Interaction Of Adipocytes Research Articles

Pathophysiology of cellulite: Possible involvement of selective endotoxemia.

The most relevant hallmarks of cellulite include a massive protrusion of superficial adipose tissue into the dermis, reduced expression of the extracellular glycoprotein fibulin-3, and an unusually high presence of MUSE cells in gluteofemoral white adipose tissue (gfWAT) that displays cellulite. Also typical for this condition is the hypertrophic nature of the underlying adipose tissue, the interaction of adipocytes with sweat glands, and dysfunctional lymph and blood circulation as well as a low-grade inflammation in the areas of gfWAT affected by cellulite. Here, we propose a new pathophysiology of cellulite, which connects this skin condition with selective accumulation of endogenous lipopolysaccharides (LPS) in gfWAT. The accumulation of LPS within a specific WAT depot has so far not been considered as a possible pathophysiological mechanism triggering localized WAT modifications, but may very well be involved in conditions such as cellulite and, secondary to that, lipedema.

Cancer-associated adipocytes promote the invasion and metastasis in breast cancer through LIF/CXCLs positive feedback loop.

Cancer-associated adipocytes (CAAs), which are adipocytes transformed by cancer cells, are of great importance in promoting the progression of breast cancer. However, the underlying mechanisms involved in the crosstalk between cancer cells and adipocytes are still unknown. Here we report that CAAs and breast cancer cells communicate with each other by secreting the cytokines leukemia inhibitory factor (LIF) and C-X-C subfamily chemokines (CXCLs), respectively. LIF is a pro-inflammatory cytokine secreted by CAAs, which promotes migration and invasion of breast cancer cells via the Stat3 signaling pathway. The activation of Stat3 induced the secretion of glutamic acid-leucine-arginine (ELR) motif CXCLs (CXCL1, CXCL2, CXCL3 and CXCL8) in tumor cells. Interestingly, CXCLs in turn activated the ERK1/2/NF-κB/Stat3 signaling cascade to promote the expression of LIF in CAAs. In clinical breast cancer pathology samples, the up-regulation of LIF in paracancerous adipose tissue was positively correlated with the activation of Stat3 in breast cancer. Furthermore, we verified that adipocytes enhanced lung metastasis of breast cancer cells, and the combination of EC330 (targeting LIF) and SB225002 (targeting C-X-C motility chemokine receptor 2 (CXCR2)) significantly reduced lung metastasis of breast cancer cells in vivo. Our findings reveal that the interaction of adipocytes with breast cancer cells depends on a positive feedback loop between the cytokines LIF and CXCLs, which promotes breast cancer invasion and metastasis.

Inhibition of CXCR2 by Glucocorticoids in Adipose Tissue

Obesity-induced chronic adipose tissue inflammation is a significant risk factor for metabolic and cardiovascular disease (CVD), which affects 30.3 million adults in the United States. Interaction of adipocytes with hormonal, metabolic and immune systems play an integral role in the underlying pathophysiological mechanisms that leads to development of obesity-related complications. Despite this association, the mechanisms that coordinate the inflammatory mediators in causing adipose tissue inflammation are not well understood. Glucocorticoids (GC) are well known for their potent anti-inflammatory actions; however, the mechanism by which GC coordinate the inflammatory response of adipocytes are unknown. From our genome-wide microarray data derived from adipocyte-specific glucocorticoid receptor (GR) knockout (AdipoGRKO) mice, we found that GR inactivation leads to a significant increase in pro-inflammatory gene in white adipose tissue (WAT). Additionally, WAT isolated from AdipoGRKO mice showed significant increase in immune cell infiltration, which correlates with our gene expression data. Among the top up-regulated genes, we found the C-X-C Motif Chemokine Receptor 2 (Cxcr2), which is a powerful mediator of chemotaxis to the sites of inflammation. Although studies have shown the presence of Cxcr2 in adipocytes and suggested the contribution of Cxcr2 signaling in adipocyte development, its role in integrating adipose tissue inflammatory response is unknown. This led us to hypothesize that GR is critical to repress Cxcr2 gene expression and its pro inflammatory effects in adipocytes. Our in vitro studies using 3T3-L1 cells derived adipocytes showed that treatment with the synthetic glucocorticoid, Dexamethasone (Dex) led to a significant repression of Cxcr2 mRNA and protein levels. Furthermore, these effects are mediated by GR acting directly to repress Cxcr2 gene expression. Systemic administration of corticosterone significantly altered Cxcr2 expression in adipose tissue compared to untreated mice further supporting our results. Together our findings suggest that administration of glucocorticoids could inhibit adipose tissue inflammation and alleviate the comorbidities that arise from inflamed adipose tissue.

