Conjugated Linoleic Acid

CLA is a naturally occurring collection of fatty acids, of where the entire group are referred to as 'CLA'. CLA stands for Conjugated Linoleic Acid, and are quite literally linoleic acid (omega-6) fatty acids that are conjugated with a double bond at some point on the fatty acid.^[1] They tend to be found in food products (including meats) from ruminant animals, and include:

• Meat, where CLA can be stored in animals that humans eat and the humans that eat the animals^[2][3][4]
• Milk^[5]
• Cheeses, in high amounts (1.5% or so) in Pecorino Cheese from sheep^[6]
• Butter, although enrichment alters the precise CLA levels^[7]
• White Button Mushrooms (Agaricus bisporus)^[8]

Estimated human intake of CLA is around 0.5-1g daily at best,^[9] with other estimates lower at 350-430mg (Germany), 151-210mg (USA) and matching the older estimate of 0.5-1g daily in Australia.^[10] Studies using low (0.5-1g) doses of CLA can potentially have their benefits replicated through foods, but higher doses would require supplementation.

When consuming food via natural sources, the c9t11 isomer (to be discussed) predominates at 75-80% of total CLA by weight.^[10] Since CLA was first isolated in ruminant animals, the c9t11 isomer is sometimes also referred to as rumenic acid. Dairy in general (milk, butter, cheeses) tend to have a range of CLA between 0.25-1.5% of total fatty acids as CLA, excluding possible CLA secondary to vaccenic acid.^[11][12]

Meat, Dairy, and white button mushrooms?

Conjugated Linoleic Acid (CLA) is a term used to refer to any linoleic acid (omega-6) fatty acid with conjugated bonds.^[13] They are found in high amounts in animal products, and can be synthesized in vivo in mammals via the delta-9-desaturase enzyme^[1] and can be synthesized from the trans fatty acid, vaccenic acid, after humans consume the trans-vaccenic acid.^[14]

Of these isomers two are more heavily researched and reported to have significantly unique effects in the body; the trans-10,cis-12 isomer (t-10,c-12) and the cis9,trans-11 isomer (c-9,t-11). These two are used mostly because of the heavier amounts of research into them, which provides more evidence for their safety.^[15]

This CLA isomer, when fed to humans via 3.4g of mixed CLA isomers for 4 weeks, has been associated with changes in 93 genes by more than 1.5-fold differences (relative to control), of which 44 genes have crossover with t10c12 and 20 genes are uniquely affected when only both isomers are considered.^[16]

When consuming food via natural sources, the c9t11 isomer (to be discussed) predominates at 75-80% of total CLA by weight.^[10] Since CLA was first isolated in ruminant animals, the c9t11 isomer is sometimes also referred to as rumenic acid. It has been shown in vitro to promote neuronal stem cell differentiation while the t10c12 isomer hindered neuron differentiation at doses ranging from 2-20uM.^[17]

This particular isomer is associated with some neuronal benefits and could potentially be neuroprotective (understudied) and is associated with increased insulin sensitivity and glucose control; it is not associated with increased lean mass, decreased fat mass, or inflammation like the other isomer is.

c9t11 is the 'natural' CLA isomer due to it being in high levels in food source relative to others, and although it may be 'healthy' it is not significantly associated with fat burning effects

This CLA isomer, when fed to humans via 3.4g of mixed CLA isomers for 4 weeks, has been associated with changes in 265 genes by more than 1.5-fold differences (relative to control), of which 44 genes have crossover with t10c12 and 20 genes are uniquely affected when only both isomers are considered.^[16] It appears to be more biologically active than the c9t11 isomer in general.

Oral supplementation of t10c12 has been demonstrated to acutely induce insulin resistance in obese men,^[18] and is theorized to do this via increasing lipid peroxidation (a form of oxidative stress) as measured by urinary isoprostanes.^[19] The latter study compared a 3.4g CLA mixture (equal parts c9t11 and t10c12) against t10c12, and found four-fold higher urinary isoprostanes with t10c12 relative to CLA (0.25+/-0.07 increase relative to baseline in CLA, 1.04±0.7 increase in t10c12) which was correlated with t10c12's greater suppression of insulin sensitivity independent of all other variables.^[19] t10c12's ability to induce lipid peroxidation, as assessed by urinary 8-iso-PGF2α, is much greater than that of c9t11; increases of up to 578% of baseline levels have been seen with isolated t10c12,^[20] yet increases of 25% noted with similar doses of c9t11.^[21] As increased 8-iso-PGF2α urinary levels may be a diagnostic problem (See Lipid Peroxidation section), these results also imply that t10c12, in vivo, is more heavily involved in peroxisomal oxidation.

t10c12 is seen as the more bioactive isomer as it pertains to fat loss; being inversely correlated with body weight in diabetics^[22] and showing preferential disposition into adipose tissue (fat mass) over skeletal muscle.^[23] Studies in animal models suggest that the t10c12 isomer causes many of the effects on adipocytes such as increased LPL expression and triglyceride release,^[24][25] and increased UCP2 expression.^[26] When investigated in rats, 0.4% of t10c12 in the diet for 8 weeks (when compared to c9t11) was the causative isomer behind a previous notion that CLA can increase insulin sensitivity by proliferating fat cells and reducing individual adipocyte size (with no overall change in fat mass)^[27] whereas c9t11 was no different than control in reducing adipocyte size.^[28] Reductions in blood pressure seen with CLA, thought to be secondary to effects in fat cells, are also exclusive to t10c12.^[24]

t10c12 appears to either be more potent or outright causative of changes in fat mass and insulin sensitivity/resistance seen with CLA, and the increase in urinary 8-iso-PGF2α levels is also greatly attributed to t10c12

