Isoleucine Amino Acids: Properties, Function, Benefits, and Sources

What is isoleucine?

Since the body is unable to produce isoleucine on its own, this amino acid must be consumed in the form of food. It plays a crucial role in protein synthesis, energy production, and muscle metabolism as one of the branched-chain amino acids (BCAAs), alongside valine and leucine. Similarly, this amino acid has a strong aversion to water. Its four possible isomers are all due to its two asymmetric carbon atoms. Proteins contain a single isomer. The average adult needs 0.7 g/day of it, and it can be broken down by the muscles in the body.

Isoleucine structure

Isoleucine (molecular formula C₆H₁₃O₂N, molecular weight 131.2), a branched-chain aliphatic amino acid, is involved in hydrophobic interactions due to its large, nonpolar R-groups. Isoleucine is an essential amino acid, just like valine and leucine. Disease of the maple syrup peeps occurs when their catabolism is flawed. There are four stereoisomers of isoleucine, with one of them being found in protein, and asymmetrical centers at the α- and β-carbons. Within water-soluble globular proteins, the large side chains often bind together. The polymer's three-dimensional structure is thereby stabilized by the hydrophobic amino acid residues.

The structure of Valine, Leucine, and Isoleucine. (Bhagavan N V., 2002)

Isoleucine pKa

The side chain of isoleucine is a hydrophobic branched alkyl group (–CH(CH₃)₂) that lacks a substantial pKa, as it does not ionize in physiological conditions. Consequently, it does not influence the ionization characteristics of the amino acid within a standard pH range. At physiological pH (~7.4), isoleucine primarily exists in its zwitterionic form. It possesses an α-Carboxyl group with a pKa of 2.35 and an α-Amine group with a pKa of 9.68.

Values of pKa for protonation of glutamine and isoleucine. (Kiani F., et al., 2014)

Isoleucine solubility

With a hydrophobic character of 1.83 and the ability to exist in solution as zwitterions, L-isoleucine is recognized as the most hydrophobic amino acid. The zwitterionic L-isoleucine molecule can be readily split into three different forms, depending on the pH of the solution: the neutral [RH^o] form, the ionized [RH⁺] form, and the [RH⁻] form. At pH 7, the preponderant species is this [RH^o] species, which possesses a negative charge from its carboxyl group and a positive charge from its amino group. The cationic [RH⁺] species is more common in acidic solutions, whereas the anionic [RH⁻] species, which possesses a negatively charged carboxyl group in its molecule, is more common in an alkaline environment.

Difference between leucine and isoleucine

While both leucine and isoleucine are basic branched-chain amino acids (BCAAs), their side chains and other characteristics set them apart from one another. Leucine has a straight side chain that branches at the second carbon. Isoleucine has a branching structure in its side chain that starts at the third carbon. Because of this subtle structural variation, leucine and isoleucine are structural isomers with distinct configurations, yet they exhibit distinct stereochemistry (three-dimensional spatial arrangement).

Leucine is well-known for its ability to activate the mTOR pathway, which is critical for the development and maintenance of muscle. It also plays a crucial role in protein synthesis. The part it plays in muscular anabolism (the process of growing muscle) is where it really shines. Many people think it's the most crucial BCAA for building muscle. While isoleucine does help with protein synthesis and muscle regeneration, it is mostly responsible for generating energy while you work out. It plays a role in the manufacturing of hemoglobin and is well-known for regulating blood sugar levels. Though it has a less role in promoting muscle growth than leucine, it is still essential for muscle metabolism.

Structural formula of leucine and isoleucine. (Giuseppe A., et al., 2022)

Pathway of branched chain amino acid catabolism. (Zhang S., et al., 2017)

Isoleucine amino acids at Creative Peptides

Isoleucine sources

The branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) are necessary amino acids that must be acquired through dietary sources. BCAAs serve as fundamental components for tissue protein, constituting 35% of the necessary amino acids in muscle, and possess other metabolic roles. In the average Polish diet, BCAAs were delivered by meat and meat products, which contributed 39.8% of leucine, 41.3% of isoleucine, and 37.4% valine. The other main food sources for BCAAs were grain products, and milk and dairy products. The detailed information indicated that processed red and poultry products, and bread, rolls and bread products were the main food group sources in the contribution of BCAAs. These findings should be taken into consideration when considering the quality of vegan, vegetarian and flexitarian diets due to the role of BCAAs in protein synthesis, which is widely described in the scientific literature. BCAA content is generally higher in animal proteins than plant proteins, with the highest level in red meat. While vegetables typically contain lower amounts of isoleucine compared to animal-based sources, some vegetables such as spinach, peas, and broccoli provide small amounts.