Anti-Inflammatory and Cytotoxicity Effects of Cudrania tricuspidata Fruits Vinegar in a Co-Culture System with RAW264.7 Macrophages and 3T3-L1 Adipocytes.

Vinegar has been found to have in vitro improvement effect on inflammatory biomarkers, and clinically used to improve inflammation and obesity-related diseases. This study was designed to analyze in vitro anti-inflammatory effects of Cudrania tricuspidata fruits vinegar (CTFV) in a co-culture system with macrophages and adipocytes. We analyzed the physicochemical properties and polyphenolic ingredients of CTFV, and investigated in vitro anti-inflammatory effects of CTFV in a co-culture system with macrophages and adipocytes. The cells were cultured in the presence of CTFV for 24 h in contact with each other, then, harvested. The levels of monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), nitric oxide (NO), and interleukin (IL)-6 were evaluated by using the Griess reagent, western blot, or enzyme-linked immunosorbent assay assays. We found that increasing levels for NO, iNOS, TNF-α, IL-6 and MCP-1 were caused by LPS treatment and co-culture using the contact method, whereas CTFV efficaciously attenuated inflammatory response by improving inflammatory parameters including NO, iNOS, TNF-α, IL-6 and MCP-1. The present study indicates that CTFV might provide a nutraceutical product or functional food resource for improving inflammation processed via the interaction of adipocytes and macrophages.

ADIPOSE TISSUE AND ITS ROLE IN MICROENVIRONMENT OF THE COLORECTAL ADENOCARCINOMA CANCER CELL

Introduction. The mechanisms of adipose-tissue’s influence on tumor progression has been studied a lot, but the way of interaction of adipocytes with tumor cells have not been well defined until now.
 Objective. The aim of this study was to evaluate the mechanisms of adipocytes and tumor cells interaction under the influence of radiation and chemo-radiation therapy in locally advanced rectal cancer (LARC) patients.
 Material and methods. A prospective randomized single-center study was conducted. It involved 110 patients with LARC and pre-obesity. The patients were randomized into a main group A (radiation therapy and oxaliplatin-based chemotherapy) and a comparison group B (radiation therapy and fluoropyrimidine-based mono-chemotherapy). Superoxide free radicals and NO levels generated by mitochondria of adipocytes were evaluated In both groups’. Also, there was estimated the indices of MMP-2, MMP-9, 8-oxoG, and free fatty acids (FFA) level.
 Results and discussion. Level of superoxide radicals in tumor-adjacent adipose tissue was 0.58±0.15 (main group) and 0.70±0.12 nmol/g·min (comparison group) (p<0.001). Blood levels of FFA increased in group A up to 2.05±0.15, and in group B up to 2.48±0.20 mmol/l (while in it was 0.57±0.11 mmol/L). 8-oxoG levels in tumor-adjacent adipose tissue had no statistically significant differences.
 Conclusions. The tumor-adjacent adipose tissue is an energy depot that can act as a promoter of tumor progression supplying the locally advanced rectal cancer with an energy substrate FFA. It has been established that the level MMP-2 activity significantly reduces the degree of intercellular matrix remodeling by the XELOX chemotherapy.

M1 macrophage subtypes activation and adipocyte dysfunction worsen during prolonged consumption of a fructose-rich diet

Fructose-rich diet (FRD) has been associated with obesity development, which is characterized by adipocytes hypertrophy and chronic low-grade inflammation. Interaction of adipocytes and immune cells plays a key role in adipose tissue (AT) alterations in obesity. We assessed the metabolic and immune impairments in AT in a murine obesity model induced by FRD at different periods. Adult Swiss mice were divided into groups of 6 and 10 weeks of fructose (FRD 6wk, FRD 10wk) or water intake (CTR 6wk, CTR 10wk). FRD induced increased in body weight, epidydimal AT mass, and plasmatic and liver Tg, and impaired insulin sensitivity. Also, hypertrophic adipocytes from FRD 6wk-10wk mice showed higher IL-6 when stimulated with LPS and leptin secretion. Several of these alterations worsened in FRD 10wk. Regarding AT inflammation, FRD mice have increased TNFα, IL-6 and IL1β, and decrease in IL-10 and CD206 mRNA levels. Using CD11b, LY6C, CD11c and CD206 as macrophages markers, we identified for first time in AT M1 (M1a: Ly6C+/−CD11c+CD206− and M1b: Ly6C+/−CD11c+CD206+) and M2 subtypes (Ly6C+/−CD11c−CD206+). M1a phenotype increased from 6 weeks onward, while Ly6C+/− M1b phenotype increased only after 10 weeks. Finally, co-culture of RAW264.7 (monocytes cell line) and CTR or FRD adipocytes showed that FRD 10wk adipocytes increased IL-6 expression in non- or LPS-stimulated monocytes. Our results showed that AT dysfunction got worse as the period of fructose consumption was longer. Inflammatory macrophage subtypes increased depending on the period of FRD intake, and hypertrophic adipocytes were able to create an environment that favored M1 phenotype in vitro.