Interestingly, t10c12 appears to also benefit muscle mass in rats when compared to c9t11 when both are 0.5% of the diet in mice although all groups (both isomers, and a mixture) were better than control.^[29] The mixture (both isomers at 0.25%) was the best, suggesting synergism between the two isomers in this regard.^[29] These effects may have been secondary to t10c12's better benefits to anti-oxidant enzymes in the muscle cell. Other studies implicate t10c12 as the active isomer in increasing endurance running capacity in mice due to partitioning energy usage to fatty acids rather than glucose, causing an indirect preservation of glycogen.^[30]

A similar study as the above (0.5% t10c12 against c9t11 and a CLA mix group) found t10c12 to be more effective at preventing osteoporosis in mice, possibly secondary to effects on adipocytes; the effects noted with t10c12 were significantly better than c9t11, but not significantly better than the mixture.^[31]

t10c12 also appears to upregulate the LDL receptor in liver cells (receptor that takes up low-density lipoprotein from the body) whereas c9t11 had no effect in vitro.^[32]

Other effects of CLA, benefits to bone health and muscle metabolism, may also be more effectively accredited to t10c12 rather than c9t11

Other isomers that exist are 9trans, 11trans CLA (9t11t),^[10] as well as c11t13 and 8t10c.^[15]

A unique CLA isomers known as 9-hydroxy10-trans-12-cis CLA (9-HODE) from Valeriana fauriei and adlay seeds alongside another hydroxylated CLA known as 13-HODE, both of which were able to inhibit fat accumulation in adipocytes with EC₅₀ values ranging from 0.17-0.40ug/mL and IC₅₀ values of 0.29-0.41ug/mL; effects about 8-fold more potent than the basic CLA isomer t10c12.^[33] 9-HODE and related hydroxylated CLA molecules have previously been shown to be a ligand of PPARy (similar to CLA)^[34] with a potency similar to 10uM troglitazone (pharmaceutical) when at 20uM, and the stress-inducible GPCR G2A.^[35][36]

An oxygenated metabolite of CLA found in tomato products, 13-oxo-CLA (c9t11 isomer), was found to be almost twice as potent as the c9t11 CLA isomer at 20uM, and slightly more potent than 9-oxo-CLA (an isomer); additionally, 13-oxo-CLA was able to improve metabolism of obese mice when fed at 0.02% of the diet.^[37]

Some interesting configurations of linoleic acid here for future research, no practical application of these at this moment in time; however

Both isomers have been found to increase anti-oxidant enzyme expression in vitro via modulation of NF-kB expression^[38] and the combination, when put into macrophages, appears to be suppressive of NF-kB activation (an anti-inflammatory effect).^[39]

In a study on 22 healthy Japanese persons with a slightly overweight BMI of 20+/-0.4, 2.2g of CLA daily for 3 weeks (47.3% c9t11, 50.7% t10c12) was able to significantly increase CLA content of red blood cells and plasma about four-fold relative to 2.2g linoleic acid.^[13] With RBCs having 0.06% total fatty acids as CLA in control, and at 4 weeks the test group between 0.31-0.5%; plasma increased from 0.12% to between 0.26-0.92% of total fatty acids.^[13] An increase of CLA content of lipoproteins (HDL, LDL, vLDL) was also observed after 4 weeks, despite these lipoproteins not significantly changing in their concentrations in the blood.^[13] A similarly dosed study^[40] found the same trends, but slightly lower changes in the blood of Irishmen.

Seems to take more than 1 week to build up body stores, and stays in the body at least one week after cessation of supplementation; begins to be cleared out 2 weeks after cessation.

2.2g of CLA, with about 1g of each main isomer (c9t12, t10c12) was able to elevate blood triglyceride values from 65.6+/-8.7mg/dL to 79.9+/-7.6mg/dL (121% of baseline value) after 3 weeks of supplementation, with the effects lasting 2 weeks after cessation, in young healthy humans.^[13]

2.2g of CLA, with about 1g of each main isomer (c9t12, t10c12), in young healthy Japanese persons was unable to significantly influence HDL, LDL, or vLDL concentrations after 3 weeks of supplementation.^[13]

The c9t11 isomer has been demonstrated to be anti-diabetic, being able to reduce the occurrence of diet-induced obesity in animal models.^[41][42] Possibly secondary to increased insulin sensitivity, c9t11 is also associated with improvements in lipid biomarkers.^[43][44]

The t10c12 isomer is known to be pro-diabetic, causing inflammation in a fat cell which is linked to the fat loss effects of CLA (by hindering glucose and fatty acid uptake into fat cells) as well as effects on insulin sensitivity (by preventing glucose entry into a fat cell, it circulates for greater time). In vitro, the link between inflammation in a fat cell and diabetic effects is appears to ultimately be due to cytokines (inflammatory signals)^[45][46] and initially dependent on calcium release in fat cells.^[47] t10c12 is also known to be an inhibitor of PPARy,^[48] which although is an anti-obesity mechanisms (by preventing differentiation of fat cells) is also pro-diabetic by reducing glucose uptake into fat cells.^[49]

The difference is demonstrated when feeding each isomer to rats, where c9t11 at 0.5% prevents diet induced insulin resistance while t10c12 at 0.5% increased insulin resistance while also increasing lean mass and decreasing fat mass^[41] while a mixture somewhat lessens the impact of each isolated isomer.^[42]

Both isomers appear to have different effects on insulin sensitivity, with c9t11 being insulin sensitizing and t10c12 being able to induce insulin resistance in fat cells; the fat loss effects of CLA, however, appear to be dependent on this

Insulin sensitivity is a relation of how effective insulin is in reducing blood sugar levels or in activating cells to induce 'insulin-like effects', with a more insulin sensitive person requiring less insulin units to do X amount of work and a more resistant person requiring more units of insulin to do the same amount of work.

