Imaging Immune and Metabolic Cells of Visceral Adipose Tissues with Multimodal Nonlinear Optical Microscopy

Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface

Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.

Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems

Evidence for lipid mobilization from adipocyte and uptake of exogenous lipid by ATMs.

<p>(A) Free fatty acids (FFA) concentration of the VAT conditioned medium as a function of incubation time. Error bars represent standard deviation across triplicate measurements. (B) Evidence of lipid droplet microvesiculation revealed by CARS imaging. (C) An autofluorescent and lipid-rich ATM was observed in proximity of adipocytes with lipid droplet microvesiculation. (D) Monitoring the trafficking of exogenous deuterated palmitic acid with spontaneous Raman microspectroscopy. Absence and presence of C-²H peak in the lipid droplets of adipocytes and ATMs, respectively, were observed.</p

Potential source of autofluorescence and lipid in macrophages.

<p>(A) Widefield fluorescence images of RAW 264.7 macrophages (left panel) and RAW264.7 macrophages in co-culture with explanted VATs (right panel). (B) Peptide array to identify adipose tissue secreted pro-inflammatory cytokines. (C) Immunofluorescence (IF) imaging and (D) Western blot analysis revealed increased expression of iNOS in RAW264.7 macrophages co-cultured with explanted VATs as compared to control RAW264.7 macrophages. (E) RAW264.7 macrophages co-cultured with explanted VATs accumulated intracellular lipid droplets observable with ORO staining (upper right panel) and CARS imaging (lower right panel). (F) Real time PCR analysis of gene expression of fatty acid binding proteins (encoded by Fabp1-6 genes) and fatty acid transport proteins (encoded by Slc27a1-6 genes) in control RAW264.7 macrophages and RAW264.7 macrophages co-cultured with VATs.</p

Quantitative analysis of VAT composition in lean and obese mice.

<p>(A) Average number of lipid-rich ATMs per analysis volume with xyz dimensions of 250 µm×250 µm×50 µm. (B) Average diameter of lipid droplets of ∼200 adipocytes for lean VATs and ∼100 adipocytes for obese VATs. (C) Normalized level of collagen fibrils type I, as measured by SHG intensity, in the VATs of lean and obese mice. Data was normalized to 1 for lean VATs and respectively for obese VATs. Error bars represents standard deviation across 81 volumes analyzed.</p

Imaging the visceral adipose tissues (VATs) of lean and obese mice with NLO microscopy.

<p>(A) Histology of lean (upper panel) and obese (lower panel) VATs. (B) CARS imaging of lipid droplets of adipocytes (red) and two-photon fluorescence (TPF) imaging of preadipocytes (green) immunolabeled with FITC-conjugated antibodies to Pref-1 of a lean VAT. (C) CARS imaging of lipid droplets (red) and two-photon autofluorescence imaging of unidentified cells (green) surrounding an adipocyte of an obese VAT.</p

Lipid-rich adipose tissue macrophages (ATM) identified with Oil Red O (ORO) staining and immunofluorescence imaging.

<p>(A) Phase contrast and bright field images of isolated VAT stromal cells stained with ORO. (B) Immunofluorescence images of isolated VAT stromal cells stained with conjugated antibodies to CD4 and CD68 cell surface markers for macrophages.</p

Imaging the kinetics of lipid accumulation in ATMs of explanted VATs.

<p>Steady accumulation of lipid droplets in ATMs was observed with CARS microscopy as a function of time in explanted VATs. Collagen fibrils visualized with second harmonic generation were displayed in blue.</p