Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking.

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.

Differential effects of sugar and fat on adipose tissue inflammation.

Obese individuals experience low grade inflammation initiated within their adipose tissue. However, the early events that lead to the release of these inflammatory factors from adipose tissue are poorly characterized. To separate glucose effects from lipid effects on adipose tissue, we used an adipose-specific TXNIP knockout model where excess basal glucose influx into adipocytes led to modest increase in adiposity without using high fat diet. We found an uncoupling of two events that are generally presumed to be coregulated: (1) an increase of adipose tissue macrophage (ATM) number; and (2) pro-inflammatory activation of ATMs. These two events are associated with different triggering signals: elevated free fatty acids output and extracellular matrix remodeling with increased ATM number, whereas decreased adiponectin level with activated ATM. This separation reflects non-overlapping pathways regulated by glucose and lipids in adipocytes, and neither group alone is sufficient to elicit the full inflammatory response in adipose tissue.

Optimal HSF1 activation in response to acute cold stress in BAT requires nuclear TXNIP.

While TXNIP (thioredoxin interacting protein) in the plasma membrane and vesicular location is known to negatively regulate cellular glucose uptake by facilitating glucose transporter endocytosis, the function of TXNIP in the nucleus is far less understood. Herein, we sought to determine the function of nuclear TXNIP in vivo, using a new HA-tagged TXNIP knock-in mouse model. We observed that TXNIP can be found in the nucleus of a variety of cells from different tissues including hepatocytes (liver), enterocytes (small intestine), exocrine cells (pancreas), and brown adipocytes (BAT). Further investigations into the role of nuclear TXNIP in BAT revealed that cold stress rapidly and transiently activated HSF1 (heat shock factor 1). HSF1 interaction with TXNIP during its activation is required for optimal HSF1 directed cold shock response in BAT.

PPARγ and C/EBPα response to acute cold stress in brown adipose tissue.

Brown adipose tissue (BAT) has the ability to burn calories as heat. Utilizing BAT thermogenesis is thus an attractive way to combat obesity. However, the transcriptional network resulting in the lipid synthesis to oxidation shift during thermogenesis is not completely understood. Here, we report the regulation of two master regulators of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), during acute cold stress in BAT. We found PPARγ dissociates from DNA in a fifth of its binding sites and these include Cebpa enhancers, leading to decreased C/EBPα expression. This dissociation requires PPARγ binding to activating ligands and is thus modulated by diet. Meanwhile, PPARα also detaches from DNA, and co-activator PGC1α associates with ERRα as part of a transcriptional network regulating lipid metabolism. Subsequent global replacement of C/EBPα by C/EBPß and its associated transcriptional machinery is required for upregulation of structural lipid synthesis despite general upregulation of fatty acid oxidation.

TXNIP interaction with GLUT1 depends on PI(4,5)P2.

GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1-4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).

Excess dietary carbohydrate affects mitochondrial integrity as observed in brown adipose tissue.

Hyperglycemia affects over 400 million individuals worldwide. The detrimental health effects are well studied at the tissue level, but the in vivo effects at the organelle level are poorly understood. To establish such an in vivo model, we used mice lacking TXNIP, a negative regulator of glucose uptake. Examining mitochondrial function in brown adipose tissue, we find that TXNIP KO mice have a lower content of polyunsaturated fatty acids (PUFAs) in their membrane lipids, which affects mitochondrial integrity and electron transport chain efficiency and ultimately results in lower mitochondrial heat output. This phenotype can be rescued by a ketogenic diet, confirming the usefulness of this model and highlighting one facet of early cellular damage caused by excess glucose influx.

Mouse long-chain acyl-CoA synthetase 1 is active as a monomer.

Fatty acids are essential cellular building blocks and a major energy source. Regardless of their metabolic fate, fatty acids first need to be activated by forming a thioester with a coenzyme A group. This reaction is carried out by acyl-CoA synthetases (ACSs), of which ACSL1 (long-chain acyl-CoA synthetase 1) is an important member. Two bacterial homologues of ACSL1 crystal structures have been solved previously. One is a soluble dimeric protein, and the other is a monomeric peripheral membrane protein. The mammalian ACSL1 is a membrane protein with an N-terminal transmembrane helix. To characterize the mammalian ACSL1, we purified the full-length mouse ACSL1 and reconstituted it into lipid nanodiscs. Using enzymatic assays, mutational analysis, and cryo-electron microscopy, we show that mouse ACSL1 is active as a monomer.

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin.

Growth factors, such as insulin, can induce both acute and long-term glucose uptake into cells. Apart from the rapid, insulin-induced fusion of glucose transporter (GLUT)4 storage vesicles with the cell surface that occurs in muscle and adipose tissues, the mechanism behind acute induction has been unclear in other systems. Thioredoxin interacting protein (TXNIP) has been shown to be a negative regulator of cellular glucose uptake. TXNIP is transcriptionally induced by glucose and reduces glucose influx by promoting GLUT1 endocytosis. Here, we report that TXNIP is a direct substrate of protein kinase B (AKT) and is responsible for mediating AKT-dependent acute glucose influx after growth factor stimulation. Furthermore, TXNIP functions as an adaptor for the basal endocytosis of GLUT4 in vivo, its absence allows excess glucose uptake in muscle and adipose tissues, causing hypoglycemia during fasting. Altogether, TXNIP serves as a key node of signal regulation and response for modulating glucose influx through GLUT1 and GLUT4.