A mix of papers from the past weeks: Affimer proteins might be the new antibodies, crosstalk between ERK and PI3K/AKT/mTORC1 signalling, the power of GWAS and much more

Horribly busy these days so no chance to write extensively about these, but in case they are of relevance to others, here is a list of some potentially interesting reads:

eLife: Affimer proteins are versatile and renewable affinity reagents (Tiede et al.) ➡ a potential future alternative to antibodies.  https://elifesciences.org/articles/24903

Nature: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer (Zabala-Letona et al.). Quite surprised that the mechanisms only rely on inhibitor treatment (encouraging in a way if this is “all” that is required…). Mechanism suggested to be important for proliferating cells in general. https://www.nature.com/nature/journal/vaop/ncurrent/full/nature22964.html#ref6
Scientific Reports: Inhibition of ERK1/2 restores GSK3b activity and protein synthesis levels in a model of tuberous sclerosis (Pal et al.): https://pdf.nature.com/redirect-nature?ddsId=art:10.1038/s41598-017-04528-5&originUrl=https://www.nature.com/articles/s41598-017-04528-5&contentType=pdf ➡ relevant for insulin signalling via ERK. Had a quick skim of this paper. Not entirely convinced this is robust, HEK293s + MEFs used, high concentrations of insulin, drugs etc.
Scientific Reports: Direct binding of MEK1 and MEK2 to AKT induces Foxo1 phosphorylation, cellular migration and metastasis (Procaccia et al.). https://www.nature.com/articles/srep43078.pdf
British Journal of Cancer: Akt as a target for cancer therapy: more is not always better (lessons from studies in mice) (Wang et al.). Highlights how deletion of AKT2 in the liver can lead to increased cancer incidence, thus emphasising the importance of considering tissue-specific effects when inhibiting PI3K/AKT signalling in the clinic.   https://www.nature.com/bjc/journal/vaop/ncurrent/pdf/bjc2017153a.pdf
American Journal of Human Genetics: 10 Years of GWAS Discovery: Biology, Function, and Translation (Visscher et al.) – it is a good read.
JCI Insight: Independent tissue contributors to obesity-associated insulin resistance (Kusters et al.https://insight.jci.org/articles/view/89695
Cell Metabolism: How does fat transition from white to beige? (Marc L. Reitman) http://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30353-4?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1550413117303534%3Fshowall%3Dtrue ➡ a commentary on some recent surprising findings.
Cell Metabolism: Leptin’s Physiologic Role: Does the Emperor of Energy Balance Have No Clothes? (what a title; by Jeffrey Flier and Eleftheria Maratos-Flier) http://www.cell.com/cell-metabolism/fulltext/S1550-4131(17)30302-9?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS1550413117303029%3Fshowall%3Dtrue
Cell: Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms (Rusu et al.http://www.cell.com/cell/fulltext/S0092-8674(17)30694-3?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867417306943%3Fshowall%3Dtrue

Neoplastic changes in stem cells, nutrient acquisition strategies of mammalian cells and much, much more

After a couple of weeks in cell culture and RNAseq land, finally some time for a bit of science reading! However, not much time for writing it all up as I usually do, so here is detailed coverage of two of my recent reads. Enjoy!

Due to some unexpected results, I have had to search the stem cell literature for information on adaptive changes due to selection pressure. Came across one important paper from 2009 in Nature Biotechnology by Werbowetski-Ogilvie et al.: Characterization of human embryonic stem cells with features of neoplastic progression ➡ based on some of these points, it is really important to characterise stem cell models thoroughly, especially if you study a cancer pathway!!

  • Variant pluripotent stem cells with neoplastic changes may arise in culture and be mistaken for “better” stem cells because of higher expression of stem cell markers.
  • Paper demonstrates that such variant stem cells (2 clones studied) have acquired expression of FGFR1 and IGF1R and have become refractory to differentiation in vitroIn vivo, such cells exhibited a better capacity for teratoma formation, but were not deemed to be malignant because they didn’t metastasise (but mice kept for 6 weeks, so perhaps this could happen eventually?).

 

Nature just published a very interesting review “Nutrient acquisition strategies of mammalian cells” by Wilhelm Palm and Craigh Thompson. A few key points that I have got out of it:

  • Metabolic flexibility in cells achieved by using different nutrient sources: prepared for periods of starvation.
  • Proliferating cells: growth factor-induced increases in nutrient uptake required to support biomass accumulation. Proliferating cells need to reorganise metabolism so that nutrients are not oxidised fully to CO2 and ATP, but used in anabolic processes.
  • Bulk of ATP production in mammalian cells + generation of non-essential metabolites: glucose, fatty acids, glutamine
  • Cancer cells have changes that enable them to survive and grow in poorly vascularised environments by using extracellular nutrients, including low-molecular-mass nutrients and macromolecules as well as cellular debris.
  • Class IA PI3K and RAS signalling pathways are central regulators of cellular nutrient acquisition. Activated by various growth factor signals. Such cell-extrinsic regulation of nutrient uptake constitutes a fundamental barrier for cellular transformation. Oncogenic changes that result in hyperactivation of Ras/PI3K signalling ➡ cellular autonomy for both cell cycle entry and nutrient acquisition.
  • mTORC1 as a central coordinator of amino acid availability and cell growth. Interplay with AMPK is central to cellular metabolic homeostasis in a variable nutrient environment. Because mTORC1 is also modulated downstream of class IA PI3K, the cell is able to integrate metabolic status and growth factor signals. Activation of AKT/mTORC1 signalling defines a cellular state of high rates of glucose and amino acid uptake through transporters with concomitant suppression of lysosomal catabolism of macromolecules.
  • PI3K and Ras pathways increase glucose uptake and glycolysis. Myc increases expression of amino acid transporters. Together, Myc, Ras and PI3K stimulate ribosomal biogenesis and mTORC1-dependent translational initiation.
  • Expression of various cell surface receptors that mediate the uptake of certain nutrients (e.g. iron or cholesterol) is ultimately modulated by the very same pathways listed above.
  • Macropinocytosis: non-selective endocytic pathway for bulk ingestion of extracellular solutes. Driven by actin-based protrusions. Actin-driven protrusions are immediate downstream responses of growth factor stimulation. Accordingly cells with oncogenic mutation  in Ras pathways exhibit high levels of macropinocytosis. It remains to be established if the same is true for cells with oncogenic PI3K mutations.
  • Epithelial cells can internalise whole neighboring cells via a non-phagocytic process ➡ process known as entosis ➡ digestion of the internalised cell. This behaviour is enhanced by oncogenic mutations in Ras.
  • Autophagy is also important for tumour survival.
  • Glutamine is the second most-consumed nutrient in proliferating cells ➡ major carbon source for anaplerotic reactions that replenish TCA cycle intermediates. Glutamine also provides nitrogen for synthesis of amino acids, nucleotides and hexosamines. In vivo, tumours also replenish the TCA cycle from glucose-derived pyruvate. Some cancers also express high levels of branched-chain amino acid transferases ➡ enables them to use leucine, isoleucine and valine as nitrogen sources. These are example metabolic adaptations that give cancer cells a survival advantage.
  • Evidence for paracrine signalling based on observations that non-transformed cells often undergo phenotypic changes in the tumour microenvironment. Mechanisms exist that allow tumour cells to acquire their nutrients from neighbouring cells. Tumours can also signal to distant organs.
  • More research required to identify how signalling pathways regulate cellular nutrient uptake and what factors determine differences in nutrient usage.

