Obesity is associated with increased recurrence and reduced survival of breast cancer. Adipocytes constitute a significant component of breast tissue, yet their role in provisioning metabolic substrates to support breast cancer progression is poorly understood. Here, we show that co-culture of breast cancer cells with adipocytes revealed cancer cell-stimulated depletion of adipocyte triacylglycerol. Adipocyte-derived free fatty acids were transferred to breast cancer cells, driving fatty acid metabolism via increased CPT1A and electron transport chain complex protein levels, resulting in increased proliferation and migration. Notably, fatty acid transfer to breast cancer cells was enhanced from “obese” adipocytes, concomitant with increased stimulation of cancer cell proliferation and migration. This adipocyte-stimulated breast cancer cell proliferation was dependent on lipolytic processes since HSL/ATGL knockdown attenuated cancer cell responses. These findings highlight a novel and potentially important role for adipocyte lipolysis in the provision of metabolic substrates to breast cancer cells, thereby supporting cancer progression.

A major challenge of the post-genomics era is to define the connectivity of protein phosphorylation networks. Here, we quantitatively delineate the insulin signaling network in adipocytes by high-resolution mass spectrometry-based proteomics. These data reveal the complexity of intracellular protein phosphorylation. We identified 37,248 phosphorylation sites on 5,705 proteins in this single-cell type, with approximately 15% responding to insulin. We integrated these large-scale phosphoproteomics data using a machine learning approach to predict physiological substrates of several diverse insulin-regulated kinases. This led to the identification of an Akt substrate, SIN1, a core component of the mTORC2 complex. The phosphorylation of SIN1 by Akt was found to regulate mTORC2 activity in response to growth factors, revealing topological insights into the Akt/mTOR signaling network. The dynamic phosphoproteome described here contains numerous phosphorylation sites on proteins involved in diverse molecular functions and should serve as a useful functional resource for cell biologists.

GLUT4 is an insulin-regulated glucose transporter that is responsible for insulin-regulated glucose uptake into fat and muscle cells. In the absence of insulin, GLUT4 is mainly found in intracellular vesicles referred to as GLUT4 storage vesicles (GSVs). Here, we summarise evidence for the existence of these specific vesicles, how they are sequestered inside the cell and how they undergo exocytosis in the presence of insulin. In response to insulin stimulation, GSVs fuse with the plasma membrane in a rapid burst and in the continued presence of insulin GLUT4 molecules are internalised and recycled back to the plasma membrane in vesicles that are distinct from GSVs and probably of endosomal origin. In this Commentary we discuss evidence that this delivery process is tightly regulated and involves numerous molecules. Key components include the actin cytoskeleton, myosin motors, several Rab GTPases, the exocyst, SNARE proteins and SNARE regulators. Each step in this process is carefully orchestrated in a sequential and coupled manner and we are beginning to dissect key nodes within this network that determine vesicle–membrane fusion in response to insulin. This regulatory process clearly involves the Ser/Thr kinase AKT and the exquisite manner in which this single metabolic process is regulated makes it a likely target for lesions that might contribute to metabolic disease.

Insulin-stimulated glucose uptake into muscle and adipose tissue is vital for maintaining whole-body glucose homeostasis. Insulin promotes glucose uptake into these tissues by triggering a protein phosphorylation signalling cascade, which converges on multiple trafficking processes to deliver the glucose transporter GLUT4 to the cell surface. Impaired insulin-stimulated GLUT4 translocation in these tissues underlies insulin resistance, which is a major risk factor for type 2 diabetes and other metabolic diseases. Despite this, the precise changes in insulin signalling and GLUT4 trafficking underpinning insulin resistance remain unclear. In this review, we highlight insights from recent unbiased phosphoproteomics studies, which have enabled a comprehensive examination of insulin signalling and have transformed our perspective on how signalling changes may contribute to insulin resistance. We also discuss how GLUT4 trafficking is disrupted in insulin resistance, and underline sites where signalling changes could lead to these trafficking defects. Lastly, we address several major challenges currently faced by researchers in the field. As signalling and trafficking alterations can be examined at increasingly high resolution, integrative approaches examining the two in combination will provide immense opportunities for elucidating how they conspire to cause insulin resistance.

Daniel J. Fazakerley, James R. Krycer, Alison L. Kearney, Samantha L. Hocking, David E. James