A few human interventions have been conducted on insulin sensitivity in response to CLA supplementation, and two have demonstrated how hetereogeneous the results can be. The first study of 10 sedentary and lean men given 3.2g of a 50:50 isomer mix,^[50] found 2 subjects with an increase in insulin sensitivity and 6 with a decrease, with 2 not having any significant influence. Another study, which had an average 29% decrease in insulin sensitivity (as assessed by the insulin sensitivity index; mathematical model) found that out of 9 subjects, three noted an increase in insulin sensitivity (ranging from 9-13% increases) and the other 6 noted decreases (ranging from 9-79%).^[51] Both of these studies were done by the same research group, and the authors hypothesized that age may play a role (older at more risk) and that genetic predisposition to diabetes may play a role.^[51]

The typical results are more mixed, with some leaning towards lowering insulin sensitivity^[21][52][51] but most research suggesting any effects on insulin sensitivity or resistance are not statistically reliable.^{[53][54][55][56][7][57][58]}

At the outset, it appears more of the data is leaning towards CLA being somewhat inert or irrelevant when concerning insulin sensitivity and resistance. However, some data do suggest it can cause insulin resistance; when insulin resistance is found, the clinical significance of which is unreliable but potentially of concern

In the studies noting increased insulin resistance, doses of CLA used were either 3-3.2g CLA of mixed isomers^[52][51] or the same dose of the lesser potent c9t11;^[21] Two studies were conducted in obese persons^[52][21] and one in overweight,^[51] but the state of overweight (as well as the state of type II diabetic or those with metabolic syndrome) doesn't appear to be relevant as at least four studies in obese or overweight persons have shown no effects on insulin resistance at the same oral dose of CLA,^{[54][53][56][58]} and two studies noting insulin resistance increases have been done in diabetics^[52] and non-diabetics,^[51] which is matched with studies showing null results in type II diabetics^[58] and non-diabetics.^[53][7] As insinuated prior, the oral dose does not appear to be related as the majority of studies mentioned in this section use doses of 2.5-3.2g active CLA isomers. Insulin resistance was calculated by HOMA,^[52] hyperinsulinemic euglycemic clamp,^[21] and by mathematical modelling of kinetics of glucose and insulin;^[51] as studies assessing insulin sensitivity and finding null results also use a variety of analytical methods, it is unlikely that the cause for discrepancy in the data is due to research error.

The discrepancy appears to come from whether or not insulin resistance was assessed by a glucose challenge, or the insulin sensitivity in the face of experimentally induced high blood glucose to replicate a meal. All three studies noting increases in insulin resistance used glucose challenges^[52][21][51] and another study noting variability but no change used a glucose challenge.^[50] In these studies, changes in insulin resistance were 14.4%,^[21] 19%,^[52] and 29%.^[51] The latter study demonstrated a range of 9-79%, however, showing large variability.^[51] Other studies noting no significant influence used fasting blood glucose and insulin readings, which are indicative of a chronic change in glucose metabolism.

The state of the body prior to CLA usage does not seem to be well correlated with how CLA influences insulin sensitivity, but it appears CLA inducing insulin resistance relies greatly on co-ingested (or co-injected) carbohydrate. It is possible CLA induces short term changes in insulin resistance, that are reversible upon cessation of supplementation and only noticeable during carbohydrate consumption

Still no current sufficient explanation as to the variance seen in persons who get a glucose spike

2.2g CLA daily for 3 weeks was not able to significantly influence circulating levels of liver enzymes in otherwise healthy Japanese adults.^[13]

In animals, particularly mice, CLA supplementation and particularily the t10c12 isomer lead to hepatic steatosis; otherwise known as fatty liver build-up which tends to precede metabolic abnormalities.^{[59][60][61][62]}

Human studies investigating hepatic steatosis (fatty liver buildup) do not note the same results found in animals, suggesting the difference may be species dependent.^[63] One review study summarizing 64 interventions across four species^[64] concluded that humans were less affected by CLA than were hamsters and rats, but mice hyperrespond to CLA supplementation and are sensitive to hepatic steatosis from CLA.^[64]

The notion that CLA induces fatty liver does not appear to be of concern to humans, and is associated with mice for some reason

In a study comparing the kinetics of the c9t11 isomer against the t10c12 isomer, it appears the t10c12 isomer of CLA has a greater affinity for being stored in triglycerides in adipose (body fat) tissue whereas the c9t11 has relative affinity for skeletal muscle.^[23]

Aside from potency on the soon to be mentioned mechanisms, t10c12 seems to go to body fat more than c9t11

The main mechanism that is touted to CLA isomers is their ability to bind to and activate the Peroxisome Proliferator activated Receptor alpha (PPARa) which is highly expressed in the liver but also kidney and heart, with the c9t11 isomer having the most potency on the receptor, followed by t10c12 and then other isomers.^[65] IC₅₀ values observed were 140+/-90uM for c9t11 and 200+/-30uM for t10c12. c9t11 CLA is about 8-fold more potent than linoleic acid (parent non-conjugated omega-6) at inducing PPARa activity.^[65] The biological effects of PPARa activation have been seen after oral administration in rats^[66] and is hypothesized to increase fat burning in the liver.

Additionally, CLA has been demonstrated in vitro and in vivo in humans^[67] to inhibit PPARy, the PPAR isomer that is found in fat cells and moderates fat cell proliferation and accumulation of triglycerides (a PPAR that, although obesogenic, may also protect from diabetes) and this inhibition is attributed to the t10c12 isomer of CLA.^[48] Interestingly, while t10c12 was demonstrated to inhibit PPARy c9t11 was found to activate PPARy in human fat cells in vitro.^[48][68][69] Interestingly, genetic variations in PPARy are associated with variations in genetic response to CLA supplementation in humans and may be a research avenue for explaning inter-individual differences.^[16]

When looking at the third main type of PPAR receptor (PPARb/d), the metabolite of CLA known as furan-CLA appears to be a weak agonist.^[66] No evidence has established this as biologically relevant.