 

Other bits and pieces to check out:

In Science this week: mTORC1 activity repression by late endosomal phosphatidylinositol 3,4-bisphosphate (Marat et al. http://science.sciencemag.org/content/356/6341/968)

Identification and characterization of a supraclavicular brown adipose tissue in mice (JCI Insight): https://insight.jci.org/articles/view/93166 

Feng Zhang and his crew with another CRISPR tool paper in Nature: Engineered Cpf1 variants with altered PAM specificities; https://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3900.html

Hepatic Diacylglycerol-Associated Protein Kinase Cε Translocation Links Hepatic Steatosis to Hepatic Insulin Resistance in Humans (Horst et al., Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717306770 

Phosphorylation of TXNIP by AKT Mediates Acute Influx of Glucose in Response to Insulin (Waldhart et al., Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717306836 

MYO6 Regulates Spatial Organization of Signaling Endosomes Driving AKT Activation and Actin Dynamics (Masters et al. Cell Reports): http://www.sciencedirect.com/science/article/pii/S2211124717307052

Interesting paracrine signalling mechanism: Intercellular transmission of the unfolded protein response promotes survival and drug resistance in cancer cells (Rodvold et al. Science Signaling) http://stke.sciencemag.org/content/10/482/eaah7177 

A Nature Reviews Endocrinology review on why people should study mice at thermoneutrality: https://www.sparrho.com/item/warming-the-mouse-to-model-human-diseases/1385ea1/

A Diabetes review on the global T2D Epidemic and the importance of early β-cell failure: http://diabetes.diabetesjournals.org/content/66/6/1432

An Expanded Genome-Wide Association Study of Type 2 Diabetes in Europeans (Scott et al. Diabetes) http://diabetes.diabetesjournals.org/content/early/2017/05/25/db16-1253

 

 

Some interesting reading: bits and pieces of recent research

Nature: Blocking FSH induces thermogenic adipose tissue and reduces body fat (Liu et al.) ➡ apparently, this could be used to treat osteoporosis and obesity associated with menopause…  Looks like a nice study, but haven’t scrutinised it properly.

Nature: Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling (Brockmann et al.). Skimmed this through, and basically the group applies high-throughput technology and protein abundance as a readout for phenotypes arising from random mutagenesis in haploid cells to identify novel regulators of signal trasnduction. They identify a new mechanisms whereby Gβγ is targeted for degradation by KCTD5 to limit the ability of GPCRs to trigger PI3K/AKT activation.

Nature resource-type paper that is important for mining -omics datasets / hypothesis generation: Architecture of the human interactome defines protein communities and disease networks (Huttlin et al.) ➡ the resource is called BioPlex 2.0; this is the abstract of the paper describing what it’s all about:

“The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein– protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease1–3. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification–mass spectrometry methodology4 to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering5 of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness6,7 are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.”

Nature News & Views on the ATP-independent mitochondrial regulation of cell fate decisions in haematopoietic stem cells which was demonstrated by two independent studies recently (linked to within the news and views). It is very interesting because it is linked back to mTOR signalling and also demonstrates the importance of mitochondria in cells that are otherwise very glycolytic and don’t rely on mitochondria to generate energy. Title to search for if interested: Mitochondria link metabolism and epigenetics in haematopoiesis (Schell, J. & Rutter, J.).

Nature News & Views (Cell forces meet cell metabolism) on another interesting mechanism relating to how cell obtain their energy during the costly process of cell-cell adhesion, with link to the original paper that reported this (. Very interesting because it is relevant for understanding how cell-cell interactions link to intracellular metabolism regulation. In the described case, force exerted through E-cadherins links to AMPK activation and increased glycolysis in epithelial cells. Also remindes me of the recent paper linking PI3K activation and remodelling of the actin cytoskeleton to release Aldolase and trigger glycolysis in preparation for cell migration (by Lewis Cantley’s group). Must admit that I haven’t read the original paper discussed in this News and Views and after skimming through the quality of the figures and methods, I already noted wrong statistics (t tests instead of 1-Way ANOVA or 2-Way ANOVA!!).

eLife: Synergistic interactions with PI3K inhibition that induce apoptosis (Zwang et al.). This group looked for genes that promote breast cancer cell survival in the face of PI3K inhibition (the initial cells they use have PIK3CA H1047R and ERBB2 amplification, they then confirm in additional cell lines). They performed a shRNA-based apoptosis screen and identified PIM2ZAK, TACC1, ZFR and ZNF565 as genes whose inhibition in the presence of the p110α/δ inhibitor GDC0941 (625 nM). It is interesting to note that from the initial shRNA screen where 54 candidates were identified, only 5 were confirmed upon orthogonal validation. Goes to show the importance of validation experiments, which they also do for the 5 final candidates by overexpressing them to check that the phenotype is rescued – a method which validated 3/5 candidates. All 5 eventually validated in vivo. I like the fact that they drug concentrations are not ridiculously high (which is the case for many papers, confounding their findings), and they also test additional inhibitors in the context of the proposed interactions and find that the effect is p110α-dependent and occurs throught he canonical signalling pathway (AKT etc.).They also use z-score filtering following the initial screen to account both for the magnitude of the effect and the consistency of replicates. Another interesting thing in this paper: they use a so-called BH3 profiling assay to determine mitochondrial priming (i.e. the proportion of anti-apoptotic and apoptotic proteins). In brief, you permeabilise the cells and incubate them with increasing concentrations of synthetic BIM peptide; you then fix the cells and stain for endogenous cytochrome c, then perform flow cytometry to quantify loss of cytochrome c (measure of mitochondrial depolarisation).

