Wednesday, June 10, 2015

Exploring the Biological Nature of Brown and Beige Fat

Over two years ago this blog discussed the possibility of incorporating a specialized preparation routine before exercise in an attempt to stimulate both brown and beige adipose tissue in order to increase the efficiency and overall calorie and fat burning potential of standard exercise. However, that post did not seek to fully understand or discuss the specific biological mechanisms that govern the behavior of brown or beige adipose tissue. This lack of knowledge limits the efficiency for exercise programs as individuals could either be consuming certain foods or performing certain warm-up tasks to increase exercise potential in addition to those suggested in the past blog post. Increasing exercise efficiency could be an easy means to increase the overall health of society without having to devote more precious time to exercise; therefore it would prove useful to better understand the processes that activate these types of fat.

At the most basic level there are two key elements to the fat burning capacity of brown fat. First, brown fat has multiple mitochondria versus the single mitochondria possessed by white fat; these additional mitochondria allow for greater rates of metabolism along with an increased lipid concentration. Also brown fat releases norepinephrine which reacts with lipases to breakdown fat into triglycerides and later to glycerol and non-esterified fatty acids finally producing CO2 and water, which can lead to a positive feedback mechanism.1,2 Second, brown fat contains significant expression rates of uncoupling protein 1 (UCP-1).1 UCP-1 is responsible for dissipating energy, which leads to the decoupling of ATP production and mitochondrial respiration.1 Basically UCP-1 returns protons after they have been pumped out of the mitochondria by the electron transport chain where these protons are released as heat instead of producing energy (i.e. leaking).

It is important to understand that there are two types of brown fat: natural brown fat and intermediate brown fat commonly known as beige fat. Natural brown is typically exemplified by the fat located in the interscapular region and contains cells from muscle-like myf5+ and pax7+ lineage.3 Natural brown fat is typically isolated from white fat and almost entirely synthesized in the prenatal stage of development as a means to produce heat apart from shivering.4 Beige fat is commonly interspaced within white fat, do not have these muscle-like cells (although Myh11 could be involved),5 and can be activated by thermogenic pathway and the strain of exercise. Beige fat also has the potential to influence the conversion of white fat to beige fat through a process commonly called “browning”.6,7

Natural brown fat is thought to have larger concentrations of UCP1-expression because they constitutively express it after differentiation versus beige, which expresses large amounts of UCP-1 in response to thermogenic or exercise cues.1,5 Therefore, natural brown fat is more effective at energy expenditure. However, it may not be possible to develop more natural brown fat after development; therefore, any positive progression in brown fat development will come from beige fat.

Early understanding of brown fat activation involved non-discriminate increases in the activity of the sympathetic nervous system (SNS). The standard pathway governing brown fat activation uses a thermogenic response involving the release of norepinephrine, which initiates cAMP-dependent protein kinase (PKA) and p38-MAPK signaling leading to the production of free fatty acids (FFA) through lipolysis due to UCP-1 induced proton uncoupling.4 UCP-1 concentrations are further increased through secondary pathways involving the phosphorylation of PPAR-gamma co-activator 1alpha (PGC1alpha), cAMP response element binding protein (CREB) and activating transcription factor 2 (ATF2).8 Among these three elements PGC1alpha appears to be the most important co-activating many transcription factors and playing an important role in linking oxidative metabolism and mitochondrial action.9

However, due to the complicated nature of SNS activation and its other downstream activators the attempt to replicate it in the form of weight loss drugs like Fenfluoramine or Ephedra resulted in severe negative cardiovascular side effects like elevated blood pressure and heart rate.10 While some argue that either increasing the sensitivity or the rate of simulation to the SNS can improve upon these results, the underlying elements associated with downstream activation of the SNS makes facilitating direct influence too complicated. Therefore, from a biological perspective it makes more sense to focus on a downstream element that interacts with brown fat at a more localized level.

Just a side note based on the differing interactivity between brown/beige and white fat from the SNS, white fat appears to represent long-term energy storage and brown fat is shorter-term energy, an unsurprising conclusion. However, frequent energy expenditure, like exercise, may condition the body to produce more beige fat versus white fat viewing short-term energy needs as more valuable than long-term energy needs. Basically if the above point is accurate then it stands to reason that a person would see more benefit from 20 minutes of exercise 6 days a week versus 40 minutes of exercise 3 days a week.