CLA mixtures are known as PPAR modulators, being able to activate PPARa (located mostly in the liver, related to lipid reducing effects and possible some body fat loss effects) and to both activate and inhibit PPARy in body fat cells (depending on isomer) and to 'moderate' the PPARy receptors and subsequent body fat regulation

CLA is claimed to reduce fat via suppressing expression of lipogenic (fat gaining) enzymes such as fatty acid synthase, acetyl CoA Carboxylase, and inhibiting Lipoprotein Lipase (LPL).^[70][71] These effects have been demonstrated to be a result of PPARy inhibition from the t10c12 isomer.^[48]

Then CLA is further touted to increase energy expenditure via increases in Carnitine Palmitoyltransferase-1 (CMPT-1) and acyl-CoA oxidase,^[72][73] and these have been linked to the t10c12 isomer even when investigating fat burning effects in the liver.^[72][73]

The enzyme Fatty Acid Synthase (FAS) has been a locus of research as CLA appears to interact with it, but studies are mixed with either a decrease of activity in this enzyme via less mRNA (good for fat burning)^{[74][71][75][62]}, no significant effect,^[76][77] or a paradoxical increase in activity.^[76]

Some protein changes in the body are due to the aforementioned PPARa or PPARy modulation, whereas others may be influenced either directly or by other means by CLA supplementation; t10c12 seems to be more relevant in these mechanisms

Regardless of mechanisms, in vitro studies consistently note it has the ability to release glycerol from adipocytes (fat cells), indicative of increased fat release from triglycerides and subsequent fat burning.^{[78][79][80][81][82][83]}

There appears to be significant differences in results between humans and research animals in regards to the effects of CLA.^[64][84] It is routinely noted that animal studies constantly have better results in fat loss than do human studies, which report lacklustre effects of CLA; this may be secondary to animals tending to respond more to PPARa activation; a hypothesized mechanism of CLA.^[85]

Interestingly, mice are a good research animal if purposely seeking out an animal polar opposite of humans. Mice routinely experience significant fat loss in response to CLA in the range of 60-80%^[64][86][87] yet also are the only species to note hepatomegaly (growth of the liver) and fatty acid buildup in the liver (hepatosteatosis) in response to CLA.^[64]

Appears to be species differences, with laboratory animals more responsive to the mechanisms of CLA; thus, extrapolation from animal studies is most likely not valid if looking for clinical significance or potency of CLA

Studies investigating CLA and metabolic rate are mixed. At least one study has noted an increase in metabolic rate when 3.76g of CLA, with a 35% c9t11 and 35% t10c12 content was used via yoghurt for 14 weeks.^[67] Via indirect calorimetry, metabolic rate was found to be increased by 4% although no significant weight loss was recorded over the 14 weeks in obese subjects; diet not controlled.^[67] One other study has noted increases in metabolic rate, but attributed this to a gain in lean mass induced by refeeding (in a study aimed to see whether CLA could suppress weight regain after weight loss, which it failed to do but induced nutrient partitioning to lean mass); an indirect form of increasing the metabolic rate.^[88]

Several studies have concluded no differences in metabolic rate including 4g of CLA for 12 weeks in overweight but healthy persons,^[51] no overall difference in metabolic rate despite altered fat oxidation during sleep 4g CLA daily,^[89] or 12 weeks of CLA at 3.9g daily in exercising and normal weight persons.^[90]

Some studies note an increase in metabolic rate, but either lack practical significance or are otherwise confounded; for the most part, CLA does not appear to increase nor suppress metabolic rate

CLA has been implicated in fat loss in several trials. In Chinese persons at 1.7g CLA (50/50 isomers) daily for 12 weeks by 0.69kg relative to placebo's 0.07kg and no changes in lean body mass,^[63] in overweight and obese (BMI 25-35) with 3.4g of 50/50 isomer CLA for 12 weeks causing fat loss without weight loss (increased lean mass) with doses below 3.4g being ineffective,^[91] at 0.6g CLA thrice a day in obese humans with exercise, able to cause a shift towards fat loss indepedent of weight,^[92]4.5g of supplementation with 3.4g of CLA isomeric mixture (50/50) in 85 persons of mostly obese and metabolically unwell (metabolic syndrome) with a 1.13kg reduction of body weight over 4 weeks,^[93] 0.5+/-2.1% body fat over 6.5-7.5 months in obese children given 3g CLA daily,^[94] a −1.25+/-0.71kg loss over 16 weeks in postmenopausal and obese diabetic women (relative to Safflower Oil as a control, losing 0.11+/-0.55kg) and −0.86 ± 0.59kg over another 16 weeks during study crossover, relative to a 0.90 ± 0.79kg gain in Safflower,^[95] 6 months of 3.2g CLA daily losing 0.6+/-2.5kg, relative to the placebo group (Safflower Oil) gaining 1.1+/-3.2kg,^[89] a loss of 0.6kg fat mass after 3g CLA (Tonalin) via milk for 12 weeks in overweight and obese persons with pre-metabolic syndrome,^[56] a 2.6% greater loss (of whole fat mass) relative to placebo when consuming either CLA mixtures or the t10c12 isomer at 4.2g for 12 weeks,^[96] a 1.0+/-2.2kg fat loss over 6 months with no dietary controls with 3.6g CLA daily,^[53] and a loss of either 1.7 ± 3.0kg fat mass with CLA fatty acids at 3.6g for a year, or 2.4 ± 3.0kg loss with CLA triglycerides for the same time period where placebo gained 0.2kg.^[97] A recent study using microencapsulated CLA noted reductions in weight of −2.68%+/-0.82% within 30 days, although no more reductions appeared to occur up to 90 days (with placebo reaching −1.97%+/-0.60%).^[98]

