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 –

1. van Marken Lichtenbelt, W, et Al. “Cold-activated brown adipose tissue in healthy men.” The New England Journal of Medicine. 2009. 360:1500-08.

2. Lowell, B, and Spiegelman, B. “Towards a molecular understanding of adaptive thermogenesis.” Nature. 2000. 404:652-60.

3. Seale, P, et Al. “PRDM16 controls a brown fat/skeletal muscle switch.” Nature. 2008. 454:961–967.

4. Sidossis, L and Kajimura, S. “Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis.” J. Clin. Invest. 2015. 125(2):478-486.

5. Long, J, et Al. “A smooth muscle-like origin for beige adipocytes.” Cell Metab. 2014. 19(5):810–820.

6. Kajimura, S, and Saito, M. “A new era in brown adipose tissue biology: molecular control of brown fat development and energy homeostasis.” Annu Rev Physiol. 2014. 76:225–249.

7. Harms, M, and Seale, P. “Brown and beige fat: development, function and therapeutic potential.” Nat Med. 2013. 19(10):1252–1263.

8. Collins, S. “β-Adrenoceptor signaling networks in adipocytes for recruiting stored fat and energy expenditure.” Front Endocrinol (Lausanne). 2011. 2:102.

9. Handschin, C, and Spiegelman, B. “Peroxisome proliferatoractivated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism.” Endocr. Rev. 2006. 27:728–735.

10. Yen, M, and Ewald, M. “Toxicity of weight loss agents.” J. Med. Toxicol. 2012. 8:145–152.

11. Alvarez, R, et Al. “A novel regulatory pathway of brown fat themogenesis, retinoic acid is transcriptional activator of the mitochondrial uncoupling protein gene.” J. Biol. Chem. 270:5666-5673.

12. Mercader, J, et Al. “Remodeling of white adipose tissue after retinoic acid administration in mice.” Endocrinology. 2006. 147:5325–5332.

13. Kiefer, F, et Al. “Retinaldehyde dehydrogenase 1 regulates a thermogenic program in white adipose tissue.” Nat. Med. 2012. 18:918–925.

14. Nguyen, K, et Al. “Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis.” Nature. 2011. 480(7375):104–108.

15. Qiu, Y, et Al. “Eosinophils and type 2 cytokine signaling in macrophages orchestrate development of functional beige fat.” Cell. 2014. 157(6):1292–1308.

16. Stanya, K, et Al. “Direct control of hepatic glucose production by interleukins-13 in mice.” The Journal of Clinical Investigation. 2013. 123(1):261-271.

17. Pedersen, B, and Febbraio, M “Muscle as an endocrine organ: focus on muscle-derived interleukin-6.” Physiological Reviews. 2008. 88(4):1379–406.

18. Bostrom, P, et Al. “A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.” Nature. 2012. 481(7382):463–468.

19. Lee, P, et Al. “Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans.” Cell Metab. 2014. 19(2):302–309.

20. Erickson, H. “Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?” Adipocyte. 2013. 2(4):289-293.

21. Timmons, J, et Al. “Is irisin a human exercise gene?” Nature. 2012. 488(7413):E9-11.

22. Albrecht, E, et Al. “Irisin - a myth rather than an exercise-inducible myokine.” Scientific Reports. 2015. 5:8889.

23. Ivanov, I, et Al. “Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences.” Nucleic Acids Research. 2011. 39(10):4220-4234.

24. Rao, R, et Al. “Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis.” Cell. 2014. 157:1279-1291.

25. Ye, L, et Al. “TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis.” Cell. 2012. 151:96-110.

26. Gao, X, Wu, L, and O’Neil, R. “Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways.” J. Biol. Chem. 2003. 278:27129–27137.

27. Thodeti, C, et Al. “TRPV4 channels mediate cyclic strain-induced endothelial cell reorientation through integrin-to-integrin signaling.” Circ. Res. 2009. 104:1123–1130.

28. Masuyama, R, et Al. “TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts.” Cell Metab. 2008. 8:257–265.

29. Phelps, C, et Al. “Differential regulation of TRPV1, TRPV3, and TRPV4 sensitivity through a conserved binding site on the ankyrin repeat domain.” J. Biol. Chem. 2010. 285:731–740.

30. Bordicchia, M, et Al. “Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes.” The Journal of Clinical Investigation. 2012. 122(3):1022-1036.

31. Sarzani, R, et Al. “Comparative analysis of atrial natriuretic peptide receptor expression in rat tissues.” J Hypertens Suppl. 1993. 11(5):S214–215.

32. Sarzani, R, et Al. “Expression of natriuretic peptide receptors in human adipose and other tissues.” J Endocrinol Invest. 1996. 19(9):581–585.

33. Sarzani, R, et Al. “Fasting inhibits natriuretic peptides clearance receptor expression in rat adipose tissue.” J Hypertens. 1995. 13(11):1241–1246.

34. Sengenes, C, et Al. “Natriuretic peptides: a new lipolytic pathway in human adipocytes.” FASEB J. 2000. 14(10):1345–1351.

35. Potter, L, and Hunter, T. “Guanylyl cyclase-linked natriuretic peptide receptors: structure and regulation.” J Biol Chem. 2001. 276(9):6057–6060.

36. Sengenes, C, et Al. “Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes.” J Biol Chem. 2003. 278(49):48617–48626.

37. Illum, L. “Transport of drugs from nasal cavity to the central nervous system.” Eur. J. Pharm. Sci. 11:1-18.

38. Koopmann, A, et Al. “The impact of atrial natriuretic peptide on anxiety, stress and craving in patients with alcohol dependence.” Alcohol and Alcoholism. 2014. 49(3):282-286.

A Theory Behind the Relationship Between Processed Foods and Obesity

While there has been a general slowing in the progression of global obesity, especially in the developed world, there has yet to be a reversal of this detrimental trend. A recent study has suggested that one aspect of influence regarding obesity progression lies with the consumption of foods that have incorporated emulsifiers and how they interact with intestinal bacteria including increasing the probability of developing negative metabolic syndromes in mice.1 Based on this result understanding the digestive process may be an important element to understanding how emulsifiers and emulsions may influence weight outcomes.

An emulsion is a mixture of at least two liquids where multiple components are immiscible, a characteristic commonly seen when oil is added to water resulting in a two-layer system where the oil floats on the surface of the water before it is mixed to form the emulsion. However, due to this immiscible aspect most emulsions are inherently unstable as “similar” droplets join together once again creating two distinct layers. When separated these layers are divided into two separate elements: a continuous phase and a droplet phase depending on the concentrations of the present liquids. Due to their inherent instability most emulsions are stabilized with the addition of an emulsifier. These agents are commonly used in many food products including various breads, pastas/noodles, and milk/ice cream.

