My Favorite Papers of 2012

These are the papers that I thought were the most interesting in my world this year. It was a big year for hormonal messengers involved in obesity and diabetes.  These include Fgf21 (Kim et. al) and VEGF (Hagberg et. al).  From a biochemistry perspective there was a lot of great work on the role of SREBP1 (Moon et. al) and its regulation both from a dietary (Haas et al.) perspective and from a mechanistic perspective (Owen et al.).  Finally both Kim et al. and Kusminski et al. highlighted the importance of mitochondrial function in the systemic response to obesity.



Haas, J., Miao, J., Chanda, D., Wang, Y., Zhao, E., Haas, M., Hirschey, M., Vaitheesvaran, B., Farese, R., Kurland, I., Graham, M., Crooke, R., Foufelle, F., & Biddinger, S. (2012). Hepatic Insulin Signaling Is Required for Obesity-Dependent Expression of SREBP-1c mRNA but Not for Feeding-Dependent Expression Cell Metabolism, 15 (6), 873-884 DOI: 10.1016/j.cmet.2012.05.002

Hagberg, C., Mehlem, A., Falkevall, A., Muhl, L., Fam, B., Ortsäter, H., Scotney, P., Nyqvist, D., Samén, E., Lu, L., Stone-Elander, S., Proietto, J., Andrikopoulos, S., Sjöholm, A., Nash, A., & Eriksson, U. (2012). Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes Nature, 490 (7420), 426-430 DOI: 10.1038/nature11464

Kusminski, C., Holland, W., Sun, K., Park, J., Spurgin, S., Lin, Y., Askew, G., Simcox, J., McClain, D., Li, C., & Scherer, P. (2012). MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity Nature Medicine, 18 (10), 1539-1549 DOI: 10.1038/nm.2899

Moon, Y., Liang, G., Xie, X., Frank-Kamenetsky, M., Fitzgerald, K., Koteliansky, V., Brown, M., Goldstein, J., & Horton, J. (2012). The Scap/SREBP Pathway Is Essential for Developing Diabetic Fatty Liver and Carbohydrate-Induced Hypertriglyceridemia in Animals Cell Metabolism, 15 (2), 240-246 DOI: 10.1016/j.cmet.2011.12.017

Kim, K., Jeong, Y., Oh, H., Kim, S., Cho, J., Kim, Y., Kim, S., Kim, D., Hur, K., Kim, H., Ko, T., Han, J., Kim, H., Kim, J., Back, S., Komatsu, M., Chen, H., Chan, D., Konishi, M., Itoh, N., Choi, C., & Lee, M. (2012). Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine Nature Medicine DOI: 10.1038/nm.3014

Owen, J., Zhang, Y., Bae, S., Farooqi, M., Liang, G., Hammer, R., Goldstein, J., & Brown, M. (2012). From the Cover: Insulin stimulation of SREBP-1c processing in transgenic rat hepatocytes requires p70 S6-kinase Proceedings of the National Academy of Sciences, 109 (40), 16184-16189 DOI: 10.1073/pnas.1213343109

My Favorite Papers of 2012 by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

How is PtdIns(5)P Made?

For most phosphatidylinositides, the routes of synthesis and degradation have been largely elucidated.  However, due to difficulty in detecting PtdIns(5)P, only recently have investigators been able to assess the synthesis and degradation of this phospholipid.  The major stumbling block has been the separation of PtdIns(5)P from PtdIns(4)P, which migrate quite closely on HPLC/TLC based separations.  The two major advances in the field have been improved separation of these monophosphorylated lipids (for example see Sarkes and Rameh 2010 and Zolov et al. 2012) and separation-independent identification of PtdIns(5)P by an enzyme based phosphorylation assay. (see Jones et al., 2012).  I am a co-author on the Zolov paper and work closely with that group.

Which enzymes are involved?

Potential Routes for PtdIns(5)P Synthesis.

