Insulin signaling requires glucose to promote lipid anabolism in adipocytes.

Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilization based on nutritional status. This is coordinated by insulin, which triggers kinase signaling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, we sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting that distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed that glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signaling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilizes protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism.

Serine 474 phosphorylation is essential for maximal Akt2 kinase activity in adipocytes.

The Ser/Thr protein kinase Akt regulates essential biological processes such as cell survival, growth, and metabolism. Upon growth factor stimulation, Akt is phosphorylated at Ser474; however, how this phosphorylation contributes to Akt activation remains controversial. Previous studies, which induced loss of Ser474 phosphorylation by ablating its upstream kinase mTORC2, have implicated Ser474 phosphorylation as a driver of Akt substrate specificity. Here we directly studied the role of Akt2 Ser474 phosphorylation in 3T3-L1 adipocytes by preventing Ser474 phosphorylation without perturbing mTORC2 activity. This was achieved by utilizing a chemical genetics approach, where ectopically expressed S474A Akt2 was engineered with a W80A mutation to confer resistance to the Akt inhibitor MK2206, and thus allow its activation independent of endogenous Akt. We found that insulin-stimulated phosphorylation of four bona fide Akt substrates (TSC2, PRAS40, FOXO1/3a, and AS160) was reduced by ∼50% in the absence of Ser474 phosphorylation. Accordingly, insulin-stimulated mTORC1 activation, protein synthesis, FOXO nuclear exclusion, GLUT4 translocation, and glucose uptake were attenuated upon loss of Ser474 phosphorylation. We propose a model where Ser474 phosphorylation is required for maximal Akt2 kinase activity in adipocytes.

Evaluation of computational programs to predict HLA genotypes from genomic sequencing data.

Motivation: Despite being essential for numerous clinical and research applications, high-resolution human leukocyte antigen (HLA) typing remains challenging and laboratory tests are also time-consuming and labour intensive. With next-generation sequencing data becoming widely accessible, on-demand in silico HLA typing offers an economical and efficient alternative. Results: In this study we evaluate the HLA typing accuracy and efficiency of five computational HLA typing methods by comparing their predictions against a curated set of > 1000 published polymerase chain reaction-derived HLA genotypes on three different data sets (whole genome sequencing, whole exome sequencing and transcriptomic sequencing data). The highest accuracy at clinically relevant resolution (four digits) we observe is 81% on RNAseq data by PHLAT and 99% accuracy by OptiType when limited to Class I genes only. We also observed variability between the tools for resource consumption, with runtime ranging from an average of 5 h (HLAminer) to 7 min (seq2HLA) and memory from 12.8 GB (HLA-VBSeq) to 0.46 GB (HLAminer) per sample. While a minimal coverage is required, other factors also determine prediction accuracy and the results between tools do not correlate well. Therefore, by combining tools, there is the potential to develop a highly accurate ensemble method that is able to deliver fast, economical HLA typing from existing sequencing data.

Oxysterol-binding protein-related protein 5 (ORP5) promotes cell proliferation by activation of mTORC1 signaling.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large family of proteins that mainly function in lipid transport and sensing. ORP5 is an endoplasmic reticulum (ER)-anchored protein implicated in lipid transfer at the contact sites between the ER and other membranes. Recent studies indicate that ORP5 is also involved in cancer cell invasion and tumor progression. However, the molecular mechanism underlying ORP5's involvement in cancer is unclear. Here, we report that ORP5 promotes cell proliferation and motility of HeLa cells, an effect that depends on its functional OSBP-related domain (ORD). We also found that ORP5 depletion or substitutions of key residues located within ORP5-ORD and responsible for interactions with lipids interfered with cell proliferation, migration, and invasion. ORP5 interacted with the protein mechanistic target of rapamycin (mTOR), and this interaction also required ORP5-ORD. Of note, whereas ORP5 overexpression induced mTOR complex 1 (mTORC1) activity, ORP5 down-regulation had the opposite effect. Finally, ORP5-depleted cells exhibited impaired mTOR localization to lysosomes, which may have accounted for the blunted mTORC1 activation. Together, our results suggest that ORP5 expression is positively correlated with mTORC1 signaling and that ORP5 stimulates cell proliferation, at least in part, by activating mTORC1.

Lipid droplet biogenesis and functions in health and disease.

Ubiquitous yet unique, lipid droplets are intracellular organelles that are increasingly being recognized for their versatility beyond energy storage. Advances uncovering the intricacies of their biogenesis and the diversity of their physiological and pathological roles have yielded new insights into lipid droplet biology. Despite these insights, the mechanisms governing the biogenesis and functions of lipid droplets remain incompletely understood. Moreover, the causal relationship between the biogenesis and function of lipid droplets and human diseases is poorly resolved. Here, we provide an update on the current understanding of the biogenesis and functions of lipid droplets in health and disease, highlighting a key role for lipid droplet biogenesis in alleviating cellular stresses. We also discuss therapeutic strategies of targeting lipid droplet biogenesis, growth or degradation that could be applied in the future to common diseases, such as cancer, hepatic steatosis and viral infection.

PI(3)P and DFCP1 regulate the biogenesis of lipid droplets.