FEBS Journal: The energy sensing LKB1-AMPKα1 pathway regulates IGF1 secretion and consequent activation of the IGF1R-PKB pathway in primary hepatocytes (Chen et al.) – it is a small study limited to primary hepatocytes, and the effects are not striking, but might be interesting to read anyway. Looks like metformin reduces IGF1 secretion.

EMBO News & Views covering the 2 most recent Mitofusin papers: Let’s burn whatever you have: mitofusin 2 metabolically re-wires brown adipose tissue (Scheideler, M. & Herzig, S.)

On the biophysical/hardcore biochemistry side of things, some studies looking at molecular Cas9 mechanisms:

PNAS: Mechanisms of dupex DNA destabilisation by RNA-guided Cas9 nuclease during target interrogation (Mekler et al.)

PNAS: High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding (Boyle et al.)

Also on the techy side from Jennifer Lippincott-Schwartz’ lab in Nature: Applying systems-level spectral imaging and analysis to reveal the organelle interactome (Valm et al.)

Out of general interest and haven’t read beyond abstract, but in Science this week: Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation (Zhang, X. & van den Pol, A)

Science news

Cancer Cell: A Pan-Cancer Proteogenomic Atlas of PI3K/AKT/mTOR Pathway Alterations (Zhang et al.) – an important contribution to the cancer literature; this study takes advantage of the recent completion of the data generation stage of the The Cancer Genoem Atlas (TCGA) and performs a systematic analysis of the PI3K/AKT/mTOR pathway in over 10,000 cancer, covering 32 different major cancer types. It examines mutated genes via whole-exome/genome sequencing, transcriptomics data (RNAseq) and candidate signalling data with reverse-phase protein arrays. It is a data-dense study, but I will just highlight a few points that were interesting to me. Although activities in the PI3K/AKT and mTOR pathways were highly correlated in multiple cancer, there were also instances with evidence for some decoupling. Moreover, this study provides some high-throughput functional characterisation of a larger set of activating PIK3CA mutations in two different immortalised cell lines; one caveat with this functional analysis, however, is the fact that it relies on overexpression of the mutated proteins. Nevertheless, as the setting is the same for all tested variants, it allows for direct comparisons among them to be made – and such comparisons are quite useful as there is very limited data on the functional significance of different PIK3CA mutations beyond the well-known hotspot variants. Overall, the large sample size used to generate this data provides substantial power to detect meaningful patterns that can be used to stratify variants in the clinical setting.

Nature: TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling (Wang et al.) – a key paper and really worth a read (or at least of key points as it is quite data dense!!). Probably one of the most comprehensive papers on mTORC2 that I have seen that also links it to relevant (patho)physiology. Very interesting that growth factors (incl. insulin) tip the balance between mTORC2 and mTORC1 formation, favouring mTORC2 which then drives increased AKT activation. Evidence provided that this is relevant in conditions of PI3K/AKT hyperactivation. Very interesting!! Also, noted that they use HEK293s in multiple experiments and manage to look at PI3K/AKT signalling after serum starving these for 16h.. Usually these cells exhibit hyperactivation of this pathway, but the control conditions here show no sign of this?

Science: A subcellular map of the human proteome (Thul et al.) – an important atlas-like resource, essentially an image-based map of the subcellular proteome based on transcriptomics, immunofluorescence and mass spectrometry. It is quite impressive, the data covers 12,0003 proteins using a panel of 22 human cell lines and 13,993 antibodies. Also, they have managed to get the images annotated through a Citizen Science approach via massive multiplayer game with participation from over 180,000 worldwide players! This dataset is actually quite important as it allows for more refined interaction networks to be constructed. The interactive resources can be accessed here: http://www.proteinatlas.org

Science: ATP as a biological hydrotrope (Patel et al.) – haven’t read beyond abstract, but interesting because it seems to suggest a role for ATP in protein solubilisation within cells. (Hydrotorope = amphiphilic molecules with low cooperativity and millimolar working concentrations, differentiating them from surfactants ➡ act to solubilise hydrophobic molecules in acqueous solutions).

Science Signalling: p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM (Stewart-Ornstein, J. and Lahav, G.) ➡ this study highlights an issue that is worth keeping in mind whenever a cell biology paper is examined: signalling dynamics do differ across cell lines, hence using a single cell lines as model system for a major phenomenon might not yield results that are broadly applicable. This paper has also got some mathematical modelling for those interested in that. I must admit that I have not read it in much detail and I am unable to comment on the computational approach.

Diabetes: Mechanisms of Insulin Resistance in Primary and Secondary Non-Alcoholic Fatty Liver (Jelenik et al.) ➡ can’t usually access, but here is the abstract:

“Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance and may result primarily from increased hepatic de novo lipogenesis (PRIM) or secondarily from adipose-tissue lipolysis (SEC). We studied mice with hepatocyte- or adipocyte-specific sterol regulatory-element binding protein-1c (SREBP-1c) overexpression as models of PRIM and SEC. PRIM mice featured increased lipogenic gene expression in liver and adipose tissue. Their selective, liver-specific insulin resistance was associated with increased C18:1-diacylglycerol (DAG) content and protein kinase C (PKC)ε translocation. SEC mice had decreased hepatic ChREBP-mediated lipogenesis and featured portal/lobular inflammation along with total, whole-body insulin resistance. Hepatic mitochondrial respiration transiently increased and declined with aging along with higher muscle reactive oxygen species production. In conclusion, hepatic insulin resistance originates from lipotoxicity but not from lower mitochondrial capacity, which can even transiently adapt to increased peripheral lipolysis. Peripheral insulin resistance is prevented during increased hepatic lipogenesis, only if adipose tissue lipid storage capacity is preserved.”

Nature Cell Biology Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling (Jin et al.)

Cell Reports: Widespread Mitotic Bookmarking by Histone Marks and Transcription Factors in Pluripotent Stem Cells (Liu et al.) – interesting paper that demonstrates that one of the mechanisms whereby the core stemness factor OCT4 maintains pluripotency is by bookmarking stemness genes during mitosis, i.e. a memory mechanisms that allows for re-expression of these genes when mitosis is completed. This is important in stem cells due to their unusual cell cycle characteristics (10-12h cycling, no G0 phase and very short G1 phase). I find one of their approaches quite cool – testing the effect of specifically degrading OCT4 during mitosis by fusing OCT4 to a Cyclin destruction box.