Overall, 10 studies collected showing statistically significant reductions in fat mass. The most dramatic loss was 1.13kg (2.48lbs) over 4 months, which is not an impressive rate of weight loss (comparing this to ephedrine, Ephedrine can induce twice the fat mass loss in a single month). The ranges of fat loss frequently cross over the zero point (ie. 1.1+/-3.2kg weight loss means that somebody gained 2.1kg while another lost 4.3kg) and CLA's wide range of potency and poor reliability spans all studies. CLA does have the ability to be a fat burner, but even in studies where it exerts clinical signifiance its reliability and potency are poor

Conversely, no effect has been noted over 8 weeks with 2.7g active CLA of either a 50/50 isomer blend or pure c9t11 in obese hyperlipidemic men,^[57] No effect of milk enriched with 1.3g CLA daily either as c9t11 or a mixed isomer blend for 4 weeks,^[99] no (0+/-0.9kg) effect of 20g CLA on overall weight over 9 weeks when compared to an isocaloric amount of oleic acid (main fatty acid in olive oil),^[100] no significant effect when 4.2g of CLA isomers are added to the daily diet via butter in food products,^[7] no significant fat loss over 14 weeks using yoghurt as a medium to deliver 3.76g of CLA (35% c9t11, 35% t10c12) when diet is not controlled,^[67] no effect different than placebo of CLA at 2.4g Tonalin oil (brand name) when paired with chromium at 400mg in exercising women,^[55] no effect in healthy exercising men and womena t 4g for 12 weeks,^[90] 3.2 and 6.4g of CLA daily for 12 weeks in obese persons showing a trend towards (-0.17kg fat mass after 12 weeks, relative to 0.11kg gain in placebo) but was statistically insignificant in reducing fat mass,^[101] a loss of 0.65kg body fat over 6 months after daily ingestion of 3.2g CLA of supplementation that was not statistically significant relative to placebo,^[54] 3.4g CLA daily for 2 years reduced fat mass by 1.7+/-2.4kg in obese healthy persons,^[102] and no significant effect of 1.5 or 3g of either isolated isomer on fat mass over 18 weeks.^[103]

A recent double-blinded, placebo-controlled trial on the fat-loss effects of CLA on exercising women with obesity casts further doubt on the efficacy of CLA for fat loss. 28 obese women received 3.2 g/day CLA or 4 g/day olive oil as a placebo while performing an 8-week aerobic exercise regimen. Although both the CLA and placebo groups showed improvements in oxygen uptake, trunk fat, leg fat, and total body fat, the CLA group did not show statistically different changes from the placebo group.^[104] While it is possible that this study was not of sufficient duration to produce a notable effect in fat loss, if taken at ‘face value', the results indicate that CLA supplementation was no better than a placebo in the study population. This suggests that at best, the effects of CLA take a long time to manifest and may be subtle. At worst, CLA is not effective for increasing fat loss in response to exercise.

More studies (12) have been conducted showing no statistically significant effects of CLA on fat loss than there have showing statistically significant fat losses, no common motifs or leads to separate the studies showing positive results and the studies showing null results

As assessed by human interventions with no regard to animal studies (due to species differences), CLA just does not appear to be a good fat burner relative to many other options out there. CLA does not show dose-response, has questionable influences on parameters of lipid and glucose metabolism, and is unreliable as well as not being overly potent

CLA, at an oral dose of 3.4g daily for a year, has also been shown to not be able to suppress weight regain after weightloss any more than placebo,^[105] and in smaller trials a suppression of appetite is associated with CLA, that does not seem to reduce caloric intake.^[106]

A study investigating the kinetics of the c9t11 isomer against the t10c12 isomer found that the c9t11 isomer appears to have affinity for skeletal muscle, being preferentially stored in the phospholipid bilayer of skeletal muscle; t10c12 had affinity for triglyceride storage in adipose tissue.^[23] This was also seen in another study measuring muscle CLA levels where supplementation of 4g CLA oil daily (38% c9t11) led to an increase from 0.46+/-0.08% to 0.56+/-0.06% total fatty acids^[51] and t10c12 increased from not being a component, to 0.09% at the same oral load.^[51]

c9t11 appears to favor disposition in skeletal muscle tissue while t10c12 gets diverted to adipose to a greater degree

At least one study using 4g of CLA daily (38.8% c9t11, 38% t10c12) for 12 weeks in overweight but otherwise healthy men and women found a decrease in insulin sensitivity as assessed by the glucose insulin index (glucose AUC x insulin AUC) and a mathematical model known as the 'insulin sensitivity index'.^[51] Glucose AUC was increased 39% following an oral glucose tolerance test, and insulin AUC by 20%, and this was attributed to changes in myocyte fatty acid composition, particularily ceramide (which increased from 401.3nmol/g to 660.3nmol/g dry weight).^[51]

Several studies giving CLA to persons have noted changes in lean mass (defined as total weight after subtracting body fat).

Studies that come back positive note that in young obese men, 3g CLA isomers paired with 3g fish oil could increase lean mass by 2.4% over 12 weeks while not affecting young and lean men or older men,^[107] an increase of 0.64kg after 12 weeks in response to 6.4g CLA but not 3.2g CLA isomers in otherwise healthy, obese humans,^[101] an 1.8+/-4.3% average increase in lean mass from CLA at 3.4g mixed isomers for 1 year,^[97] and was able to beneficially influence lean mass during a period of weight regain (after weight loss was induced by very low calorie diets), as the weight regained was 12-13.7% lean mass with 1.6-3.2g CLA (relative to 8.6-9.1% increase in placebo) over 13 weeks.^[88]

