Gut Microbiota-Derived Conjugated Linoleic Acid (CLA) in Health and Disease

Abstract

Dietary fatty acids have a major role to play in human health and disease conditions as they are now recognized as the major biologic regulators. Conjugated linoleic acid (CLA) is a generic term denoting a group of isomers of linoleic acid (C18:2, n-6) with a conjugated double bond. CLA is produced as a result of the biohydrogenation of other fatty acids and has attracted increased research interest because of its health-promoting benefits and biological functions. CLA has been shown to impact immune function and has protective effects against cancer, obesity, diabetes, and atherosclerosis that are evident from both preclinical and clinical studies. Studies investigating the mechanisms involved in the biological functions of CLA are emerging with results from both in vivo and in vitro studies. The most commonly used isomers of CLA which have a prominent effect on metabolic and homeostatic regulation are mostly concerned with two of its isomers i.ec9,t11-CLA and t10,c12-CLA. In this study, the role of CLA on various forms of cancers has been given priority along with its mechanism to enable the future research more translational. However, we believe that more intense research is required to further evaluate its efficacious nature and future implication in therapeutics.

1. Introduction

Conjugated linoleic acid (CLA) is a group of naturally occurring fatty acids synthesized from linoleic acid by bacteria present in the alimentary tract of ruminant animals [1]. CLA may also be synthetically produced by partial hydrogenation of linoleic acid or oils rich in linoleic acid (e.g., sunflower or safflower oils) or by alkali isomerisation of the same [2, 3]. CLA is a mixture of positional and geometric isomers of linoleic acid (LA) with double bonds between carbon atoms 7 and 9, 8 and 10, 9 and 11, 10 and 12, or 11 and 13. They occur in cis or trans configuration. The most prevalent form is the cis-9, trans-11 CLA isomer found in food derived from ruminants such as milk, cheese, and meat [4]. Since the discovery of CLA, it has shown many health benefits which include antiadipogenic [5, 6, 7], anticarcinogen [8, 9], antiatherogenic [10], antidiabetogenic, [11], and anti-inflammatory [11] effects. Linoleic acid, known as an element of the geometric isomers of CLA, is a class of fatty acids with 18 carbon atoms. This is the common term for a class of position isomers that has two double bonds with a methylene group between both of them. The double bond in this conjugation is often in areas 9 and 11 or 10 and 12, and it could be in either a cis or trans form (Figure 1). To a lesser extent, pigs, chickens, and turkeys also produce CLA naturally in their digestive tracts. This synthesis is caused by the fermentative bacteria Butyrivibrio fibrisolvens, which isomerizes the linoleic acid in CLA, or by synthesis via 9-desaturase of 11-trans octadecanoic acid. The CLA content of beef fat ranges from 1.7 to 10.8 mg/g, with 9-cis and 11-trans isomers. Dairy products (milk and derivatives) also contain it. The enzyme 9-desaturase, which encourages the desaturation of the 11-trans octadecanoic acid, can be used to obtain CLA. The most well-known CLA isomers are 11-trans and 9-cis as they can be found in food (Figure 2). Additionally, it is feasible to obtain CLA in an industrial form through the partial hydrogenation of linoleic acid or through thermal processing, with the goal of creating a substance with the highest level of biological activity and a specific chemical makeup. Regarding the oxidative degradation intracellular framework, which defines many biological features of fatty acids, CLA plays a significant role in lipid metabolism [12]. With relation to the antioxidant intracellular system, which describes many biological aspects of fatty acids, CLA plays a significant role in the metabolism of lipids. Inhibition of the Peroxisome Proliferator-Activated Receptors (PPARs), particularly PPARγ, mediates the effects of CLA on lipid and glucose metabolism on body composition. CLA isomers inhibit PPAR, leading to inhibiting cell differentiation and modifying the function of proteins that regulate lipogenesis and lipolysis of fat which are two ways that gene expression modification might reduce fat in the body. Evidence suggests that PPAR activation can slow the progression of cholesterol as well as enhance insulin sensitivity, which makes it an appealing treatment option for the management of a variety of illnesses, including diabetes mellitus [13]. The expression of genes involved in lipid metabolism, such as acyl CoA syntheses and lipoprotein lipase, is regulated by PPAR in adipocytes. PPAR additionally controls the production of the fatty acid transport protein involved in adipocytes intake of lipids [14]. The decrease in body fat is caused by a decrease in adipocyte quantity than by a decrease in the adipocyte size. The triglyceride level of adipose cells directly correlates with their size, thus lowering the concentration decreases cell size. Smaller adipocyte, caused by reduced triacylglycerol, is caused by enhanced oxidation of mitochondrial lipids induced by CLA [15].

Figure 1.

Composition of both elementary conjugated metabolites of linoleic acid. In food supplements, cis-9,trans-11-CLA and trans-10, cis-12-CLA arethe primary isomers.In the gastrointestinal tract of ruminant species, symbiotic bacteria typically produce CLA. In dietary sources, cis,trans-11-CLA (c-9, t-11-CLA) (second panel) predominates as an isomer and makes for up to 90% of the total CLA. Another prominent isomer, trans-10, cis-12-CLA (t-10, c-12-CLA) (bottom panel), makes up 1–10% of the total CLA found in dietary sources.

Figure 2.

Hypothetical mechanism of action of CLA isomers. An assumption has been made for possible role of CLA isomers in liver cancer and obesity. The peroxiredoxin 3 (PRDX3) is a member of conserved proteins having role in proliferation, apoptosis inhibition, pro-inflammatory activity, and the response to oxidative stress. Similarly iron protein ferritin light chain (FTL1) may have crucial role in dysregulation of iron metabolism and initiation of breast cancer. Regucalcin (RGN) is involved in regulation of genes involved in fatty acid synthesis.