Emulsifier-based stabilization occurs by reducing interfacial tension between immiscible phases and by increasing the repulsion effect between the dispersed phases through either increasing the steric repulsion or electrostatic repulsion. Emulsifiers can produce these effects because they are amphiphiles (have two different ends): a hydrophilic end that is able to interact with the water layer, but not the oil layer and a hydrophobic end that is able to interact with the oil layer, but not the water layer. Steric repulsion is born from volume restrictions from direct physical barriers while electrostatic repulsion is based on exactly its namesake electrically charged surfaces producing repulsion when approaching each other. As previously mentioned above some recent research has suggested that the consumption of certain emulsifiers in mice have produced negative health outcomes relative to controls. Why would such an outcome occur?

A typical dietary starch, which is one of the common foods that utilize emulsifiers is composed of long chains of glucose called amylose, a polysaccharide.2 These polysaccharides are first broken down in the mouth by chewing and saliva converting the food structure from a cohesive macro state to scattered smaller chains of glucose. Other more complex sugars like lactose and sucrose are broken down into their glucose and secondary sugar (galactose, fructose, etc.) structures.

Absorption and complete degradation begins in earnest through hydrolysis by salivary and pancreatic amylase in the upper small intestine with little hydrolyzation occurring in the stomach.3 There is little contact or membrane digestion through absorption on brush border membranes.4 Polysaccharides break down into oligosaccharides that are then broken down into monosaccharides by surface enzymes on the brush borders of enterocytes.5 Microvilli in the entercytes then direct the newly formed monosaccharides to the appropriate transport site.5 Disaccharidases in the brush border ensure that only monosaccharides are properly transported, not lingering disaccharides. This process differs from protein digestion, which largely involves degradation in gastric juices comprised of hydrochloric acid and pepsin and later transfer to the duodenum.

Within the small intestine free fatty acid concentration increases significantly as oils and fats are hydrolyzed at a faster rate than in the stomach due to the increased presence of bile salts and pancreatic lipase.3 It is thought that droplet size of emulsified lipids influences digestion and absorption where the smaller sizes allow for gastric lipase digestion in the duodenal lipolysis.6,7 The smaller the droplet size the finer the emulsion in the duodenum leading to a higher degree of lipolysis.8 Not surprisingly gastric lipase activity is also greater in thoroughly mixed emulsions versus coarse ones.

Typically hydrophobic interactions are responsible for the self-assembly of amphiphiles where water molecules react to a disordered state gaining entropy as the hydrophobes of the amphiphilic molecules are buried in the cores of micelles due to repelling forces.9 However, in emulsions the presence of oils produce a low-polarity interaction that can facilitate reverse self-assembly10,11 with a driving force born from the attraction of hydrogen bonding. For example lecithin is a zwitterionic phospholipid with two hydrocarbon tails that form reverse spherical or ellipsoidal micelles when exposed to oil.21 Basically emulsions could have the potential to significantly increase the hydrogen concentration of the stomach.

This potential increase in free hydrogen could be an important aspect to why emulsions produce negative health outcomes in model organisms.1 One of the significant interactions that govern the concentrations and types of intestinal bacteria is the rate of interspecies hydrogen transfer between hydrogen producing bacteria to hydrogen consuming methanogens. Note that non-obese individuals have small methanogen-based intestinal populations whereas obese individuals have larger populations where it is thought that the population of methanogen bacteria expands first before one gains significant weight.13,14 The importance behind this relationship is best demonstrated by understanding the biochemical process involved in the formation of fatty acids in the body.

Methanogens like Methanobrevibacter smithii enhance fermentation efficiency by removing excess free hydrogen and formate in the colon. A reduced concentration of hydrogen leads to an increased rate of conversion of insoluble fibers into short-chain fatty acids (SCFAs).13 Proprionate, acetate, butyrate and formate are the most common SCFAs formed and absorbed across the intestinal epithelium providing a significant portion of the energy for intestinal epithelial cells promoting survival, differentiation and proliferation ensuring effective stomach lining.13,15,16 Butyric acid is also utilized by the colonocytes.17 Formate also can be directly used by hydrogenotrophic methanogens and propionate and lactate can be fermented to acetate and H2.13

Overall the population of Archaea bacteria in the gut, largely associated to Methanobrevibacter smithii, is tied to obesity with the key factor being availability of free hydrogen. If there is a lot of free hydrogen then there is a higher probability for a lot of Archaea, otherwise there is a very low population of Archaea because there is a limited ‘food source’. Therefore, the consumption of food products with emulsions or emulsion-like characteristics or components could increase available free hydrogen concentrations, which will change the intestinal bacteria composition in a negative manner that will increase the probability that an individual becomes obese. This hypothesis coincides with existing evidence from model organisms that emulsion consumption has potential negative intestinal bacteria outcomes. One possible methodology governing this negative influence is how the change in bacteria concentration influences the available concentration of SCFAs, which could change the stability of stomach lining.

In addition to influencing hydrogen concentrations in the gut, emulsions also appear to have a significant influence on cholecystokinin (CCK) concentrations. CCK plays a meaningful role in both digestion and satiety, two components of food consumption that significantly influence both body weight and intestinal bacteria composition. Most of these concentration changes occur in the small intestine, most notably in the duodenum and jejunum.18 The largest influencing element for CCK release is the amount and level of fatty acid presence in the chyme.18 CCK is responsible for inhibiting gastric emptying, decreasing gastric acid secretion and increased production of specific digestive enzymes like hepatic bile and other bile salts, which form amphipathic lipids that emulsify fats.

When compared against non-emulsions, emulsion consumption appears to reduce the feedback effect that suppresses hunger after food intake. This effect is principally the result of changes in CCK concentrations versus other signaling molecules like GLP-1.19 Emulsion digestion begins when lipases bind to the surface of the emulsion droplets; the effectiveness of lipase binding increases with decreasing droplet size. Small emulsion droplets tend to have more complex microstructures, which produce more surface area that allow for more effective digestion.

This higher rate of breakdown produces a more rapid release of fatty acids as the presences of free fatty acids in the small intestinal lumen is critical for gastric emptying and CCK release.20 This accelerated breakdown creates a relationship between CCK concentration and emulsion droplet size where the larger the droplet size the lower the released CCK concentration.21 One of the main reasons why larger emulsions produce less hunger satisfaction is that with the reduced rate of CCK concentration and emulsion breakdown there is less feedback slowing of intestinal transit. Basically the rate at which the food is traveling through the intestine proceeds at a faster rate because there are fewer cues (feedback) due to digestion to slow transit for the purpose of digestion.

As alluded to above the type of emulsifier used to produce the emulsion appears to be the most important element to how an emulsion influences digestion. For example the lipids and fatty acid concentrations produced from digestion of a yolk lecithin emulsion were up to 50% smaller than one using polysorbate 20 (i.e. Tween 20) or caseinate.7 Basically if certain emulsifiers are used the rate of emulsion digestion can be reduced potentially increasing the concentration of bile salts in the small intestine, which could produce a higher probability for negative intestinal related events.

