Fat Tissue Regulation ~ Part V: C5L2KO - Meet the New Droid, Kinda Like the Old Droid

It seems that our friend C3KO city mouse has found his country mouse cousin:  C5L2KO.  In keeping with the Star Wars saga, albeit stretching things a bit with this one, I've found the depiction of our new friend!

To catch up, C3KO is a knockout mouse lacking the ability to produce Complement 3 (C3) protein which is a precursor for production of acylation stimulating protein, ASP.  Therefore C3KO is ASP deficient.  The result of this genetic mutation is to produce a mouse that is resistant to obesity, and essentially an ASP equivalent of insulin deficiency -- Type 1 diabetes.  If you've not read about C3KO, here are the links to the two relevant installments in this series:
  Fat Tissue Regulation ~ Part II: Meet C3KO
  Fat Tissue Regulation ~ Part III: C3KO Meets Obi No Leptinobi

It is known that fat tissue expresses insulin receptors.  Indeed this has been exploited to more clearly elucidate the roll of insulin acting on fat tissue in the form of insulin receptor knockout mice, the FIRKO mouse (F = fat specific, IR = insulin receptor) to be exact.  As it is less well characterized, ASP action on fat tissue is not universally well known or acknowledged.  Indeed ASP is pretty much a TWICHOOB's worst nightmare.  It seems silly to me to even be having this part of the discussion because it is absolutely not controversial that ASP plays a critical role in clearing dietary fat into the adipose tissue.  Or, we are to believe the fat tissue itself manufactures such a hormone for no purpose related controlling its own function.  Huh?  In any case, one piece of the puzzle that was missing was how ASP works and the absence of an identified receptor.  This piece of the puzzle was found in 2005 by Katherine Cianflone's group.

C5L2 Is a Functional Receptor for Acylation-stimulating Protein

J. Biol. Chem., Vol. 280, Issue 25, 23936-23944, June 24, 2005

David Kalant, Robin MacLaren, Wei Cui, Ratna Samanta, Peter N. Monk, Stephane A. Laporte, and Katherine Cianflone

From the Introduction:

The function of acylation-stimulating protein (ASP,¹ also known as C3a des-Arg) as a stimulator of triglyceride synthesis (TGS) has been well documented in human adipocytes, 3T3-L1 preadipocytes,and human skin fibroblasts (HSF) (2-5). The activity of ASP also is shared by C3a (6). In addition to stimulation of TGS, ASP stimulates glucose transport (7-9) and reduces triglyceride lipolysis (10), enhancing fat storage overall. Furthermore, high affinity binding of both ligands (ASP and C3a) to HSF and 3T3-L1 cells (1, 3, 11) and of ASP to adipose tissue plasma membranes (12) has been demonstrated. On the other hand, both ASP (C3a des-Arg) and C3a have other functions in common in addition to TGS stimulation (13). ASP and C3a influence secretion of a number of hormones, including interleukin-6 and tumor necrosis factor  (14, 15), as well as prolactin, growth hormone, and adrenocorticotropin in pituitary cells (16). ASP has also been shown to influence insulin secretion in cultured pancreatic islet cells and in vivo (17).

By contrast, no binding of ASP to the C3a receptor (C3aR) has been observed in cells that bind C3a, including U937 macrophage, polymorphonuclear monocytes, transfected RBL cells, or transfected HEK293 (11, 18, 19). Furthermore, ASP does not exhibit the anaphylatoxic functions of C3a: (i) stimulation of eosinophil chemotaxis (20), (ii) prostanoid production by guinea pig macrophages or rat Kupffer cells (21), or (iii) degranulation of U937 cells (18). The division between ASP and C3a, in binding and function, may be explained by the existence of two receptors: the C3aR, which binds only C3a, and another that binds both ligands.

