Perilipin 2 downregulation in β cells impairs insulin secretion under nutritional stress and damages mitochondria
Akansha Mishra, Siming Liu, Joseph Promes, Mikako Harata, William Sivitz, Brian Fink, Gourav Bhardwaj, Brian T. O’Neill, Chen Kang, Rajan Sah, Stefan Strack, Samuel Stephens, Timothy King, Laura Jackson, Andrew S. Greenberg, Frederick Anokye-Danso, Rexford S. Ahima, James Ankrum, Yumi Imai
Akansha Mishra, Siming Liu, Joseph Promes, Mikako Harata, William Sivitz, Brian Fink, Gourav Bhardwaj, Brian T. O’Neill, Chen Kang, Rajan Sah, Stefan Strack, Samuel Stephens, Timothy King, Laura Jackson, Andrew S. Greenberg, Frederick Anokye-Danso, Rexford S. Ahima, James Ankrum, Yumi Imai
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Research Article Endocrinology Metabolism

Perilipin 2 downregulation in β cells impairs insulin secretion under nutritional stress and damages mitochondria

Abstract

Perilipin 2 (PLIN2) is a lipid droplet (LD) protein in β cells that increases under nutritional stress. Downregulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was downregulated in mouse β cells, INS1 cells, and human islet cells. β Cell–specific deletion of PLIN2 in mice on a high-fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Downregulation of PLIN2 in INS1 cells blunted GSIS after 24-hour incubation with 0.2 mM palmitic acid. Downregulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Downregulation of PLIN2 decreased specific OXPHOS proteins in all 3 models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2-deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress, as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells has an important role in preserving insulin secretion, β cell metabolism, and mitochondrial function under nutritional stress.

Authors

Akansha Mishra, Siming Liu, Joseph Promes, Mikako Harata, William Sivitz, Brian Fink, Gourav Bhardwaj, Brian T. O’Neill, Chen Kang, Rajan Sah, Stefan Strack, Samuel Stephens, Timothy King, Laura Jackson, Andrew S. Greenberg, Frederick Anokye-Danso, Rexford S. Ahima, James Ankrum, Yumi Imai

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Figure 5

Mitochondrial dysfunction in PLIN2-deficient INS1 cells.

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Mitochondrial dysfunction in PLIN2-deficient INS1 cells. (A–E) Oxygen co...
(A–E) Oxygen consumption rate (OCR) corrected for DNA contents in Scr- and SiPLIN2-treated INS1 cells. (A) OCR profile representative of 4 experiments each performed in quadruplicates. (B–E) Glucose response (B), ATP production (C), maximal respiration (D), and percentage of proton leak (E). Each dot is an average value of 1 experiment. n = 4. (F) Insulin secretion measured with or without 0.5 mM D-Mannoheptulose overnight as in Methods and corrected for total insulin contents. n = 7, representative of 3 experiments. (G) Insulin secretion measured with or without 1 μM nifedipine for 2 hours as in Methods, corrected for total insulin contents, and expressed taking average of Scr 2.5 mM glucose as 1. n =12 for Scr 2.5 mM, 6 for Scr 12 mM, and 9 for SiPLIN2. Representative of 3 experiments. (F and G) #P < 0.05 versus Scr 2.5 mM glucose and &P < 0.05 versus Scr 12 mM glucose by 1-way ANOVA with Sidak’s post hoc test. (H) Western blot of OXPHOS complex proteins were performed in protein lysate of INS1 cells transfected by Scr and SiPLIN2, treated with or without 0.2 mM palmitic acids (PA) for 24 hours prior to harvest. Densitometry data were corrected for Ponceau S staining and expressed taking average value of Scr without PA as 1 for each complex. n = 8 for no PA and 4 for PA-treated INS1 cells. #P < 0.05 versus Scr without PA. A representative blot is shown in Supplemental Figure 4E. (I) qPCR probed DNA from Scr- and SiPLIN2-treated INS1 cells for mitochondrial DNA (mt-ND6) and nuclear DNA (β actin). Expression of mt-ND6 was corrected for β actin, and the average of value for Scr was taken as 100%. n = 8 combined from 3 independent experiments. All data are mean ± SEM. *P < 0.05 by Student’s t test.