Furthermore studies using low-molecular mass emulsifiers (two non-ionic, two anionic and one cationic) demonstrated three tiers of TG lipolysis governed by emulsifier-to-bile salt ratio.3 At low emulsifier-bile ratios (<0.2 mM) there was no change in solubilization capacity of micelles whereas at ratios between 0.2 mM and 2 mM solubilization capacity significantly increased, which limited interactions between the oil and destabilization reaction products reducing oil degradation.3 At higher ratios (> 2 mM) emulsifier molecules remain in the adsorption layer heavily limiting lipase activity, which significantly reduces digestion and oil degradiation.3

Another possible influencing factor could be change in glucagon concentrations. There is evidence suggesting that increasing glucagon concentration in already fed rats can produce hypersecretory activity in both the jejunum and ileum.22-24 It stands to reason that due to activation potential of glucagon-like peptide-1 (GLP-1) in consort with CCK, glucagon plays some role. However, there are no specifics regarding how glucagon directly interacts with intestinal bacteria and the changes in digestion rate associated with emulsions.

The methodology behind why emulsions and their associated emulsifiers produce negative health outcomes in mice is unknown, but it stands to reason that both how emulsions change the rate of digestion and the present hydrogen concentration play significant roles. These two factors have sufficient influence on the composition and concentration of intestinal bacteria, which have corresponding influence on a large number of digestive properties including nutrient extraction and SCFA concentration management. SCFA management may be the most pertinent issue regarding the metabolic syndrome outcomes seen in mice born from emulsifiers.

It appears that creating emulsions that produce smaller drop sizes could mitigate negative outcomes, which can be produced by using lecithin over other types of emulsifiers. Overall while emulsifiers may be a necessary element in modern life to ensure food quality, instructing companies on the proper emulsifier to use at the appropriate ratios should have a positive effect on managing any detrimental interaction between emulsions and gut bacteria.



Citations –

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2. Choy, A, et Al. “The effects of microbial transglutaminase, sodium stearoyl lactylate and water on the quality of instant fried noodles.” Food Chemistry. 2010. 122:957e964.

3. Vinarov, Z, et Al. “Effects of emulsifiers charge and concentration on pancreatic lipolysis: 2. interplay of emulsifiers and biles.” Langmuir. 2012. 28:12140-12150.

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7. Hur, S, et Al. “Effect of emulsifiers on microstructural changes and digestion of lipids in instant noodle during in vitro human digestion.” LWT – Food Science and Technology. 2015. 60:630e-636.

8. Armand, M, et Al. “Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract.” American Journal of Clinical Nutrition. 1999. 70:1096e1106

9. Njauw, C-W, et Al. “Molecular interactions between lecithin and bile salts/acids in oils and their effects on reverse micellization.” Langmuir. 2013. 29:3879-3888.

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19. Adam, T, and Westerterp-Plantenga, M. “Glucagon-like peptide-1 release and satiety after a nutrient challenge in normal-weight and obese subjects.” Br J Nutr. 2005. 93:845–51.

20. Little, T, et Al. “Free fatty acids have more potent effects on gastric emptying, gut hormones, and appetite than triacylglycerides.” Gastroenterology. 2007. 133:1124–31.

21. Seimon, R, et Al. “The droplet size of intraduodenal fat emulsions influences antropyloroduodenal motility, hormone release, and appetite in healthy males.” Am. J. Clin. Nutr. 2009. 89:1729-1736.

22. Young, A, and Levin, R. “Diarrhoea of famine and malnutrition: investigations using a rat model. 1. Jejunal hypersecretion induced by starvation.” Gut. 1990. 31:43-53.

23. Youg, A, Levin, R. “Diarrhoea of famine and malnutrition: investigations using a rat model. 2. Ileal hypersection induced by starvation.” Gut. 1990. 31:162-169.

24. Lane, A, Levin, R. “Enhanced electrogenic secretion in vitro by small intestine from glucagon treated rats: implications for the diarrhoea of starvation.” Exp. Physiol. 1992. 77:645-648.

Discussion of the Inherent Nature of the Sugar/Fat Tax

In recent years the question of whether or not society has a duty to the health of her citizens has arisen taking the form of the implementation of a ‘sin’ tax against unhealthy food and drink. The tax can typically take two forms: first, a flat fee assigned to a group of products (all sodas for example regardless of overall sugar content) or a progressive fee assigned to a given product based on sugar or fat content over a certain baseline amount. Most of these types of taxes fall into the former category. Not surprisingly regardless of category manufacturers and suppliers of products which would fall under this tax do not want it and incessantly lobby against implementation of such policy.

The principle argument made by these suppliers is that the populace should reject government implementing policy which creates favoritism among existing choices. Of course the suppliers use more buzz and emotional language like ‘keep the government from restricting your food freedom’ or ‘the government thinks you are too stupid to make sound nutritional decisions’.

The interesting thing about this mindset is that it is wrong. Government already constantly, through the use of various subsidies and taxes in a number of industries, create this ‘distortion of choice’ that food suppliers are so concerned about when talking about a fat tax, but could care less about when talking about oil subsidies. With regards to the ‘too stupid’ attack angle, with over 33% of the U.S. population obese and another 33% classified as overweight and the basic biochemical understanding that a significant portion of weight is determined by one’s energy balance, it can easily be argued that yes most people appear to be lacking in the ability to effectively manage their own nutrition.

Despite these realities food suppliers would counter-argue that unlike smoking and alcohol government still should not be involved with food consumption favoritism because it is an individual’s decision; whether or not an individual is obese does not affect society as second-hand smoke or drunk driving do, instead only the individual in question is affected. This argument is foolish because obesity rates certainly influence society largely via the economy through work productivity rates and healthcare costs. With Medicare and Medicaid participation in a percentage of these healthcare costs clearly it is in the prerogative of the government to act. While one obese individual may not provide any significant influence, numerous obese individuals do and numerous is the scenario that society is currently facing.

The final strategy that suppliers use to combat the fat/sugar tax is to use a form of divide and conquer. These suppliers cite that the supporters of these types of taxes come from one of two camps. The first camp encompasses individuals who genuinely want to make society healthier and have determined that one way to accomplish that goal is to increase the cost of less nutritious options. The second camp encompasses individuals, typically lawmakers (according to the suppliers), who are looking for additional revenue streams. Suppliers seek to create division between these two groups by citing that if people reduce consumption of their products due to the higher costs due to extra taxes, then the revenue stream anticipated by the second group will shrink. Basically suppliers try to make the argument that if one group gets what it wants the other group will not get want it wants.

The inherent logic of this proposal is flawed because there is no significant ‘investment’ cost in creating the tax. For example suppose person A invested in project A for 5 dollars after an analysis concludes that person A should receive 100 dollars from the project. Would person A regret investing if the return was only 35 dollars? Of course not; person A may be disappointed that the return was not what was originally anticipated, but to regret making an investment which results in a 30 dollar profit with little associated opportunity cost is foolish.

This situation is akin to a fat tax, there is little investment cost thus regardless of how much is acquired due to the probability of reduced consumption lowing actual revenues whatever revenue is acquired will still be meaningful. However, this analysis does demand that lawmakers look at this issue through a logical mindset, which may be asking too much based on the behavior of lawmakers in the past.

