Dataset Information

Perilipin 5-mediated lipid droplet-mitochondria coupling determines mitochondrial capacity

ABSTRACT: Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity requiring tight regulation of intracellular lipid metabolism and fatty acid (FA) flux. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LD) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with Adipose triglyceride lipase (ATGL) and the ATGL co-activator Comparative gene identification-58 (CGI-58). Furthermore, PLIN5 anchors mitochondria to the LD membrane via its carboxyl-terminal domain. However, the role of this LD-mitochondria coupling (LDMC) in cellular FA flux and energy catabolism is less established. In this study, we investigated the impact of abolished LDMC on cellular FA flux, mitochondrial respiration and protein interaction. To do so, we established novel PLIN5 truncation variants lacking LDMC while maintaining normal interactions with lipolytic key players. Radiotracer studies with transgenic cell lines stably overexpressing either wild type or truncated PLIN5 revealed that LDMC has no significant impact on FA esterification upon lipid loading or TG catabolism during stimulated lipolysis. Moreover, we found that LDMC is not essential for FA shuttling into mitochondria, which was in line with only minor effects of disrupted LDMC on FA oxidation. In contrast, LDMC significantly improved the mitochondrial respiratory capacity and metabolic flexibility of lipid-challenged cardiomyocytes, which was corroborated by LDMC-dependent interactions of PLIN5 with mitochondrial proteins involved in mitochondrial respiration, dynamics and cristae organization. Taken together, this study suggests that PLIN5 preserves mitochondrial function by adjusting FA supply via the regulation of TG hydrolysis and that LDMC is a vital part of mitochondrial integrity.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Heart, Cardiocyte

SUBMITTER: Natalia Kunowska

LAB HEAD: Urlich Stelzl

PROVIDER: PXD028541 | Pride | 2022-02-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	A_L1_dia_RD1_1_1897.d.zip	Other
	A_L2_dia_RD2_1_1898.d.zip	Other
	A_L3_dia_RD3_1_1899.d.zip	Other
	A_P1_dia_RD4_1_1901.d.zip	Other
	A_P2_dia_RD5_1_1902.d.zip	Other

Items per page:

1 - 5 of 40

Publications

Lipid droplet-mitochondria coupling via perilipin 5 augments respiratory capacity but is dispensable for FA oxidation.

Kien Benedikt B Kolleritsch Stephanie S Kunowska Natalia N Heier Christoph C Chalhoub Gabriel G Tilp Anna A Wolinski Heimo H Stelzl Ulrich U Haemmerle Guenter G

Journal of lipid research 20220121 3

Disturbances in lipid homeostasis can cause mitochondrial dysfunction and lipotoxicity. Perilipin 5 (PLIN5) decorates intracellular lipid droplets (LDs) in oxidative tissues and controls triacylglycerol (TG) turnover via its interactions with adipose triglyceride lipase and the adipose triglyceride lipase coactivator, comparative gene identification-58. Furthermore, PLIN5 anchors mitochondria to the LD membrane via the outermost part of the carboxyl terminus. However, the role of this LD-mitocho ...[more]

PMID: 35065923

Similar Datasets

Project description:Methionine adenosyltransferase (MAT) enzymes generate SAMe (S-adenosylmethionine), the main biological methyl donor. There are two MAT encoding genes in mammals (Mat1a and Mat2a), which show different activities and cellular distribution. Mat1a encodes the enzyme mainly expressed in normal liver. Mat1a ablation in mice results in the spontaneous development of non-alcoholic steatohepatitis (NASH). We observed that SAMe depletion in Mat1a KO mice had three main effects on hepatic lipid metabolism: 1) impaired TG (triglyceride) export via VLDL; 2) impaired mitochondrial FA (fatty acid) oxidation (as evidenced by membrane depolarization, downregulation of Phb1 (prohibitin 1, a mitochondrial chaperone protein) and Mcj/Dnajc15 (endogenous mitochondrial repressor of respiratory chain), and accumulation of long-chain acylcarnitines); and 3) increased FA uptake. The convergence of these three factors induced TG accumulation in LD (lipid droplets). LD expansion confronts hepatocytes with a high demand of PC (phosphatidylcholine) molecules to cover the LD surface since other phospholipids, such as PE (phosphatidylethanolamine), cannot stabilize LD and prevent coalescence. In Mat1a KO this situation is aggravated, since SAMe-dependent PC synthesis via PE methylation is decreased, the PC/PE ratio reduced and mitochondrial FA oxidation impaired. To put a brake to this drain of PC molecules to LD, FA are rerouted in Mat1a KO mice liver to other catabolic (endoplasmic reticulum and peroxisome oxidation) and biosynthetic (ceramides synthesis) pathways, causing oxidative stress, inflammation and fibrosis. SAMe treatment for two months in 8-9 month old Mat1a KO mice ameliorated mitochondrial dysfunction (reduces membrane depolarization, improves Phb1 and Mcj expression, and increases SAMe transport to mitochondria) improving FA oxidation efficiency (FA and acylcarnitine levels decrease), which results in a drastic reduction in TG accumulation. SAMe treatment in Mat1a KO mice resulted in more PC available for proper membrane function, improving liver lipid homeostasis, histology (H&E, Sudan red, Sirius red) and liver injury (ALT, AST).