Moving away from direct SNS stimulation perhaps the appropriate method of increasing browning involves increasing transcription and translation of UCP1. Interestingly enough empirical evidence exists to support the idea that reinoic acid could be an effective inducer of UCP-1 gene transcription in mice and operates through a non-adrenergic pathway.11,12 However, a more focused study using loss of function techniques involving retinaldehyde dehydrogenase, which is responsible for converting retinal to retinoic acid, determined that retinal, not retinoic acid is the major inducer of brown fat activity.13 Unfortunately there is no direct understanding regarding the proportional response of brown fat to retinal or retinoic acid. Therefore, the general fat-soluble nature of vitamin A will probably make it difficult to utilize its derivatives as biological stimulants for brown fat activation or browning.

Another possible strategy to stimulate browning is through activated (type 2/M2) macrophages induced by eosinophils which are commonly triggered by IL-4 and IL-13 signaling. When activated this way these macrophages recruit around subcutaneous white fat and secrete catecholamines to facilitate browning in mice.14,15 A secondary means by which both IL-4 and IL-13 may influence fat conversion is their direct interaction with Th2 cytokines.16 Unfortunately while on its face this strategy looks promising, in a similar vein to vitamin A, it might not be effective due to unknown long-term side effects associated with IL-4 and IL-13 activation. Due to this lack of knowledge, if IL-4 or 13 is thought to be a viable biochemical strategy for inducing weight loss, long-term proper time lines for effects and dosages must be explored in humans, not just short-term studies in mice.

A more controversial agent in browning is fibronectin type III domain-containing protein 5 or more frequently known as irisin. Due to its significantly increased rate of secretion from muscle under the strain of exercise, some individuals believe that irisin is a key mediator in browning acting as a myokine;17 if this characterization is accurate then irisin could be a significant player in the biological benefits produced by exercise including weight loss, white fat conversion and reduced levels of inflammation.18,19 However, other parties believe that because human studies with irisin have produced results that do not demonstrate benefits similar to those studies using mice, irisin is another molecule that cannot scale-up its effectiveness when faced with the added biological complexity of humans versus a mouse.20-22

The key element within this controversy could be that irisin expression is augmented by the increased expression of PGC1alpha, but PGC1alpha increases the expression of many different proteins and other molecules, so the expression of irisin may not be relevant to the positive changes associated with exercise. Another factor may be that a key difference between mice and humans is the mutation in the start codon of the human gene involved in the production of irisin, which significantly reduces irisin availability.23 Thus this mutation could be the limiting factor to why despite a very conserved genetic sequence, humans do not see anywhere near the benefit mice do. If this explanation is correct it does potentially still leave the door open to directly inject irisin into the body to increase concentrations in an attempt to aid exercise derived results, but if PGC1alpha is the key, then this increased concentration of irisin could be of minimal consequence.

Another potential element that demonstrates a significant concentration increase in accordance to increased PGC1alpha is a hormone known as meteorin-like (Metrnl).24 The concentration of this hormone increases in both skeletal muscle and adipose tissue during exercise and exposure to cold temperatures in accordance to increases in PGC1alpha concentrations. When Metrnl circulates in the blood it seems to produce a widespread effect that induces browning resulting in a significant increase in energy expenditure.24 The influence of Metrnl on white fat does not appear due to direct interaction with the fat, but instead indirect action on various immune cells most notably M2 macrophages via the eosinophil pathway, which then interact with the fat through activation of various pro-thermogenic actions.24 As discussed above this interaction with eosinophil appears to function through IL-4 and IL-13 signaling indicating a common pathway purpose between IL-4/IL-13 and the original SNS pathway. Not surprisingly blocking Metrnl has a negative effect on the biological thermogenic response.24

Another potential strategy for browning may be targeting appropriate receptors instead of specific molecules; with this strategy in mind one potential target could be transient receptor potential vanilloid-4 (TRPV4). TRPV4 acts as a negative regulator for browning through its negative action against PGC1a and the thermogenic pathway in general.25 In addition TRPV4 appears to activate various pro-inflammatory genes that interact with white adipose tissue making it more difficult to facilitate browning even if the appropriate signals are present. TRPV4 inhibition and genetic ablation in mice significantly increase resistance to obesity and insulin resistance.25 The link between inflammation and thermogenesis is highlighted by the activity of TRPV4, which is one of the early triggers for immune cell chemoattraction.25