The supplier analysis conveniently excludes the savings that will develop on Medicare and Medicaid from healthier individuals having to take less medication due to chronic health problems. Also the concern that individuals will simply move from one unhealthy food item to another is only applicable under narrow product specific taxes. For example such behavior could be expected if a soda tax is implemented with students drinking more fruit juice (which is mostly sugar), energy drinks, coffee and water. However, if the tax is applied (either progressively or flatly) in a broad way in the way a sugar/fat tax should be this concern is mitigated.

There are two immediate moral concerns with a sugar/fat tax. One concern is that these types of taxes are inherently unfair to healthy individuals. Individuals who properly manage their weight and health are punished by such a policy if they ever wish to ‘reward’ themselves for completing a workout or some other accomplishment. It is the not the fault of these individuals that other individuals cannot maintain a healthy weight and yet they face the same penalty when purchasing certain foods.

Another concern is how ‘food deserts’ would respond to such policy. Regrettably there are still a number of areas in the United States where food choice is scarce. In most of these regions the majority of available food is of lower quality and nutritional value, foods that would be more likely to fall under these types of fat/sugar taxes. Thus in these areas such a tax would put undue burden on these individuals who do not have a viable choice to select a healthier dining option.

Another real problem with the sugar/fax tax is a lack of public support. The campaign of taxation demonization in general has heavily crippled any public support for taxes which may influence those respondents. In general support for taxes boils down to whether or not that tax will affect the particular individual usually regardless of what benefits are derived from it. Additional taxes on millionaires have drawn large public support because they do not affect most of the public; however, most polling data on sugar/fat taxes have revealed significant resistance to implementation.

Recall that relying on polling data to make any decision is stupid because polls almost never have large enough sample sizes to properly represent the public. Although polling data should not be trusted, it seems reasonable to suggest that general attitudes will demonstrate resistance to sugar/fat taxes generally based on consumption patterns and most people consume the targeted type(s) of products. Therefore, the large-scale application of these taxes is unlikely outside of strong government actors willing to go against ‘popular’ opinion and certain elements in the business community.

The ironic aspect of the sugar/fax tax debate is that unhealthy foods already have an inherent tax associated with their consumption, that being poorer health. However, most individuals do not have the foresight to associate numerous choices characterized with little consequence in the present has having a significant influence on the future until too late. Thus the shortening of lifespan due to obesity typically ‘sneaks’ up on a person more than anything and it is frequently eliminated through cognitive dissidence. While the psychological element of health is muted individually, the inherent near-term financial tax of these foods is masked by the lack of transparency and price separation in the healthcare insurance industry. Basically how insurance companies ‘score’ their customers through the underwriting process creates an environment where it is difficult to understand the significance of compounding food decisions.

For example when one applies for health insurance as an individual, they agree in principle to a given rate which can be changed after an ‘independent’ underwriting of the applying individual. This underwriting is undertaken to determine the financial ‘risks’ associated with insuring that particular individual. After the process, if the applicant is still acceptable for coverage, the applicant is quoted a final price for the policy which he/she can either accept or decline. However, over the course of this process the applicant is not given information to why any change was made (if one was made) in the premium payment rate. Not providing this information leaves the applicant wondering about the rationality of the change, due to weight, cholesterol, smoking, family history, etc.

One step forward from this concern would be if health insurance companies could provide a detailed ‘invoice’ of sorts analyzing what aspects of an individual’s health profile were influential in producing a given premium price. In addition a benchmark range list could be provided showing price addition/subtraction relative to other values for given health indicators.

For example suppose an applicant’s blood pressure is 150/110, a higher than recommended value. The invoice would state that such a blood pressure cost the applicant 45 dollars a month towards the overall premium. However, supplemental material in the invoice would indicate that if blood pressure were 120/80 instead the associated cost towards the premium from blood pressure would only be 29 dollars a month. Providing information in such a format would allow applicants to see in full detail how aspects of their health influenced the direct financial cost of purchasing health insurance. Note how this issue of transparency is different from the more popularly known push for transparency in the insurance industry where insurance companies need to reveal the rationality behind sudden changes in premiums for large groups of customers.

This system would also need to have significant divergence between different health indicators to be truly effective. For example if a healthy individual paid 1,000 dollars a year in health insurance costs and an obese individual paid 1,200 dollars a year it can be reasoned that the transparency of such a system will do little to drive the obese individual to change his/her eating habits, especially when that 200 dollars is spread over 12 months. Therefore, it is important that health insurance premiums, which have risen significantly in the last few decades, are lowered for healthier individuals.

It can be argued that creating this significant gap between non-obese and obese individuals is appropriate because it represents a risk-associated interest in the insurance industry as obese individuals have a higher probability of needing medical attention. Note that with the high costs associated with health insurance premiums, it is not advisable that health insurance companies raise premium prices for the obese, instead the decrease for non-obese individuals is appropriate to create a sufficient separation and incentive.

The transparency in underwriting method outlined above may draw criticism from the health insurance industry. Some may argue there is too much additional work required to create such ‘personalized’ invoices. Without associated genetic screening, which is not included in this invoice because individuals can do little to positively change their existing genetics, this argument is reaching. The bulk of the invoice is either based in a flat ranged price based on existing parameters that are created by the insurance companies which exist in almost a form letter format or information provided by the applicant that is scored anyways, thus any concern of ‘more work’ is unfounded (in theory). Basically the insurance company simply needs to make a master list and then apply it to all individuals.

Some may argue that the transparency opens up how different prices are offered to different races, ethnicities or sexes. This should not be a concern at all because either a company has scientific evidence to back treating a male with 140/100 blood pressure different from a woman with the same blood pressure or they are being bias and that bias should be brought to light. Finally some may argue that the transparency is unfair because it could expose the company to competition. Such a concern is insulting as an industry which cites capitalism as the reason it should be allowed to make millions of dollars in profit a year would deny one of the principle tenets of capitalism when it does not suit them.

A genuine concern about this method is the reaction of employers. What type of legal ramifications will exist, if any, when employers who still provide health insurance elect to fire certain individuals because of the additional costs those employees are adding to the company? Could people sue for discrimination or would that reason be countered by the fact that weight is a controllable element which is affecting the profit potential of the company in question?

The final element is a continuation in nutrition education must be undertaken so individuals realize what foods can be consumed frequently, what foods consumed on occasion and what foods should be eliminated from the diet. Obviously without the proper education linking certain unhealthy food options to an increased probability of gaining weight and perspective additional health problems such a transparency program in the health insurance industry would be less effective.

Overall while a sugar/fat tax looks to establish a direct means to reduce unhealthy food consumption, such policy has met with resistance from the general public and industry. One alternative option is to create more transparency in providing health insurance. Health insurance is a large financial investment and to see a direct comparison of how premium costs affect individuals of poor health vs. healthy individuals could provide significant incentive for an individual to become healthier. It stands to reason that this strategy may be superior to a sugar/fat tax (for those who have health insurance at least) because of the magnitude of the premium difference in a single setting vs. the ‘nickel and dime’ nature of the tax. A sugar/fat tax could be a useful element in the fight against obesity, but society needs to investigate other means as well because at this moment there is still no ‘silver’ bullet.