Nature Protocols: FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry (Arrigucci et al.) note that this is only applicable for non-adherent cell types at the moment)

eLife: Addressing the ethical issues raised by synthetic human entities with embryo-like features (Aach et al.) – interesting read, I think…

 

 

 

This week’s interesting science

Nature: Common genetic variation drives molecular heterogeneity in human iPSCs (Kilpinen et al.). The first results form the large-scale HipSci initiative funded by Wellcome.

A very interesting Nature study on vascular development and how it is regulated by FGF-dependent control of metabolism (glycolysis) via Myc: FGF-dependent metabolic control of vascular development (Yu et al. 2017). Here is a copy of the final conclusion of the paper: The FGF/MYC/HK2-dependent regulation of vascular development is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic is unexpected. Previously, FGF activity has been linked to prevention of endothelium-to-mesenchymal transition both in the lymphatic16 and in the systemic vasculature17, injury response18 and maintenance of endothelium-to-mesenchymal transition both in the lymphatic and in the systemic vasculature, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors and in the systemic vasculature17, injury response18 and maintenance of vascular integrity19. While the FGFR1 and FGFR3 are the receptors involved, which of the 22 FGF family members is responsible for the required FGF signalling input is not known. In summary, FGF signalling regulates blood and lymphatic vascular development through control of endothelial metabolism driven by MYC-dependent regula-tion of HK2 expression. Therapeutic targeting of this FGF–MYC–HK2 pathway may open new possibilities for treatment of diseases associated with insufficient or excessive vascular growth.

Just out in Nature Medicine: Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients (Zehir et al. 2017) ➡ Interesting recurrence of certain well-known oncogenes (e.g. PIK3CA) across a range of tumour types, irrespective of lineage, but also very interesting to see how mutations in certain oncogenes are absent from specific tumour histotypes. TP53 mutations top the list and correlate positively with more aggressive cancers. Most of the TP53 mutations were inactivating due to truncation or altered splicing. Second on the list is KRAS G12 followed by PIK3CA H1047R and E545K.

Cell Metabolism: PPARδ Promotes Running Endurance by Preserving Glucose (Fan et al. 2017). Just skimmed through, looks quite comprehensive. PPARd orchestrates the transcriptional switch to fat utilisation and glucose sparing in response to exercise.

Cell Metabolism: DNA-PK Promotes the Mitochondrial, Metabolic, and Physical Decline that Occurs During Aging (Park et al. 2017) – a mechanism that involves phosphorylation of Hsp90 and inhibition of AMPK.

An interesting paper in Cell (Stem Cell Lineage Infidelity Drives Wound Repair and Cancer by Ge et al.) that uncovers how tumour cells hijack normal wound repair processes which under normal circumstances allow for transient stem cell lineage infidelity. In contrast, a pre-cancerous stem cells are locked into this plastic state, giving rise to excessive growth and ultimately full-blown cancer.

Nature Protocols: Assessment of engineered cells using CellNet and RNA-seq (Radley et al. 2007) – a how-to-guide for the computational platform CellNet, which allows one to upload one RNAseq data from a particular cell type / stage and compare it to large datasets on different cell types. This should be very useful for estimation of cell fate transitions in response to different differentiation perturbations.

 

eLife: Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering (Lee et al. 2017). These guys test different modifications of sgRNAs and ssODN repair templates and show that they can be tolerated. For instance, they tag the ssODN with an Alexa-647 which allows them to enrich for cells that have been successfully transfected with the repair template. They also manage to fuse the sgRNA and ssODN template to each other, complex these with Cas9 (RNP), and show that this improved cellular delivery based on cationic polymers. However, note that despite this improval the efficiency of gene editing is still lower compared to nucleofection/electroporation under comparable conditions.

Nature Methods: CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets (Tsai et al. 2017). A new genome-wide method is described for the assessment of CRISPR off-target effects in vitro and following tag integration into host cell DNA. Much more sensitive compared to previous methods that achieved whole-genome assessment of off-target effects. A second study in Nature Methods reports on a different method (SITE-seq) that also aims to profile genome-wide off-target effects with high sensitivity: Mapping the genomic landscape of CRISPR–Cas9 cleavage (Cameron et al. 2017). However, the specificity of Cas9-mediated gene editing in a cell will also be highly cell-specific and depend  on the concentration of enzyme and additional components that are delivered for gene editing (sgRNA, ssODN etc.). The effect of increasing sgRNA and Cas9 concentrations in vitro is assessed in the biochemical assay reported by Cameron et al. Both of the above methods achieve a much higher sensitivity of off-target site detection because they specifically enriched for DNA fragments associated with Cas9 cleavage prior to sequencing (i.e. all the random background is removed). Once such off-targets are identified genome-wide, they can be interrogated in any cell line that has been edited – many will, however, remain intact in the cell because of the effects of chromatin on target accessibility as well as cell-specific DNA repair mechanisms.

 

The science round-up of the week

Nature & Nature++

The implications of this Nature paper from last week are major for the stem cell community: Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations (Merkle et al. 2017).

Nature Genetics: Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci (Stender et al. 2017)

Nature Genetics: Pathogenic variants that alter protein code often disrupt splicing (Soemedi et al. 2017). Copied from abstract: “We analyzed 4,964 published disease-causing exonic mutations using a massively parallel splicing assay (MaPSy), which showed an 81% concordance rate with splicing in patient tissue. Approximately 10% of exonic mutations altered splicing, mostly by disrupting multiple stages of spliceosome assembly. We present a large-scale characterization of exonic splicing mutations using a new technology that facilitates variant classification and keeps pace with variant discovery.” I can’t even imagine how expensive this study must have been…

Nature Reviews Endocrinology: Metric for glycaemic control – from HbA2c to continuous glucose monitoring. Copied from abstract so you can decide if relevant or not: “I focus on markers of average glycaemia and the utility and/or shortcomings of HbA1c as a ‘gold-standard’ metric of glycaemic control; the notion that glucose variability is characterized by two principal dimensions, amplitude and time; measures of glucose variability that are based on either self-monitoring of blood glucose data or continuous glucose monitoring (CGM); and the control of average glycaemia and glucose variability through the use of pharmacological agents or closed-loop control systems commonly referred to as the ‘artificial pancreas’. I conclude that HbA1c and the various available metrics of glucose variability reflect the management of diabetes mellitus on different timescales, ranging from months (for HbA1c) to minutes (for CGM).”

Nature Cell Biology: Cell Competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes (Kon et al. 2017). This very interesting for the benign-malignant paradox that refers to the occurrence of cancerous mutations in benign disease without further progression to cancer. Here is a copy of the abstract (I read the paper, and it seems solid experimentally although they perform all the wrong statistics throughout!!): “Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.” There is a news & and views as well: “Metabolic changes promote rejection of oncogenic cells.”