Studies that come back negative note no changes in lean mass in response to 1.7g CLA daily for 3 months in overweight and obese persons,^[63] 8 weeks usage of either an isomer mix of CLA at 3.5g daily or 3.5g pure c9t11 isomer in overweight men with high blood lipids,^[57] no gain in lean mass after 16 weeks of 6.4g mixed CLA isomers in post-menopausal diabetic women,^[95] no gain of lean mass in young and lean men, or older men, despite a gain in lean mass seen in obese youth after 3g CLA with 3g fish oil daily,^[107] no effect of CLA at 3.9g on lean mass in non-obese persons over 12 weeks,^[90] no effects from 14 weeks of CLA supplementation at 3.76g via yoghurt in healthy persons,^[67] 24 months of CLA supplementation at 3.4g mixed isomers daily in overweight humans,^[102] and no effect of CLA on lean mass with varying doses from 1.7-6.8g daily of mixed isomers for 12 weeks.^[108]

Some studies do note a discord, with either fat loss occurring without lean mass accrual^{[63][95][102][108]} or lean mass accrual occurring without fat mass loss.^[107][101] It is plausible that lean mass and fat mass are regulated by CLA in vivo for humans by different mechanisms.

In studies that investigated fat mass or weight loss, lean mass (total weight after subtracting fat mass) appears to increase in some but not the majority of studies. Not enough evidence to suggest that this effect is potent or reliable (it looks like it isn't), but it appears to be unrelated to the effects of CLA isomers on fat loss

One study investigated a combination of whey protein and creatine monohydrate, at 36g and 9g respectively, with or without an additional 6g CLA. After 5 weeks of resistance training, these novice lifters had greater power and lean mass gains when CLA was combined with whey and creatine.^[109] Whereas Whey + Creatine increased strength as assessed by bench press by 9.7% +/- 17.0% over 5 weeks, the addition of CLA enhanced these increases to 16.2% +/- 11.3%; lean mass increased 1.3% +/-4.1% in the whey protein and creatine group, and by 2.4% +/- 2.8% in the group using CLA.^[109] CLA by itself, at 5g daily for 7 weeks and paired with a resistance training program, is associated with a 1.3kg increase in lean mass while placebo was associated with an 0.2kg gain; and a concurrent fat loss of 0.8kg existed with CLA, while placebo gained 0.4kg; muscular gains were only significant for males tested, and although there was some benefit from CLA on bench press strength, leg press strength was only affected by exercise.^[110]

When tested in non-novice athletes, young (23yr) males with an average of 5.6 years training experience and with the ability to, on average, bench more than their body weight took CLA at 6g daily with 3g other fatty acids (with placebo being 9g olive oil) no significant effects on lean mass or fat mass were observed after 28 days of training.^[111]

Not too many studies investigating CLA in athletic populations rather than obese weight-loss populations, and due to the unreliability seen in the other human studies it is hard to draw conclusions from 3 studies

6g of CLA daily for 3 weeks in resistance trained men who were subject to blood tests before and after each workout did not significantly increase circulating testosterone levels in vivo.^[112] However, when tested in vitro (Leydig cells) CLA appeared to have the ability to increase testosterone synthesis at a concentration of 30uM.^[112]

A white button mushroom extract with a high dose of c9t11 CLA was shown in vitro was shown to be a non-competitive inhibitor of aromatase, with similar potency and mechanisms to linolenic acid (basic omega-6 fatty acid).^[8] White button mushrooms do contain other aromatase inhibitors however,^[113] so the aforementioned study is slightly confounded.

Two human studies have investigated as to whether CLA can affect appetite, and the results are mixed; one study noted a decrease in subjective appetite with 1.8 and 3.6g mixed CLA isomers without affecting caloric intake^[106] while the other noted no influence on feelings of appetite.^[90]

When investigating as to whether endogenous Oleoylethanolamide (an intrinsic appetite suppressant) is affected by dietary CLA, a study in mice comparing 3% CLA in the feed against control (3% linoleic acid) found no differences.^[114]

One in vitro study looking at the effects of c9t11 and t10c12 isomers on NPC differentiation found that, via manipulating the protein content of Cyclin D1, the c9t11 isomer had dose independent benefits to neuronal growth with the best response at 5uM concentration while t10c12 showed dose-dependent inhibition of NPC differentiation.^[17] These mechanisms of promotion are different than those seen with DHA from Fish Oil.^[115]

CLA appears to protect neurons from glutamate-induced excitotoxicity (3uM) in concentrations of 10-30uM (and is able to reduce cell death from 73.6+/-6.5% to 31.7+/-7.2% at 30uM),^[116] and this is seen with both a CLA mixture^[117] yet has been attributed to the c9t11 isomer.^[116] This protective effect has also been noted after glutamate induced toxicity and was then removed with CLA introduced 1-5 hours later, suggesting that co-ingestion may not be a pre-requisite.^[116] CLA does not appear to enhance cell survival on its own.^[116]

The mechanism was hypothesized to be via Bcl-2 induction, which stabilizes the mitochondria and protects the mitochondria from releasing self-destructive cytokines when damaged. CLA was found to not influence the mitochondria on its own, but via Bcl-2 induction preventing mitochondria from becoming damaged from glutamate.^[116]

A study on mice (not the best model for human effects of CLA) fed 3% CLA instead of linoleic acid decreased endogenous levels of 2-AG (2-Arachidonoylglycerol), an endocannabinoid, in the cerebral cortex.^[114] Levels of 2-AG were unaffected in the hypothalamus, and the other endocannabinoid (anandamide) was unaffected in both locations.^[114]

In a population of overweight and obese persons, 76.5% of which had metabolic syndrome, it was found that 3.4g CLA for 28 days was able to benefit blood vessel health in the fasted state as assessed by peripheral arterial tonometry, with fed state being statistically insignificant.^[93] These results are opposite those found in a previous study, using overweight but otherwise healthy persons and using Flow-mediated Dilation (FMD) where 3.4g CLA at for 12 weeks was found to decrease blood flow.^[118] Both studies had decreases in body weight (-1.13+/-1.65kg,^[93] -1.1+/-1.2kg^[118]) thus the effects on blood flow seem independent of the effects on weight loss.