2. CLA as a probiotic metabolite and its implications

According to the World Health Organization (WHO) and Food and Agricultural Organization (FAO), Probiotics are “live microorganisms which when administered in adequate amounts confers a health benefit to the host” [16]. The probiotics mainly belong to two types of lactic acid-producing microorganisms, Bifidobacteria, and the low GC (Guanine- Cytosine) content lactic acid bacteria (LAB) that include Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus genera. The most widely used probiotics belong to the genera Lactobacillus and Bifidobacterium [17, 18]. The probiotic properties of the genus Lactobacillus are important because of their use in various food manufacturers such as fermented milk, fruit juice, cheese, fermented cereal, or vegetable-based foods [19]. In humans, Lactobacillus is reported to have various roles such as stimulation of the immune system, prevention and reduction of the intensity and duration of diarrhea, and reduction of lactose intolerance [20]. The other properties which are associated with the Lactobacillus are synthesis of vitamin B, improvement of mineral and nutrient absorption, degradation of anti-nutritional factors and modulation of intestinal physiology, and reduced pain perception [20]. In this way, Lactobacillus is considered to be effective against certain diseases and confers health benefits. The properties of lactic acid bacteria considered to be potent probiotics are the ability to survive and grow in the in vivo conditions that resemble the site of administration [21]. The probiotic strain should be tolerant toward bile and acidic environment present inside the gut. The strain should have antimicrobial properties along with antibiotic resistance toward selected antibiotics [21]. Cell adherence along with the viability must be taken into account for the strain along with the beta galactosidase activity [21]. The LAB is supposed to produce various metabolites, but the primary metabolites of LAB are acetoacetate, acetate, and lactate [22]. The major short chain fatty acid (SCFA) found in human feces is acetate. The bacteria isolated from the human intestine are capable of producing butyrate from acetate in colon [23]. The other members of the flora use substrates such as acetoacetate and lactate for producing other SCFAs [24]. Within the gut, butyrate is considered to have a potent anti-carcinogenic effect. Butyrate has very clearly demonstrated the anti-carcinogenic effect of SCFA [25]. Butyrate is supposed to control cellular machineries like apoptosis and cell differentiation. It has got some effects at the molecular level such as regulating gene expression via phosphorylation and acetylation of histone protein [26, 27, 28]. The production of CLA along with other metabolites similarly has proved to be very useful in the modulation of certain disease conditions. The role of CLA in obesity, asthma, colon-rectal cancer, inflammatory bowel disease (IBD), and certain other diseases is well established [29]. LAB such as Lactobacillus or Lactococcus along with Bifidobacterium are most commonly used in the production of fermented food and can be a potential probiotic [29]. Similarly, some of these microorganisms also have the ability to produce CLA from the linoleic acid (LA) [29, 30, 31]. In a study conducted by Luis M. Rodríguez-Alcalá et al., 22 probiotic bacteria were tested for the production of CLA using the UV (ultra violet) screening method and high preference liquid chromatography (HPLC) technique. Out of 22 tested strains, five microorganisms, two strains of genera Bifidobacterium, two Lactobacillus, and one Lactococcus were selected for their ability to produce CLA after incubation in skim milk with free LA (lactic acid) as a substrate [32]. The cells of the immune system upon any stimulus such as cell injury or infection secrete an array of immune responses toward the stimulus. Dendritic cells (DC) are at the front of the innate immune response. They present antigens to the naive T cells along with the secretion of two cytokines IL (interleukins)-10 and IL-12 [33]. The immunomodulatory potential of c9, t11 CLA was examined by Loscher et al. on bone marrow-derived dendritic cells (BMDC). CLA reduced both protein and mRNA concentrations of IL-12p70 and IL-12p40 which is a pro-inflammatory cytokine; meanwhile, the concentration of both protein and m RNA concentration was increased for IL-10 increasing surface expression of the IL-10 receptor. This study also suggested that c9, t11-CLA reduced nuclear factor kappa B (NF-kB) and increased cytosolic nuclear factor kappa B suggesting prevention of degradation IkBa, a protein responsible for sequestering of NF-kB in the cytosol, whereas this effect was reversed in presence of IL-10 neutralizing antibody [34]. Therefore, this study suggests the role of CLA in the treatment of Th1-mediated disease. The potential anti-inflammatory effect of CLA has also been shown in many animal models such as IBD, 2.5% dextran sodium sulphate-induced colitis in mice, and other models [35]. The CLA-fed mice were shown to be protected from colitis-induced weight loss, rectal bleeding, and colonic tissue damage. The mechanism responsible for CLA-mediated protection against colitis was the upregulation of PPARγ and PPARδ and the reduction in NF-kB-mediated inflammation [35]. Rectal bleeding and colitis-induced weight loss were reduced in CLA-fed animals. The anti-inflammatory effect of CLA was also studied in response to lipopolysaccharide (LPS) by Zhao et al. The result demonstrated that both TNF-α and mRNA expression in peripheral blood mononuclear cells (PBMC) was significantly reduced in CLA-fed animals, and this reduction was attributed to the attenuation of NF-kB signaling [36]. In the past 30 years, obesity has become one of the major challenges in the society. There has been a substantial increase in the number of patients. The anti-obesity mechanism of CLA was reviewed by Wigham et al. in the year 2007 [37]. The two major isomers of CLA, that is, 10, 12 and 9, 11 when provided in a mixture or the isomer 10, 12 alone have proved to have some anti-obesity effect in many animal and human studies [37, 38]. The major mechanism through which CLA regulates includes a reduction in energy intake, increase in energy expenditure, inhibition of adipogenesis by CLA, increase in inflammation, suppression of lipogenesis, and stimulation of lipolysis (Figure 3) [37, 38]. Resistance to insulin and diabetes mellitus type 2 is a result of obesity’s challenging impact on the liver, pancreas, and additional metabolic organs as well as on adipose metabolism [39]. The blood flow to the adipocytes declines with obesity and the adipocytes boosting expansion, resulting in hypoxia [40]. Adipocytokines, also called active metabolites, and inflammatory mediators like tumor necrosis factor-alpha and interleukin-6 (TNF- and IL-6), as well as free fatty acids, have been overproduced as a result of the expansion of adipocytes and hypoxia [41].

Figure 3.

Depicts the role of CLA in obesity. CLA plays an important part in reducing body fat. Although it is unclear if this results in less food consumption, CLA may also lessen feelings of hunger. The majority of an individual’s weight or BMI does not appear to be reduced by CLA. However, it also can inhibit adipogenesis (This is a potential treatment strategy for overweight).