How the gang of three, leptin, ghrelin and amylin, influence obesity

Obesity is commonly defined as an excessive amount of body fat relative to lean mass.1 Individuals who are classified as obese typically suffer from additional health problems which either do not afflict or have dramatically lower rates of affliction in those who are not obese. Unfortunately in the United States excess weight gain, which commonly leads to obesity, has reached what some are calling epidemic levels with more than 60% of American adults either overweight or obese.1 In addition to the dramatic rise in adult obesity even more concerning is the even more rapid rise in childhood obesity.1

One of the more practical rationalities explaining this dramatic increase in obesity is a change in energy homeostasis between energy intake versus energy expenditure. Energy intake is almost entirely derived from food consumption where energy expenditure is derived from biological thermogenesis which includes basal metabolism, adaptive thermogenesis and physical activity.2 Based on this information it makes sense to argue that an increase in food consumption joined with a corresponding decrease in physical activity due to modern conveniences creates an imbalance resulting in weight gain and obesity.

This explanation can address most of the new increases in adult obesity, but it seems to breakdown when applied to children and infants. Surprisingly, in addition to children and adults, some research demonstrates that even infants have not escaped this obesity epidemic.3 Despite changing food consumption and exercise patterns in adults and young children to teens, little has changed in the diet of infants, either breast milk from mother or bottle formula. One possible explanation is that obese mothers are influencing these changes in their infants, which does have some backing from empirical evidence,3 but at the moment it appears to be a stretch to presume such an explanation to be the principle reason behind this increase.

This change in obesity rate with no definitive explanation stemming from energy intake has lead others to question if the above theory regarding disruption in energy balance is the most influential element behind the obesity rate increase. Make no mistake that an increase in energy imbalance in favor of intake is a significant component in increased obesity rate, but due to the infant ‘question’ it may not be the only significant component. Two other popular explanations have emerged to explain this gap: toxic compounds in the environment and increased viral infections.

These two additional explanations have generated certain levels of empirical backing which add to their legitimacy. However, these elements will not be addressed in this particular blog post, but in a separate post in the future. This blog post will focus on the biochemistry behind how the increasing energy imbalance induces such a significant level of weight gain when under normal circumstances adult body weight is relatively constant despite large variations in daily food intake and energy expenditure.4 Clearly the reaction of the body to hormones and other agents, which govern appetite and energy expenditure change in obese individuals versus non-obese individuals.

Among the hormones that direct energy homeostasis, leptin is widely viewed as one of the most important, if not the most important. Leptin is secreted by adipocytes at a rate which generates serum concentrations in similar proportion to existing adipose tissue.5-7 The principle role of leptin is to provide a measurement of energy to the central nervous system to determine what types of adjustments are required to properly maintain energy homeostasis.8,9 Under normal conditions higher than normal leptin concentrations result in limited to no appetite whereas lower than normal leptin concentrations result in increased appetite as well as reduced energy expenditure and an initialization of the starvation response.10

Most work involving leptin utilize mice as the model organism all which have six distinct leptin receptors (LR) that have been designated a through f and are sub-divided into three classes: short, long and secreted.10 The secreted receptor (LRe) is created via products of alternatively spliced mRNA or proteolytic cleavage of membrane bound LRs.10,11 Secreted forms only contain extracellular domains which bind circulating leptin acting almost like a leptin inhibitor of sorts.10,11 Short receptor (LRa, LRc, LRd and LRf) function is not fully clear, but they are thought to aid leptin transport across the blood brain barrier.11,12 Finally long receptor (LRb) is crucial for leptin action as it is the principle binding receptor, which initiates leptin influence on a given neuron.10

While leptin does act in various portions of the brain, the principle region of action is in the hypothalamus, acting on thyroid and growth hormones along with sex steroids13,14 largely due to high LRb mRNA expression and associated receptor expression.13,15-16 On a side note in addition to appetite, leptin also regulates glucose homeostasis independently of adiposity and regulates glycemia.17-20

The chief area of action for leptin in the hypothalamus is the arcuate nucleus (ARC). In the ARC LRb acts on two different types of neurons: first neuropeptide Y (NPY) and agouti-related peptide (AgRP) synthesizers and second pro-opiomelanocortin (POMC) synthesizers.13,16 For signaling POMC is processed to a-melanocyte-stimulating hormone (aMSH) which binds to melanocortin-3 or 4 receptors (MC3R or MC4R) inducing appetite suppression.21-24 NPY is an orexigenic (promotes food consumption) hormone which incorporates a negative feedback mechanism suppressing growth and reproductive elements as a means to reduce energy expenditure25-27 whereas AgRP is an inverse agonist for MC4R, which also decreases cAMP production.28 Basically both NPY and AgRP act to increase appetite and reduce energy expenditure in times when the body believes a low energy state exists. Leptin binding to LRb inhibits NPY and AgRP secretion16,29 and promotes aMSH secretion in a dose-dependent fashion30 due to promotion of POMC. So overall leptin actively influences appetite by inhibiting appetite promoting NPY and driving POMC synthesis.

When individuals become obese leptin levels do not decrease. In obese individuals leptin levels are actually still represented in somewhat proper proportion to corresponding adipose tissue.7,31-32 The inability of higher leptin concentrations to reduce weight or appetite has lead to the conclusion that obese patients develop some form of leptin resistance.

A vast majority of the studies associated with leptin resistance have taken place in mice, but is also believed to properly translate to humans as well. For the purpose of experimentation mice are commonly divided into a control group and a secondary group that becomes obese due to increased food consumption. This secondary group is commonly referred to as diet-induced obesity (DIO) mice because the obesity is developed through food consumption not genetic knockout or mutation.

In DIO mice the development of leptin resistance has been hypothesized to develop over three stages: first, mice gain weight increasing the amount of adipose tissue and corresponding levels of leptin, but appear to maintain a normal response to leptin. Second, associated leptin interacting peripheral neuronal areas suffer from diminished capacity to respond to leptin. Third, associated leptin interacting central neuronal areas suffer from diminished capacity to respond to leptin.33-35

Evidence for leptin resistance has built considerably over the years. For example between non-obese and DIO mice there are no changes relative to NPY, AgRP and POMC mRNA expression with no leptin exposure.29,36 After leptin is injected decreases NPY and AgRP mRNA expression occur in control mice, but did not change significantly in DIO mice.29 Also DIO mice appear to have less activation of MC4R compared to control mice,29 which makes little sense when considering that DIO mice have higher concentrations of leptin. Some believe that the lack of MC4R activation coupled with no additional reduction in AgRP expression drives leptin resistance downstreatm in the melanocortin system.29

