Garrett R. Mullins

Diacylglycerol kinase alpha is a critical signaling node and novel therapeutic target in glioblastoma and other cancers

Although diacylglycerol kinase α (DGKα) has been linked to several signaling pathways related to cancer cell biology, it has been neglected as a target for cancer therapy. The attenuation of DGKα activity via DGKα-targeting siRNA and small-molecule inhibitors R59022 and R59949 induced caspase-mediated apoptosis in glioblastoma cells and in other cancers, but lacked toxicity in noncancerous cells. We determined that mTOR and hypoxia-inducible factor-1α (HIF-1α) are key targets of DGKα inhibition, in addition to its regulation of other oncogenes. DGKα regulates mTOR transcription via a unique pathway involving cyclic AMP. Finally, we showed the efficacy of DGKα inhibition with short hairpin RNA or a small-molecule agent in glioblastoma and melanoma xenograft treatment models, with growth delay and decreased vascularity. This study establishes DGKα as a central signaling hub and a promising therapeutic target in the treatment of cancer.

Phosphorylation of Lipin 1 and Charge on the Phosphatidic Acid Head Group Control Its Phosphatidic Acid Phosphatase Activity and Membrane Association

Background: Phosphorylation controls intracellular localization of lipin 1 and has been proposed to regulate activity. Results: Lipin 1 preferentially binds di-anionic phosphatidic acid, and this is eliminated by phosphorylation. Conclusion: Lipin 1 association with phosphatidic acid is regulated by phosphorylation and electrostatic charge of substrate. Significance: Phosphorylation and the local membrane environment both significantly contribute to the regulation of lipin 1 PAP activity. The lipin gene family encodes a class of Mg2+-dependent phosphatidic acid phosphatases involved in the de novo synthesis of phospholipids and triglycerides. Unlike other enzymes in the Kennedy pathway, lipins are not integral membrane proteins, and they need to translocate from the cytosol to intracellular membranes to participate in glycerolipid synthesis. The movement of lipin 1 within the cell is closely associated with its phosphorylation status. Although cellular analyses have demonstrated that highly phosphorylated lipin 1 is enriched in the cytosol and dephosphorylated lipin 1 is found on membranes, the effects of phosphorylation on lipin 1 activity and binding to membranes has not been recapitulated in vitro. Herein we describe a new biochemical assay for lipin 1 using mixtures of phosphatidic acid (PA) and phosphatidylethanolamine that reflects its physiological activity and membrane interaction. This depends on our observation that lipin 1 binding to PA in membranes is highly responsive to the electrostatic charge of PA. The studies presented here demonstrate that phosphorylation regulates the ability of the polybasic domain of lipin 1 to recognize di-anionic PA and identify mTOR as a crucial upstream signaling component regulating lipin 1 phosphorylation. These results demonstrate how phosphorylation of lipin 1 together with pH and membrane phospholipid composition play important roles in the membrane association of lipin 1 and thus the regulation of its enzymatic activity.

Catecholamine-induced lipolysis causes mTOR complex dissociation and inhibits glucose uptake in adipocytes

Significance Adipose tissue maintains metabolic homeostasis during fasting and fed conditions. When nutrients are plentiful, anabolic signaling is mediated by insulin, stimulating adipocytes to take up glucose for energy storage. In the absence of nutrients, catabolic signaling initiates lipolysis, or the release of lipids for energy use, and is mediated by catecholamines. These opposing pathways are evolutionarily conserved and prevent futile cycling, but can lead to metabolic disorders such as insulin resistance if not properly regulated. Here we define a novel mechanism whereby lipolysis inhibits insulin-stimulated glucose uptake in adipocytes. This signaling mechanism likely contributes to insulin resistance when lipolysis is active, such as during high stress or obesity, and this new understanding may lead to novel treatment approaches for hyperglycemia. Anabolic and catabolic signaling oppose one another in adipose tissue to maintain cellular and organismal homeostasis, but these pathways are often dysregulated in metabolic disorders. Although it has long been established that stimulation of the β-adrenergic receptor inhibits insulin-stimulated glucose uptake in adipocytes, the mechanism has remained unclear. Here we report that β-adrenergic–mediated inhibition of glucose uptake requires lipolysis. We also show that lipolysis suppresses glucose uptake by inhibiting the mammalian target of rapamycin (mTOR) complexes 1 and 2 through complex dissociation. In addition, we show that products of lipolysis inhibit mTOR through complex dissociation in vitro. These findings reveal a previously unrecognized intracellular signaling mechanism whereby lipolysis blocks the phosphoinositide 3-kinase–Akt–mTOR pathway, resulting in decreased glucose uptake. This previously unidentified mechanism of mTOR regulation likely contributes to the development of insulin resistance.

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase β-dependent (IKKβ-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.