3. Biological activities of CLA

3.1 Anticancer effects by CLA and prevention of cardiovascular disease

CLA has been shown effective against a number of cancer models such as skin, forestomach, mammary, colon, and liver [11, 42, 43]. It is stated not only to reduce initiation, promotion, progression, and development of cancer but also to reduce metastasis of cancer [11, 42, 43]. The suggested methods for CLA action are reducing eicosanoid production, interfering with cell signaling pathways, inhibiting DNA synthesis, enhancing apoptosis, and inhibiting angiogenesis as shown in reduced matrix metalloproteinase and vascular endothelial growth factors [11, 42, 43, 44]. Modern potential anticancer treatments rely on lifestyle modifications, including dietary changes. CLA, for example, can reduce the risk of developing cancer [45]. Necroptosis was induced by several PPAR ligands, which prevented the development of cells with lung cancer in humans. However, no studies have yet looked into how CLA affects PPAR mRNA and nutrient levels in non-small cell lung cancer cell lines [46].

It has been reported that CLA reduces atherosclerotic lesions in rabbits and hamsters [47, 48, 49]. It reduces total cholesterol triacylglycerides low density lipoprotein (LDL), holesterol and increased High Density Lipoprotein (HDL) cholesterol in various animal models [49, 50, 51]. Various factors are involved such as reduction in blood pressure, involvement of PPAR key role in lipogenesis, sterol regulatory element binding protein (SREBPs, a key role in fatty acid synthesis and elongation), and/or stearoyl Co-A desaturase (SCD, a key role for triglyceride and cholesterol formation) [48, 51, 52, 53]. Between the bloodstream and the supporting connective tissue, the endothelium is an overlay of flat epithelial cells. It describes important processes that, when they are dysfunctional, cause endothelial dysfunction. The development of vascular disorders is attributed to this syndrome. A mouse ovariectomized model has been utilized to explore the consequences brought on by the lack of sex hormones because it is well-known that they have a cardio-protective impact. The impact of dietary fats like CLA on cardiovascular illnesses has been researched as a means of maintaining vascular health. Antioxidant, anti-atherogenic, and anti-inflammatory properties are some of the benefits of CLA that have been scientifically demonstrated. It increased the antioxidant activity and stopped the orchiectomy’s effects on prostacyclin, nitric oxide (NO), and cholesterol oxidation products (COPs). These results might help us comprehend how things work. The impact of an eight-week CLA diet supplement on parameters linked to aortic and mesenteric artery vascular dysfunction as well as the quantity of lipid oxidative released after orchidectomy. The inclusion of CLA in the diet prevented the production of proteinoids from rising and preserved the physiological parameters of NO with enhanced antioxidative activity. Also, it stopped the vascular wall’s cholesterol and COPs from rising. The antioxidant activity was increased, and the orchidectomy-induced changes to prostacyclin, NO, and COPs were avoided by a CLA-supplemented diet. These results may aid in our understanding of the processes by which CLA prevents cardiovascular illnesses [54].

3.2 Body fat reduction by CLA

In 1995, for the first time, it was reported that CLA has some effect on the reduction of body fat. The multiple effects through which CLA acts on the reduction of body fat include increasing energy expenditure and reducing lipid accumulation in adipose tissue [55, 56]. It may also work by increasing adipocyte differentiation or by adipocyte apoptosis. CLA has also some modulatory effects on adipokines and cytokines such as leptin, TNF-α, adiponectin, or interleukins and increasing fatty acid β oxidation in skeletal muscle [56]. Ruminant fats contain a lot of conjugated linoleic acid which appears to enhance body composition and risk factors for cardiovascular disease. Experimental models have already demonstrated CLA’s ability to lower body fat levels and to have positive effects on the glycemic index, atherosclerosis, and cancer. Moreover, CLA supplements may enhance bone mineralization, assist in the resynthesizing of glycogen, and alter immunological function. Additionally, CLA supplementation may boost lipolysis and decrease the build up of fatty acids in adipose tissue. The putative factors responsible may be its ability to decrease lipase lipoprotein activity and increase carnitine-palmitoyl-transferase-1 (CAT-1) activity, interact with PPAR, and increase mitochondrial uncoupling protein-1(UCP-1) expression. While human studies have indicated some advantages of CLA supplementation, such as weight loss, the findings are still inconclusive. Additionally, some have demonstrated detrimental effects, such as harm to lipid profiles and glucose metabolism. This article’s goal is to summarize the information that is now available regarding the advantages of CLA for energy consumption and muscle mass, with a focus on the mechanisms of action [57]. Although most research on CLA’s impact on obesity has concentrated on how much body fat it reduces, other studies have shown that it also boosts muscle mass and improves athletic performance. These effects have been attributed to altered signal transduction pathways in metabolism or energy metabolism, changes in the type transformation of muscle fibers, and other physiological modifications in the skeletal muscle. The exact mechanism by which CLA affects cardiometabolic risk is not fully understood. This review’s objective is to provide an overview of what is presently known about how CLA affects skeletal muscle metabolism. There is a lot of potential for using CLA because it not only enhances lean mass but also reduces body fat [58]. When supplemented over a duration of 6 to 16 weeks, CLA may have a somewhat anti-obesity effect on women’s body weight (BW), body mass index (BMI), and TBF, especially in post-menopausal and overweight women. This meta-finding analysis needs to be supported by more well-designed studies [59].