With this information it makes sense to conclude that the leptin resistance is being driven by the leptin concentration itself, not by another element. Studies have appeared to rule out reduced LRb expression as a reason for resistance because LRb mRNA expression typically does not demonstrate a significant difference between control and DIO mice.29,37 While there are many other hypotheses regarding the mechanism of action, the two that receive the most study are the failure of extracellular leptin to reach its appropriate receptors in the brain and the failure of the leptin activated LRb signaling mechanism.38-41

After leptin binds to LRb a number of secondary steps occur. LRb binding initiates the activation of intracellular Jak2 tyrosine kinase. Activation of Jak2 triggers autophosphorylation of the four tyrosine residues on Jak2 as well as phosphorylation of the three tyrosine residues on the intracellular tail of LRb: 985, 1077 and 1138.10 With respect to positive action LRb residue 1138 is the most important as its phosphorylation recruits signal transducer and activator of transcription (STAT) 3, for activation which is directly responsible for transcription of POMC.10 Residue 1077 recruits transcriptional activation of STAT5, which is thought to increase energy expenditure.10

However, to ensure that the effect of leptin does not permeate to a dangerous level, leptin has an inherent negative feedback element. While leptin activation of Jak2 leads to phosphorylation on residue 1138 activating STAT3 leading to the transcription of POMC, STAT3 also results in the transcription of inhibitory suppressor of cytokine signaling 3 (SOCS3).10 SOCS3 acts as the principle negative feedback inhibitor of LRb by binding to residue 985 inhibiting STAT3 recruitment and possibly activation.42,43 Tyrosine phosphatase SHP-2, which is recruited by the phosphorylation of residue 985, is also thought to be a secondary inhibitor, but SOCS3 is considered more important (if not simply based on more confirmed function).42-47 The negative inhibitory ability of SOCS-3 is supported by SOCS-3 deficiencies resulting in significant increased leptin action.29,45 A third LRb inhibitor is tyrosine phosphatase PTP1B, but because this element appears to operate independently of leptin interaction it will not be discussed further.

The rationality behind a feedback mechanism seems straightforward. For obese individuals it may be appropriate that the body limit food consumption and supplement itself through the breakdown adipose tissue, but this process does not supply all necessary nutrients for long-term survival. Therefore, the leptin inhibition feedback mechanism seems designed so that the obese do not overeat, but do eat once and a while in attempt to acquire these essential nutrients. Also the feedback mechanism could be related to necessary changes in behavior which demand greater levels of short-term energy largely derived from food consumption. However, the feedback mechanism may overcompensate in its role in obese individuals.

Relating back to the second theory behind leptin resistance focusing on the activation of the signaling mechanism after the initial binding event, leptin resistance may revolve around an inhibitory desensitization event (similar to nicotinic acetylcholine receptors). For this theory an initial leptin binding event starts the positive secondary mechanism and extended binding begins the inhibitory mechanism. Thus, due to anticipated increases in leptin binding rate and absolute binding times as adipose tissue increases due to an individual becoming obese, leptin response rates at higher leptin concentrations will decrease due to desensitization of LRbs.

Higher baseline SOCS-3 mRNA in the ARC in DIO mice29,48 support the idea of desensitization as if leptin was losing affinity for LRb then SOCS-3 levels would not change significantly. However, what is the cause of this increase? One possibility could be suppose phosphorylation of residue 1138 occurs at a faster rate than phosphorylation of residue 985 (this makes sense if under normal circumstances a sustainable positive effect is seen), but the effect or SOCS-3 has a longer residence time than POMC (recall that POMC is cleaved into aMSH); therefore, the longer leptin stays bound to LRb the more inhibition begins to overtake leptin action. Under this scenario, the longer leptin stays bound to LRb the greater the inhibitory effect on residue 1138 reducing STAT3 activity. Overall the higher concentrations of leptin in obese individuals do not allow for a sufficient period of time for the LRb receptor to “deactivate”, thus leptin action is significantly more inhibited.

Another possibility may be that leptin could bind to the short receptors (LRa, c, d and f), possibly present on the neuron, which could trigger an inhibitory mechanism separate from the initial mechanism associated with LRb. The theory here is that the long receptors have a higher expression rate in neurons which allow for more binding opportunities with leptin, but the short receptors have a higher binding affinity for leptin. As long as there is free leptin the high frequency short-term binding to LRb wins out over the low frequency long-term binding to short LRs, thus leptin represses appetite. However, suppose in situations of high leptin concentrations (obese individuals) more LRe is synthesized and secreted (as a secondary mechanism of inhibition) which binds a lot of the free leptin changing the pattern of influence so that long-term binding short LRs start to have more influence than LRb which leads to leptin resistance. Overall this theory seems more unlikely relative to desensitization as increasing leptin concentrations, no significant reduction in LRb expression and increased SOCS-3 expression all support a form of desensitization and this secondary theory relies on some unknown functionality of certain leptin receptors.

One interesting side note on this element is that STAT3 recruitment decreases profoundly in the ARC, but small recruitment decrease is seen in the ventromedial hypothalamus (VMH) or the dorsomedial hypothalamus (DMH).48 One reason for this suggestion is that cytokine signaling-3 (SOCS-3) expression is also localized to the ARC over other regions49,50 as SOCS-3 reduces the effectiveness of leptin action, probably through inhibition in the secondary pathway. Another reason is that leptin concentration in the VMH or the DMH is not high enough to induce the desensitization element associated with leptin resistance and resulting prolonged decreased STAT3 recruitment. Finally a third possibility may be a more prominent associated helper effect of amylin in the VMH over the ARC.

One of the principle reasons for regionalized increase in SOCS-3 (the first explanation above) could simply be a volume difference due to ARC being localized in a region more conducive to transport of leptin across the blood brain barrier.51 This argument is further supported by a delay in the time course for LRb signaling in non-ARC neurons relative to ARC neurons when exposed to leptin peripherally injected, but this delay does not exist when leptin is centrally injected (central injection skips passage through the blood brain barrier).51

Recall that the concentration of leptin is tied to adipose tissue, thus when exploring the nature of obesity and the role of leptin it is appropriate to investigate the role of saturated fat. Experience with cell culture studies have shown that increasing levels of saturated fat induces increased insulin resistance both in signaling and gene expression due to inhibition of the receptor, receptor substrate 1 and 2 tyrosine phosphorylation and activation of insulin antagonists phosphoinositide-3 (PPI3) kinase and Akt.52-54 There are some conflicting studies regarding how dangerous saturated fat is with regard to catalyzing obesity in different individuals.55,56 A reason behind these opposing results is probably drawn from the complexity of gene response with regards to saturated fat.