The biogenesis of lipid droplets (LDs), key organelles for cellular lipid storage and homeostasis, remains poorly understood. Seipin is essential to normal LD biogenesis but exactly how it regulates LD initiation remains to be elucidated. Our previous results suggested that seipin may bind anionic phospholipids such as PI(3)P. Here, we investigate whether PI(3)P is functionally linked to seipin and whether PI(3)P can also impact LD biogenesis. In seipin-deficient cells, there were enlarged PI(3)P puncta where its effector, DFCP1, also appeared to congregate. Reducing cellular PI(3)P partially rescued the defective LD initiation caused by seipin deficiency. Increasing PI(3)P impeded the lipidation of nascent LDs. We further demonstrated that DFCP1 localized to LDs and facilitated the efficient lipidation of nascent LDs. However, the normal function and localization of DFCP1 were disrupted when cellular PI(3)P homeostasis was perturbed. Our results thus identify PI(3)P as a novel regulator of LD initiation and suggest that PI(3)P may impact the biogenesis of LDs through DFCP1.

ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets.

Lipid droplets (LDs) are evolutionarily conserved organelles that play important roles in cellular metabolism. Each LD is enclosed by a monolayer of phospholipids, distinct from bilayer membranes. During LD biogenesis and growth, this monolayer of lipids expands by acquiring phospholipids from the endoplasmic reticulum (ER) through nonvesicular mechanisms. Here, in a mini-screen, we find that ORP5, an integral membrane protein of the ER, can localize to ER-LD contact sites upon oleate loading. ORP5 interacts with LDs through its ligand-binding domain, and ORP5 deficiency enhances neutral lipid synthesis and increases the size of LDs. Importantly, there is significantly more phosphatidylinositol-4-phosphate (PI(4)P) and less phosphatidylserine (PS) on LDs in ORP5-deficient cells than in normal cells. The increased presence of PI(4)P on LDs in ORP5-deficient cells requires phosphatidylinositol 4-kinase 2-α. Our results thus demonstrate the existence of PI(4)P on LDs and suggest that LD-associated PI(4)P may be primarily used by ORP5 to deliver PS to LDs.

ABHD15 regulates adipose tissue lipolysis and hepatic lipid accumulation.

OBJECTIVE: Insulin suppresses adipose tissue lipolysis after a meal, playing a key role in metabolic homeostasis. This is mediated via the kinase Akt and its substrate phosphodiesterase 3B (PDE3B). Once phosphorylated and activated, PDE3B hydrolyses cAMP leading to the inactivation of cAMP-dependent protein kinase (PKA) and suppression of lipolysis. However, several gaps have emerged in this model. Here we investigated the role of the PDE3B-interacting protein, α/ß-hydrolase ABHD15 in this process. METHODS: Lipolysis, glucose uptake, and signaling were assessed in ABHD15 knock down and knock out adipocytes and fat explants in response to insulin and/or ß-adrenergic receptor agonist. Glucose and fatty acid metabolism were determined in wild type and ABHD15-/- littermate mice. RESULTS: Deletion of ABHD15 in adipocytes resulted in a significant defect in insulin-mediated suppression of lipolysis with no effect on insulin-mediated glucose uptake. ABHD15 played a role in suppressing PKA signaling as phosphorylation of the PKA substrate Perilipin-1 remained elevated in response to insulin upon ABHD15 deletion. ABHD15-/- mice had normal glucose metabolism but defective fatty acid metabolism: plasma fatty acids were elevated upon fasting and in response to insulin, and this was accompanied by elevated liver triglycerides upon ß-adrenergic receptor activation. This is likely due to hyperactive lipolysis as evident by the larger triglyceride depletion in brown adipose tissue in these mice. Finally, ABHD15 protein levels were reduced in adipocytes from mice fed a Western diet, further implicating this protein in metabolic homeostasis. CONCLUSIONS: Collectively, ABHD15 regulates adipocyte lipolysis and liver lipid accumulation, providing novel therapeutic opportunities for modulating lipid homeostasis in disease.

How difficult is inference of mammalian causal gene regulatory networks?

Gene regulatory networks (GRNs) play a central role in systems biology, especially in the study of mammalian organ development. One key question remains largely unanswered: Is it possible to infer mammalian causal GRNs using observable gene co-expression patterns alone? We assembled two mouse GRN datasets (embryonic tooth and heart) and matching microarray gene expression profiles to systematically investigate the difficulties of mammalian causal GRN inference. The GRNs were assembled based on > 2,000 pieces of experimental genetic perturbation evidence from manually reading > 150 primary research articles. Each piece of perturbation evidence records the qualitative change of the expression of one gene following knock-down or over-expression of another gene. Our data have thorough annotation of tissue types and embryonic stages, as well as the type of regulation (activation, inhibition and no effect), which uniquely allows us to estimate both sensitivity and specificity of the inference of tissue specific causal GRN edges. Using these unprecedented datasets, we found that gene co-expression does not reliably distinguish true positive from false positive interactions, making inference of GRN in mammalian development very difficult. Nonetheless, if we have expression profiling data from genetic or molecular perturbation experiments, such as gene knock-out or signalling stimulation, it is possible to use the set of differentially expressed genes to recover causal regulatory relationships with good sensitivity and specificity. Our result supports the importance of using perturbation experimental data in causal network reconstruction. Furthermore, we showed that causal gene regulatory relationship can be highly cell type or developmental stage specific, suggesting the importance of employing expression profiles from homogeneous cell populations. This study provides essential datasets and empirical evidence to guide the development of new GRN inference methods for mammalian organ development.