Nature Cell Biology: Metabolic control of primed human pluripotent stem cell fate and function by the miR-200c-SIRT2 axis (Cha et al. 2017). After a quick skim, seems OK overall, except that I don’t trust the qPCR results because of the way they are represented. Would be great if people could stop normalising to samples where a gene is not expressed anyway (we have all been taught basic maths and know that you can’t divide by 0)… Not an attack on this study only, but on most qPCR studies these days. There is also a news&views on this paper in Nature Cell Biology as well (SIRT2 and glycolytic enzyme acetylation in pluripotent stem cells). What is interesting, though, is this whole concept of a glycolytic-acetyl-coA switch that could interact with the epigenetics of stem cells and their cell fate decisions.

Nature Cell Biology: SWELL1 is a regulator of adipocyte size, insulin signalling and glucose homeostasis. Can’t actually access the actual article 😦 It says aop, not sure what it means, though.

Cell Metabolism 

For those of us with a sweet tooth (a pathological tendency to snack!!),  FGF21 Is a Sugar-Induced Hormone Associated with Sweet Intake and Preference in Humans (Søberg, Sandholt et al. 2017). The gist of it is that there are human genetic variants in FGF21 that increase sweet preference, but surprisingly they do not correlate with obesity/diabetes. I found some of the points in the Introduction interesting – those regarding the difference between mouse and human FGF21 biology. So a word of caution when it comes to metabolism and species-specific differences!

From the Japanese iPSCs experts on the effect of metabolism on pluripotency states, published in Cell Metabolism: Hybrid Cellular Metabolism Coordinated by Zic3 and Esrrb Synergistically Enhances Induction of Naive Pluripotency (Sone et al. 2017). Currently, a hot topic in the stem cell field is the notion of naive vs primed pluripotency. Human iPSCs and ESCs are thought to be “primed”, i.e. more lineage-restricted and corresponding to the post-implantation epiblast state, compared to mouse counterparts which correspond to pre-implantation embryonic cells. Mouse ESCs can be induced to become even more naive (so can human ESCs/iPSCs as of recently) with certain small molecules or by genetic perturbation. Interestingly, whereas more primed stem cells rely on glycolysis, and low rates of oxidative phosphorylation, naive pluripotency seems to require both glycolysis and oxidative phosphorylation. This balance is now shown to depend on the synergy between the transcription factors Zic3 and Esrrb which both activate glycolysis genes, but have opposite effects on OXPHOS. It is really interesting to follow the metabolic theme in stem cells – the ability of cells to shift their metabolism is emerging as an important prerequisite for appropriate cell fate regulation. Also, although this study determines that both glycolysis and OXPHOS need to be ON for efficient reprogramming of mouse fibroblasts to naive stem cells, it still doesn’t have the answer as to why this is the case. Technically, this study is also quite neat – done well! Although quite interesting to see raw Ct values being reported as opposed to log-transformed derivatives (BUT great that the spread of the data is shown properly and SDs are used!).

A review on “Autophagy and Tumour Metabolism” (Kimmelman & White, 2017) ➡ interesting, examining both cell-autonomous and non-cell-autonomous effects. This field is also seeing a shift in perception, recognising that autophagy may be tumour-suppressive at the early stages of tumour initiation, but promote tumour growth at later stages (possibly due to systemic interactions of the cancer with non-cancerous tissues).

A review of “Metabolic Flexibility in Health and Disease” (Goodpaster & Sparks, 2017) that will interest quite a few people as it examines fasting/refeeding paradigms and emphasises the role of skeletal muscle and adipose tissue. It also briefly examines evidence for and against the importance of individual tissues for the development of insulin resistance.

What I have read of interesting papers this week

Some useful reviews

One in Nature Medicine “Refining strategies to translate genome editing to the clinic” (Cornu et al. 2017). It covers some of the recent gene editing clinical trials and outlines key standardisations required to translate CRISPR and similar techniques into effective therapeutic strategies.

Brendand Manning and Alex Toker have written a comprehensive Cell review “AKT/PKB Signaling: Navigating the Network”. Looking forward to giving it a proper read!

Insulin signalling

Key paper in Cell: The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover (Tawo et al. 2017)

Apparently, previous studies have shown that although CHIP-null mice exhibit normal embryonic development, lack of CHIP (ubiquitin ligase, key for cellular protein quality control) accelerates ageing. Also, the livers of these mice develop insulin resistance. Using C. elegans and D. melanogaster, this study demonstrates that under non-stress conditions, CHIP ubiquitylates INSR and targets it for endocytic (not proteasomal) degradation; however, under proteotoxic stress, CHIP is “busy” sorting out other damaged proteins and this results in increased INSR levels and enhanced downstream activation of PI3K/AKT signalling. Interestingly, they make a link to severe INSR due to mutations in the receptor by mentioning K1095E; apparently, they have identified this residue to be ubiquitylated. So the findings are extrapolated to speculations about the effects of metabolic insults on INSR degradation and therefore glucose homeostasis.

Metabolic

Chad Cowan and co. on “Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease” (Friesen et al. 2017, Stem Cell Reports)

From Jens Brüning’s lab on IL-6: IL-6 improves energy and glucose homeostasis in obesity via enhanced central IL-6 trans-signaling (Timper et al. 2017 Cell Reports) – not read in detail, but abstract suggests that similar to leptin, central IL-6 signalling suppresses feeding and improves glucose tolerance, but its actions are actually enhanced upon challenging mice with HFD. And the mechanism of signalling is interesting – the soluble IL-6R complexes with gp130 on the target cell surface, hence why they call this trans-signalling.

Obesity-induced hepatic steatosis is mediated by endoplasmic reticulum stress in the subfornical organ of the brain (Horwath et al. 2017, JCI Insight) – not read beyond abstract, but might be of relevance. Apparently reporting an uncoupling of hepatic steatosis from other obesity-linked conditions – i.e. reducing brain stress can reverse NAFLD, but without altering body weight, food intake, adiposity, or hypertension.

β-Klotho deficiency protects against obesity through a crosstalk between liver, microbiota, and brown adipose tissue (Somm et al. JCI Insight) – also only given this one a brief skim of the abstract and the introduction.

Some “old” stem cell signalling knowledge

An 10-year-old paper, but nonetheless interesting for me…

Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling (Wang et al. 2007, Blood)

The abstract of this paper rightly starts out by stating that little is known about the cell-surface receptors (and implicit signalling pathways) that are activated under conditions that support stem cell self-renewal – despite improvements in developing defined conditions. Advances have been made in the past 10 years, but I would still argue that a lot remains to be learned.