The reference links were preserved so I left them in.  One of the complicating mysteries in all of this is that ASP is virtually identical to this C3a protein which is integrally involved in the immune system.  Basically C3a seems to act like ASP in fat cells, but ASP seems to be inactive and unable to mimic C3a systemically.  No doubt some of the other ASP functions discussed in the first paragraph will be the subject of future posts here at the Asylum.  They continue:

We recently have reported that the orphan G protein-coupled receptor (GPCR) C5L2 binds ASP and C3a with high affinity in transfected RBL cells and exhibits saturable binding of ASP in transfected HEK293 cells (1). Furthermore, we demonstrated the expression of C5L2mRNA in human adipose tissue, HSF, and 3T3-L1 by reverse transcription-PCR and expression of the protein on the cell surface of HSF using anti-C5L2 polyclonal antiserum. These data suggested that C5L2 might mediate the TGS-stimulating effect of ASP. Although C5L2 also binds C5a and C5a des-Arg, these ligands do not stimulate triglyceride synthesis in 3T3-L1 cells (1), and in fact, it has recently been demonstrated that C5L2 is a nonfunctional receptor for C5a (22).

In the present study, we provide evidence, through both gain-of-function and loss-of-function assays in physiologically relevant cells, that C5L2 not only binds ASP but also is a functional ASP receptor.

By gain-of-function assays they are referring to experiments where the C5L2 receptors are introduced and the ASP associated functions of glucose uptake and triglyceride synthesis are enhanced, while loss-of-function would refer to, well, loss of same when C5L2 are absent or inactivated.  I'm not going to delve deeply into all of the experiments and their meaning.  It is sufficient to quote from their concluding paragraph:

Together these studies demonstrate clearly that C5L2 is a functional receptor for ASP.... Whereas there is much left to learn about the physiological role of ASP in humans, the information gained to date suggests that ASP is not only an important regulator of energy storage and adipose tissue metabolism but also may play a role in obesity and related diseases. The demonstration of a functional ASP receptor, through gain-of-function and loss-of-function assays, is a critical step in understanding the mechanisms of ASP action. Further investigation of C5L2 activation and signaling, particularly ligand binding, may aid in finding C5L2 antagonists and exogenous agonists, which could be used to modulate the ASP pathway. Together these studies will place the ASP pathway, including C5L2, in its proper physiological context.

One of the next steps for Cianflone's group was to create a C5L2 knockout mouse, dubbed C5L2KO.  C5L2KO is a new droid similar to our old droid C3KO, because if we would see similar outcomes in fat tissue from knocking out either the precursor of ASP or by knocking out the receptor integral to its action. 

Reduced adipose tissue triglyceride synthesis and increased muscle fatty acid oxidation in C5L2 knockout mice   

Journal of Endocrinology (2007) 194, 293–30

Sabina Paglialunga, Patrick Schrauwen, Christian Roy, Esther Moonen-Kornips, Huiling Lu, Matthijs, K C Hesselink, Yves Deshaies, Denis Richard and Katherine Cianflone

As we can see by the introduction, much progress was made in the couple of years leading up to this study and its publication.  I'm going to bulletpoint  and paraphrase much of the introduction and include references. and leave the reference links intact but shrunk them.

• ASP binds and activates the C5L2 receptor.  
• The ASP-C5L2 pathway stimulates adipocyte triglyceride synthesis (esterification) by increasing  diacylglycerol acyltransferase (DAGAT) activity.  DAGAT is the last enzyme in the esterification process converting a diglyceride to a triglyceride (Yasruel et al. 1991, Kalant et al. 2005) , and is believed to be the rate-limiting step in the esterification process.
• Both ASP and its arginated precursor C3a increase triglyceride synthesis in preadipocytes and adipocytes.  ( Walsh et al. 1989, Cianflone et al. 1994, 1999). 
• ASP stimulates glucose transport in adipose tissue by increasing glucose transporter 4 (GLUT-4) translocation ( Maslowska et al. 1997, Tao et al. 1997) 
• ASP has been shown to inhibit lipolysis
• The GLUT-4 and lipolytic actions of ASP are independent and additive to insulin ( van Harmelen et al. 1999).
• ASP increases clearance of dietary triglycerides (chylomicrons)  ( Murray et al. 1999a, Xia et al. 2004).
• C5L2 is a G-protein-coupled receptor highly expressed in fat depots, liver, and spleen ( Kalant et al. 2005). 
• C5L2 is similar to the family of receptors activated in immune attacks to recruite macrophages and neutrophils, however C5L2 is not involved this activity. ( Ohno et al. 2000, Cain & Monk 2002). 
• C5L2 binds other ligands, but only ASP and C3a have been shown to activate the C5L2 receptor.  (See text for more detail.)
• C5L2 is necessary and sufficient for ASP action ( Kalant et al. 2005).

