Getting to the Heart of the Matter: The role of PKA in Plin-5 regulated lipolysis in cardiac muscle

Figure 7
Figure 7. Proposed scheme depicting the impact of PKA phosphorylation of Plin5 on the regulation of TG breakdown from Plin5-containing LDs.  In the nonfasted state, Plin5 recruits and binds CGI-58. Upon fasting and the concomitant rise in cAMP, active PKA phosphorylates Plin5, leading to the release of CGI-58 from Plin5, a prerequisite required for the CGI-58 mediated stimulation of the TG hydrolytic activity of ATGL. Results from this study strongly suggest that PKA phosphorylation of Plin5 at serine 155 triggers the release of CGI-58 from Plin5 binding. (Pollak et al., 2015)

Kelly Cann

ChemBio Spotlight Week 3

Paper:The Interplay of Protein Kinase A and Perilipin 5 Regulates Cardiac Lipolysis

Getting to the Heart of the Matter: The role of PKA in Plin5 regulated lipolysis in cardiac muscle

Lipid Droplets (LD) are cellular organelles present across many organisms. LDs play a crucial roll by storing triglycerides as an energy source for the cell and participating in a variety of cellular processes such as the generation of signaling molecules. Lipid mobilization from LDs is regulated by the association and phosphorylation of peripilin proteins which recruit and release lipases and cofactors necessary for triglyceride catabolism to the LD surface. LDs in adipose tissue, where triglycerides are frequently stored, are known to be under the regulation of peripilin 1 which is phosphorylated by protein kinase A (PKA), but there have been very few studies done on the regulation of peripilin 5 and LDs in cardiac muscle (CM). In CM the energy demand is continuously high and fatty acids are frequently lipolysed from LDs to meet this demand. An inability to break down cardiac LDs due to a lack of adipose triglyceride lipase (ATGL) or an overexpression of cardiac specific peripilin 5 (CM-Plin5) causes abnormal retention of lipids in the cell (a condition known as steatosis). However, there is a discrepancy in the effect of this cardiac steatosis that is not yet understood: in individuals lacking ATGL, steatosis results in heart dysfunction and shortened life span, but individuals with steatosis due to lack of CM-Plin5 have heart function and life spans compatible with individuals lacking steatosis entirely. Pollak et al. hypothesized that cardiac lipolysis was not constantly inhibited by cardiac Plin5 overexpression, as is the case for individuals lacking ATGL, but instead was somehow regulated. They sought to determine the currently unknown impact of PKA on the regulation of Plin5 as a potential explanation for this incomplete lipolytic barrier.

Several experiments get at the heart of their findings. An increase in cardiac triglyceride levels in CM-Plin5 transgenic mice in both fasted and non-fasted states as compared to wild type (WT) mice indicates that the overexpression of CM-Plin5 is causing the increased triglyceride level, rather than diet. Immunoblot and densitometric analysis of Plin5 protein levels in heart homogenates from fasted and non-fasted mice reveal no significant difference in the test groups, indicating that cardiac triglyceride breakdown is not controlled by CM-Plin5 protein expression levels. Incubation of CM-Plin5 and AGTL-deficient cardiac tissue with and without PKA displays a marked increase in fatty acid (FA) release from Plin5-enriched LDs as compared to the AGTL-deficient cardiac tissue, indicating that PKA incubation decreases the lipolytic barrier caused by Plin5 overexpression. Similar experiments in mouse models display consistent results. The authors show that PKA is active by examining immunoblot analysis in vitro in cardiac tissues and in vivo in mouse models which both display increased levels of phosphorylated versus non-phosphorylated hormone-sensitive lipase (HSL). The authors investigate Ser155 as a possible PKA phosphorylation site on Plin5 by performing a single amino acid mutation at the suspected phosphorylation site and observing a decrease in induced FA release as compared to non-mutated Plin5. Finally, the authors investigate literature suggestions that Plin5 overexpression may promote association of LDs and the mitochondria by measuring FAO in mitochondria and mitochondrial LD content from various CM-Plin5 mice and find that the mitochondria display increased content and impaired FAO capacity. The authors propose a mechanism for PKA phosphorylation regulation of TG breakdown from Plin5 LDs where a rise in cAMP activates PKA which phosphorylates Plin5 at Ser155, releasing CGI-58 which stimulates the hydrolytic activity of ATGL (Figure 7, Stimulated). This is in contrast to the LD in a basal, non-fasted state where cAMP is never elevated and Plin5 does not get phosphorylated (Figure 7, Basal).