Some studies note that CLA trends towards reductions in blood pressure relative to controls such as Safflower Oil,^[93] but in general do not reach statistical signifiance.^{[119][120][55]} Diastolic blood pressure tends to decrease more than Systolic in many of the above noted studies.

Statistically insignificant trends towards a reduction in blood pressure, or no effect whatsoever

One study assessing CLA and its effects on oxidation noted that free CLA fatty acids, as well as CLA methyl esters, exhibited dose-dependent pro-inflammatory effects in vitro while the triglycerides had no effect.^[121] The mechanism may be through being oxidized (as CLA is a polyunsaturated fatty acid) and then turning into a lipid peroxide, which has been seen in other studies on rats and lambs^[122] where CLA was more prone to oxidative stress than other polyunsaturated fatty acids.

In an in vitro study on low density lipoprotein (LDL), it was found that levels of CLA at 2umol/L exerted a pro-oxidative effect, but lower doses were anti-oxidative; suggesting a dose-response relation.^[123]

Pairing CLA (2% of diet by weight for 21 days in rats) with Vitamin E, the standard anti-oxidative agent for dietary lipids, was able to further reduce levels of malondialdehyde (MDA, biomarker for DNA damage) whereas CLA was already able to do so; it also added to the reduction in catalase noted acutely, suggesting that both molecules additively (not synergistically) reduce oxidation as assessed by MDA and Catalase.^[124] The interaction of Vitamin E and CLA as it pertains to urinary 8-iso-PGF2α (biomarker for lipid peroxidation) is insignificant.^[125]

Interactions with oxidation are complex, and no consistencies are noted in humans at this moment in time

A urinary biomarker, 8-iso-PGF2α, is increased in the urine as a result of lipid peroxidation induced by free radicals in the body and sometimes 8-iso-PGF2α is used as a way to assess lipid peroxidation in vivo. It has been noted to be increased after CLA consumption by 170% after 3 weeks of 7% CLA by dietary intake,^[126] by 25% after 3 months of 3g daily intake,^[21] 83% after 5 weeks of 5.5g CLA via enhanced butter,^[7] and a 48% increase after 16 weeks of 5.5g CLA enriched milk.^[127] There doesn't appear to be studies measuring 8-iso-PGF2α and not noting an increase, so it is seen as quite a reliable change after CLA supplementation.^{[128][129][93]} When isoprostane levels are measured in the blood, they appear to reflect urinary levels.^[118]

These lipid peroxidative effects may be mostly due to the t10c12 molecule, as 3.4g pure t10c12 can cause a 578% increase in urinary 8-iso-PGF2α levels while the same dose of mixed isomers caused a four-fold less increase^[19] and similar doses of pure c9t11 cause a 25% increase.^[21] One study to compare a mixed CLA blend (50:50 ratio) against t10c12 found the blend to increase 8-iso-PGF2α by 171% after 3.5g daily for 6 weeks, and t10c12 by 463% after 3.5g daily.^[125] The first study noting 578% may be an overestimate, as obese subjects tend to have greater increases in 8-iso-PGF2α relative to leaner persons.

Reliable increases in circulating and serum 8-iso-PGF2α as a response to dietary or supplemental CLA ingestion are seen in human interventions, and the t10c12 isomer appears to be more potent than the c9t11 isomer

This increase in lipid peroxidation seen from CLA does not appear to cause endothelial distress per se,^[93] and do not appear to deplete circulating levels of Vitamin E, while returning to normal levels 2 weeks after cessation of CLA supplementation.^[130] Although the increase in urinary 8-iso-PGF2α has correlated to an increase in insulin resistance as assessed by euglycemic clamp.^[19]

When looking at the mechanisms, it is possible that CLA could merely inhibit the degradation of 8-iso-PGF2α into its metabolite 2,3 dinor by competition. Both molecules are preferentially oxidized in peroxisomes, and an influx of CLA is able to suppress formation of 2,3 dinor while causing a backlog of 8-iso-PGF2α in vitro, and these trends are noted in rats as well.^[131] Adding to this notion is how the t10c12 isomer, previously shown to be more effective in raising 8-iso-PGF2α, is more effectively and likely to be oxidized in peroxisomes relative to the c9t11 isomer.^[131][132] As the only currently demonstrated method of lipid peroxidation seen in vivo is due to 8-iso-PGF2α, the notion that everything in this subsection is a false diagnosis (similar to creatinine and creatine) cannot be ruled out.

The possibility that all the above information on pro-oxidative effects is merely due to poor use of a diagnostic marker and not actually indicative of increased lipid peroxidation is plausible

t10c12, the more pluripotent isomer of CLA, appears to be pro-inflammatory. t10c12 can cause an increase in MEK/ERK signalling^[46] with downstream effects on NF-kB, the nuclear transcription factor that mediates activation of cytokines.^[45] t10c12 appears to work, in part, through activating the JNK receptor as inhibiting this action reduces the effects of t10c12 on increasing cytokines such as COX-2 and Interleukins.^[133] Both ERK as well as NF-kB activation from CLA t10c12 are associated with decreased PPARy activation, and the cumulative effect is more inflammation and less glucose and fat uptake into adipocytes; inflammation in fat cells and PPARy activation are quite negatively correlated.^{[134][135][136][137][138]} This reduction of glucose uptake into fat cells is also mechanistically associated with increased insulin resistance, as increasing inflammation (and thus decreasing PPARy activity) is seen as pro-diabetic.