3.3 CLA inimmune and inflammatory responses

The major anti-inflammatory response of CLA reported is reducing colonic inflammation, decreasing antigen-induced cytokine production in immune-competent cells, and modulating the production of cytokines prostaglandins and interleukin B4 [60, 61, 62, 63, 64]. The immune-related response of CLA has been shown by modulating tumor necrosis factor α (TNF- α), cytokines (i.e., interleukin 1, 4, 6, or 8), prostaglandins, or nitric oxide while reducing allergic-type immune response [65, 66, 67]. In Holstein cow, the enhancement of n-3 fatty acid in EM with the use of essential fatty acid (EFA) prevented n-3 FA dehydration. The combined EFA + CLA treatment led to small further alterations in the anti-inflammatory response, and the enhanced n-3 FA(Fatty Acid) status and decreased n-6:n-3 ratio by the EFA treatment showed a more distinct influence on the inflammatory process during the period of transition than the single CLA treatment [68]. Many studies have confirmed the rumen microbiota’s vital role in maintaining the rumen epithelium’s innate immunity equilibrium. Information on the immunoprotective impacts of various CLA isomers on rumen respiratory epithelium is scarce as they are the natural microbial metabolites of the rumen. The 100 M trans-10,cis-12-CLA in this study reduced dramatically the gene expression associated with inflammation, cell proliferation, and migration in RECs after activation with lipopolysaccharide, exerting greater anti-inflammatory actions than the cis-9,trans-11-CLA. Trans10,cis-12-CLA significantly decreased, when compared to cis-9,trans-11-CLA with enhanced supervision of signaling pathways and cytokine production involving KEGG pathways NF-B, chemokine, NOD-like binding site, Hippo, PI3K-Akt, TGF- and Rap1 signaling. Also, compared to the LPS group, pre-treatment with trans-10,cis-12-CLA dramatically decreased the activation of lipogenic genes and the production of the unsaturated fat pathway in RECs; however, cis-9,trans-11-CLA showed the opposite results. These findings point to different CLA isomer variations in RECs’ control of systemic inflammation and work in different signaling, and they will serve as crucial references for figuring out their target application in the future (Figure 4) [69].

Figure 4.

A possible explanation for how CLA alters carcinogenesis. The chemical breakdown of arachidonic acid is impacted by CLA, which also reduces Lipoxygenases (LOX) and Cyclooxygenase (COX), Prostaglandin E2 (PGE2), and TB2 levels, and inhibits the activation of NF-B. significantlythe Bcl-2 and Ki-67 proteins, phosphorylating ErbB3 and Akt, and upregulatingcaspases and CLA affect apoptosis.

3.4 Bone health and CLA

According to the reports, CLA improves bone mass as reported by ash weight, bone density, bone mineral contents, bone dry weight, bone length, or calcium, magnesium, or phosphate content [70]. However, the effect of CLA on body ash and bone mass has not been consistent. This inconsistency has been reported due in part to the interaction between CLA and calcium in diet [70]. However, there have been mixed reports of bone resorption by CLA. Some suggest that CLA decreases the osteoclast activity, thus enhancing bone resorption, while others suggest CLA has no effect on bone resorption [71, 72]. Rats receiving a dietary supply of CLA showed increased levels of CLA isomers in many tissues, including bone compartments. Improvements in the cis-9, trans-11 CLA monomeric concentration of rat bone tissue lipids were linked to changes in bone indicators and bone production rates in developing rats. In contrast to polar lipids, CLA had a stronger impact just on the fatty acid structure of neutral lipids in rat tissues. Rats’ bones with dietary CLA supplementation have higher concentrations of CLA isomers. Dietary CLA caused variances in CLA concentration, with the brain showing the lowest quantities of isomers while having the highest concentrations in certain bone tissues. The examination of CLA benefits will yield fresh data on the interactions between fatty acids and osteoblasts [73].

4. CLA and cancer

The anticancer effect of CLA has been studied in both in vitro cell and in vivo animal models. They can be found in the food that ruminant animals produce as well as in the seeds of some plants [74]. As specified both the isomer of CLA, that is, c9 t11 and t10 and c12 have shown to inhibit both the promotion and initiation stages of carcinogenesis, suggesting the chemopreventive effect of CLA (Table 1) [86]. The anticarcinogenic effect of CLA was discovered accidentally by researchers at the University of Wisconsin in 1979. Although the anticancer effect of CLA is well known, it has other known properties such as reduction of cardiovascular disease and inflammation along with the regulation of body weight, reducing body fat, and increasing lean body mass [87]. The mechanism behind the anti-cancer properties of CLA is the activation of PI3/Akt signaling and stimulation of the tumor suppressor gene, p53 [86, 87]. These pathways increase cancer cell apoptosis, decrease cancer cell proliferation, and inhibit tumor growth. Cyclin C and cyclin D modulation is carried by CLA which participates in cell cycle progression. The expression level of PPAR gamma is also increased by CLA suggesting its role in metastasis and decrease of the tumor size [86, 87].

Cancer type	Model	CLA Source	Mechanism	Outcome	Ref.
Colon	SD Rats	CLA Mixture	Inducing TGF-β- producing macrophages and T cell	This decreased the epithelial integrity and proinflammatory influx in the conjugated sodium sulfate-induced colon model.	[75]
Colon	SD Rats	C9, t11- CLA	Reduced tumor frequency, aggregate apoptosis	CLA could have anticancer activity by causing apoptosis in the rectal epithelial cell by alteration of the transmitter.	[76]
Colon	BALB/c mice	CLA Mixture	Diminished metastatic foci, stimulation PPRn	The proportion including both cells’ abdominal canal malignant regions was reduced significantly, As MKN28 and Colo320 were pretreated with LA, and glycogen replenishment indicated a decline in the antibody for epithelial stimulating elements and a rise in Bax.	[77]
Mammary	SD Rats	C9, t11 - CLA	Reduce the weight and volume of the tumor	In empirical mammary antitumor, the leading CLA isomer found in the human diet shows antineoplastic properties.	[78]
Mammary and Breast	SD Rats	CLA Mixture	Impede PUFA oxidation	CLA’s efficacy for reducing simultaneous non-infectious disorders	[79]
Breast	SCID Mice	CLA Mixture	Inhibit the growth of cancer cell	Dietary polyunsaturated fats may alter a prostate cancer patient’s survival, presenting novel therapy possibilities.	[80]
Prostate	Big Blue Rats	CLA Mixture	Inhibit the formation of PhIPin DNA	The first study to demonstrate CLA’s protection from PhIP-induced mutagenesis in the prostatic in terms of mutation frequency and gene mutation diversity. It may be used in human cancer prevention studies because of human chemoprevention because of CLA’s antagonistic effect against PhIP-induced genotoxicity.	[81]
Prostate	Copenhagen Rats	CLA Mixture	Cytotoxicity	Without performing a fatty acid compositional study, the optical properties can be utilized as a quick and easy way to manage the hydroformylation activity of vegetable oils to achieve the desired conjugated linoleic concentration.	[82]
Liver	Male F344 Rats	C9, t11 - CLA	Inhibit Tumor formation	Pomegranate seed oil high in c9,t11, and c13-CLN can reduce AOM-induced colonic carcinogenesis and that this inhibition is partly mediated by increased liver and colon CLA content and/or colon stromal PPARgamma protein expression.	[83]
Liver	Donryu Rats	CLA Mixture	Helps to generate hepatic lipid peroxidation and aggregation	Conjugated palmitoyl hydroxylase II, a percentage protein in the energy metabolism beta-oxidation pathway, wasn’t really activated by dietary CLA. Although injected rats tended to have increased levels of natural killer cell function in the spleen, no detectable effect of nutritional CLA was found.	[84]
Breast	BALB/c mice	C9, t11 - CLA	Inhibited the growth of the tumor when combined with chemotherapy drugs.	A nucleotide analog drug against a wide range of cancers is gemcitabine. Fast decomposition in vivo, which causes a short circulatory period and reduced anticancer activity, is a limitation that has to be resolved.	[85]