Although it was previously known that Plin5 played some role in cardiac muscle LD lipolysis, Pollak et al. provides evidence for the role of PKA phosphorylation of Plin5 at Ser155 in the regulation of cardiac lipolysis and explains the discrepancy with cardiac steatosis and normal heart health and life span in individuals with Plin5 overexpression. In the future, investigation of other potential PKA phosphorylation sites on Plin5 and uncovering other molecules potentially at play in the regulation of cardiac LD lipolysis will be of primary interest.

20 Replies to “Getting to the Heart of the Matter: The role of PKA in Plin-5 regulated lipolysis in cardiac muscle”

  1. Nice job, Kelly! You mentioned that the cardiac muscle has a high level of lipid breakdown because of the high energy demands of the cell. I wonder if there is some way for the body to “know” that lipids should travel to the heart so that cardiac muscle can get more access to energy. What do you think? Also, it seems that phosphorylating Plin5 helps to propagate the pathway of lipid breakdown based on the mechanism that the authors suggest (phosphorylation of Plin5 → releasing CGI-58 → stimulation of ATGL). I’m curious about what the phosphorylation of Plin5 does to the structure of Plin5 to allow this to happen. Just a few thoughts…Thanks! ☺

    1. Hi Zach! I think that your thought regarding increased energy demand is right on track with an earlier trend in the field. I think perhaps this is what provoked the earlier line of reasoning that the authors cited which indicated the previous belief that the majority of FAs taken up by the heart tissue were immediately activated and taken up by the mitochondria for fatty acid oxidation. It makes sense that if the heart has high energy demands, there are two possible ways to accommodate those demands: either provide it with lipids for the dynamic storage and subsequent utilization of LDs, or forgo the storage process entirely and use the majority of incoming lipids for FAO (which, at first glance, seems like the most energy efficient method for the cell, but is perhaps not evolutionarily consistent). The authors indicate that recent research is in line with the storage and utilization of LD model. There is no indication that normal, healthy cardiac tissue is experiencing an energy deficit at basal metabolic rate due to lack of available lipids, so I don’t know if increased lipid trafficking to the heart would actually prove beneficial. In many ways, it makes sense that the LD pool in the heart is dynamic, as it does not seem to be the best place to store fats long term. In looking at your question regarding what phosphorylation does to the structure of Plin5, it seems that is currently unknown for this system (but would be a great direction for further research!). The authors currently suspect over 6 possible phosphorylation sites on Plin5 (not just the one Ser-155 which they identified), so we could infer that there may be an additive effect. Although we are not aware of the exact structural changes for this mechanism, we see phosphorylation as a theme of regulation throughout biochemistry, so we may be able to apply our understanding of potential shifts in other systems here (like altering charge and structure due to the addition of such a large, negative substituent).

  2. Kelly – kudos on tackling a paper published two weeks ago! Respect. My question has to do with the scope of the paper. By the time the reader gets to the discussion, the authors are bragging about how they “…demonstrated in vivo and in vitro that Plin5 is a substrate for PKA phosphorylation…,” yet by the end of the paper they are only committing to “several lines of evidence” that their data “suggest”. At a first pass, this paper seemed to have everything: classic technique (immunoblotting) assessing in vivo phenotypes of mice hearts, in silico analysis to implicate the phosphorylation site, and an intriguing observation of the two different steatosis effects. These are all aspects we praised when we read the Science paper in class, so my question is this: what do you think held this paper back from being published in a higher impact journal? Could you suggest a result or experiment that would have elevated the study past the ranks of JBC?