The simplest mechanism is through phosphorylation of PI directly by a PtdIns-5-Kinase.  There are two known classes of Ptdins-5-Kinases in mammalian cells, Pikfyve and three isoforms in the PtdIns(4)P-5-Kinase family (Pip5k1a, Pip5k1b and Pip5k1c).  Classically, Pikfyve is thought to convert PtdIns(3)P into PtdIns(3,5)P₂ wheras the other classes phosphorylate PtdIns(4)P into PtdIns(4,5)P₂.  I think that the strongest evidence is that Pifkve is essential for PtdIns(5)P levels in the cell, either directly or indirectly.

Biochemically, there seems to be three potential ways by which PtdIns(5)P could be made, through direct phosphorylation of PtdIns, or through dephosphorylation of either PtdIns(3,5)P₂ or PtdIns(4,5)P₂.  Of course, it is possible that in different contexts, each of these pathways could be involved.

Route 1: Direct Phosphorylation of PtdIns

Although there is limited evidence that the PtdIns(4)P-5-Kinases can phosphorylate PI, there is substantial evidence that PI(5)P can be generated by Pikfyve, in vitro (Sbrissa et. al, 1999).  Inside cells, it is less clear whether this is the case.  There is rapid and tightly correlated turnover of both PtdIns(3,5)P₂ and PtdIns(5)P in most cells (Zolov et. al, 2012, Sbrissa et al., 2012) to the point that it is difficult to tell if changes in PtdIns(3,5)P₂ preceed changes in PtdIns(5)P or correlate with them independently.  

Route 2: De-Phosphorylation of PtdIns(3,5)P₂

Two main lines of evidence support the possibility that PtdIns(3,5)P₂ could be the source of some or all of the PtdIns(5)P in the cell:  

1. Myotubularins, which are 3-phoshphatses leads to increased PtdIns(5)P and their deletion may lead to reductions in PtdIns(5)P (Vaccari, et al., 2011, Oppelt et al., 2012).
2. The kinetics of acute PtdIns(5)P synthesis or degradation may lag slightly behind the synthesis or degradation of PtdIns(5)P.  In any case, the levels of PtdIns(5)P and PtdIns(3,5)P₂ are very tightly correlated (Zolov et al., 2012).

The killer experiment here would be to test whether ablation of PtdIns(3)P levels would have direct effects on PtdIns(5)P levels, but since it is not clear whether PI3K inhibitors such as Wortmannin would affect Pikfyve in vivo that experiment may not be interpretable without ruling out direct effects first.

Route 3: De-Phosphorylation of PtdIns(4,5)P₂

An alternate theory has suggested that some or all of PtdIns(5)P is derived by the activity of a 4-Phosphatase which convertes PtdIns(4,5)P₂ into PtdIns(5)P.  The exact identity of this 4-phosphatase is not yet known.  Jones et al. show that peroxide increases PtdIns(5)P levels, and propose a role for PtdIns(4,5)P₂ dephosphorylation in that process.  However, in contrast to our findings (Zolov et al., 2012), this paper finds no role for Pikfyve in the synthesis of PtdIns(5)P, using similar approaches but a different assay to measure PtdIns(5)P (see below).

What is the Best Way to Measure PtdIns(5)P?

Regarding the role of Pikfyve, there seems to be a controversy here.  I've summarized the assays and their results in the table below.

Assay	Inositol Labelling	Mass Assay
Summary	Cells are grown in inositol depleted media with radioactive inositol. Cells are lysed and lipid headgroups are separated by HPLC based on charge.	Cells are grown in any condition, lipids are extracted and phosphorylated with PIP4K and radioactive ATP. Only PtdIns(5)P can be phosphorylated by this enzyme, so all hot PIP2 (based on TLC and counting) is derived from PtdIns(5)P.
Normalization	Total phosphatidylinosotol	Total cellular phospholipids
Result	Pikfyve knockdown/inhibition nearly completely decreases PtdIns(5)P levels.	Pikfyve knockdown/inhibition does not affect PtdIns(5)P levels.

Setting aside the role of peroxide in PtdIns(5)P as potentially a special case, you could make arguments for both methods.  Hopefully this can be resolved quickly since knowing where this lipid comes from is the first step in figuring out what it does.

