Diabetes Metab J.  2020 Dec;44(6):919-927. 10.4093/dmj.2019.0181.

Hypoxia Increases β-Cell Death by Activating Pancreatic Stellate Cells within the Islet

• Kim JJ
• Lee E
• Ryu GR
• Ko SH
• Ahn YB
• Song KH

• KMID: 2513055
• DOI: http://doi.org/10.4093/dmj.2019.0181

Abstract

Hypoxia can occur in pancreatic islets in type 2 diabetes mellitus. Pancreatic stellate cells (PSCs) are activated during hypoxia. Here we aimed to investigate whether PSCs within the islet are also activated in hypoxia, causing β-cell injury.

Islet and primary PSCs were isolated from Sprague Dawley rats, and cultured in normoxia (21% O₂) or hypoxia (1% O₂). The expression of α-smooth muscle actin (α-SMA), as measured by immunostaining and Western blotting, was used as a marker of PSC activation. Conditioned media (hypoxia-CM) were obtained from PSCs cultured in hypoxia.

Islets and PSCs cultured in hypoxia exhibited higher expressions of α-SMA than did those cultured in normoxia. Hypoxia increased the production of reactive oxygen species. The addition of N-acetyl-L-cysteine, an antioxidant, attenuated the hypoxia-induced PSC activation in islets and PSCs. Islets cultured in hypoxia-CM showed a decrease in cell viability and an increase in apoptosis.

PSCs within the islet are activated in hypoxia through oxidative stress and promote islet cell death, suggesting that hypoxia-induced PSC activation may contribute to β-cell loss in type 2 diabetes mellitus.

Keyword

Hypoxia; Insulin-secreting cells; Islets of Langerhans; Oxidative stress; Pancreatic stellate cells

Figure

• 

  Fig. 1 Activation of pancreatic stellate cells (PSCs) in islets after hypoxia. (A) Images showing pimonidazole (green) staining of islets after incubation in normoxia or hypoxia for 2 hours. The nuclei were stained with 4′,6-diamidino-2-phenyl-indole (DAPI, blue). (B) Images showing α-smooth muscle actin (α-SMA) staining and the percentage of α-SMA-positive cells within the islet after incubation in normoxia or hypoxia for 12 hours. Arrows indicate cells expressing α-SMA (brown). Bar, 100 µm. (C) The expression of α-SMA in Western blot analysis. Values are mean±standard error of the mean (n=3). aP<0.05 for normoxia vs. hypoxia, bP<0.01 for 12 hours vs. 24 hours, cP<0.01 for 0 hour vs. 24 hours.

• 

  Fig. 2 Activation of pancreatic stellate cells (PSCs) after hypoxia among freshly isolated PSCs. Images showing α-smooth muscle actin (α-SMA) staining and the percentage of activated PSCs after the cells were incubated in hypoxia or normoxia for 48 hours. Bar, 20 µm. Values are presented as mean±standard error of the mean (n=4). DAPI, 4′,6-diamidino-2-phenyl-indole. aP<0.05 for normoxia vs. hypoxia.

• 

  Fig. 3 Involvement of oxidative stress in pancreatic stellate cell (PSC) activation after hypoxia. (A) Generation of reactive oxygen species. Images showing dichlorodihydrofluorescein diacetate (DCF) fluorescence (green) in PSCs after exposure to hypoxia or normoxia for 48 hours. DCF fluorescence was quantified using a scanning fluorometer. Bar, 200 µm. Values are presented as mean±standard error of the mean (n=6). (B) Western blot analysis showing the expression of α-smooth muscle actin (α-SMA) in islets after hypoxia in the presence or absence of treatment with 2.5 mM N-acetyl-L-cysteine (NAC). Values are presented as mean±standard error of the mean (n=3). (C) Representative images of α-SMA staining in primary PSCs cultured in hypoxia with or without 2.5 mM NAC for 48 hours. Bar, 20 µm. DAPI, 4′,6-diamidino-2-phenyl-indole. aP<0.05 for normoxia vs. hypoxia, bP<0.01 for 0 hr (0 hour) vs. 24 hr (24 hours), cP<0.05 for 24 hours vs. 24 hours+NAC.

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  Fig. 4 Effect of pancreatic stellate cell (PSC) activation on islet viability. (A) Representative images of acridine orange (AO, green)/propidium iodide (PI, red) staining and quantification of PI-positive cells in rat islets incubated in PSC media and normoxia-conditioned media (CM) and hypoxia-CM from PSCs for 48 hours. Bar, 200 µm. (B) Representative images of AO/PI staining in rat islets incubated in PSC media and normoxia-CM and hypoxia-CM from C2C12 cells for 48 hours. Bar, 200 µm. (C) Representative images of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay and quantification of TUNEL-positive cells in rat islets incubated in PSC media and normoxia-CM and hypoxia-CM from PSCs for 48 hours. The nuclei were stained with 4′,6-diamidino-2-phenyl-indole (DAPI, blue). Bar, 100 µm. Values are presented as mean±standard error of the mean (n=3). aP<0.05 for PSC media vs. hypoxia-CM, bP<0.05 for PSC media vs. normoxia-CM, cP<0.05 for PSC media vs. hypoxia-CM and for normoxia-CM vs. hypoxia-CM.

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