7% of the diet, about 20g daily, has minimal influence on circulating IL-6 levels.^[126]

Inflammatory bowel diseases (in this section, referring to both Crohn's disease and ulcerative colitis) are associated with dysregulation of the immune system^[139] and are thought to be responsive to the diet.^[140] Persons with inflammatory bowel diseases are notorious for having a high rate of supplemental or alternative medicinal usage, with one source noting that the rate was 49.5%.^[141]

CLA supplementation is thought to be protective against inflammatory bowel diseases via PPARγ activation,^[142] similar to the drug 5-aminosalicylic acid;^[143] rosiglitazone has also noted benefit with ulcerative colitis,^[144] suggesting PPARγ is a therapeutic target. CLA has been found to upregalate the PPARγ receptor in some animal models such as bacterial-induced colitis^[145] and to suppress macrophage activity via this receptor;^[146] furthermore, the protective effects of CLA are abolished when the PPARγ receptor is deleted.^[147]

CLA appears to offer relief from symptoms associated with inflammatory bowel diseases including ulcerative colitis and Crohn's disease though increasing PPARγ signalling; this may be due to increasing the expression of the receptor

In persons with mild to moderately active Crohn's disease given 6g of CLA daily (1:1 ratio of the two main isomers; 77% CLA by weight) for 12 weeks, the inflammatory cytokines produced by T-cells (CD4+ and CD8+) were reduced while IL-2 secretion was increased and serum IL-6 was higher after CLA ingestion.^[148] The symptoms were reduced (assessed by CDAI) by 13.1% at week 6 and 23.6% at 12 weeks, and although the authors suspected that this may not be clinically significant the quality of life reported by patients increased;^[148] the overall remission rate (33% of persons reporting a 100 point reduction on CDAI) is comparable to trials using rosiglitazone.^[144][149]

May be benficial to persons with inflammatory bowel diseases, but this requires more trials to ascertain (as the trial in humans at this moment in time was not placebo controlled and patients did not discontinue their medication)

A few human studies have paired CLA with polyunsaturated fatty acids such as Fish Oil. This combination is based on supplements like Fish Oil being able to attenuate the adverse changes seen with CLA in research animals^[87] and has been seen with Flaxseed oil as well.^[150] CLA, specifically the t10c12 isomer, is able to reduce PUFA content in the liver^[151] and is suspected as being one (of possibly many) reasons why mice get fatty liver from CLA, as well as other adverse health effects, since PUFAs normally increase fat oxidation in the liver (via PPARa) and suppress fat accumulation (via SREBP-1c).^[152]

When tested in humans, a pair of 3g CLA and 3g Fish Oil was found to not influence insulin sensitivity over 12 weeks in all but one older man tested.^[153] and another study assessing young lean and obese as well as older lean and obese men (4 groups total)^[107] using 2.28g of a 50/50 CLA isomer mix paired with 1.53g EPA+DHA found that, over 12 weeks and relative to placebo (palm oil and soy oil at 80/20) the combination was able to increase lean mass and decrease fat mass of obese youth (0.88+/-0.5kg increase in lean mass, -83+/-136g fat loss) but did not reach significance in either older group or lean males^[107] and increased adiponectin in both young groups (9% lean, 12% obese) with no affect on older males.^[107] This latter study, however, was not designed to establish synergism between the nutrients.

Some biological plausibility for the combination (theoretically beneficial), but the benefits may be species dependent and no evidence exists to establish synergism in humans

Fucoxanthin, a fat burning pigment from brown seaweed, has been shown to be synergistic with Punicic Acid which is highly similar to CLA in structure. A study in rats using standardized diets and four groups of low (0.083mg/kg) or high (0.167mg/kg) fucoxanthin, and a third group was fed low fucoxanthin with 0.15g/kg CLA daily (fourth group control).^[154] exhibited synergistic effects in reducing circulating triglycerides and body weight in rats yet did not significantly alter much gene expression induced by fucoxanthin (PPARy, UCP2).^[154]

May be synergistic for fat burning, more studies in humans would be needed (due to interspecies differences on CLA)

Resveratrol and CLA have both been shown, in vitro, to reduce triglyceride build-up (during periods of caloric excess) in cultured fat cells, thus their synergism was investigated. Concentrations of 10 and 100uM of both resveratrol and the t10c12 isomer of CLA were used in mature adipocytes, and neither synergism nor additive effects were observed on triglyceride depletion, fatty acid synthase activity, or HSL activity.^[77] Trends towards negation were actually seen (with the combination being lesser than either individual part), but statistically insignificant. Another in vitro study on human fat cells noted that resveratrol (50uM) may actually work in opposition of the t10c12 isomer (50uM), incubation of resveratrol alongside t10c12 in fat cells reduced the ability of t10c12 to prevent glucose and lipid uptake and induce inflammation, increase cell stress, and increase intracellular calcium in the fat cells.^[155] Resveratrol appeared to negate suppression of PPARy by CLA, and induce activity of PPARy when incubated in isolation.^[155]

When the pair is supplemented in the diet of rats, normally responsive to CLA supplementation, 30mg/kg Resveratrol paired with 1% (mixed isomer mix) CLA in the diet for 6 weeks effectively inhibited each other. 20% fat loss was seen with resveratrol and 18% seen with CLA, but the combination led to 7% reduction in fat mass.^[156]

The two appear to be antagonistic, and at least with mechanisms related to PPARy resveratrol appears to negate CLA; other effects of resveratrol related to PDE4 may be unaffected, but many effects of CLA would be effectively negated if PPARy is negated

One year of supplementation with a high dose (7.5g CLA, 6g isomers in a 50:50 ratio) was not associated with any clinically relevant toxicology signs, although a reduction in HDL cholesterol and increase in Triglycerides were seen as statistically significant and an increase in White Blood Cell from 5.5+/-0.3 to 6.6+/-0.3K/ul was noted.^[157]

The two main isomers of CLA, c9t11 and t10c12 have been suspected, in animals, to reduce the fat output of breast milk. 750mg CLA isomers taken for 5 days in breast feeding women was not found to reduce fat content of milk, although trace amounts of CLA can be detected in breast milk.^[158]