References

Jones, D., Foulger, R., Keune, W., Bultsma, Y., & Divecha, N. (2012). PtdIns5P is an oxidative stress-induced second messenger that regulates PKB activation The FASEB Journal DOI: 10.1096/fj.12-218842

Oppelt, A., Lobert, V. H., Haglund, K., Mackey, A. M., Rameh, L. E., Liestøl, K., Oliver Schink, K., et al. (2012). Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration. EMBO reports. doi:10.1038/embor.2012.183

Sarkes, D., & Rameh, L. E. (2010). A Novel HPLC-Based Approach Makes Possible the Spacial Characterization of Cellular PtdIns5P and Other Phosphoinositides.The Biochemical journal384, 375–384. doi:10.1042/BJ20100129

Sbrissa, D., Ikonomov, O. C., & Shisheva, A. (1999). PIKfyve, a mammalian ortholog of yeast Fab1p lipid kinase, synthesizes 5-phosphoinositides. Effect of insulin. J Biol Chem, 274(31), 21589–21597. pmid:10419465

Sbrissa, D., Ikonomov, O. C., Filios, C., Delvecchio, K., & Shisheva, A. (2012). Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM201636. American journal of physiology. Cell physiology, (313). doi:10.1152/ajpcell.00105.2012

Vaccari, I., Dina, G., Tronchère, H., Kaufman, E., Chicanne, G., Cerri, F., Wrabetz, L., et al. (2011). Genetic interaction between MTMR2 and FIG4 phospholipid phosphatases involved in Charcot-Marie-Tooth neuropathies. PLoS genetics, 7(10), e1002319. doi:10.1371/journal.pgen.1002319

Zolov, S. N., Bridges, D., Zhang, Y., Lee, W., Riehle, E., Verma, R., Lenk, G. M., et al. (2012). In vivo, Pikfyve generates PI(3,5)P2, which serves as both a signaling lipid and the major precursor for PI5P. Proceedings of the National Academy of Sciences of the United States of America, 109(43), 17472–7. doi:10.1073/pnas.1203106109


How is PtdIns(5)P Made? by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

How is SREBP Regulated by Insulin?

SREBP is a transcription factor which integrates anabolic signals and drives transcription of several important lipogenic genes such as Fatty Acid Synthase, Acetyl-CoA Carboxylase and the LDL Receptor.  In liver tissue ,this serves to enhance the uptake, synthesis and storage of lipid in the postprandial state.  The biochemical mechanisms by which this happens is unclear but a recent paper from the Brown and Goldstein laboratory has shed some light on this.

Figure: SREBP Processing and Translocation (from http://en.wikipedia.org/wiki/Srebp)

Previously it had been known that insulin causes both the cleavage and nuclear translocation of SREBP in hepatocytes (see Figure). Confusing the issue was the fact that SREBP was also increased transcriptionally. Owing to the presence of a SRE element in the promoter, I thought that the transcriptional effects were likely due to a positive feedback loop where insulin causes SREBP processing, which in turn causes more transcription of the mRNA.  Adding credence to this hypothesis was the fact that inhibitors of the PI3K->mTORC1 pathways (Wortmannin and Rapamycin) inhibited both transcription and processing of SREBP1.

In the Owen et al. paper, a transgenic rat is generated which puts SREBP1c under the control of a non-insulin responsive promoter, allowing for examination of the processing of SREBP1c independent of the SREBP1c promoter.  Consistent with previous findings, they show that both Wortmannin and Rapamycin block processing and mRNA synthesis, but that another inhibitor LYS6K2 which is specific for S6K (a target of mTORC1) blocks only processing and not mRNA levels.



This not only suggests that S6K is the proximal effector of the PI3K-mTORC1 pathway with respect to processing, but that S6K plays no role in the transcriptional regulation.  This also, for the most part, excludes a role for the SREBP -> SRE positive feedback loop, since under LYS6K conditions, SREBP cleavage is blocked but mRNA levels are unchanged.  Put another way, if the SREBP positive feedback loop was important, then this would suggest that mRNA of SREBP would be reduced under all conditions in which SREBP processing is blocked.