The hESCs used in this study are maintained in MEF-conditioned medium on Matrigel or in defined medium supplemented with Heregulin-1B, Activin A, IGF1 & FGF2. Following various stimulations, the cell lysates are profiled with RTK arrays (42 human RTKs) to identify activated receptors. Hits are subsequently validated with inhibitor studies.

There is a strong phosphorylation of IGF1R and IR upon addition of conditioned medium, and the study then goes on to determine that it is IGF1R specifically that maintains self-renewal and prevents differentiation in stem cell maintenance conditions. Interestingly, they find that the stem cells express high levels of IGF1R (flow cytometry) and very low expression of insulin receptor (IR), which is consistent with my observations of absence of a response to insulin stimulation, but a clear activation of PI3K/AKT signalling in response to IGF1 stimulation. Looks quite solid and they also show a requirement for ERBB2/3 signalling. One thing that surprises me, though, is that they perform GF-depletion studies (DMEM/F12 + 0.5 % BSA) overnight which is really harsh for stem cells – mine start to day after 3h.

Another study was published in Nature at the same time as this paper, supporting the notion that IGF1R signalling in stem cells is essential for their self-renewal potential (Bendall et al. 2007, Nature).

Delayed Accumulation of H3K27me3 on Nascent DNA Is Essential for Recruitment of Transcription Factors at Early Stages of StemCell Differentiation (Petruk et al. 2017a, Molecular Cell) – there is an accompanying paper by the same group Petruk et al. 2017b in Cell Reports as well.

This is quite cool, by the group who originally developed a chromatin assembly assay relying on PLA (proximity ligation assay) to study the structure of nascent chromatin. Here, they show that immediately following DNA replication in stem cells, there is a delay in H3K27me3 deposition on the DNA, which allows transcription factors induced by differentiation signals to bind to their target sites. This is a very finely tuned time window that allows for exquisite control of differentiation only when the right transcription factors are induced and when the chromatin is open. Because the time window is limited, it also prevents spurious binding of other transcription factors later on, essentially allowing the cell to reconfigure itself in the process of differentiation once the primary transcription factors have done their job. There is an accompanying news and views as well: A Determined “Hesitation” on H3K27me3 Empowers Stem Cells to Differentiate (Huang and Wang 2017, Molecular Cell). In the Cell Reports paper, which I haven’t read, they apparently show that the same is true for HSCs.

Of general interest

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity (Saleheen et al. 2017, Nature)

Moving beyond non-human models to understand the complete  loss of certain genes in human. This is possible due to large scale sequencing of inbred populations that are likely to harbour two copies of rare mutations that result in complete loss of function for a particular gene. Really interesting and a more comprehensive catalogue of possible human knockouts is soon to come out. In this study, the authors link the discovered gene knockouts to more than 200 phenotypic traits, providing a detailed functional analysis. Interesting to go back and link the phenotypes to those observed in mice engineered to lack some of the same genes – you might be surprised at some of the discrepancies, cautioning us against thinking of mice as mini-humans. An accompanying news and views is also available at Nature.

 

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency (Yang et al. 2017, Cell)

This is quite ground-breaking in the developmental world (if it stands the test of independent replication). The study reports the first ever derivation of pluripotent stem cells (both mouse and human) with both extraembryonic (i.e. can generate placenta etc.) and embryonic capacity, terms extended pluripotent stem (EPS) cells.

Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation (Slobodin et al. 2017, Cell)

Quite cool both on the biological and techy side of things. Very, very nice approach to screen for differential promoter effects by using a barcoded reporter, essentially designing a new technique “Barcoded polysomal profiling” (BPP). Biologically, the study reports that mA6 of RNA is an epigenetic mechanism that determines the future activity and fate of the transcribed mRNA, thereby indirectly coupling transcription and translation.

Not read in detail, but this sounds a bit cool (apparently, you can change the sex of C. elegans with fatty acids): Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation (Tang et al. Cell 2017 quite impressive with only 2 authors!).

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a (Swarts et al. 2017, Molecular Cell –  from the Jinek lab) – just one of those papers that are good to have; Cas12a = Cpf1, the cousin of Cas9, also used for gene editing. Different from Cas9 by being able to process its own guide RNAs + different PAM requirement + Cpf1 generates staggered cuts.

 

Science this week

Stem cell research

Modeling Developmental and Tumorigenic Aspects of Trilateral Retinoblastoma via Human Embryonic Stem Cells (Avior et al. 2017, Stem Cell Research)

I love reading good papers reporting modelling of early-onset human diseases in human stem cells. There is one in Stem Cell Research that caught my attention this week, by Avior et al., modelling ablation of retinoblastoma protein (Rb) in human embryonic stem cells (hESCs). This is a well-known tumour suppressor and an inherited loss of Rb protein leads to retinoblastoma development, the most common primary intraocular pediatric cancer (95 % of cases diagnosed before the age of 5). In 6 % of cases, patients develop trilateral retinoblastoma characterised by neuroectodermal tumour occurence in additional to retinal tumours in the eye. Combined with results from conditional knockout mice, the Rb protein has been suggested to play a role in neural development. In the current study,  following CRISPR/Cas9 gene editing to knockout the Rb protein, modified hESCs are subjected to an array of assays to study the resulting phenotype. After observing normal neural stem cell generation in a 2D directed differentiation protocol, despite expectations of a phenotype compared to wildtype hESCs, the authors conclude that a more complex differentiation paradigm is needed. As a result, they move on to generate teratomas in immunocomprised mice and observe a substantial expansion of neural structures in Rb-null teratomas. This highlights the importance of using multiple methods to assess a phenotype reliable. I have come to the same conclusion in my studies as 2D differentiation protocols often rely on non-physiological concentrations of various drugs and peptides, whereby a phenotype may remain masked. This study also reveals mitochondrial dysfunction in Rb-null hESCs and establishes a framework for high-throughput testing of hundreds of FDA-approved chemotherapies in this human cell models.

 

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells (D’Antonio et al. 2017, Stem Cell Research)

This paper made me aware of fluorescent cell barcoding as an efficient way to pool multiple stem cell samples together and save on antibodies during initial characterisation. To make pluripotency and multilineage analysis across multiple stem cell lines available, the authors provide an Excel file which users can use to generate their own scores and heatmaps using Ct values of a reference gene of interest and any chosen marker genes.