And so, C5L2KO mice were born.  The 2007 study looked at C5L2KO mice vs. wild type (WT) mice placed on either a low fat (LF) diet or a high fat/high sugar (HF/HS) diet.   It is noted that the LF diet results in an upregulation of de novo lipogenesis, while it is downregulated when dietary fat is readily available for storage.

LF Diet: Compared to WT, the C5L2KO

• increased food intake and reduced food efficiency
• demonstrated a small delay in postprandial TG clearance with markedly decreased capacity for adipose tissue TG storage and re-esterification
• had reduced respiratory quotient, RQ, indicative of increased fatty acid oxidation as a proportion of the fuel mix, increased ex vivo muscle fatty acid oxidation, and increased expression of proteins involved in skeletal muscle energy metabolism.

HF/HS Diet:  It was noted that challenging the mice with this diet masked some of the observations with the LF diet, but highlighted other differences between the mice.  

• Food efficiency was enhanced in both WT and C5L2KO mice.
• C5L2KO exhibited increased skeletal muscle protein content (cytochrome c, CD36, UCP3) than WT mice,  but the differences were less marked vs. on the LF diet.
• A marked increase in AMPK activity in C5L2KO relative to WT mice was observed.
• The delayed postprandial triglyceride clearance in C5L2KO mice was "strikingly exaggerated" vs. WT mice, and fatty acid re-esterification was further suppressed.

Direct quotations from Discussion:

... increased in vivo fat oxidation, ex vivo tissue fat oxidation, muscle phospho-ACC and expression of key oxidative proteins (CD36, UCP3, and cytochrome c) are all evidence of elevated fat utilization in the muscle.

... in spite of the adaptation of muscle in C5L2KO mice, the compensation does not appear to be sufficient, and the C5L2KO mice still manifest disturbed insulin metabolism (increased fasting plasma insulin, delayed GTT and decreased insulin stimulation of adipose TGS [triglyceride synthesis]).

I find that bolded line very interesting.  Previously the fact that insulin seems to stimulate ASP has been suggested to minimize the possible role of ASP in fat tissue regulation.  However in this mouse, knocking out the ASP interfered with insulin's action.  Together with another paper I'll be addressing soon, this suggests that ASP can perhaps stimulate insulin and/or alter the insulin sensitivity of the adipocytes.  

... ASP inhibits the uptake of lipoprotein lipase derived fatty acids in muscle, although it stimulates this process in adipose tissue ( Faraj & Cianflone 2004).

... We hypothesize that in the absence of C5L2, the effect of ASP on muscle may be removed, contributing to the increase in muscle fat oxidation found in the C5L2KO mice. C5L2 is also expressed in brain and liver ( Gao et al. 2005, Kalantet al. 2005) and this may be linked to changes in food intake, RQ, and hepatic lipid metabolism demonstrated here. However, the direct effect of ASP on fat oxidation in muscle, as well as direct effects on brain and liver remains to be evaluated in future studies.

As with the C3KO, these C5L2KO mice ate more but did not become obese indicating a clear compensatory change in metabolic rate.  This is indication of this adipose-derived hormone -- ASP -- acting remotely, e.g. in skeletal muscle and perhaps the brain, to alter non-adipose function.  ASP is, thus, secreted by the fat tissue itself to regulate it's accumulation and mass!!

There is some discussion on somewhat conflicting results in different strains of C3KO mice that I'll hopefully get to address in the near future.  Since this is a 2007 paper, and I have some more recent offerings by this research group on my reading list, I also hope that some of these hypotheses and conjectures have been tested so we have more answers.

But there is no doubt that C5L2KO combined with its cousin C3KO provide powerful evidence as to the role of ASP in the regulation of fat tissue mass.  In likely the next installment, I'll be comparing C5L2KO with it's insulin cousin, FIRKO.

And yet, in his latest book, Gary Taubes assures us in a footnote that insulin reigns supreme.  Because ... "A hormone...known as acylation stimulating protein is almost assuredly an insignificant exception. It is secreted by the fat tissue itself, a process that is regulated at least in part by insulin."

In my opinion, the results of this study cast serious doubt on such a statement by a science journalist who has admitted repeatedly to not having the time (nor apparently the inclination) to read any new studies in the area of fat metabolism.

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For Part VI, click here.