For instance one study found that high dietary saturated fat intake significantly increased the risk of obesity by 32% in those carrying 2 or more risk alleles vs. those carrying 0 or 1 risk alleles based on the relationship between STAT3 and saturated fat.52 This study determined that a vast majority of individuals are presumed to have 2 risk alleles making them more susceptible to obesity due to saturated fat consumption.52 The interaction between STAT3 and saturated fat is thought to involve saturated fat activation of toll-like receptor-4 (TLR4) and cross-talk between that activation and the JAK-STAT3 pathway.52,57 This interaction may explain why mice feed diets higher in saturated fat appear to have more aggravated cases of leptin resistance. For example some research demonstrates that for diets equally high in total fat the one comprised predominately of poly-unsaturated fat does not significantly change NPY or AgRP mRNA levels in the ARC unlike diets comprised predominately of poly-saturated fat.58 Based on this result leptin levels should be inhibited less for unsaturated fat as opposed to saturated fat.

However, an unexpected side result is that POMC mRNA expression does not change regardless of principle fat component of the diet.58 This result is unexpected because with more leptin resistance there should be some corresponding drop in POMC mRNA expression. One explanation for this result could be that there was a decrease, but the decrease was not observed because the time frame of the experiment was not long enough. Overall ARC-based NPY and AgRP also seem tied to saturated fat content largely stemming from the increases in leptin concentration derived from saturated fat instead of just total fat content.

Another issue regarding leptin resistance involves how it influences overall appetite. For the purpose of this discussion take two individuals one who is not obese and one who is obese. The overall influence of leptin resistance can be viewed in two ways. First, when an individual moves from not obese to obese leptin loses effectiveness, but not in a negative fashion; the overall leptin effect is higher than that seen in the non-obese individual, but the leptin influence ratio decreases. For example a non-obese individual with 100 ug of leptin would eat 900 calories instead of 1000 calories whereas an obese individual with 200 ug of leptin would eat 850 calories instead of 1000 calories. Second, when an individual moves from not obese to obese leptin effectiveness becomes negative both overall and as a ratio. In this scenario an individual with 200 ug of leptin would eat 930 calories. The second scenario seems supported by conclusions that food intake is increased in obese individuals ranging from 14 to 17%58 as well as decreased STAT3 phosphorylation and other LRb signaling.10 Thus if this result accurately depicts the second scenario leptin resistance must be fairly significant, creating a positive feedback loop expanding the probability that an individual remains obese.

One question from this second scenario is that even while food intake increases in obese individuals, in the short-term NPY and AgRP mRNA (and protein) levels drop.58 There does not appear to be any significant information pertaining to whether or not these mRNA levels rebound in the presence of long-term leptin resistance. If the NPY/AgRP values do rebound then it would be significant evidence that leptin was the defining element connecting NPY/AgRP and saturated fat. If not, then there most likely needs to be a secondary element which links NPY/AgRP decrease and saturated fat apart from leptin. If this is the case the most promising element for this secondary linkage is STAT3 and its TLR4 saturated fat link.

Recent information has determined that leptin may not only regulate appetite, but may also interact with dopamine neurons stimulating the reward pathway in the brain.59-62 Leptin is now thought to act in consort with the mesolimbic dopamine system which is made up of dopamine releasing neurons in the ventral tegmental area (VTA) which innervate into the striatum, amygdala and prefrontal cortex.59 In addition to some neurons expressing LRb in the VTA, other studies have identified LRb expressing neurons in the lateral hypothalamic area (LHA) which project to the VTA.10 Therefore, it appears that there are two separate leptin based systems which could influence the dopamine based reward pathway in some context. This effect would especially be prominent if leptin limited the pleasure received from consuming food because then the development of leptin resistance would neutralize this limiting characteristic increasing the probability of greater food consumption in obese individuals.

With regards to leptin resistance and its overall influence on obesity it may have an important partner in amylin. Amylin is a 37 amino acid peptide hormone commonly co-secreted with insulin from pancreatic b-cells,64 which principally binds to receptors in the hindbrain area postrema (AP).65,66 The importance of amylin relative to leptin and obesity is a number of experiments have demonstrated that increasing concentrations of amylin in both non-obese and obese individuals induces weight loss with an increased reduction when combined with leptin.66,67 One lingering question is whether or not this weight loss can be principally attributed to amylin directly or if amylin reduces leptin resistance allowing leptin to be more effective.

One piece of evidence which supports the latter explanation and ties to the issue of leptin resistance is that amylin-augmented weight loss in DIO rats was observed with an increase in POMC expression in the ARC.68,69 This increase in POMC expression implies that weight loss seen in obese individuals due to increased levels of amylin would eventually be realized as reduced food intake due to aMSH binding, an increase probably stemming from leptin derived POMC increases.

While leptin activity in the ARC is important it appears, not surprisingly, that it not the exclusive operating area for leptin influence with regards to appetite control. Selective lesioning of leptin receptors on ARC POMC neurons does not completely replicate the development of obesity which accompanies general leptin deficiency (from knockouts).48 Also leptin resistance demonstrates a decrease in STAT3 signaling throughout the VMH and AP which support the notion of leptin resistance beyond the ARC.70,71 Therefore, the mechanism behind amylin induced reduction of leptin resistance could stem from amylin increasing signal effectiveness in the VMH and AP.66

Action in the VMH is supported by an approximate 43% increase in leptin signaling (derived from STAT3 comparisons) in the VMH without any direct increase in the ARC.66,72 An increase in STAT3 concentration coincides with other studies.67 The important targets for leptin in the VMH appear to be glucose-sensitive neurons and transcription factor SF-1.73-76 Transcription factor SF-1 could be important because it induces DIO in mice that lack it.77 One reason explaining this result is that co-localization studies identify SF-1 VMH neurons linked to leptin-induced activation of STAT3.66

No changes in leptin response were seen in the caudal nucleus of the solitary tract (NTS) with direct amylin treatment.66 Nor did amylin influence leptin, LRb or SOCS-3 concentrations via gene expression changes.66 Once again the lack of amylin influence on leptin, LRb, SOCS-3 could be a timecourse issue due to measurements not being long enough or taken at the appropriate time, but overall this is unlikely. Moreover it is unlikely that the AP is a site of cooperative action between leptin and amylin due to the low level of overall expression of leptin receptors and low leptin-derived STAT3 expression.66,78 Also amylin receptors are limited in the ARC and amylin administration does not activate c-Fos in the hypothalamus.79,80 Finally amylin functions through calcitonin receptor binding using receptor activity-modulating protein (RAMP) via a second messenger cGMP81,82 not through Jak kinases lke leptin. Therefore, if the principle area of action for amylin is the AP its action must propogate from the AP to an area more conducive for leptin action like the VMH.

Based on all of the results amylin influence seems to be localized to its respective receptors in the AP and through neuronal connections (probably polysynaptic) act upstream on the VMH enhancing leptin response through the NTS and the lateral parabrachial nucleus (lPBN).66,79,84,85 This conclusion is supported by amylin binding in the AP activating the NTS, IPBN and central nucleus in the amygdala to inhibit fasting derived activation of the lateral hypothalamic (LH) area65 where LH afferents are thought to influence VMH signaling.86,87 Also lesions on the lPBN blocked amylin-induced c-Fos expression.83 There is also some evidence to suggest that leptin binding is also increased in the DMN.66 In addition leptin may have a feedback effect on amylin in that amylin effectiveness is reduced in LRb knockouts.88

So how does amylin binding aid leptin? Amylin knockout mice exhibited overall reduced leptin signaling in response to exogenous leptin, largely patterned with a reduced residence time of action.66 This reduction in operational time (initially the leptin was able to function and later become less effective) is thought to involve a reduction in LRb expression.66 A smaller amount of LRb would result in faster desensitization of available receptors and loss of leptin activity.