Project description:Aims/hypothesis: While lipid deposition in skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like skeletal muscle. Here we investigated the effects of PLIN5 overexpression M-bM-^@M-^S in comparison with effects of PLIN2 M-bM-^@M-^S on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in skeletal muscle promoted expression of a cluster of genes under control of PPARM-NM-1 and PGC1M-NM-1 involved in FA catabolism and mitochondrial oxidation. Rats received a high fat diet for 3 weeks; 2 weeks after start of the diet intervention Plin5 (OXPAT) or Plin2 (ADRP) were overexpressed in either the right or left tibialis anterior muscle. One week later pooled tibialis anterior muscle samples were analysed on microarrays.

Project description:Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in tissues that are active in fatty acid oxidation such as the heart, oxidative muscle, the liver, and brown adipocytes. PLIN5 shares homology with PLIN1: it prevents lipolysis in basal state but promotes lipolysis upon the activation of protein kinase A (PKA). In vitro studies expressing exogenous PLIN5 have shown that PLIN5 is phosphorylated at serine 155 (S155) by PKA. In addition to the regulation of lipolysis, phosphorylation of PLIN5 at S155 has been shown to regulate expression of genes in lipid metabolism, mitochondria, inflammation, and autophagy by traveling to the nucleus and activating peroxisome proliferater-activated-receptor-gamma-coactivator-1-α (PGC1a). However, a role of PLIN5 phosphorylation at S155 has been primarily studied in vitro with exception of a few studies expressing exogenous wild type (WT) and phosphorylation resistant PLIN5 (S155A) in the heart or the liver of mice. Here, we aim to determine the extent of endogenous PLIN5 phosphorylation in vivo and its role in the regulation of lipid metabolism and gene expression in the liver using PLIN5 S155A knock in mice (S155A). First, we enriched LDs from the liver of fed and fasted C57BL6 WT mice to measure non-phosphorylated and S155 phosphorylated PLIN5 by LC-MS/MS. While peak area of non-phosphorylated PLIN5 was similar, S155 phosphorylation was increased in the liver from fasted mice compared with fed mice confirming that endogenous PLIN5 is phosphorylated at S155 when PKA is activated during fasting (3.0-fold increase, p<0.05). Then, we utilized Phos-tag gel as a simple method to assess the extent of PLIN5 phosphorylation. When the heart and the liver from WT and S155A mice were run on Phos-tag gel and immunoblotted for PLIN5, a phosphorylated band was significantly reduced in S155A tissues confirming that S155 is the dominant phosphorylation site in vivo. Next, we assessed phenotypes of S155A mice to gauge the necessity of S155 phosphorylation for metabolism and gene expression in the fasted liver. For the glucose tolerance test, 4-month-old male and female S155A mice showed no differences, but one year-old S115A males had impaired glucose tolerance. There was not a significant difference in weight or blood glucose in fasted young S155A females compared to wild type, but aged male S155A mice had a higher fasted blood glucose (p<0.05) without difference in weight. There was not a significant difference in serum insulin, triglycerides (TG), beta-hydroxybutyrate, or non-esterified fatty acids levels between WT and S155A mice in either males or females. The liver TG contents did not differ between WT and S155A mice of both genders. When qPCR tested the liver of fasted WT and S155A mice, Pgc1a target genes upregulated by fasting and proposed to be regulated by PLIN5 phosphorylation did not show striking difference in expression. Pgc1a, Ppara, Acot1, Pdk4, and Ascl1 expression was similar between WT and S155A liver of fasted mice. Cpt1a and G6pc showed trend of reduction in S155A liver but did not reach statistical significance (n=5 to 7). Inflammation markers such as IL1b and Ccl2 did not differ between WT and S155A liver of fasted mice either. We performed unbiased RNA sequencing of WT and S155A liver from fasted female mice to identify genes differentially regulated by phosphorylation of PLIN5 at S155. There were limited number of genes differentially expressed in S155A female livers with 3 genes < 0.05 for adjusted p value. Interestingly, RNA seq identified insulin receptor substrate 1 (Irs1) as a gene with significantly decreased expression in the liver of female S155A mice (Log2 fold change -0.74, adjusted p value 0.003). The difference was confirmed in qPCR of fasted liver for the female mice (p<0.05), but not for males. Interestingly, insulin receptor substrate 2 (Irs2) measured by qPCR was not different in the liver of female S155A mice but was reduced in male S155A mice (p<0.05). The reduction of IRS2 protein in the liver of S155A male mice was confirmed by Western blot. In conclusion, endogenous PLIN5 in the liver increases phosphorylation at S155 upon fasting in mice. However, the loss of S155 phosphorylation does not affect serum lipids or liver TG. Also, changes in the expression of PGC1a target genes were subtle in the liver of fasted S155A mice compared with WT mice indicating that the loss of PLIN5 S155 phosphorylation can be readily compensated by other mechanisms. Unexpectedly, IRS2 showed decreased expression in the liver of S155A male mice that may explain glucose intolerance in aged male mice. Thus, PLIN5 phosphorylation may impact the insulin signaling in the liver by supporting IRS2 expression in male mice.