Owen JL, Zhang Y, Bae SH, Farooqi MS, Liang G, Hammer RE, Goldstein JL, & Brown MS (2012). Insulin stimulation of SREBP-1c processing in transgenic rat hepatocytes requires p70 S6-kinase. Proceedings of the National Academy of Sciences of the United States of America PMID: 22927400


How is SREBP Regulated by Insulin? by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

Rab5 Knockdown In Vivo

This paper from the Zerial Laboratory describes a study in which the three mammalian isoforms of Rab5 are reduced in whole livers or cultured hepatocytes by a lipid nanoparticle mediated knockdown treatment. They then analyse images from these cells and liver sections and determine that there are reduced early endosomes, late endosomes and lysosomes in cells depleted of Rab5.

They also identify two functional defects in these tissues; reduced LDL uptake and impaired apical polarity of the hepatocytes. They also generate a simplified model of Rab5/early endosomal dynamics and suggest a role for Rab5 in vesicle fission as well as fusion. Although this model fits the available data quite well, it ignores several other key components of endosomal formation and maturation.

The authors also present the curious finding that while Rab5 depletion reduces both LDL uptake and endosome numbers, dynamin depletion only affects LDL uptake but not the number of endosomes. One possible explanation for this defect is that they quantify the presence of endosomes in this assay largely by EEA1 puncta. Since EEA1 is a Rab5 effector, it is possible that in the Rab5 depleted cells there is still a population of early endosomes, but that these are not detected by EEA1 staining.

Together this study presents a convincing picture for the role of Rab5 in endosomal dynamics, and highlight the important role of Rab5 in both endocytic pathways and in the establishment of polarity in vivo.

Citation

Zeigerer A, Gilleron J, Bogorad RL, Marsico G, Nonaka H, Seifert S, Epstein-Barash H, Kuchimanchi S, Peng CG, Ruda VM, Del Conte-Zerial P, Hengstler JG, Kalaidzidis Y, Koteliansky V, & Zerial M (2012). Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature, 485 (7399), 465-70 PMID: 22622570

Disclosure: I have received constructs and yeast strains from the Zerial laboratory in the past.



Rab5 Knockdown In Vivo by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

Why Blog

Based on the twitter meme #whydoiblog. This is a good question, seeing as I do this infrequently and without a ton of focus. I guess I'd say to get something off my chest that I think other people might want to hear. I aspire towards being able to talk about research in a more public and open way, but until I am independent that is unfair to people who might not want to operate that way. Until then I'll continue to use this as a sporadic forum for me to speak out to the ether.

Why Blog by Dave Bridges is licensed under a Creative Commons Attribution 3.0 Unported License.

IRS Serine Phosphorylation and Insulin Sensitivity?

This is part of my last year in science series. Click here for the rationale.

Insulin signalling passes through a cascade of signaling proteins starting with the recruitment of the Insulin Receptor Substrate (IRS) to the Insulin Receptor.  Insulin resistance, or impaired insulin signaling is a hallmark of obesity and diabetes.  One of the ways in which was thought to happen was through phosphorylation of Serine 307 on IRS, a phosphorylation event which has been proposed to be inhibitory to insulin signaling.  As an example, it has been proposed that inflammation (via JNK or IKKbeta), overnutrition (via S6K) or several other factors can lead to the phosphorylation of this protein.  Since several of these factors correlate with reduced insulin signaling, and the ablation of these factors leads to both increased insulin signaling and reduced serine phosphorylation, the obvious hypothesis was that serine phosphorylation of IRS is causative of insulin signaling.

Correlation does not equal causation, so in order to test this hypothesis in vivo, Morris White's group at Harvard Medical School generated knockin mice, in which Serine 307 of IRS1 is mutated to an alanine (and is therefore unable to be phosphorylated).  The idea would be that these mice would be unable to phosphorylated IRS1 on Serine 307, and therefore would be resistant to the deleterious effects of this phosphorylation.  This serine to alanine knockin model is considered the gold standard for translating an observational protein phosphorylation site into an in vivo phenotype.  Unfortunately for the prevailing hypothesis, the opposite was true.