The reverse of this thought process makes sense to why amylin may ‘rescue’ leptin action in obese individuals. Recall that STAT3 expression does not appear significantly reduced in non-ARC areas of leptin action like the VMH (this is probably the reason that most obese individuals do not incessantly eat). Increased amylin concentrations could increase the amount of LRb expression creating more leptin binding targets. More leptin binding targets would reduce the overall binding time a given LRb had with leptin generating more ‘deactivation’ time which could reduce the rate of desensitization. This theory is further supported by other data demonstrating a 70% increase in ARC leptin binding with amylin/leptin sustained infusion.67 However, there is little information regarding how long-term the weight loss produced by amylin augmentation may be. Overall it is difficult to conclude that amylin by itself can result in enough weight loss to reduce leptin concentration enough to eliminate leptin resistance in combination with greater LRb expression.

Overall leptin and amylin appear to have cooperative effect in managing energy intake and expenditure in humans. While leptin appears to have a greater overall influence, especially in non-obese individuals due to a lack of resistance, both molecules are able to reduce weight in non-obese and obese individuals individually, but their effects are significantly augmented when both are present. Like leptin, amlyin is also thought to increase energy expenditure.69,89,90 The nature of leptin resistance and the general decrease in weight in response to increased amylin levels due to injection or other means of administration leads to two conclusions. First, it seems reasonable to suggest that amylin concentrations in obese individuals could also be negatively affected either in a manner of overall availability or its overall effectiveness (similar to leptin). Second, amylin as an outside therapeutic target (endogenous administration) does not appear to have the necessary influence for use as a stand-alone treatment for obesity because while weight is lost, the overall obesity does not appear reversed.

Ghrelin is another element which may play a meaningful role in energy regulation affecting the probability of becoming obese. The principle role of ghrelin is to stimulate growth hormone release91-93 it is also thought to play a role in regulation of body weight.94,95 Its role in regulation comes from the response of increasing ghrelin concentrations stimulating appetite with a corresponding decrease in levels after eating, more than likely induced by insulin and leptin.96-99 Ghrelin exerts its effect by binding to NPY/AgRP neurons. The reason behind this appetite stimulation aspect of ghrelin could stem from its relationship with growth hormones as more available energy is typically advantageous to augmenting the effectiveness of growth hormones.

A vast majority of synthesized ghrelin is produced in the stomach so to act in the brain it needs to cross the blood brain barrier, similar to leptin. Studies have shown that due to the non-saturated, saturated transport structure used by ghrelin normal individuals typically have an inverse relationship between weight and ghrelin transport.100 Unlike what is believed for leptin, not surprising given the general opposition of their roles, triglycerides promote ghrelin transport across the blood brain barrier rather than inhibit.100 The ghrelin promotion effect makes sense if one believes that triglycerides are a signal to the brain for starvation.101 However, the loss of transport in obese mice is not accounted for by triglycerides leading others to conclude that there must be a second secreted molecule which drives this transport inefficiency. One possibility may be obestatin.

From a synthesis standpoint leptin inhibition of ghrelin seems to make sense both in the opposing effects on appetite as well as obese individuals having lower baseline concentrations of ghrelin than non-obese individuals.102,103 However, the interesting element to this lower baseline is that it does not appear to be uniform (steady-state). Instead the relationship between ghrelin concentrations in obese and non-obese individuals differed based on time of day with daylight levels being somewhat similar with obese individuals having much lower concentrations at night.94

Currently the reason behind the significant change in ghrelin concentration at night is unknown, but a consequence of this change may relate to obesity from a standpoint of energy expenditure more than energy intake. Despite significant increases in ghrelin concentration during the night in healthy individuals, there is little food consumption or need for food consumption to correspond to this increase; therefore, these concentration increases probably have more to do with growth hormone regulation than energy intake. Growth hormone activation typically results in greater energy expenditure. However, in obese individuals if nocturnal ghrelin concentrations are not increasing then there is less growth hormone activity which can result in stunted growth, less efficient cell repair and less overall energy consumption. Thus this change in ghrelin concentration could be another positive feedback associated with obesity in that the more obese an individual is the less energy he/she expends during the night through the use of growth hormones, thus increasing the probability of maintaining that obesity.

One final issue is that there is some question whether or not leptin transport across the blood-brain barrier is obstructed in obese individuals compared to non-obese individuals. If leptin transport activity is negatively influenced it could offer an alternative explanation to why greater concentrations of leptin may induce less leptin activity. Unfortunately there are two immediate problems with this theory. First, the methodology explaining how obesity negatively influences leptin transport is unknown and little viable explanations exist to why obesity would negatively affect transport. The best explanation could be that triglycerides, which typically increase in concentration as an individual becomes more obese, reduce leptin transport, but the demanded volume of reduction seems too high from an intuitive standpoint. Second, decreases in leptin transport are counter to the demonstrated increases in SOCS-3 concentrations in DIO mice. Therefore, even if leptin transport across the blood-brain barrier is reduced in obese individuals, which is still questionable,71,104-106 it is difficult to suggest that this reduction is significant enough to act as a meaningful explanation for ‘leptin resistance’.

Overall the process of moving from a non-obese individual to an obese individual from an energy balance perspective appears to lean heavily on hormone leptin. Leptin functions not only to influence appetite, but also reward pathways inducing a secondary biochemical means to affect food consumption and a possible interaction with saturated fat which may change leptin secretion patterns. The biggest energy balance element driving maintenance of obesity is the development of leptin resistance which probably follows a desensitization pathway with leptin saturation relative to LRb in the ARC. In addition to leptin, amylin and ghrelin also play important roles in appetite regulation and energy expenditure.

At the moment there does not appear to be a ‘silver’ bullet treatment for obesity on a biochemical level. However, there are opportunities for co-therapies to be successful. For example as previously mentioned it does not appear that increasing amylin concentration will be sufficient to overcome leptin resistance in obese individuals; however, co-therapy using an amylin mimic and an aMSH agnoist could work together to overcome a significant potion of the leptin resistance. Further study of obestatin could lead to a better understanding of its role, if any, in ghrelin function and whether or not it would be useful in treatment. Another option may be cholecystokinin which has shown to indirectly increase STAT3 concentration in low-dose leptin environments.107 Finally there is the lingering option of addressing SOCS-3 concentration changes through inhibition. Therefore, while addressing obesity appears to be a difficult mountain to climb, by better understanding the biochemical realities and changes between obese individuals and non-obese individuals both new drug therapies and food consumption strategies can be developed to combat obesity in a safe, cost-effective and appropriate manner.

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