Adipose tissue: The dysfunctional adipocyte - a cancer cell's best friend.

The interactions of adipocytes with tumour cells have been extensively scrutinized. The adipocyte's mitogenic influence on tumour cells is attributed to several hormonal, growth factor and adipokine effects. A recent study has expanded our understanding of how adipocytes promote tumour progression by demonstrating that adipocytes actively catabolize the chemotherapeutic agent daunorubicin.

IRS-1 pY612 and Akt-1/PKB pT308 Phosphorylation and Antiinflammatory Effect of Diindolylmethane in Adipocytes Cocultured with Macrophages.

3,3'-Diindolylmethane (DIM) is a condensation product of indole-3-carbinol, a glucosinolate naturally occurring in Brassica genus vegetables. The antiinflammatory properties of DIM through the inhibition of NF-κB, as well as its ameliorating effects on glucose tolerance and hyperglicemic states, have been described. A subclinical proinflammatory profile resultant from the interaction of adipocytes and macrophages has been reported in obesity, affecting the insulin signaling pathway, contributing to insulin resistance. The aim of this study was to evaluate the effect of DIM on proinflammatory cytokines and phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 in an obesity-induced inflammation model. Differentiated 3T3-L1 adipocytes were co-cultured with RAW 264.7 macrophages and exposed to 20 µM, 40 µM and 60 µM DIM for 24 h followed by 100 nM insulin for 20 min. MCP-1, IL-6 and TNFα were quantified in the supernatant through individual ELISAs. Adipocyte lysates were used to determine the relative expression of the proinflammatory mediators by qPCR, and the phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308 proteins by western blot analysis. DIM significantly (p<0.05) reduced the production and mRNA expression of MCP-1, IL-6, and TNFα in a DIM concentration dependent manner, concomitantly increasing the abundance of IRS-1 pY612 and Akt-1/PKB pT308. Our results suggest that DIM influences the insulin transduction pathway by exerting an antiinflammatory effect. The potential therapeutic benefits of DIM in the treatment of glucose metabolic disorders deserve further studies.

Abstract 1096: Investigating the role of the adipocyte-macrophage interaction in breast cancer metastasis

Abstract Obesity is linked with a higher incidence of hormone-receptor positive breast cancer in post-menopausal women. Obesity is also associated with a worse outcome after the diagnosis of breast cancer, including greater tumor burden and higher grade tumors. Although several theories exist to explain these relationships, the proposed mechanisms are still being investigated. Obesity and the resulting expansion of adipose tissue is associated with aberrant cytokine production and a chronic inflammatory state, characterized by the recruitment of macrophages. Macrophages have been shown elsewhere to promote breast cancer progression. Therefore, we hypothesize that the interaction between adipocytes and macrophages promotes metastasis of breast cancer. Our data suggest that oxidized lipids released from adipocytes may have a role in mediating this interaction. We used a co-culture system to determine whether adipocytes could promote a pro-tumor phenotype in the macrophages. THP-1 macrophages were co-cultured with primary human visceral adipocytes using a Transwell system for 8 hours, then macrophage mRNA was analyzed for changes in transcription of markers for increased metastatic potential. Vascular Endothelial Growth Factor A (VEGF-A) mRNA levels were significantly increased in macrophages after exposure to adipocytes in comparison to control macrophages that were cultured alone. Co-culture experiments were repeated using human breast adipocytes to more directly model the conditions in the breast microenvironment. Again VEGF-A mRNA levels were significantly increased in macrophages after co-culture with adipocytes. Obesity is associated with stressed and necrotic adipose tissue in the breast. Therefore, to mimic obesity related dysfunction and cell death, we treated breast adipocytes with Tumor Necrosis Factor-alpha and Interleukin 6 prior to co-culturing with macrophages. The cytokine treatment enhanced the effects of adipocyte co-culture on macrophage VEGF-A mRNA levels. The increased inflammation and adipocyte dysfunction seen in obesity has been proposed to favor the generation of biologically active oxidized lipid mediators such as 4-hydroxynonenal (4-HNE). We therefore treated THP-1 macrophages with 4-HNE as a model oxidized lipid, hypothesizing that 4-HNE may convey some of the effects on macrophage gene expression seen in co-culture. We found that transcription of VEGF-D and platelet derived growth factor (PGF), both members of the VEGF family of cytokines, were significantly enhanced in 4-HNE-treated THP-1 cells. Together, our data show that the interaction of adipocytes and macrophages in obese breast tissue can stimulate pro-angiogenic macrophage signaling. This is a novel mechanism whereby the obese microenvironment can promote breast cancer metastasis. Citation Format: Nalini V.S. Yadav, Aaron T. Jacobs, Linda Connelly. Investigating the role of the adipocyte-macrophage interaction in breast cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1096. doi:10.1158/1538-7445.AM2014-1096