The paper, from Copps et al. published in January of 2010 show that on a normal diet S307A mice showed modest reductions in insulin sensitivity, and increased fasted insulin levels.  Both of these effects were amplified by high fat diet, and were associated with a reduction in weight gain, in contradiction to the previous hypothesis, that these effects would be ameliorated.  Mechanistically, when on a high fat diet (or coupled with liver specific IRS2 knockout) the S307A mouse had decreased tyrosine phosphorylation of IRS, but no effect of the downstream insulin targets Akt and S6K. Therefore it is unclear exactly how the insulin intolerance is propagated into effects on glucose homeostasis.

In the past year this article has been cited 10 times (according to Google Scholar), mostly in review articles, but the major upshot here, is that the models which showed effects on IRS Serine 307 phosphorylation and insulin resistance, and concluded that insulin resistance was mediated by increased phosphorylation may need re-interpretation.  IRS is phosphorylated on several other sites, so the general hypothesis that serine phosphorylation of IRS causes insulin resistance could still be true, but that again might need to wait until such a knockin model can be generated.  This work also points out the risks of correlating phenotypes with incompletely characterized phosphorylation sites.



Copps KD, Hancer NJ, Opare-Ado L, Qiu W, Walsh C, & White MF (2010). Irs1 serine 307 promotes insulin sensitivity in mice. Cell metabolism, 11 (1), 84-92 PMID: 20074531 DOI

Last Year in Science

I hope to put together a series of posts on papers from about a year ago. Quite often the context of a paper can get lost in the flurry surrounding the initial release of a paper. My hope is that I can provide a little bit of insight on these papers with a little bit more since publication. If you have any ideas for things that might be interesting to go over (again) just let me know. For now I'll try to read some of the glamor mags in my field (Cell, Cell Metabolism, Nature Cell Biology, Nature and Science) and see if anything strikes my interest.

Inositol Phosphates and Insulin Signaling

When most people think of the role of inositols in Akt signaling, they immediately think about the role of PIP3 in the PDK1-Akt signaling axis.  A recent paper published in Cell by Solomon Snyder's group at John's Hopkins highlights the role of soluble inositol phosphates in insulin signaling.

Soluble Inositol Phosphates

Inositol is best known as a lipid head group, that can be phosphorylated to form 8 potential phosphorylated phosphatidylinositols. These membrane bound signaling lipids have many important roles in cell biology, including in signal transduction. In addition to these 8 membrane bound lipids, the inositol headgroup can be solubilized from the lipid tails by phospholipases.  This leads to the important second messenger IP3, which can then be further phosphorylated to yield IP4, IP5 and IP6.  Adding even more to the complexity, these rings can be pyrophosphorylated to yield even more species including IP7 and IP8 among others. The functions of these phosphorylated inositol rings are largely unknown.

Role of IP7 in Akt Activation

The IP6-Kinase 1 phosphorylates IP6 to form IP7 (pyrophospho-IP5). The current paper, Chakraborty et al. (2010), describes insulin signaling in cells in which IP6 is knocked out. As expected, IP7 concentrations are reduced in these cells, but the major finding is that Akt phosphorylation and activation is increased. The proposed mechanism for this effect is that IP7 acts as an endogenous, physiological inhibitor of Akt, likely by competitively inhibiting the ability of PIP3 to bind to the same site in its PH domain. Once IP7 is reduced, this inhibition is released, and Akt can be activated more easily.

Consistent with hyperactivation of Akt, these knockout mice exhibit increased insulin sensitivity and a reduction in diet-induced obesity. Akt and its downstream targets are known to be major mediators of insulin signaling, and so increased insulin signaling through the Akt pathway leads to increased glucose disposal and a resistance to diet-induced weight gain, insulin resistance, hyperinsulinemia and hyperglycemia. These data are consistent with a role of IP7 as a negative regulator of insulin signaling and the authors propose that IP6K1 may be a novel potential therapeutic target to improve insulin sensitivity.



Chakraborty, A., Koldobskiy, M., Bello, N., Maxwell, M., Potter, J., Juluri, K., Maag, D., Kim, S., Huang, A., & Dailey, M. (2010). Inositol Pyrophosphates Inhibit Akt Signaling, Thereby Regulating Insulin Sensitivity and Weight Gain Cell, 143 (6), 897-910 DOI: 10.1016/j.cell.2010.11.032