5
Figure 5
Mammalian target of rapamycin (mTOR) knockdown in Kupffer cells (KCs) alleviates thioacetamide (TAA)‐induced acute liver injury. (a–c) Serum alanine aminotransferase (sALT), aspartate aminotransferase (sAST) levels (n = 6 mice/group) and liver histopathology (representative of six experiments) were used to evaluate liver injury in diabetic mice and controls after treatment with mTOR‐siRNA and TAA. (d) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of liver sections (200× magnification, representative of six experiments). (e) The ratio of TUNEL‐positive cells in different experimental groups (n = 6 mice/group). (f) The levels of Bcl‐2, Bcl‐xL and β‐actin proteins were measured by western blot (representative of three experiments). *P < 0.05. HPF, high‐power field; sALT, serum alanine aminotransferase; sAST, aspartate aminotransferase; siRNA, small interfering RNA; STZ, streptozotocin.
Figure 6
Figure 6
The inhibition of 5′ AMP‐activated protein kinase (AMPK) under hyperglycemic conditions suppresses mammalian target of rapamycin (mTOR)‐dependent autophagy and promotes the expression of the NLRP3 inflammasome in Kupffer cells (KCs). (a) The levels of intracellular p‐AMPK and β‐actin proteins were measured by western blot (representative of three experiments). Diabetic mice and controls were subjected to AMPK activator (AICAR, 100 mg kg−1, intraperitoneally) treatment once a day for 7 days prior to thioacetamide (TAA) administration. (b) The levels of intracellular p‐AMPK, AMPK, p‐mTOR, mTOR, LC3B, p62 and β‐actin proteins were detected by western blot (representative of three experiments). (c) The detection of autophagic microstructures in KCs by transmission electron microscopy; the areas enclosed within black squares were further amplified (1200× and 5000× magnification; scale bars, 5 and 2 μm; representative of three experiments). (d and e) Immunofluorescence staining of NLRP3 and LC3B in KCs (200× magnification; representative of three experiments). (f) The levels of intracellular NLRP3, cleaved caspase‐1, procaspase‐1, ASC, interleukin‐1β (IL‐1β), pro‐IL‐1β and β‐actin proteins were measured by western blot (representative of three experiments). (g) The expression of proinflammatory genes in KCs was detected by quantitative real‐timePCR (n = 6 mice/group). (h) Isolated KCs from different experimental groups were cultured for 6 h. IL‐1β and IL‐18 protein levels were measured in the culture supernatant by ELISA (n = 6 mice/group). *P < 0.05. AICAR, 5‐aminoimidazole‐4‐carboxamide ribonucleotide; CON, control; DAPI, 4′,6‐diamidino‐2‐phenylindole; mRNA, messenger RNA; NLRP3, NOD‐like receptor family pyrin domain‐containing 3 protein; STZ, streptozotocin.
Figure 7
Figure 7
5′ AMP‐activated protein kinase (AMPK) activator (AICAR) treatment alleviates thioacetamide (TAA)‐induced acute liver injury in hyperglycemic mice. (a–c) Serum levels of aspartate transaminase (sAST) and alanine aminotransferase (sALT; n = 6 mice/group) and liver histopathology (representative of six experiments) were used to evaluate liver injury in diabetic mice and controls post‐AICAR and TAA treatment. (d) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of liver sections (200× magnification, representative of six experiments). (e) The relative ratio of TUNEL‐positive cells in different groups (n = 6 mice/group). (f) The levels of Bcl‐2, Bcl‐xL and β‐actin proteins were measured by western blot (representative of three experiments). *P < 0.05. AICAR, 5‐aminoimidazole‐4‐carboxamide ribonucleotide; CON, control; NLRP3, NOD‐like receptor family pyrin domain‐containing 3 protein; STZ, streptozotocin.

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