Anti‐adipogenic and anti‐inflammatory mechanism of nobiletin in co‐culture of adipocyte and macrophage (1045.13)

Adipose tissues are characterized by the enhanced infiltration of macrophages. It is considered that the paracrine loop between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes and insulin resistance in obese adipose tissues. Nobiletin is abundantly present in the fruit peel of Citrus and has several health beneficial effects including antioxidant, anti‐inflammation and anticancer activities. However, not much is known about the anti‐adipogenic and anti‐inflammatory effect of nobiletin in co‐culture system of adipocyte and macrophage. The objective of this study was to evaluate the inhibitory effect of nobiletin on the interaction between adipocytes and macrophages. The results revealed that nobiletin markedly inhibited NO, TNF‐α and MCP‐1 secretion in contact and trans‐well systems of adipocytes and macrophages in a dose‐dependent manner. In addition, nobiletin blocked the expression of adipogenic transcription factors, including PPARγ, C/EBPα and aP2 in 3T3‐L1 cells of transwell system. Also, nobiletin inhibited the expression of iNOS in RAW264.7 cells. These results indicate that nobiletin exhibits anti‐adipogenic and anti‐inflammatory properties in the interaction of adipocytes and macrophages. Nobiletin may be useful for ameliorating the adipogenic and inflammatory changes in obese adipose tissues.

Comprehensive proteomic datasets for studying adipocyte–macrophage cell–cell communication

Cellular communication is a fundamental process in biology. The interaction of adipocytes with macrophages is a key event in the development of common diseases such as type 2 diabetes. We applied an established bilayer cell co-culture system and comprehensive mass spectrometry analysis to detect proteome-wide the paracrine interaction of murine adipocytes and macrophages. Altogether, we identified 4486 proteins with at least two unique peptides of which 2392 proteins were informative for 3T3-L1 adipocytes and 2957 proteins for RAW 264.7 macrophages. Further, we observed over 12,000 phosphorylation sites of which we could assign 3,200 informative phosphopeptides with a single phosphosite for adipocytes and 4,514 for macrophages. Using protein set enrichment and phosphosite analyses, we deciphered regulatory protein pathways involved in cellular stress and inflammation, which can contribute to metabolic impairment of cells including insulin resistance and other disorders. The generated data sets provide a holistic, molecular pathway-centric view on the interplay of adipocytes and macrophages in disease processes and a resource for further studies.

Butyrate Attenuates Inflammation and Lipolysis Generated by the Interaction of Adipocytes and Macrophages

Paracrine interaction between macrophages and adipocytes in obese visceral fat tissues is thought to be a trigger of chronic inflammation. The immunomodulatory effect of the short chain fatty acid, butyric acid, has been demonstrated. We hypothesize that sodium butyrate (butyrate) attenuates inflammatory responses and lipolysis generated by the interaction of macrophages and adipocytes. Using contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the production of tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and the release of free glycerol, free fatty acids (FFAs) into the medium. We also examined the activity of nuclear factor-kappaB (NF-κB) and the phosphorylation of mitogen-activated protein kinases (MAPKs) in co-cultured macrophages, as well as lipase activity and expression in co-cultured adipocytes. We found increased production of TNF-α, MCP-1, IL-6, and free glycerol, FFAs in the co-culture medium, and butyrate significantly reduced them. Butyrate inhibited the phosphorylation of MAPKs, the activity of NF-κB in co-cultured macrophages, and suppressed lipase activity in co-cultured adipocytes. Lipase inhibitors significantly attenuated the production of TNF-α, MCP-1 and IL-6 in the co-culture medium as effectively as butyrate. Butyrate suppressed the protein production of adipose triglyceride lipase, hormone sensitive lipase, and fatty acid-binding protein 4 in co-cultured adipocytes. Pertussis toxin, which is known to block GPR41 completely, inhibited the antilipolysis effect of butyrate. Butyrate suppresses inflammatory responses generated by the interaction of adipocytes and macrophages through reduced lipolysis and inhibition of inflammatory signaling.