Table 1.

Preclinical evidence of CLA against various forms of cancer.

4.1 CLA and breast cancer

Breast cancer is the second most common cause of cancer death among women worldwide. Cell proliferation is supposed to be downregulated by CLA in breast cancer [88]. CLA induces apoptosis via Liver X Receptor (LXR) in the MCF-7(Michigan Cancer Foundation-7) cancer breast line [89]. Cell growth and invasions inhibited through PI3/Akt signaling in the breast cancer cell line. It has also been shown that apoptosis is stimulated by a mixture of CLA through p-53-mediated cell signaling in the MCF-7 breast cancer cell line [90]. Intravenous injection of CLA in nude mice-xenografted human breast MCF-7 tumors showed an enhanced tumor growth inhibition effect. Many clinical studies have also suggested the anticancer effect of CLA in stages I–III of breast cancer. According to one study, the breast cancer tissue expression of S14 was decreased after a short course of treatment of 7.5gm CLA/day [90, 91]. Another study showed a reduction in proliferative marker Ki-67 in primary invasive breast cancer tissue on the supplementation of 7.5 gm CLA for 10 days. The limitations of these two studies were a small population and less duration of dose. But the safety and tolerability of CLA were measured to be 7.5 gm/day for a period of 20 days [91, 92]. In breast cancer, CLA was proven to reduce cell proliferation [74]. Certain in vivo experimentation on rats, fed on proportions of CLA with linoleic acid and simultaneous tumor induction showed The protective benefits of CLA. However animals fed with HFD and varying concentration of CLA showed no impact on tumor growth. Along with that neither linoleic acid nor arachidonic acid replaced CLA in the mammary tissue [93].

4.2 CLA and colon cancer

Colon cancer also called colorectal cancer is cancer due to uncontrolled growth in the colon or rectal region [94]. Rectal bleeding and anemia are the typical symptoms associated with colon cancer. The major cause of colorectal cancer can be associated with lifestyle and increasing age with only a very few due to genetic disorders. It is more common in developed countries, and it is the third most commonly diagnosed cancer in the world [94]. CLA has been shown to have an anti-proliferative effect on colon cancer. Trans 10, cis 12 isomer of CLA induces cell death through reactive oxygen species (ROS)-mediated endoplasmic reticulum stress in the human colon cell line [95]. This isomer of CLA exhibited an anti-proliferative effect in the Caco-2 cancer cell line. It induces cell cytotoxicity via both PPAR gamma and APC/beta-catenin signaling pathways [95, 96]. Cell proliferation by CLA has been carried out through p53-dependent mechanism, and it is also supposed to regulate the expression of G1 restriction point in colon cancer cells [97]. In animal in vivo studies, it has been demonstrated that CLA mixture ameliorates inflammation-induced colorectal cancer through activation of the PPAR gamma signally pathway. Similarly, the mixture of CLA isomers was effective in inhibiting colon cancer metastasis in BALB/c (Bagg and Albino) mice [96, 97]. The inhibitory effect of CLA on growth and metabolism in the human HT29 colon cancer cell line has been demonstrated with special regard to the conversion of trans 11 trans 13 CLA isomer. CLA has also been shown to reduce colon carcinogenesis in azoxymethane-pretreated rats [97, 98]. Shiraishi et al. mentioned research on malignant rats fed beef tallow over an extended period. Carcinogenic rats fed beef tallow over an extended period of time. The combination of CLA isomers was employed to prevent the development of colon cancer. After administering azoxymethane to the animal model first, the impacts of the CLA combination were examined. According to the findings, a combination of 1% CLA in the triglyceride form and 1% CLA in the free lipid form reduced the risk of colon cancer. Although CLA-FFA was better at colony carcinogenesis, its exact apoptotic mechanism remained unknown [74]. Kim et al. also considered the implicit parcels of the two main CLA isomers against colon cancer cells Caco-2, in vitro. The results indicated that t10, c12-CLA cure dropped the situations of the insulin growth factor stashing and IGF-binding proteins, thus reserving the Caco-2 cell growth. The study concluded that only one isomer t10, c12-CLA convinced cell apoptosis and dropped DNA conflation, whereas c9, t11-CLA had no effect [99].