Another paper from the same lab: Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics (Panopoulus et al. 2017 Cell Stem Cell)

A lot of effort is invested into understanding how iPSCs differ from hESCs and whether somatic memory may limit their utility in disease modelling and as potential treatment strategies. It is not well understood whether aberrant methylation of CpG sites in iPSCs is mainly caused by genetic variability or are artefacts of the reprogramming factors. The current study reports the following: “Here we generated 22 iPSC clonal lines from six individuals (three pairs of older monozygotic twins). We profiled the 22 iPSC lines, at early (passages 5 [p5] and 9 [p9]) and late (passage 20 [p20]) passages, as well as fibroblasts (tissue of origin) using genome-wide methylation arrays and RNA sequencing (RNA- seq) data.We estimate aberrantmethylation of the iPSCs relative to ESCs, and we show that aberrant methylation affects gene expression and is enriched for CpGs associated with MYC and MYC-related protein motifs. We then identify genome-wide associations between CpG methylation variation and genetic background, clone, and passage, andwe showthat these associ- ationslikely result fromrelevant biological processes.Weexamine whether aberrant CpGs are enriched for CpGs associated with genetic and non-genetic effects, and we show that aberrant methylation preferentially occurs at CpGs showing clone-associ- ated effects and is less enriched at sites associated with genetic background. Our study shows that non-genetic regulatory mechanisms associated with clone-specific effects most strongly underlie iPSC aberrancy.”

My immediate reservations without having read the study in detail is the limited number of twin pairs and therefore cells examined as well as the fact that they are all female.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types (Panopoulus et al. 2017 Stem Cell Research)

An iPSC resource from the same lab  (very productive!). Will be useful for examining phenotypic diversity across individual induced pluripotent stem cell lines.  Pertinent to the same topic, a study in Cell Stem Cell examines the influence of genetic variation on gene expression in human iPSCs: Large-Scale Profiling Reveals the Influence of Genetic Variation on Gene Expression in Human Induced Pluripotent Stem Cells (DeBoever et al. 2017 Cell Stem Cell). Actually, this seems to be a theme in the current Cell Stem Cell issues and multiple other papers examine the same topic from different perspectives. For instance, Warren et al. provide proof-of-concept that large cohorts of human iPSCs can be used to performed GWAS studies in a dish, here in the context of metabolic disease.

 

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells (Guo et al. 2017 Protein & Cell)

A variation on a system that has been introduced in the past – the ability to activate genes from their endogenous locus via a Dox-inducible expression of dCas9 fused to a transcriptional activation domain.

Other bits and pieces that I have not read in detail, but that caught my interest – mostly metabolism-related

Mitochondrial Patch Clamp of Beige Adipocytes Reveals UCP1-Positive and UCP1-Negative Cells Both Exhibiting Futile Creatine Cycling (Bertholet et al. 2017 Cell Metabolism) – up until now all beige adipocytes were assumes to function like brown adipocytes, utilising UCP1 dissipate energy as heat; this paper suggests that there is a subset of beige adipocytes in mice, in epididymal fat in particular, that are UCP-negative yet perform thermogenesis by engaging in futile creatine cycling. Quite biophysical, by the way! If of interest, there is a Preview commentary on this article as well: Now UCP(rotein), Now You Don’t: UCP1 Is Not Mandatory for Thermogenesis (Szabo and Zoratti 2017, Cell Metabolism).

Another paradigm-shaking paper in Cell Metabolism modifies our understanding of the incretin effect mediated by GLP1. I have actually only read the Preview by Habener and Stanojevic (Pancreas and Not Gut Mediates the GLP-1-Induced Glucoincretin Effect) and it is always best to have a look at the actual paper and its data instead, so acknowledging the absence of appropriate assessment on my own, it might still be worth a read for those interested in this field!

Surprising results are the theme in Cell Metabolism this week; another one: FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms by BonDurant et al., providing evidence that adiponectin is dispensable for the metabolic effects of FGF21.

A paper in PNAS by Steptoe and Wardle (Life skills, wealth, health, and wellbeing in later life) looks at the importance of 5 key life skills – conscientiousness, emotional stability, determination, control, and optimism – in later life (cross-sectionally and longitudinally), concluding that (after adjustment of multiple confounding factors) these life skills are associated with wealth, income, subjective wellbeing, less depression, low social isolation and loneliness, more close relationships, better self-rated health, fewer chronic diseases and impaired activities of daily living, faster walking speed, and favorable objective biomarkers (concentration of high-density lipoprotein cholesterol, vitamin D and C-reactive protein, and less central obesity). They were also associated with greater psychological well-being and less loneliness, and a lower incidence of new chronic disease and physical impairment over a 4-y period. I like this study – a good reason to practice mindfulness throughout life!

 

 

 

 

The weekly science round up – 5 April

Actually, some of  the papers already came out last week, but only just got round to having a proper look at them.

Science

Lysosomal cholesterol activates mTORC1 via an SLC38A9–Niemann-Pick C1 signaling complex (Castellano et al.)

Copied from the final bit of the paper: “This work identifies dedicated machinery that couples cholesterol trafficking through the lyso- some to regulation of cellular growth signaling. LDL-derived cholesterol affects mTORC1 through the combined action of a positive regulator, SLC38A9, and a negative regulator, NPC1 (Fig. 4G). SLC38A9 conveys increases in lysosomal cholesterol through its conserved CARC and CRAC motifs, leading to lysosomal recruitment and activation of mTORC1. In contrast, NPC1 associates with the mTORC1 scaffolding complex and translates cholesterol depletion into mTORC1 inhibition.”

Nature

Structural insights into adiponectin receptors suggest ceramidase activity (Vasiliauskaité-Brooks et al.)

Discovery that ADIPORs (1+2) have ceramidase activity that can be enhanced upon adiponectin stimulation. Overall, this enzymatic activity is low so future studies are required to understand whether it bears relevance to the function of adiponectin in vivo and its link to metabolic improvements.

 

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue (Lynes et al.) 

Cold-induced activation of brown adipose tissue (BAT) is known to result in increased energy expenditure, and it is believed that BAT energy expenditure can be harnessed therapeutically in metabolic disorders. Using global lipodmics, this study identifies a BAT-specific lipokine 12,13-diHOME which is released in response to cold exposure and activates BAT fuel utilisation. Consistent with a decrease in BAT mass with obesity, 12,13-diHOME levels were negatively correlated with obesity and other phenotypes characteristic of the metabolic syndrome. It is impressive that this lipokine is first identified in human participants exposed to cold, followed by mechanistic studies in mouse models, where the effects of 12,13-diHOME on free fatty acid uptake are comparable to noradrenaline-mediated BAT activation. Overall, an elegant study advancing the field with a new candidate for pharmacologic BAT activation.