Adipocytes from obese subjects increase NF‐kB activation in invasive breast cancer cells

Obesity in post‐menopausal women doubles the breast cancer risk and metastasis. Concomitantly, nuclear factor kappa B (NF‐κB) is known to play a significant role in breast cancer cell metastasis. We used a novel three dimensional (3D) co‐culture system with breast cells (non‐cancerous, MCF10A, cancerous, MCF7 and invasive, MDA231) and adipocytes from pre‐ and post‐menopausal women in lean and obese groups. Comparison of cytokine profiles of co‐cultures revealed two‐way interaction of adipocytes and breast cells. Highly significant cytokine changes were reported for post‐menopausal adipocytes and MDA MB 231 co‐culture. Therefore, we assayed the NF‐κB activation effects of lean and obese post‐menopausal adipocytes on MDA MB 231 cells. Media transfer from lean/obese post‐menopausal adipocyte cultures in to growing MDA MB 231 breast cells resulted significant changes in NF‐κB activation pathway genes. This further verified our previous data that obese conditioned media increased the levels of NF‐κB family members (P65, P50, P52, Rel B and Rel C) compared to the lean counterpart. Therefore, our data indicates that adipocytes from post‐menopausal obese women can increase the NF‐κB activation in MDA MB 231 cells. In conclusion, this study suggests that increased breast cancer risk in obese post‐menopausal women may be at least in part due to obesity mediated NF‐κB activation. Support: UTORC, AgResearch and UTGSM.Grant Funding Source : Internal

Diosgenin attenuates inflammatory changes in the interaction between adipocytes and macrophages

Obese adipose tissues are characterized by the enhanced infiltration of macrophages. It is considered that the paracrine loop involving monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and the free fatty acid between adipocytes and macrophages establishes a vicious cycle that aggravates inflammatory changes and insulin resistance in obese adipose tissues. Diosgenin, a saponin aglycon found in a variety of plants, has anti-inflammatory properties. In the present study, we examined the effect of diosgenin on the inflammatory changes in the interaction between adipocytes and macrophages. A coculture of 3T3-L1 adipocytes and RAW 264 macrophages markedly enhanced the production of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nitric oxide compared with the sum of their single cultures; however, treatment with diosgenin inhibited the production of these proinflammatory mediators. Diosgenin also suppressed the inflammation in RAW 264 macrophages that was induced by the conditioned medium derived from 3T3-L1 adipocytes. Furthermore, diosgenin inhibited the conditioned medium-induced degradation of inhibitor kappaB and the phosphorylation of c-jun N-terminal kinase in macrophages. These results indicate that diosgenin exhibits anti-inflammatory properties in the interaction of adipocytes and macrophages by inhibiting the inflammatory signals in macrophages. Diosgenin may be useful for ameliorating the inflammatory changes in obese adipose tissues.

Alterations of insulin sensitivity by the implantation of 3T3-L1 cells in nude mice. A role for TNF-alpha?

Visceral adipocytes have different functions than those from the subcutaneous fat area. These differences could contribute to the pathological significance of excessive visceral fat accumulation for accompanying insulin resistance and hyperinsulinaemia. This study addresses this hypothesis and describes a unique method to clarify whether the functional differences between visceral and subcutaneous adipocytes depend on their anatomical location. 3T3-L1 cells or TNF-alpha overexpressing CHO cells were implanted into subcutaneous fat area or mesenteric area as visceral fat area in athymic mice of BALB/C strain. Then, serum insulin, glucose, TNF-alpha, and several markers of lipid metabolism were measured in the fasting condition. OGTT was also analysed. During the course of glucose loading, the mice which had 3T3-L1 cells implanted into mesenteric area but not into subcutaneous fat area showed remarkably increased serum insulin and TMF-alpha concentrations, compared to the control mice. Moreover, serum insulin concentrations of the mice, implanted with TNF-alpha overexpressing cells into subcutaneous fat area, were apparently higher than that of control mice. This method of implanting adipose cells into subcutaneous or visceral fat area showed high TNF-alpha concentration and insulin resistance by the adipose cells in visceral area of nude mice. Furthermore, we found that the functional significance of visceral fat accumulation for TNF-alpha-induced insulin resistance is partly caused by the interaction of adipocytes with surrounding conditions in mesenteric area.