    1. Hi Zach! I’m so gad you pointed this out! I think there are several potential reasons that this paper was not published in a higher impact journal. The first being that this paper did not flip a paradigm or bridge disciplines and that it still has a ways to go before it really gets to the level of transnational medicine. The authors followed along an already existing trend line investigating links between LDs and Plins, they just did so in a new tissue (cardiac muscle) and with a new Plin (Plin5). Additionally, the authors paint an incomplete picture. As Zach pointed out above, the authors are not able to provide the structural information necessary to determine what the phosphorylation is actually doing to release CGI-58. Additionally, they have only identified one of many phosphorylation sites on Plin5. I think that for this study to have been published in a higher impact journal, they should have identified all of the phosphorylation sites and provided the structural information. Without this information, you can’t really fully target, alter, or inhibit the process for applications in translational medicine either.

  3. Hi Kelly, Good job with the review! As I was reading through your summary, I was particularly interested in the energy requirements for cells that are unable to break down LDs; normally CM cells use lipids as a main source of energy, but the CM-Plin5 and ATGL cells would obviously be unable to use lipids for energy. Perhaps unsurprisingly, the authors note that these CM-Plin5 mouse cells require huge quantities of glucose in order to fulfill the energy requirements of highly active cardiac cells because they cannot utilize the lipids. Thus, glucose catabolism must occur at a rapid rate, and it seems possible that the body would simply be unable to provide such large quantities of glucose for extended periods of time, particularly during fasting. Do you think such a high rate of glucose catabolism, and the resulting possibility of exhausting stores of glucose, could worsen the condition of mice that suffer from an inability to hydrolyze LDs, and perhaps increase mortality rates as well? For example, if energy stores were low, the heart might contract more slowly and less effectively, which might result in macroscopic damage and plaque buildup. Do you think that is possible?

    1. Hi Mike! Thanks for this interesting idea. While it is certainly possible that the role of glucose in this study could be a hidden variable (perhaps also contributing to Zach’s point about this research not being up to snuff for publication in a higher impact journal), the authors do show that CM-Plin5 mice still have normal heart function despite their increased glucose uptake. This seems to imply that the animals are able to meet the energy demands of the heart without experiencing decreased function or slowed contractions like you suggest. However, it is possible that if studied over a longer period of time the mice may experience greater macroscopic damage. It is also worth noting that CM-Plin5 mice can break down LDs in the presence of activated PKA, which seemed to be the author’s focus for this study. Perhaps future studies will more closely address the role of glucose as an energy source in cardiac tissue.

  4. Hey Kelly, great review of a recent paper! I felt like you accurately summarized the major findings, especially clearing up the stated relationship between Protein Kinase A –mediated phosphorylation of peripilin-1 and cardiac lipolysis. Given that perilipin levels are known to have a linear relationship with obesity prevalence in both mice and humans (1), I am curious about the specific roles fasting and non-fasting might have upon the expression of Perilipin-5, especially in relationship to other Perilipins. To this end, I am curious as to the .8 fold decrease in cardiac TG levels as compared against wild type mice in non fasting states, and what possible results would exist in TG level comparison to Perilipin-5 knockout mice. I feel like this would allow for discrete measurement of the specific effects Perilipin-5 has upon cardiac TG levels, as it has already been evidenced that overexpression of Perilipin-5 in cardiac tissue leads to massive increases in cardiac TG level (2) . As such, with such an apparently wide-ranged relationship in regards to lean muscle tissue, it stands to reason that there might be similarly dramatic variances in the expression of TG within other tissue types. Do you feel that such experimentation, both under conditions isolating Perlipin-5 in nonfasting populations, and in other forms of lean muscle tissue, would be beneficial in enhancing understanding of Perilipin’s role? Alternatively, do you feel that such scope-limited experimentation runs the risk of introducing too many confounding variables?