4.3 CLA and endometrial cancer

In developed countries, particularly among post-menopausal women, endometrial cancer is the most common malignancy found. Endometeroid adenocarcinoma which is the most common subtype occurs within a few decades of menopause, and it is mainly associated with obesity and excessive estrogen exposure which often develops in endometrial hyperplasia [100]. In the endometrial cancer cell line, RL 95–2 when treated with cis 9 trans 11 isomers of CLA, the expression of caspase 3 and Bax/bcl ratio was significantly increased, but no change was observed in Akt and p-Akt [100, 101]. According to one of the studies, the expression of total ERα level was unchanged in the treatment of cis 9 trans 11 isomers in the RL 95–2 cell line. Also, the protein expression level of p- ERα was downregulated upon treatment with 80 Mm of cis 9 trans 11 isomers [101]. From this study, it can be concluded that cis 9 trans 11 isomer of CLA induces apoptosis by estrogen receptor alpha (ERα)-mediated signaling pathway in the RL 95–2 endometrial cancer cell line. As there is not much information available on the possible role of CLA in endometrial cancer, it opens an aspect for further research in this direction [102]. Additionally, it was revealed that ERα was mediated by a number of signaling pathways, including phosphoinositide 3-kinase/Akt and extracellular signal-regulated protein kinase 1/2 [103]. The impact of isomer-specific CLA on RL 95–2 cells is evident from the fact that as the cells were exposed to c9, t11-CLA at doses of 10–160 M for 24, 48, and 72 h, respectively, the viability of the RL 95-2 cells was decreased. When the cells were exposed to c9, t11-CLA at doses of 10–160 M for 24, 48, and 72 h, respectively, the viability of the RL 95-2 cells was decreased [103]. It is demonstrated that the formation of total ERα was unaltered in RL 95-2 cells treated with the cis 9-trans 11 CLA isomer, but that at an 80 mM concentration, the isomer downregulated the level of p-ERα protein expression. According to this finding, cis 9-trans 11 CLA isomer causes apoptosis in RL 95-2 endometrial cancer cell lines via an ERα-mediated signaling mechanism [104]. In a research paper, it is observed that ATP-CVA was previously used to assess the effectiveness of chemotherapy in uterine cancer cell lines. In order to identify the inherent therapeutic efficacy of cytotoxic medications, researchers conducted the ATP-CVA on patients with endometrial cancer as part of this study to examine the viability of doing so. The SF50 (survivability percentage at 50%) of the PPC of 32 of the 33 ovarian cancer samples could be evaluated using ATP-CVA. In comparison, cisplatin (0.71), topotecan (0.93), paclitaxel (0.68), doxorubicin (1.0), etoposide (0.70), 4-epidoxorubicin (0.88), and carboplatin’s median SF50 (0.33) was significantly lower [105].

4.4 CLA and renal cell carcinoma

Cancer which starts in the cells of the kidney is said to be associated with kidney cancer. Renal cell carcinoma (RCC) and urothelial cell carcinoma (UCC) are the two most common types of kidney cancer of the renal pelvis [106]. These different types of cancer develop in a different way, and their outcome is also different and need to be staged and treated in different ways [107]. A lipid class distribution study was performed by Hoffman et al. showing that CLA was incorporated in the neutral lipid compared with the phospholipid class [108]. The content of the trans 10 cis 12 isomers of CLA was found to be different in normal and cancerous cells of the kidney suggesting a key role for the above-said isomer of CLA in kidney cancer. Much work needs to be done due to the lack of mechanistic study in this regarding the effect of CLA on kidney cancer [109]. Significant reductions in tumor weight, fluid volume, and live Ehrlich cells were seen in TTM that had been treated with CLA. Histopathology showed negligible effects, whereas hematological and biochemical profiles returned to more or less normal levels. Due to the synergistic and additive effects of its isomers, the current study demonstrated the safety and cytotoxic efficacy of milk protein CLA and provided a scientific foundation for its therapeutic usage as an anticancer agent [110].

4.5 CLA and pancreatic cancer

Pancreas is a glandular organ located behind the stomach. Pancreatic cancer is a cancer within the pancreas. The signs and symptoms of pancreatic cancer include abdominal or back pain, yellow skin, unexplained weight loss, light-colored stool, dark-colored urine, and loss of appetite [111]. Ductal pancreatic adenocarcinoma is the most prevalent form of pancreatic carcinoma, and it is said to be the fifth leading cause of death in Western countries. The relationship between dietary fat intake mainly n-6 polyunsaturated fatty acids (PUFA) and cancer was studied by many researchers [111]. CLA is an essential fatty acid with known anticancer potential. However, LA is reported to promote tumor growth caused by high sensitivity to nonenzymatic lipid peroxidation [111, 112]. The impact of dietary LA and CLA on liver metastasis and lipid peroxidation (LPO) was examined by Kilian et al. in N-nitroso bis-2-oxo-propylamine (BOP)-injected Syrian hamster for 12 weeks [112]. No difference was observed between tumor groups in liver metastasis and lipid peroxidation. Therefore, these results suggested that there was no difference between the LA and CLA-fed groups according to the impact on liver metastasis in ductal pancreatic cancer. Again Kilain et al. in 2003 investigated the impact of the high content of CLA along with dietary intake on ductal pancreatic carcinoma. The result suggested that CLA increased the weight of the pancreas in the tumor and non-tumor group compared to LA [112]. It was also found that LA and CLA do not influence lipid peroxidation or activity of anti-oxidative enzymes GSH-px (glutathione) and SOD in tumor-free control groups. Therefore, according to the study, it can be confirmed that the content of CLA in the diet does not influence pancreatic tumor growth. However, further research is required by increasing the content of the CLA dietary model [112, 113]. The frequency of pancreatic cancer in relation to CLA and LA dietary consumption of CLA and LA was examined in a subsequent investigation. Four groups of 60 male hamsters each were chosen. While the third and fourth groups received injections for 12 weeks, the initial group had 0.9% NaCl once per week. The findings demonstrated that neither CLA nor LA had an impact on the prevalence of pancreatic cancer. However in both the groups, pancreatic intratumoral tissue had higher lipid peroxidation and enhanced glutathione peroxidase activity [114].