Lipin 2 binds phosphatidic acid by the electrostatic hydrogen bond switch mechanism independent of phosphorylation

Background: Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for DAG formation at the ER membrane during lipogenesis. Results: A combination of biochemical approaches is used to characterize lipin 2 phosphatase activity and regulation. Conclusion: The electrostatic charge of PA regulates activity, but phosphorylation does not. Significance: These findings demonstrate differential regulation of PAP activity within the lipin family. Lipin 2 is a phosphatidic acid phosphatase (PAP) responsible for the penultimate step of triglyceride synthesis and dephosphorylation of phosphatidic acid (PA) to generate diacylglycerol. The lipin family of PA phosphatases is composed of lipins 1–3, which are members of the conserved haloacid dehalogenase superfamily. Although genetic alteration of LPIN2 in humans is known to cause Majeed syndrome, little is known about the biochemical regulation of its PAP activity. Here, in an attempt to gain a better general understanding of the biochemical nature of lipin 2, we have performed kinetic and phosphorylation analyses. We provide evidence that lipin 2, like lipin 1, binds PA via the electrostatic hydrogen bond switch mechanism but has a lower rate of catalysis. Like lipin 1, lipin 2 is highly phosphorylated, and we identified 15 phosphosites. However, unlike lipin 1, the phosphorylation of lipin 2 is not induced by insulin signaling nor is it sensitive to inhibition of the mammalian target of rapamycin. Importantly, phosphorylation of lipin 2 does not negatively regulate either membrane binding or PAP activity. This suggests that lipin 2 functions as a constitutively active PA phosphatase in stark contrast to the high degree of phosphorylation-mediated regulation of lipin 1. This knowledge of lipin 2 regulation is important for a deeper understanding of how the lipin family functions with respect to lipid synthesis and, more generally, as an example of how the membrane environment around PA can influence its effector proteins.

Smartphones Can Monitor Medical Center Pneumatic Tube Systems

To the Editor: The pneumatic tube system (PTS) has become a common means of transportation of specimens in medical centers. Although the PTS provides convenience and speed of transport, hemolysis of blood specimens and preanalytical variation have been related to excessive acceleration forces and prolonged time/distance traveled in the PTS (1–5). As a result, regular assessment of 3-axis acceleration (i.e., forces) in PTSs has been recommended in an article in this journal (5). An editorial related to that article suggested that products designed for PTS assessment may become commercially available and capable of recording g -forces in PTSs (2). To date, however, we have found no products that are available in the US designed to record forces in the PTS used in our health system (Swisslog). Many modern smartphones are equipped with an accelerometer that measures acceleration forces. The devices also contain a chronometer, and they are nearly ubiquitous and are portable, …

Smartphone monitoring of pneumatic tube system-induced sample hemolysis.

BACKGROUND Pneumatic tube systems (PTSs) are convenient methods of patient sample transport in medical centers, but excessive acceleration force and time/distance traveled in the PTS have been correlated with increased blood-sample hemolysis. We investigated the utility of smartphones for monitoring of PTS-related variables. METHODS Smartphones were sent through the PTS from several hospital locations. Each smartphone used 2 apps as data-loggers to record force of acceleration vs time. To relate the smartphone data to sample integrity, blood samples were collected from 5 volunteers, and hemolysis of the samples was analyzed after they were transported by hand or via 1 of 2 PTS routes. Increased sample hemolysis as measured by plasma lactate dehydrogenase (LD) was also related to the amount of transport in the PTS. RESULTS The smartphones showed higher duration of forceful acceleration during transport through 1 of the 2 PTS routes, and the increased duration correlated with significant increases in hemolysis (H)-index and plasma LD. In addition, plasma LD showed a positive linear relationship with number of shock forces experienced during transport through the PTS. CONCLUSIONS Smartphones can monitor PTS variables that cause sample hemolysis. This provides an accessible method for investigating specific PTS routes in medical centers.

The phosphatidic acid–binding, polybasic domain is responsible for the differences in the phosphoregulation of lipins 1 and 3

Lipins 1, 2, and 3 are Mg2+-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA in vitro. The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation in vitro.

Air bubbles and hemolysis of blood samples during transport by pneumatic tube systems

BACKGROUND Transport of blood samples through pneumatic tube systems (PTSs) generates air bubbles in transported blood samples and, with increasing duration of transport, the appearance of hemolysis. We investigated the role of air-bubble formation in PTS-induced hemolysis. METHODS Air was introduced into blood samples for 0, 1, 3 or 5min to form air bubbles. Hemolysis in the blood was assessed by (H)-index, lactate dehydrogenase (LD) and potassium in plasma. In an effort to prevent PTS-induced hemolysis, blood sample tubes were completely filled, to prevent air bubble formation, and compared with partially filled samples after PTS transport. We also compared hemolysis in anticoagulated vs clotted blood subjected to PTS transport. RESULTS As with transport through PTSs, the duration of air bubble formation in blood by a gentle stream of air predicted the extent of hemolysis as measured by H-index (p<0.01), LD (p<0.01), and potassium (p<0.02) in plasma. Removing air space in a blood sample prevented bubble formation and fully protected the blood from PTS-induced hemolysis (p<0.02 vs conventionally filled collection tube). Clotted blood developed less foaming during PTS transport and was partially protected from hemolysis vs anticoagulated blood as indicated by lower LD (p<0.03) in serum than in plasma after PTS sample transport. CONCLUSIONS Prevention of air bubble formation in blood samples during PTS transport protects samples from hemolysis.

Undetectable Alanine Aminotransferase during Hospitalization

A 58-year-old man with chronic systolic heart failure and type 2 diabetes mellitus presented with bacteremia, acute exacerbation of chronic heart failure, and acute renal failure. He described poor appetite and drinking beer for several days. Alanine aminotransferase (ALT)2 was 16 U/L initially but decreased until it became undetectable (<6 U/L) on the Architect 16200 (Table 1). ALT was …