The transcription factor Foxp1 regulates aerobic glycolysis in adipocytes and myocytes.

In recent years, lactate has been recognized as an important circulating energy substrate rather than only a dead-end metabolic waste product generated during glucose oxidation at low levels of oxygen. The term "aerobic glycolysis" has been coined to denote increased glucose uptake and lactate production despite normal oxygen levels and functional mitochondria. Hence, in "aerobic glycolysis," lactate production is a metabolic choice, whereas in "anaerobic glycolysis," it is a metabolic necessity based on inadequate levels of oxygen. Interestingly, lactate can be taken up by cells and oxidized to pyruvate and thus constitutes a source of pyruvate that is independent of insulin. Here, we show that the transcription factor Foxp1 regulates glucose uptake and lactate production in adipocytes and myocytes. Overexpression of Foxp1 leads to increased glucose uptake and lactate production. In addition, protein levels of several enzymes in the glycolytic pathway are upregulated, such as hexokinase 2, phosphofructokinase, aldolase, and lactate dehydrogenase. Using chromatin immunoprecipitation and real-time quantitative PCR assays, we demonstrate that Foxp1 directly interacts with promoter consensus cis-elements that regulate expression of several of these target genes. Conversely, knockdown of Foxp1 suppresses these enzyme levels and lowers glucose uptake and lactate production. Moreover, mice with a targeted deletion of Foxp1 in muscle display systemic glucose intolerance with decreased muscle glucose uptake. In primary human adipocytes with induced expression of Foxp1, we find increased glycolysis and glycolytic capacity. Our results indicate Foxp1 may play an important role as a regulator of aerobic glycolysis in adipose tissue and muscle.

Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling.

Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.

TLCD1 and TLCD2 regulate cellular phosphatidylethanolamine composition and promote the progression of non-alcoholic steatohepatitis.

The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.

Extensive transcription mis-regulation and membrane defects in AdipoR2-deficient cells challenged with saturated fatty acids.

How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we used transcriptomics, lipidomics, growth and respiration assays, and membrane property analyses in human HEK293 cells or human umbilical vein endothelial cells (HUVEC) to show that the function of AdipoR2 is to respond to membrane rigidification by regulating many lipid metabolism genes. We also show that AdipoR2-dependent membrane homeostasis is critical for growth and respiration in cells challenged with saturated fatty acids. Additionally, we found that AdipoR2 deficiency causes transcriptome and cell physiological defects similar to those observed in SREBP-deficient cells upon SFA challenge. Finally, we compared several genes considered important for lipid homeostasis, namely AdipoR2, SCD, FADS2, PEMT and ACSL4, and found that AdipoR2 and SCD are the most important among these to prevent membrane rigidification and excess saturation when human cells are challenged with exogenous SFAs. We conclude that AdipoR2-dependent membrane homeostasis is one of the primary mechanisms that protects against exogenous SFAs.

Evolutionarily conserved long-chain Acyl-CoA synthetases regulate membrane composition and fluidity.

The human AdipoR1 and AdipoR2 proteins, as well as their C. elegans homolog PAQR-2, protect against cell membrane rigidification by exogenous saturated fatty acids by regulating phospholipid composition. Here, we show that mutations in the C. elegans gene acs-13 help to suppress the phenotypes of paqr-2 mutant worms, including their characteristic membrane fluidity defects. acs-13 encodes a homolog of the human acyl-CoA synthetase ACSL1, and localizes to the mitochondrial membrane where it likely activates long chains fatty acids for import and degradation. Using siRNA combined with lipidomics and membrane fluidity assays (FRAP and Laurdan dye staining) we further show that the human ACSL1 potentiates lipotoxicity by the saturated fatty acid palmitate: silencing ACSL1 protects against the membrane rigidifying effects of palmitate and acts as a suppressor of AdipoR2 knockdown, thus echoing the C. elegans findings. We conclude that acs-13 mutations in C. elegans and ACSL1 knockdown in human cells prevent lipotoxicity by promoting increased levels of polyunsaturated fatty acid-containing phospholipids.

FOXK1 and FOXK2 regulate aerobic glycolysis.

Adaptation to the environment and extraction of energy are essential for survival. Some species have found niches and specialized in using a particular source of energy, whereas others-including humans and several other mammals-have developed a high degree of flexibility1. A lot is known about the general metabolic fates of different substrates but we still lack a detailed mechanistic understanding of how cells adapt in their use of basic nutrients2. Here we show that the closely related fasting/starvation-induced forkhead transcription factors FOXK1 and FOXK2 induce aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the E1α regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria-instead, pyruvate is reduced to lactate. Suppression of FOXK1 and FOXK2 induce the opposite phenotype. Both in vitro and in vivo experiments, including studies of primary human cells, show how FOXK1 and/or FOXK2 are likely to act as important regulators that reprogram cellular metabolism to induce aerobic glycolysis.

Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2.

Dietary fatty acids are the main building blocks for cell membranes in animals, and mechanisms must therefore exist that compensate for dietary variations. We isolated C. elegans mutants that improved tolerance to dietary saturated fat in a sensitized genetic background, including eight alleles of the novel gene fld-1 that encodes a homolog of the human TLCD1 and TLCD2 transmembrane proteins. FLD-1 is localized on plasma membranes and acts by limiting the levels of highly membrane-fluidizing long-chain polyunsaturated fatty acid-containing phospholipids. Human TLCD1/2 also regulate membrane fluidity by limiting the levels of polyunsaturated fatty acid-containing membrane phospholipids. FLD-1 and TLCD1/2 do not regulate the synthesis of long-chain polyunsaturated fatty acids but rather limit their incorporation into phospholipids. We conclude that inhibition of FLD-1 or TLCD1/2 prevents lipotoxicity by allowing increased levels of membrane phospholipids that contain fluidizing long-chain polyunsaturated fatty acids. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A CRISP(e)R view on kidney organoids allows generation of an induced pluripotent stem cell-derived kidney model for drug discovery.

Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.

Critical differences in drug metabolic properties of human hepatic cellular models, including primary human hepatocytes, stem cell derived hepatocytes, and hepatoma cell lines.

Primary human hepatocytes (PHH), HepaRG™, HepG2, and two sources of induced pluripotent stem cell (iPSC) derived hepatocytes were characterized regarding gene expression and function of key hepatic proteins, important for the metabolic fate of drugs. The gene expression PCA analysis showed a distance between the two iPSC derived hepatocytes as well as the HepG2 and HepaRG™ cells to the three PHH donors and PHH pool, which were clustered more closely together. Correlation-based hierarchical analysis clustered HepG2 close to the stem cell derived hepatocytes both when the expression of 91 genes related to liver function or only cytochrome P450 (P450) genes were analyzed indicating the non-liver feature and a similar low P450 profile in these cell models. The specific P450 activities and the metabolic pattern of well-characterized drug substances in the cell models demonstrated that iPSC derived hepatocytes had modest levels of CYP3A and CYP2C9, while CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 were barely detectable. High expression of several extrahepatic P450s such as CYP1A1 and 1B1 detected in the stem cell derived hepatocytes may have significant effects on metabolite profiles. However, one of the iPSC derived hepatocytes demonstrated significant combined P450 and conjugating enzyme activity of certain drugs. HepaRG™ cells showed many metabolic properties similar to PHHs and will in many respects be a good model in studies of metabolic pathways and induction of drug metabolism whereas there is still ground to cover before iPSC derived hepatocytes will be seen as a substitute to PHH in drug metabolism studies.

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell-Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury.

Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems-hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids-at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans.