miR-375 enhances palmitate-induced lipoapoptosis in insulin-secreting NIT-1 cells by repressing myotrophin (V1) protein expression

Abstract

Lipoapoptosis of pancreatic beta cells caused by elevated circulating free fatty acids (FFAs) has now been recognized to be a pivotal factor contributing to beta cellular dysfunction and beta-mass lose in type 2 diabetes. Although recent studies suggested an important role for the ceramide pathway in the late destructive phase of lipid overload in the pancreatic beta cells, the overall underlying mechanisms leading to lipoapoptosis, however, remained poorly understood. mir-375 was recently characterized to be a pancreatic islet-specific miRNA implicated in the regulation of insulin secretion and beta-mass turnover. In the present study we further examined its effect on palmitate-induced lipoapoptosis in NIT-1 cells, a NOD-derived beta-cell line. It was found that NIT-1 cells with ectopic mir-375 expression were much more susceptible to palmitate-induced lipoapoptosis. In contrast, knockdown of endogenous pri-mir-375 expression by a modified antisense oligo, 2'-O-me-375, almost completely protected NIT-1 cells from palmitate-induced lipoapoptosis. We further demonstrated that mir-375 could target V1 mRNA and repress its translation. Consistent with this assumption, NIT-1 cells transfected with 2'-O-me-375 showed significant higher levels of V1 protein after palmitate induction. Together, our data suggest that mir-375 could be a potential therapeutic target for prevention and intervention of beta-cell dysfunction and beta-mass lose in type 2 diabetes.

Keywords: Lipoapoptosis; NIT-1 cells; mir-375; type 2 diabetes; β-cell dysfunction; β-mass.

Figure 1

Palmitate is potent to induce NIT-1 cell lipoapoptosis. A. A representative results for TUNEL staining of apoptotic NIT-1 cells. B. A representative results for flow cytometry analysis of apoptotic NIT-1 cells. C. A bar graph showing the average apoptosis of NIT-1 cells cultured in the presence of palmitate and control medium. The data are present as mean ± SD of three independent experiments performed.

Figure 2

2'-O-Me-375 is potent to repress endogenous pri-mir-375 expressions in NIT-1 cells. The relative expression levels for pri-mir-375 were determined by real-time PCR as described. The expression levels of pri-mir-375 in NIT-1 cells cultured with control medium (Normal) were considered as 100%, its relative expression levels in cells transfected with inhibitor-NC, 2'-O-Me-375, oligo-2 and oligo-3 are present as a ratio with that of control cells. The data were derived from three replicates.

Figure 3

Ectopic mir-375 duplexes enhance palmitate-induced NIT-1 cell lipoapoptosis. A. A representative results for TUNEL staining of apoptotic NIT-1 cells. B. A representative results for flow cytometry analysis of apoptotic NIT-1 cells. C. A bar graph showing the average apoptosis of NIT-1 cells transfected with mir-375-NC, mir-375 duplexes and 2'-O-Me-375. NIT-1 cells cultured with normal medium (Normal) or in the presence of same amount Lipofactamine 2000 (lipo2000) were served as controls. The data are present as mean ± SD of three independent experiments performed.

Figure 4

Palmitate treatment is associated with a significant reduction of V1 protein in NIT-1 cells. Left: A representative of Western blot results for V1 protein levels in NIT-1 cells treated with palmitate or control medium. Right: A bar graph showing the relative V1 expression levels in NIT-1 cells treated with palmitate. The relative intensity of each band was determined as a ratio with its corresponding β-actin band. V1 relative expression levels are present as fold changes compared with control cells. The dada are present as mean ± SD of three independent experiments performed.

Figure 5

Ectopic mir-375 duplexes significantly reduce V1 protein levels in NIT-1 cells. NIT-1 cells were harvested after 72 h of transfection, and cell lysates were prepared and subjected to Western blot analysis of V1 protein expressions. Upper panel: A representative of Western blot results for V1 protein levels in NIT-1 cells cultured with control medium or transfected with mir-375-NC, mir-375 duplexes and 2'-O-Me-375, respectively. Lower panel: A bar graph showing the relative V1 protein levels in each above indicated experimental group. Similar as Figure 4, V1 relative protein levels are present as fold changes compared with that of control cells (Normal). The dada are present as mean ± SD of three independent experiments performed.

Figure 6

Repression of endogenous pri-mir-375 expression prevents palmitate-induced V1 reduction in NIT-1 cells. NIT-1 cells after 72 h of transfection were treated with 500μM of palmitate for 48 h, followed by Western blot analysis of cell lysates for V1 protein expression. Upper panel: A representative of Western blot results for V1 protein levels in each indicated transfected NIT-1 cells after palmitate treatment. Lower panel: A bar graph showing the relative V1 protein levels in each transfected NIT-1 cells after palmitate induction. Similarly, V1 relative protein levels are present as fold changes compared with that of control cells (Normal). The dada are present as mean ± SD of three independent experiments performed. It was found that repression of endogenous pri-mir-375 expressions by 2'-O-Me-375 prevented NIT-1 cells from palmitate-induced V1 reduction.