Obesity may also produce a positive feedback effect through TRPV4 by increasing cellular swelling and stretching through the ERK1/2 pathway, which increases the rate of TRPV4 activation.26,27 However, the validity of TRPV4 as a therapeutic target remains questionable for TRPV4 expression not only influences fat/energy expenditure, but also osmotic regulation, bone formation and plays some role in brain function.25,28,29 Fortunately a number of the issues with TRPV4 mutations/mis-function appear to be developmental in influence versus post-development, thus TRPV4 therapies could still be valid.

Natriuretic peptides (NPs) are hormones typically produced in the heart on two different operational capacities: atrial and ventricular. Both of these hormones appear to play a role in browning through association with the adrenergic pathway.30 The most compelling evidence for supporting this behavior is that a lack of NP clearance receptors demonstrated significant enhanced thermogenic gene expression in both white and brown adipose tissue.30 Also direct application of ventricular NP in mice increased energy expenditure.30 In addition to the above results, NPs are an inherent attractive therapeutic possibility because appropriate receptors are located in white and brown fat of both rats and humans31,32 and these receptors go through periods of significant decline in expression when exposed to fasting,33 which may account for some of the benefits seen from low calorie diets.

Atrial NPs increase lipolysis in human adipocytes similar to catecholamines (increasing cAMP levels and activation of PKA) although whether or not this increase is induced through interaction with beta-adrenergic receptors is unclear.34 Some believe that NPs activate the guanylyl cyclase containing NPRA producing the second messenger cGMP activating cGMP-dependent protein kinase (PKG).35,36 PKA and PKG have similar mechanisms for substrate phosphorylation including similar targets in adipocytes,36 thus this interaction may explain why atrial NPs act similar to catecholamines.

Recall from above that one of the means of inducing browning, especially for those tissues that are distant from SNS-based neurons, is macrophage recruitment. This recruitment appears to be initiated by CCR2 and IL-4 for when either is eliminated from mice models the conversion no longer occurs.15 Tyrosine hydroxylase (Th) is also important in this process facilitating the biosynthesis of catecholamines and later PKA levels.

With respects to producing a biomedical agent to enhance browning there appear to be three major pathways in play: 1) the SNS pathway producing a direct activation response; 2) macrophage recruitment pathway potentially involving Metrnl, which activates IL-4 and IL-13 eventually leading to PKA activation and an indirect activation response; 3) NPs activation pathway, which eventually leads to PKG activation and an indirect activation response. As mentioned earlier SNS pathway enhancement has already been attempted by at least two drugs and failed miserably, so that method is probably out. In addition the SNS pathway does not appear to have as much browning potential as the PKA or PKG pathways due to the reliance on the location of certain nerve fibers.

Enhancing macrophage recruitment could be a good strategy, but there appears to be little information regarding negative effects associated with short-term high frequency enhancement of IL-4 or IL-13 concentrations. Some reports have suggested an increase in allergic symptoms, but any more severe consequences are unknown. This is not to say that enhancing IL-4 or IL-13 is not a valid therapeutic strategy, but its overall value is unknown. In contrast enhancement of NPs appear to be a more stable choice due to positive results in initial exploration of both the application and the expected negative side effects. First, NPs can be administrated via the nose-brain pathway enabling access to the brain avoiding some potential systemic side effects.37 Second, there appear to be few, if any significant side effects to intranasal NP application, at least in the short-term.38

Overall the above discussion has merely identified some of the more promising candidates to enhance browning white fat. One could argue that resorting to drugs to enhance the overall health of an individual versus simple diet and exercise is a regretful strategy. Unfortunately the reality of modern society is that more and more people seem to have less available time to exercise or eat right. In addition to a mounting negative weight external environment (increased pollution and industrial chemicals like BPAs) this drug enhancement strategy may be the most time and economically efficient means to ensure proper weight control and overall health for the future.

Citations –

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