    1. McManaman, James L. et al. “Perilipin-2-Null Mice Are Protected Against Diet-Induced Obesity, Adipose Inflammation, and Fatty Liver Disease.” Journal of Lipid Research 54.5 (2013): 1346–1359. ( )
    2. Pollak, Nina M., et al. “Cardiac-specific overexpression of perilipin 5 provokes severe cardiac steatosis via the formation of a lipolytic barrier.” Journal of lipid research 54.4 (2013): 1092-1102. (
    1. Hi Anthony! Thank you for your comment. While I find this line of reasoning extremely interesting, I do agree that this type of experimentation may be introducing a lot of variables. The first point you make regarding obesity is well taken, but perhaps not directly correlated here. My understanding is that profound weight gain and obesity are typically caused by accumulation of lipids in adipose tissue, which is regulated by Plin1 (which there appears to be a lot of research on, some of which can be found in the references of the paper I reviewed) . The study covered in the review is unique because it is looking at cardiac muscle tissue instead of adipose tissue and looking at a Plin which has not been so thoroughly examined (Plin5). In regards to the 0.8 fold decrease in cardiac TG levels in CM-Plin5 mice, as compared to non-fasted mice, the authors state that, “The reduction in cardiac TG levels of fasted CM-Plin5 mice suggests that Plin5 overexpression does not generally impair cardiac TG catabolism.” My understanding is that the point of this experiment is really to convince the reader that there are not any other variables at play here and set up their future experiments. The authors state that the findings from these first experiments show that heart and whole body homeostasis are altered in nonfasted vs fasted states.

  5. Hey Kelly,

    That was quite the paper. There might be more abbreviations in that bad boy than I knew existed. In the discussion section the authors delve more specifically into the overall mechanism of phosphorylation of plin5 and the subsequent release of CGI-58. The authors also say that after they mutated the serine 155 that gets phosphorylated by PKA and results in the release into an alanine, FA release is actually blunted instead of increasing in the presence of PKA. I was wondering if you had any thoughts as to why this happened? Do you think there is maybe a different interactions occurring between the two proteins that results in the retention of the FA’s?

    1. Hi Tommy! Thank you for your question. The authors found that Ser-155 is the phosphorylation site on Plin5 for PKA. Part of how they determined this was via the mutation experiment yo are referencing. When they did the mutation S155A, the lack of FA release is part of what made them think that Ser155 is a valid phosphorylation site and propose the pathway they did (shown in figure 7). If the breakdown of FAs from LDs increased in the presence of the mutation, then we might infer that Ser155 is not actually a valid phosphorylation site (because breakdown seems to be occuring regardless of what amino acid is at position 155, so that residue does not seem to be critical to function). It makes sense that LD breakdown was blunted because if Ser155 is not phosphorylated, then CGI-58 would not be released from Plin5 to stimulate the TG hydrolytic activity of ATGL and the LD would not have been broken down. It seems your observation is consistent with the author’s findings and predictions! If you could clarify which two proteins you are referencing in your last sentence, I would be happy to address that point as well!

  6. Hey Kelly, fantastic job on the review. There was quite a lot going on in the paper and I was just wondering if you could clear up some confusion I am having between PKA and Plin5… I see that Plin5 over expression yields insufficient lipase activity, we see more TG in the blood, but no adverse effects. Now, do you think the major player in the lypolysis was this combination of Plin5 & PKA, or is this discovery indicative of a multitude of more significant lypolysis pathways involving PKA?

    1. Hi Ryan! Thank you for your question and the opportunity to clarify this! I think the main point here is really about the interaction of PKA and Plin5, specifically in cardiac tissue. The authors are saying that because PKA phosphorylates Plin5 (likely at multiple sites, but they were only able to provide evidence for one phosphorylation site, Ser-155), Plin5 is activated and can release CGI-58. This release is required for the stimulation of ATGL to break down the LD. It was not previously known how Plin5 was regulated. What was previously known was that CM-Plin5 overexpression impaired the break down of LDs in CM, but the authors hypothesized that this was not an ever present barrier and that Plin5 was under some form of regulation (which turns out to be PKA!). While PKA is an important player in many celluar pathways, the focus and findings here are really in regards to its relationship with Plin5.

  7. Hi Kelly! So I was curious about what the effects of multiple phosphorylation sites on Plin5 could be. Since we have not yet discovered the other phosphorylation sites, could the amount of sites be correlated to the severity of steatosis in individuals? It would be interesting to see how much the activity of LD breakdown in CM decreases with each subsequent phosphorylation on Plin5. Do you happen to know of any range in severity of steatosis caused by overexpression of Plin5, or are most individuals effected in the same way?

