Distinct pathways of cholesterol biosynthesis impact on insulin secretion

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   Figure 1

   Acute inhibition of glucose-stimulated insulin secretion (GSIS) by simvastatin (2 h) is not associated with a reduction in cholesterol content. (A) Insulin secretion from INS-1E cells after 2 h SIM treatment (n=6); (B) SIM 2 h treatment does not reduce total cellular cholesterol content (n=4). (C) Acute (2 h) ZGA treatment does not affect GSIS in INS-1E cells (n=6). *P<0.05.
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   Figure 2

   Simvastatin inhibits glucose-stimulated insulin secretion (GSIS) after an acute treatment by inhibiting a geranylgeranyl pyrophosphate-dependent mechanism. (A) Acute inhibition of GSIS by GGTi (10 μM) and its prevention in INS-1E cells pre-loaded with GGPP (20 μM) after acute SIM treatment (n=5). **P<0.01 and ***P<0.001 versus control. ^§P<0.05 versus SIM 1 μM. (B) Acute inhibition of GSIS by GGTi (10 μM) and its amelioration in mouse islets pre-loaded with GGPP (20 μM) after acute treatment with SIM (n=8). *P<0.05 and **P<0.01.
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   Figure 3

   Chronic (24 h) simvastatin and ZGA treatments reduce glucose-stimulated insulin secretion (GSIS) with concomitant reduction in total cellular cholesterol content. In INS-1E cells the chronic (24 h) treatment with SIM reduces the total cellular cholesterol content (A) (n=3) with a reduction in GSIS (B) (n=4). In INS-1E cells, the chronic (24 h) treatment with ZGA reduces cellular cholesterol (C) (n=3) with reduction in GSIS (D) (n=5). In mouse pancreatic islets, the chronic (24 and 48 h) treatment with ZGA reduces GSIS (E) (n=4) *P<0.05, **P<0.01, and ***P<0.001 versus control.
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   Figure 4

   Lipid raft membrane staining is affected by 24 h treatment with simvastatin, but not by 2 h treatment. (A) Representative images of lipid rafts in the plasma membranes of INS-1E cells stained with cholera-B toxin. Upper panel (left), lipid raft staining in control (untreated) cells; upper panel (right), lipid raft staining in cells treated with 1 μM simvastatin for 2 h; lower panel (left) lipid raft staining in cells treated with 1 μM simvastatin for 24 h; lower panel (right), lipid raft staining in cells treated with 1 mM MβCD for 1 h. (B) Mean fluorescence of lipid raft staining represented in (A) (30 ROIs each condition, from three independent experiments). Scale bars: 10 μm. ***P<0.001 vs control. The arbitrary units (AU) correspond to the sum of the gray values of all pixels in the ROI divided by the number of pixels (ImageJ User Guide, ImageJ/Fiji 1.46).
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   Figure 5

   Chronic 24-h simvastatin treatment increases membrane fluidity in INS-1E cells. (A) Mean traces are shown for two component spectra registered with 5-DSA as a probe to reflect rigid (cholesterol-rich lipid rafts) and fluid membrane microenvironments. Control (continuous black line, n=8), SIM for 2 h (dashed black line, n=8), SIM for 24 h (continuous gray line, n=8), and MβCD for 1 h (dashed gray line, n=8). (B) The rotational correlation time (T_corr) of 5-DSA was also calculated to evaluate membrane rigidity close to the hydrophilic surface of the phospholipid bilayer (n=8). (C) Analysis of the two-component spectra by calculating the rigid:fluid ratio of cell membranes (n=8). *P<0.05 vs control.
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   Figure 6

   Schematic representation of glucose-induced insulin secretion with the key points (enclosed by dashed lines) where cholesterol and the intermediates in its biosynthesis would be important. SIM, simvastatin; ZGA, zaragozic acid; GGPP, geranylgeranyl pyrophosphate; FPP, farnesyl pyrophosphate; Syn1A, syntaxin 1A.

• © 2015 Society for Endocrinology