Quantification of Hormone Sensitive Lipase Phosphorylation and Colocalization with Lipid Droplets in Murine 3T3L1 and Human Subcutaneous Adipocytes via Automated Digital Microscopy and High-Content Analysis

McDonough, Patrick M.; Ingermanson, Randall; Loy, Patricia A.; Koon, Erick D.; Whittaker, Ross; Laris, Casey; Hilton, Jeffrey M.; Nicoll, James B.; Buehrer, Benjamin M.; Price, Jeffrey H.

doi:10.1089/adt.2010.0302

Cited by 14 publications

(16 citation statements)

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“…We [25] and others [18], [19] have examined the phosphorylation of HSL in 3T3L1 adipocytes in response to FSK and L-γ-MSH. To examine the phosphorylation of perilipin 1A in response to these agents, murine 3T3L1 adipocytes were exposed to either FSK or L-γ-MSH for 5 minutes, then fixed, and labeled for nuclei and lipid droplets, along with either anti-pPeri-site 5 or anti-pPeri-site 6.…”

Section: Resultsmentioning

confidence: 99%

“…Cyclic-GMP-dependent Protein Kinase (PKG), might be a candidate for this as it shares substrate specificity with PKA and is present in 3T3L1 adipocytes [41]. Furthermore, atrial natriuretic peptide likely activates lipolysis in human adipocytes by PKG-mediated phosphorylation of HSL at serine 660 [25]. However, L-γ-MSH is not known to activate PKG.…”

Section: Discussionmentioning

confidence: 99%

“…CyteSeer®’s Colocalization Algorithm has been specifically developed for the analysis of lipid droplets and lipid droplet-associated proteins [20], [24], [25]. The Colocalization Algorithm utilizes the nuclear image from each field of view to identify the nuclei and to estimate cellular boundaries; this information is then applied to the lipid and protein images, to estimate the level of expression of lipid droplets and protein for each cell.…”

Section: Methodsmentioning

confidence: 99%

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Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

et al. 2013

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Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3′,5′-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

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“…Several automated software have been developed to measure fat cell size (17,18) but they did not aim for a more detailed analysis of individual LDs. McDonough et al convolved images with a Mexican Top-Hat filter to enhance contrast and then used an automatic threshold to create a binary mask for the LDs (19,20) while the Nuclear Analysis algorithm (semi-automatic color segmentation (21)) was used to define the nuclei mask. Dejgaard and Presley reduced incorrect LD segmentation by shape-based filtering, but at the same time losing many LDs (16).…”

mentioning

confidence: 99%

“…Dejgaard and Presley reduced incorrect LD segmentation by shape-based filtering, but at the same time losing many LDs (16). McDonough et al convolved images with a Mexican Top-Hat filter to enhance contrast and then used an automatic threshold to create a binary mask for the LDs (19,20) while the Nuclear Analysis algorithm (semi-automatic color segmentation (21)) was used to define the nuclei mask.…”

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confidence: 99%

Quantitative high‐content/high‐throughput microscopy analysis of lipid droplets in subject‐specific adipogenesis models

et al. 2017

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Neutral lipids packed in lipid droplets (LDs) are essential as a source of fuel for organisms, and specialized storing cells, the adipocytes, provide a buffer for energy variations. Many modern-society-disorders are connected with excess accumulation or deficiency of LDs in adipose tissue. Intracellular LD number and size distribution reflect the tissue conditions, while the associated mechanisms and genes rs are still poorly understood. Large-scale genetic screens using human in vitro differentiated primary adipocytes require cell samples donated from many patients. The heterogeneity appearing between donors highlighted the need for high-throughput methods robust to individual variations. Previous image analysis algorithms failed to handle individual LDs, but focused on averages, hiding population heterogeneity. We present a new high-content analysis (HCA) technique for analysis of fat cell metabolism using data from a large-scale RNAi screen including images of more than 500 k in vitro differentiated adipocytes from three donors. The RNAi-based suppression of Perilipin 1 (PLIN1), a protein involved in the adipocyte lipid metabolism, served as a positive control, while cells treated with randomized RNA served as negative controls. We validate our segmentation by comparing our results to those of previously published methods: We also evaluate the discriminative power of different morphological features describing LD size distribution. Classification of cells as containing few large or many small LDs followed by calculating the percentage of cells in each class proved to discriminate the positive PLIN1-suppressed phenotype from the untreated negative control with an area under the receiver operating characteristic curve of 0.98. The results suggest that this HCA method offers improved segmentation and classification accuracy, and can, thus, be utilized to quantify changes in LD metabolism in response to treatment in many cell models relevant to a variety of diseases. © 2017 International Society for Advancement of Cytometry.