    1. Hi Matt! I think that your comment may be related to Zach Shuler’s comment above. You can see my reply there where I addressed the potential impact of multiple phosphorylation sites! In regards to steatosis severity, I don’t believe that correlation has been studied in the available literature at this point. My understanding is that a steatosis is either macrovascular (large enough to distort the cells nucleus) microvasuclar (not large enough to do so). However, it does not seem that the authors differentiated here.

  8. Hi Kelly! So, I found this topic to be super interesting- great job on the summary. I’m curious about some of the implications of the article. I’m wondering- has anyone ever looked at mice who maybe have a mutation in the gene that codes for PKA, and seen what effect this has on fatty acid release from LDs? Perhaps this mutation may prove too toxic to study in a live model… if it were observed that a modified function or a loss-of-function PKA were correlated with fatal steatosis in mice, it would further support the author’s point that phosphorylation by PKA was a critical regulatory element of lipolysis in cardiac muscle. I’m also curious if modifying PKA in a way would have any other functional effects that may be able to be used to trace back to the other residues on Plin5 that PKA may be phosphorylating. Just an idea.

    1. Hi Gabby! Thank you for such thoughtful ideas. My literature search indicated turned up a few studies on the links between the mutation of the PKA regulatory subunit and lipolysis in adipose tissue (information below if you are interested), but not much in the way of Plin regulation, LD storage, or cardiac muscle specific studies. I wonder if perhaps this is due to toxicity or perhaps just an inability to isolate the effects of the mutation as lipolysis specific. PKA serves so many roles in the cell, it seems that it would be difficult to draw a direct cause and effect relationship or correlation between the mutation and effects on lipolysis in many cases.

      Mutation of the RIIβ Subunit of Protein Kinase A Differentially Affects Lipolysis but Not Gene Induction in White Adipose Tissue*
      Josep V. Planas‡§, David E. Cummings§¶, Rejean L. Idzerda and G. Stanley McKnight‖doi: 10.1074/jbc.274.51.36281
      December 17, 1999 The Journal of Biological Chemistry, 274, 36281-36287.

      Protein Kinase A Regulatory Subunits in Human Adipose Tissue
      Decreased R2B Expression and Activity in Adipocytes From Obese Subjects
      Giovanna Mantovani1, Sara Bondioni1, Luisella Alberti2, Luisa Gilardini2, Cecilia Invitti2, Sabrina Corbetta3, Marco A. Zappa4, Stefano Ferrero5, Andrea G. Lania1, Silvano Bosari5, Paolo Beck-Peccoz1 and Anna Spada1
      doi: 10.2337/db08-0585
      Diabetes March 2009 vol. 58 no. 3 620-626

  9. Hi Kelly, great job summarizing the article and highlighting the appropriate parts! I have a question about the proposed mechanism you mention in your second-to-last paragraph involving the cAMP-activation of PKA. I might have this wrong, but in order to get cAMP, phosphorylation would have occurred already (ATP –> cAMP somehow), so in a broad sense, is this in a way an example of positive feedback between phosphorylation events, where the first one (ATP –> cAMP) activates a second (PKA phosphorylating Plin5)? Do you think the proposed pathway inhibits cAMP production in some way to add a form of regulation (to avoid a positive-feedback loop)?

    1. Hi Besher! Thank you for your comment! PKA is dependent on cAMP levels and is activated by cAMP. My understanding is that cAMP activates PKA by binding the regulatory subunits, allowing them to dissociate from the catalytic subunits, thus activating PKA to phosphorylate its substrates. I also know that cAMP is made when adenylyl cyclase converts ATP to cAMP (which it does by removing 2 phosphates from ATP and cyclizing the remaining phosphate to the adenine). So, to the best of my knowledge, there is not actually a phosphorylation event occurring there. Since we only have the phosphorylation of Plin5’s Ser155 by PKA, I don’t believe that this represents a positive feedback of phosphorylation events. However, it is possible that there may be additional feedback regulation in this pathway that has not yet been revealed in the literature.

  10. This page has very thoughtful scientific conversations and excellent answers from the blogger! Great work everyone!

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