 

Nature Biotechnology

Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells (Weinberg et al.)

My brain gets mushy when I try to read this in detail at 10 pm in the evening, but just quite cool to see the engineering of one of the most sophisticated computational biological circuits to date. This done by combining orthogonal recombinases and various DNA sequences that control transcription. Again, I think an electrical engineer would understand this paper better than me, but still impressive…

Synergistic drug combinations for cancer identified in a crisPr screen for pairwise genetic interactions (Han et al.)

In this paper, a global CRISPR screen is performed to identify combinatorial interactions between cancer drug targets. 490,000 sgRNAs are used to screen for synthetic lethal drug target pairs in K562 leukemia cells. The approach is interesting and applicable to other areas. Worth noting the technical aspects of the screen.

A similar paper has also been published in Nature Methods: Combinatorial CRISPR–Cas9 screens for de novomapping of genetic interactions (Shen et al.)

Nature Protocols

From the Zhang lab on CRISPR screens: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening (Joung et al.)

 

Nature Genetics

Estimating the selective effects of heterozygous protein-truncating variants from human exome data (Cassa et al.)

Not the best person to understand this, but a study that looks at protein truncating variatns in ExAC if they are predicted to be consequential and estimates gene-based fitness effects and individual gene fitness cost in heterozygotes (only using data from individuals with non-Mendelian disorders). Basically useful to look at novel genes that might have important functions in development as mutations would be highly selected against. The mTOR and MAPK pathways are in the list of pathways with genes that seem to have a high gene fitness cost (unsurprising).

Nature Chemical Biology

Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase (Stanford et al.)

Worth a read – actually quite metabolic and signalling-relevant. Here is the abstract:

“Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.”

JCI Insight

Insulin’s direct hepatic effect explains the inhibition of glucose production caused by insulin secretion (Edgerton et al.)

Given the redundancy in insulin’s ability to control hepatic glucose production (direct and indirect mechanisms), this study set out to explore the contextual importance of some of these mechanisms.

An interesting excerpt from the introduction of the paper, discussing the physiological difference between peripheral insulin infusion and direct infusion into the portal vein: “It is apparent, therefore, that when insulin is infused into a peripheral vein its direct effects on the liver are underemphasized, while its indirect effects are exaggerated. Thus, the route of insulin delivery can have a major impact on both the overall response of the liver and the mechanisms by which that response is achieved. Indeed, over a range of insulin doses, peripheral insulin infusion was not as effective at suppressing HGP as compared with direct infusion into the portal vein.”

Based on this information, “the purpose of this study was to assess whether insulin’s acute indirect effects on HGP are additive to, redundant to, or synergistic with its direct hepatic effects, in the context of a physiologic increase in portal vein insulin level in a large animal model. The impact of eliminating each of insulin’s indirect effects, either alone or in combination, was determined. We focused on insulin’s ability to lower FFAs, activate brain insulin signaling, and reduce glucagon secretion since recent studies have concluded that suppression of lipolysis is the major mechanism by which insulin suppresses HGP (11), that increased brain insulin action is required for the rapid suppression of HGP (17), and because a fall in plasma gluca- gon is potentially a powerful contributor to insulin’s ability to inhibit HGP (15).”

They perform portal insulin infusion (in mice) and simulate a 6-fold increase in insulin secretion, but manage to maintain the physiologic insulin gradient between liver and the rest of the body.

This paper is very interesting given the multiple demonstrations of a key role of insulin’s indirect effect on HGP via modulation of FFA release from adipose tissue; in contrast, this study seems to imply that FFA don’t play a role in HGP suppression under what is described as more physiologic conditions. Similarly, a fall in glucagon secretion is not necessary for insulin direct ability to suppress HGP. The same is true when insulin’s brain action is inhibited. The discussion is really worth a read!

EMBO Journal

AKT-phosphorylated FOXO1 suppresses ERK activation and chemoresistance by disrupting IQGAP1-MAPK interaction (Pan et al.)

Looking forward to reading this one at some point! The accompanying News and Views sums it all up: “And Akt-ion! IQGAP1 in control of signaling pathways” (Choi et al.

Mfn2 is critical for brown adipose tissue thermogenic function (Boutant et al.)

Worth a read! Adipose tissue-specific KO of Mfn2 results in increased fat accumulation in BAT even on a low-fat diet as well as an overall decrease in energy expenditure and an inability to sustain normal body temperature via cold-induced BAT thermogenesis. This is linked to an impaired O2 flux in BAT when specifically measuring the different complexes forming the electron transport chain. At the protein level, Complex I is almost absent upon Mfn2 ablation. Mfn2 is shown to interact with perilipin and the data suggest that this enhances mitochondria-lipid droplet interactions. It seems that glycolysis is increased due to mitochondrial dysfunction. There is also an increased accumulation of FAT in subcutaneous WAT and the authors suggest that this exlpains the improved metabolic profile of HFD-fed adipose-specific Mfn2 KO mice.

USP49 negatively regulates tumorigenesis and chemoresistance through FKBP51-AKT signaling (Luo et al.)

Had a quick skim – in cancer cell lines and cancer mouse models, but the mechanism might be valid in a more physiological context, too. Basically, USP49 deubiquinates FKBP51 which results in increased stabilisation. In turn, FKBP51 promotes the interaction between AKT and PHLPP, which dephosphorylates AKT at S473 thereby reducing its activation.

The phosphorylation status of T522 modulates tissue-specific functions of SIRT1 in energy metabolism in mice (Lu et al.)

Just skimmed the abstract, but might be interesting to adipogenesis enthusiasts.

eLife

Angiopoietin-2 in white adipose tissue improves metabolic homeostasis through enhanced angiogenesis (An et al.) – only read abstract, but might be relevant. Just in press, so still not formatted properly, i.e. annoying to read.

Diabetes

 

 

Other exciting bits

I have been reading a lot of developmental biology recently and came across the following Guardian coverage of the first experiments that make it possible to break the 14-day rule for embryo research. I just find the videos and images amazing + the useful infographic of lineage specification at the bottom. The two papers giving rise to the Guardian feature can be found at Nature and Nature Cell Biology. These are landmark studies and were recently followed up by a study from the same Cambridge group, now demonstrating that embryonic and extra-embryonic stem cells can be assembled in vitroto mimic embryogenesis (effectively generating embryos in vitro, see Harrison et al. 2017 Science).

In terms of interesting metabolism reviews, it is worth having a look at Nature Reviews Endocrinology – there are good ones on adipose tissue etc.