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High content analysis of differentiation and cell death in human adipocytes

et al. 2013

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Understanding adipocyte biology and its homeostasis is in the focus of current obesity research. We aimed to introduce a high-content analysis procedure for directly visualizing and quantifying adipogenesis and adipoapoptosis by laser scanning cytometry (LSC) in a large population of cell. Slide-based image cytometry and image processing algorithms were used and optimized for high-throughput analysis of differentiating cells and apoptotic processes in cell culture at high confluence. Both preadipocytes and adipocytes were simultaneously scrutinized for lipid accumulation, texture properties, nuclear condensation, and DNA fragmentation. Adipocyte commitment was found after incubation in adipogenic medium for 3 days identified by lipid droplet formation and increased light absorption, while terminal differentiation of adipocytes occurred throughout day 9-14 with characteristic nuclear shrinkage, eccentric nuclei localization, chromatin condensation, and massive lipid deposition. Preadipocytes were shown to be more prone to tumor necrosis factor alpha (TNFa)-induced apoptosis compared to mature adipocytes. Importantly, spontaneous DNA fragmentation was observed at early stage when adipocyte commitment occurs. This DNA damage was independent from either spontaneous or induced apoptosis and probably was part of the differentiation program. V C 2013 International Society for Advancement of Cytometry Key terms adipocyte; preadipocyte; SGBS; differentiation; DNA damage; laser scanning cytometry; high content analysis OBESITY is major risk factor for a number of chronic diseases, including diabetes, cardiovascular diseases, and cancer. In physiological context, body fat mass is driven via the balance between the adipocyte's growth and removal. Adipose tissue can grow by either hyperplasia or hypertrophy or both, whereas adipocyte removal is facilitated by apoptosis. Adipogenesis, when adipocytes develop from mesenchymal stem cells, is a fairly well known process (1-3). In current studies, multiparametric model systems have been developed, where adipocyte differentiation, its nuclear, cytoskeletal, and suborganellar morphological changes can be monitored together with different drug applications in an automated manner. Among these works, Lee et al. (4) had introduced a noticeable flow cytometric assay to assess differentiation of preadipocytes, based on cytoplasmic granularity changes corresponding to lipid droplet accumulation. However, application of flow cytometry, which requires cell suspensions, strongly violates physiological optima of solid tissues. This technique also omits the possibility of in situ visualization and reanalysis of samples attached to culture dish. These latter obstacles were solved by applying image cytometry in solid tissue quantifications, as in Lin's work, where laser scanning cytometry was used to study adipocytes and measure capacity of drugs to induce adipocyte apoptosis (5,6). Yet, cell recognition of attached cells was not solved or at least not clearly explained whether single cell conto...

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Quantification of Hormone Sensitive Lipase Phosphorylation and Colocalization with Lipid Droplets in Murine 3T3L1 and Human Subcutaneous Adipocytes via Automated Digital Microscopy and High-Content Analysis

Cited by 14 publications

References 60 publications

Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

Differential Phosphorylation of Perilipin 1A at the Initiation of Lipolysis Revealed by Novel Monoclonal Antibodies and High Content Analysis

Quantitative high‐content/high‐throughput microscopy analysis of lipid droplets in subject‐specific adipogenesis models

High content analysis of differentiation and cell death in human adipocytes

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