Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Brettschneider, Jascha; Correnti, Jason; Lin, Chun-Yao; Williams, Bianca; Oranu, Amanke; Kuriakose, Amy; McIver-Jenkins, Dru; Haba, Abigail; Kaneza, Isabelle; Jeon, Sookyoung; Scorletti, Eleonora; Carr, Rotonya M.

doi:10.3791/59290

Cited by 6 publications

(6 citation statements)

References 16 publications

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“…To address this, we measured ceramides in purified lysosomes, ERs, and LDs (supplemental Fig. S3) from Etoh-Dox-fed mice using a protocol recently described by our laboratory that demonstrates our ability to isolate these organelles without contamination (26). As anticipated, induction of the lysosomal protein ASAH1 reduced lysosomal ceramide levels by approximately 25% (Fig.…”

Section: Asah Induction Decreases Ld Ceramides and Increases Lipophagymentioning

confidence: 90%

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Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Correnti

Lin

Brettschneider

et al. 2020

Journal of Lipid Research

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Alcohol’s impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol’s inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD.

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Section: Asah Induction Decreases Ld Ceramides and Increases Lipophagymentioning

confidence: 90%

“…Whole and fractionated liver samples were normalized to protein and equal serum volumes were measured. Isolation of LDs, lysosomes, and ER for ceramide analysis was performed as described (26).…”

Section: Lipid Analysesmentioning

confidence: 99%

Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Correnti

Lin

Brettschneider

et al. 2020

Journal of Lipid Research

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“…Another limitation is that the current method for LD lipid analysis focused on an individual (single LD) rather than a general view, which might also be a possible explanation for how the supplementation of oxLDL and oxHDL (especially for the longer preparation time) resulted in a considerable intragroup deviation. An effective alternative strategy is to isolate the total LD fraction from the cells using kits [ 53 ], but it is cumbersome and time consuming, which is risky for analyzing the susceptible oxidized lipids. Nevertheless, both oxLDL and oxHDL exhibited the potential to cause LD lipid peroxidation, which could therefore be proposed as prospective markers for indicating oxidative stress in CKD.…”

Section: Resultsmentioning

confidence: 99%

Oxidative Stress and Lipid Dysregulation in Lipid Droplets: A Connection to Chronic Kidney Disease Revealed in Human Kidney Cells

et al. 2022

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Chronic kidney disease (CKD), which is defined as a condition causing the gradual loss of kidney function, shows renal lipid droplet (LD) accumulation that is associated with oxidative damage. There is a possibility that an LD abnormality in quality plays a role in CKD development. This study aimed to explore the chemical composition of LDs that are induced in human kidney cells during exposure to free fatty acids as an LD source and oxidized lipoproteins as oxidative stress. The LDs were aspirated directly from cells using nanotips, followed by in-tip microextraction, and the LD lipidomic profiling was conducted using nanoelectrospray mass spectrometry. As a result, the free fatty acids increased the LD lipid content and, at the same time, changed their composition significantly. The oxidized lipoproteins caused distorted proportions of intact lipids, such as triacylglycerols (TG), phosphatidylcholines (PC), phosphatidylethanolamines (PE), and cholesteryl esters (CE). Notably, the oxidized lipids, including the hydroperoxides of TG, PC, and PE, exhibited significant elevations in dose-dependent manners. Furthermore, the dysregulation of intact lipids was paralleled with the accumulation of lipid hydroperoxides. The present study has revealed that the oxidation of lipids and the dysregulation of the lipid metabolism coexisted in LDs in the kidney cells, which has provided a potential new target for diagnosis and new insights into CKD.

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“…LDs have a low density due to their neutral lipid core allowing researchers to initially separate cell debris and nuclei from less dense cellular components using highspeed centrifugation followed by ultracentrifugation to isolate the floating, white lipid layer on top of the solution. The first method developed was to use density gradient centrifugation with sucrose [176][177][178][179][180][181] or Ficoll. 178 Recently, we demonstrated that an organelle isolation kit could be used to separate LDs from other organelles, such as the ER and lysosomes in the mouse liver.…”

Section: Ld Isolation and Characterizationmentioning

confidence: 99%

“…178 Recently, we demonstrated that an organelle isolation kit could be used to separate LDs from other organelles, such as the ER and lysosomes in the mouse liver. 181 This allows direct analysis of LD-associated cellular components within the same samples. After isolation, LDs are washed and fraction purity is checked by western blotting using antibodies that are specific to different subcellular compartments, 182 such as PLIN2 for LDs.…”

Section: Ld Isolation and Characterizationmentioning

confidence: 99%

Mechanisms of Lipid Droplet Accumulation in Steatotic Liver Diseases

Dempsey,

Ioannou,

Carr

2023

Semin Liver Dis

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The steatotic diseases of non-alcoholic fatty liver disease, alcohol-associated liver disease and chronic Hepatitis C account for the majority of liver disease prevalence, morbidity and mortality worldwide. While these diseases have distinct pathogenic and clinical features, dysregulated lipid droplet (LD) organelle biology represents a convergence of pathogenesis in all three. With increasing understanding of hepatocyte LD biology, we now understand the roles of LD proteins involved in these diseases but also how genetics modulate LD biology to either exacerbate or protect against the phenotypes associated with steatotic liver diseases. Here, we review the history of the LD organelle and its biogenesis and catabolism. We also review how this organelle is critical not only for the steatotic phenotype of liver diseases but also for their advanced phenotypes. Finally, we summarize the latest attempts and challenges of leveraging LD biology for therapeutic gain in steatotic diseases. In conclusion, the study of dysregulated LD biology may lead to novel therapeutics for the prevention of disease progression in the highly prevalent steatotic liver diseases of MASLD, ALD and HCV.

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Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Cited by 6 publications

References 16 publications

Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

Oxidative Stress and Lipid Dysregulation in Lipid Droplets: A Connection to Chronic Kidney Disease Revealed in Human Kidney Cells

Mechanisms of Lipid Droplet Accumulation in Steatotic Liver Diseases

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