4.6 CLA and liver cancer

Cancer that originates in the liver can be termed liver cancer. It is the third largest cause of cancer death in the world and fifth in the case of women due to cancer [115]. The symptoms associated with liver cancer are abdominal mass, abdominal pain, yellow skin, nausea, or liver dysfunction, and it can be detected by medical imaging equipment [115]. The liver is the most common site for metastasis, and it is mostly derived from the colorectal and neuroendocrine primary tumor [116]. The effect of CLA has been studied in hepatocellular carcinoma cell lines, and it exhibits an inhibitory effect on hypoxia-induced factor 1 (HIF 1) alpha stabilization under hypoxic conditions [117]. Along with this, the proapoptotic effect of CLA is mediated by PPAR α and PP2A signaling pathway in the SK-HEP-1 (human hepatoma cell line) [117, 118]. Moreover, mitochondria-related apoptosis and lysosomal destabilization are demonstrated in rat primary hepatoma cells by trans 10 cis 12 isomers of CLA [97]. The same isomer is known to exhibit a strong cytotoxic effect on dRLH-28 rat hepatoma cells via the activation of caspase 9 followed by cytochrome release from mitochondria [97, 119]. In animal studies, CLA inhibits azoxymethane-induced rat colon cancer by alteration of lipid composition and elevation of colonic PPAR gamma expression [119]. The cytotoxic effect of trans 10 cis 12 isomer on rat hepatoma was observed, and it was modulated by other fatty acids such as tocopherol and tocotrienol [119, 120]. The difference between in vivo and in vitro results could be attributed to the duration and isomer of CLA. In the cell line study, we mostly use the trans 10 cis 12 isomer of CLA, whereas in the animal model, CLA mixtures were used. Also, the duration of the dose is also a considerable factor; hence, further studies with purified isomers can give a better result [119, 120]. A study on anti-carcinogenic properties of pomegranate seed oil also demonstrated 70% of the c9, t11 CLA isomer. Acute otitis media (AOM) was injected directly into male F344 rats once every week for 2 weeks. The higher dietary content of c9, t11-CLA in the lipid component of the colonic mucosa and liver was related to the prevention of colonic adenocarcinomas [121]. On rat hepatoma dRLh-84 cells, CLA has been shown to possess anti-mutagenic and anti-carcinogenic activities in vitro. It has been previously established that the geometric disparity between the two main CLA isomers was the cause of their divergent anti-tumor responses. On the dRLh-84 rat hepatoma cell line, different studies revealed variations in the anticancer activity of CLA isoforms [110]. Under 1% O2 circumstances, the anti-cancer actions on the human liver cancer cell line HepG2 have been documented. Under low oxygen conditions, both the isomers c9, t11-CLA and t10, c12-CLA reduced cell proliferation and brought on apoptotic cell death (HIF-1 stabilization). This unique investigation was demonstrated to be of considerable potential for future trials because the precise process remained unknown (Table 1) [122].

5. Mechanism of action of CLA

The potential mechanisms by which CLA alters body composition include metabolic adjustments that favor a decrease in lipogenesis and potentiation of lipolysis, along with the oxidation of fatty acids in skeletal muscle, as a result of elevated carnitine in palmitoyl-transferase-1 movement and activity or perhaps due to the inhibition of adipocyte differentiation [123]. Consequently, experts have assessed how the addition of CLA to a diet affects hormonal and lipid levels as well as the functioning of oxidation-related enzymes [124]. Studies have shown that CLA isomers that are 10-trans and 12-cis, as opposed to the 9-cis 11-trans, dramatically promote lipolysis in human adipocytes and also serve to reduce triglyceride production [125]. This would substantially explain any potential techniques through which CLA affects body composition. The control of the expression of genes that regulate the transformation of this was before the adipocytes into mature adipocytes, or more specifically, the communication of these genes would lead to reducing lipogenesis, which was the foundation of metabolism hypotheses to explain the body fat-reducing action of CLA, despite the fact that various studies were in vitro [126]. The nuclear transcription factors known as peroxisome proliferator-activated receptors play an essential role in the preservation and metabolism of fatty acids (FA). They are a subset of nuclear channels that are related to the nuclear receptor family which includes the steroid, retinoid, and thyroid receptors. The nuclear receptor has already been divided into three isoforms: PPAR, PPAR, and PPAR. Lipid metabolism (particularly, the proteins associated with FA oxidase) and glucose are both regulated by the PPARs, and PPAR is also involved in the development of adipocytes. The co-activator unit (acetyltransferase capacity) and the dissolution of the co-repressor compound (histone deacetylase function) by the binding agent are required for the activation mechanism. Through transcription-competent competent structure develops when the activating PPAR:RxR complex attaches to the PPRE-responsive zones, altering the chromatin’s structural makeup. Therefore, it appears that the CLA interacts with PPAR, enhancing the transcription of genes involved in adipocyte differentiation, lipolysis (oxidation), mitochondrial biogenesis, and insulin sensitivity, all of which are associated with the weight loss impact [127]. The activity or inactivity of the PPARs, particularly PPAR, mediates the effects of CLA on the metabolism of lipids and glucose on body composition. By modulating gene expression in a way that prevents cell differentiation and changes the functioning of proteins associated with lipogenesis and lipolysis, CLA (isomer 10-trans, 12-cis)'s suppression of PPAR causes a reduction in body fat [128]. According to the available data, PPAR activation may slow the development of atherosclerosis and improve insulin sensitivity, making it an attractive target in the management of a number of conditions, particularly type 2 diabetes mellitus (DM2) and dyslipidemia. The activity of genes that regulate lipid metabolism, such as acyl CoA-synthetase and lipase, also called lipoprotein lipase (LPL), is regulated by PPAR in adipocytes. PPAR also regulates the production of the fatty acid transport protein that regulates adipocytes’ intake of lipids [129]. The decrease in fat in the body is caused less by a decrease in adipocyte quantity than by a decrease in the adipocyte size. Given that the triglyceride level of adipose cells directly correlates with their size, lowering it leads to smaller cells. Triacylglycerol synthesis may have been reduced as a result of the increased process of cellular fatty acids brought on by CLA, which did not result in their deposition in adipocytes but did result in a reduction in their size [130]. The carnitine-palmitoyl-transferase (CPT) unit carries the fatty acid into the mitochondria. CPT-1, CPT-2, and carnitine acyl carnitine translocase (CATC) are the three enzymatic players. By producing an activated complex (fatty acyl-CoA) that includes the carnitine-palmitoyl-transferase (CPT-1) enzyme, the acyl-CoA synthetase enzyme activates the fatty acids. The intermembrane gap is reached after this complex passes through the mitochondrial membrane. Following the breakdown of carnitine in the CPT-2 reaction, acyl-CoA is replenished. The large chain fatty acid (LCFA) is oxidized once it enters the mitochondrial matrix to produce adenosine triphosphate, also known as ATP through the oxidation of the fatty acids [131]. The amount of and function of CPT-1 would rise with the addition of CLA. Thus, the build up of fatty acids in the adipose and muscular tissues is decreased as a result of increased lipolysis, decreased lipase lipoprotein activity, and enhanced carnitine-palmitoyl-transferase-1 (CAT-1) activity. The researchers frequently address these action mechanisms [130]. By oxidation processes and the Krebs Cycle (CK), which take place inside the mitochondria, fatty acids are converted to H+ and e − which are then transported (via NADH+2 and FADH2) to the pulmonary chain (1). The ATP synthesis protein (2) either produces ATP (in conjunction response) or heat (uncoupling protein (3)) in response to the gradients of H+ and e − across the intermembrane gap and the matrix. The argument that CLA promotes lipolysis only by raising CPT-1 is, however, only true in circumstances where fatty acyl-CoA complex transport to the matrix of mitochondria is less effective than oxidation, which is the ability to produce ATP through the sequential breakdown of fatty acid carbon. This makes it plausible and logical to conclude that CLA supplemental intake (such as increasing CPT-1 concentration in order and stimulation) would likely only have a marginally positive impact on people who are physically active, especially for those whose oxidation is more effective compared to the movement of fatty acids to the mitochondrial matrix. The interaction of CLA with the separating enzyme of the chain of respiration, which increases its ability for oxidation, may, on the reverse, account for the weight loss that occurs when CLA administration is used [132]. A collection of transport substances found in the membrane on the inside of the mitochondria makes up the respiratory chain, also known as the electron transport chain. Cytochrome oxidase, the last among these substances, is the only one in existence that meets all requirements to transport electrons to O₂ directly. But not all of the power that comes from the electrons will end up in ATP as some of it turns into heat to preserve the unpredictability of the subsequent transfers. The respiratory chain causes the electrons to lose their inherent energy as they go through it. In order to create ATP using ADP and inorganic phosphate, some of this energy can be captured and stored. The remaining energy that is free, which is not used to re-synthesize ATP, is emitted as heat is produced, boosting the uncoupling proteins (UCP’s) function [133]. The membrane that surrounds the mitochondria contains proteins called UCPs that enable proton transport from the intermembrane gap to the mitochondria matrix. However, the protons’ return to the matrix of mitochondria does not result in ATP energy storage, which would release heat. When UCP-1, additionally referred to as thermogenin, is stimulated, it can cause weight loss because it frequently accelerates up the hydrogen return to the mitochondria matrix, causing energy from the Krebs cycle, which comes from the decomposition of energy substrates (including lipids), to be lost in the form of heat [134]. UCPs can be subdivided into UCP-1, UCP-2, and UCP-3. They differ in their distribution among tissues and possible function. UCP1 is exclusively expressed in brown adipose tissue; UCP3 is expressed in muscle and a number of other tissues; and UCP2 is expressed in a variety of tissues including white adipose tissue (WAT) and is the most highly expressed UCP. In rodents, it has been established that supplementation of a particular CLA conjunction or 10,12 CLA causes UCP2 translation in WAT [135]. Nevertheless, it is uncertain if it affects energy loss. It appears that CLA combines with PPAR, boosts CTP-1 (carnitine palmitoyltransferase-1) and UCP-1 expression, and promotes the ability of lipolysis, which stored fat for weight loss (Figure 5) [132].