Interactions of adipocytes and their precursor cells with endothelial cells in culture

Factors involved in the growth of adipose tissue were examined by testing interactions under cell culture conditions between cellular components of this tissue and plasma from overfed rats. The cellular factors were capillary fragments, endothelial cells during growth and after confluence, fibroblasts, adipocytes and adipose precursor cells before determination (adipoblasts) and after determination (preadipocytes). Multiplying adipose precursor cells stimulated markedly the multiplication of endothelial cells, while their own multiplication was inhibited. The stimulatory effect was partially transferred into the culture medium but not remaining in culture dishes conditioned by preceding cultures of adipose precursor cells, removed by Tris-EDTA buffer or mechanically. The activity was apparently not dependent on feeding conditions. Plasma from overfed rats did not affect endothelial or adipose precursor cell multiplication, but caused more rapid lipid filling of the latter. Endothelial cells facilitated lipid accumulation of preadipocytes. These results indicate that when adipose tissue is expanding by adipocyte multiplication capillarization is stimulated secondarily, being then capable of facilitating triglyceride accumulation in adipocytes.

Effect of liposome-adipocyte interaction on hexose uptake and insulin action.

The effect of unilammelar lipid vesicles composed of defined acyl chain phosphatidylcholines or binary vesicles comprised of dioleoyl phosphatidylcholine (DOPC) and a variety of other lipid species on insulin action in isolated rat adipocytes was studied. Cells were treated with vesicles and subsequently analyzed for basal and insulin-stimulated hexose uptake. Of the different vesicles tested, only those containing DOPC and phosphatidylserine (PS) markedly inhibited the effect of insulin. The inhibition was a function of PS concentration in the binary vesicle. Basal uptake, on the other hand, was stimulated by PS-containing vesicles. The changes in insulin-stimulated hexose uptake following interaction of adipocytes with DOPC-PS vesicles were accompanied by alterations in the Vmax of the uptake process and in affinity of insulin binding, although the similar ED50 values of the control and vesicle-treated groups suggest that the observed effects on insulin sensitivity may be mediated by an uncoupling of the insulin receptor from transport activation. Several lines of evidence are consistent with vesicle-cell fusion as the pathway of DOPC-PS uptake under these conditions. Fluorescence microscopic analysis of fat cells following their interaction with vesicles filled with fluorescent dye indicated that the internal contents of the adipocytes became generally labeled. Biochemical studies of cell fractionation after the interaction of adipocytes with radiolabeled vesicles and effects of metabolic inhibitors and cell fixatives on vesicle uptake also support the fusion pathway.

Affinity binding of intact fat cells and their ghosts to immobilized insulin.

Immobilized insulin, prepared by coupling insulin directly to agarose or through hydrocarbon "connecting arms," was demonstrated to be capable of firmly binding intact adipocytes and their ghosts. Various lines of evidence indicate that the insulin receptor on the plasma membrane, in addition to the insulin coupled to the agarose, was responsible for the observed binding. This evidence includes: (a) the finding that increasing the "arm" length increased the binding capacities of insulin-Sepharose affinity chromatographic columns, (b) specific inhibition and reversal by insulin and antiserum to insulin of the binding, as compared to lesser effects by other peptide hormones, (c) the indication that only the plasma membrane sacs, not the other cellular contaminants in the crude ghosts, are capable of binding, and (d) the impairment and restoration of trypsin-sensitive membrane binding sites that are also required for insulin biosensitivity. These findings support the idea that the insulin receptor is the trypsin-sensitive site. By use of the differential buoyant densities of the various cell-bead complexes that resulted from the interaction of adipocytes with insulin-Sepharose, a new procedure was developed to demonstrate and study the binding. These complexes could also be demonstrated by interference contrast microscopy. Binding readily occurred under conditions favorable for insulin stimulation of the cells. By coupling tracer amounts of [(125)I]insulin to Sepharose or insulin-Sepharose, the effects of anti-insulin antisera, free insulin, and other peptide hormones and supplemental factors on the buoyant-density distribution of the complexes could be measured, as well as the effects of other ligands coupled to Sepharose.