Spatiotemporal Heterogeneity of De Novo Lipogenesis in Fixed and Living Single Cells

Abstract

De novo lipogenesis (DNL) is a critical metabolic process that provides the majority of lipids for adipocyte and liver tissue. In cancer, obesity, type II diabetes, and nonalcoholic fatty liver disease DNL becomes dysregulated. A deeper understanding of the rates and of subcellular organization of DNL is necessary for identifying how this dysregulation occurs and varies across individuals and diseases. However, DNL is difficult to study inside the cell because labeling lipids and their precursors is not trivial. Existing techniques either can only measure parts of DNL, like glucose uptake, or do not provide spatiotemporal resolution. Here, we track DNL in space and time as isotopically labeled glucose is converted to lipids in adipocytes using optical photothermal infrared microscopy (OPTIR). OPTIR provides submicron resolution infrared imaging of the glucose metabolism in both living and fixed cells while also reporting on the identity of lipids and other biomolecules. We show significant incorporation of the labeled carbons into triglycerides in lipid droplets over the course of 72 h. Live cells had better preservation of lipid droplet morphology, but both showed similar DNL rates. Rates of DNL, as measured by the ratio of 13C-labeled lipid to 12C-labeled lipid, were heterogeneous, with differences within and between lipid droplets and from cell to cell. The high rates of DNL measured in adipocyte cells match upregulated rates of DNL previously reported in PANC1 pancreatic cancer cells. Taken together, our findings support a model where DNL is locally regulated to meet energy needs within cells.

Oleic acid differentially affectsde novolipogenesis in adipocytes and hepatocytes

“…15 The exception is isotope labelling of precursors, which is nonperturbative, nontoxic, and well metabolized. [15][16][17] While isotopic labels are easy to incorporate and have been used extensively for mass spectrometry experiments, 18,19 the cell, with external stressors stimulating condensation of metabolic components and other regulating factors. 20 This presents a need for techniques that can provide both spatially and temporally resolved metabolic information.…”

“…Vibrational microspectroscopy is a promising means of tracking metabolism throughout the cell using nonperturbative isotopic probes. [15][16][17]21 Biomolecules have unique and specific vibrational signatures and can provide information about the surrounding environment. 22 FTIR and Raman microspectroscopy have been successfully used in cellular studies, but each present its own challenges when working with live cells.…”

“…The specific geometry also leads to significantly less signal contribution from water. 17,26 OPTIR has previously been used on fixed samples to obtain information on metabolic rates and chemical compositions, 27,28 but only recently has this technique been applied to live cell imaging. 17 We previously used OPTIR to track rates of DNL by feeding 13 C glucose to differentiated 3T3-L1 cells (adipocytes) and monitoring incorporation of the labelled carbon into the ester carbonyl of triglycerides in both live and fixed cells.…”

“…17,26 OPTIR has previously been used on fixed samples to obtain information on metabolic rates and chemical compositions, 27,28 but only recently has this technique been applied to live cell imaging. 17 We previously used OPTIR to track rates of DNL by feeding 13 C glucose to differentiated 3T3-L1 cells (adipocytes) and monitoring incorporation of the labelled carbon into the ester carbonyl of triglycerides in both live and fixed cells. 17 Here, we extend this study to the hepatocyte-derived Huh-7 cancer cell line (hereafter referred to as hepatocytes) because hepatic DNL is especially relevant to NAFLD and other metabolic diseases.…”

“…17 We previously used OPTIR to track rates of DNL by feeding 13 C glucose to differentiated 3T3-L1 cells (adipocytes) and monitoring incorporation of the labelled carbon into the ester carbonyl of triglycerides in both live and fixed cells. 17 Here, we extend this study to the hepatocyte-derived Huh-7 cancer cell line (hereafter referred to as hepatocytes) because hepatic DNL is especially relevant to NAFLD and other metabolic diseases. Further, we incorporate a multiplexed imaging approach to monitor DNL and fatty-acid scavenging concomitantly (Fig.…”