Figure 5.

The overall mechanism through which CLA inhibits the formation of eicosanoids from arachidonic acid.

6. Future prospects

The role of CLA is well established in many inflammatory diseases and has drawn significant attention in the last two decades for its various biological effects. CLA reduces body fat, modulates immune and inflammatory responses, reduces cancer and cardiovascular disease, and improves bone mass. As far as obesity-related cancer is concerned, it has shown potent anti-cancer effects and management of tumor growth and metastasis. The overall result of CLA is due to its two main isomers, that is, cis 9, trans 11 and trans 10 cis 12. However, there is some controversy between in vitro and in vivo studies as most in vivo animal model uses CLA mixtures, and the effect of CLA on cancer cell proliferation and its molecular mechanism is yet to be fully understood. In the case of clinical application, the role of CLA is limited for cancer patients due to low bioavailability, that is, poor water solubility and short half-life. Moreover, when using a mixture of CLA, the result is varying, and it is lacking in scientific basis. The different isomers are shown to have different molecular mechanisms, and some are shown to have opposite effects on each other. Hence, direct extrapolation of the result needs to be more studied, and further investigation is needed to show the short- and long-term effect of CLA in a human trial in order to determine the safety for application to human. Therefore, these drawbacks are yet to be looked into for the CLA to be implemented as for potent remedy for many inflammation-related disorders.

7. Conclusion

CLA research has drawn much attention in the last two decades ranging its effect from cancer to obesity. Even being a relatively simple structure, the more diverse effect of CLA in different diseases such as cancer, obesity, inflammation, asthma, cardiovascular disease, and bone health is intensively studied. The different effect of CLA is attributed to its different isomers, namely, cis 9 trans 11 and trans 10, cis 12. Both the isomers have different molecular mechanisms which are yet to be examined more intensely. CLA is known to have a potent anti-cancer effect which is studied extensively, especially in the case of obesity-related cancer such as colon cancer, endometrial cancer, liver cancer, kidney cancer, and breast cancer. The mechanism by which the CLA acts are activation of the PI3K/Akt pathway, stimulation of the tumor suppressor gene, p53, followed by activation of caspase 9. As far as human consumption is concerned, the safety parameters are also been evaluated in a number of human clinical trials. About 90% of the studies were conducted with either of the two isomers of CLA. The daily consumption of 3–6 g/day in human beings is tested, but no such adverse effect has been reported to date. However, there are still concerns regarding the potential safety of CLA as a therapeutic agent. Therefore, better experimental designs are required to clarify the CLA activity’s mechanism.

Acknowledgments

The authors would like to sincerely thank the Lovely Professional University for its suppocrt throughout the research.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contribution

TD wrote the paper and analyzed data. AK designed the study and carried out proof reading. AKM designed the study, conducted proof reading and editing, and had primary responsibility for the final content.