Food category sources of leucine, isoleucine and valine contribution to the average Polish diet. (Górska-Warsewicz H., et al., 2018)

What does isoleucine do?

Growing data suggests a significant correlation between amino acids and plasma glucose concentrations. Branched-chain amino acids have been shown to significantly improve glucose uptake and utilization. In an animal oral glucose tolerance test, both isoleucine and leucine inhibited an increase in plasma glucose levels, with isoleucine exhibiting a more pronounced impact than the other branched-chain amino acid. In a C2C12 myotube assay, both leucine and isoleucine enhanced glucose absorption. Nishitani's team and Doi's team obtained analogous results indicating that isoleucine facilitated plasma glucose absorption in rats. A hypothesis on the process by which isoleucine and leucine modulate serum glucose levels may involve an enhancement of muscle glucose uptake, increased whole-body glucose oxidation, and a reduction in hepatic gluconeogenesis. BCAAs have been extensively shown to improve glucose absorption by activating or up-regulating glucose transporters. Leucine enhances glucose absorption by upregulating the translocation of GLUT4 and GLUT1 in rat muscle tissue. Another experiment indicated that leucine promotes the expression of the GLUT4 glucose transporter and the absorption of 2-deoxyglucose in C2C12 cells. Researchers propose two ideas to elucidate the process by which leucine modulates muscle glucose transporters. Leucine promotes the translocation of GLUT1 and GLUT4 by increasing insulin levels. Secondly, leucine enhances glucose absorption in skeletal muscle through the PI3K and PKC signaling pathways, both of which are linked to GLUT4 translocation. Research on the mechanism of isoleucine's action is limited in comparison to that of leucine. Recent laboratory investigations indicate that administering an isoleucine-deficient diet to weanling pigs reduces the protein production of GLUT1 in red muscle and GLUT4 in red, white, and intermediate muscle. Moreover, an isoleucine-deficient diet inhibits the expression of the intestinal glucose transporter SGLT-1 in the duodenum, jejunum, and ileum, as well as GLUT2 in the duodenum and jejunum. The investigation further established the role of isoleucine in augmenting glucose absorption and the expression of muscle glucose transporters (GLUT1 and GLUT4) in C2C12 myotubes.

Isoleucine up-regulates intestinal and muscular transporters. (Zhang S., et al., 2017)

Isoleucine function

(1) Regulate immune function

Isoleucine consumption levels can influence the development of immunological organs. Hale et al. noted that an isoleucine-deficient diet could diminish the relative weight of the thymus in growing broilers. Aschkenasy concluded that a deficit of dietary isoleucine will hinder the development of immunological organs, such as the thymus, in growing rats. Petro and Bhattacharjee discovered that dietary isoleucine restriction in developing mice resulted in the atrophy of lymphoid organs, particularly the thymus, and a reduction in cell count inside the spleen. Conversely, optimal isoleucine levels in diets will enhance the development of immunological organs. Gong's team demonstrated that rats on a diet supplemented with 0.5-2.5% isoleucine had larger splenic follicles, a thicker lymphatic sheath encasing the splenic artery, and a greater density of lymphocyte arrangements in the spleen compared to those on a standard diet. Further studies suggest that isoleucine influences the development of immunological organs by serving as a substrate and energy source for protein synthesis. Isoleucine is a vital vitamin for the growth and functionality of immune cells. Burns indicated that isoleucine is essential for the maturation of many human leukocytes, including lymphocytes and neutrophils, primarily because it may be integrated into cellular proteins and lipids and oxidized via the Krebs cycle following decarboxylation. The in vitro experiments have demonstrated comparable outcomes. Chuang's team demonstrated that the exclusion of isoleucine from RPMI 1640 culture media significantly impedes the phytohaemagglutinin (PHA)-induced proliferation of lymphocytes generated from human mononuclear cells. Waithe et al. also discovered that the exclusion of isoleucine from RPMI 1640 culture media inhibits the PHA-induced cell cycle progression and proliferation in human lymphocytes. In broilers, an isoleucine-deficient diet reduced the percentage of CD8+ T lymphocytes in the blood and inhibited cutaneous basophil hypersensitivity to PHA-P, which could be restored by optimal dietary isoleucine levels. The inhibition of proliferation due to isoleucine deficit, as indicated by isotope use and analysis in in vitro investigations, mostly results from reduced protein synthesis caused by diminished substrate and energy levels in immune cells.

Isoleucine maintains the immunity in vivo. (Gu C., et al., 2019)

Effects of isoleucine on immunoreactive substances. (Gu C., et al., 2019)

(2) Regulate gut function

Alongside glutamine and asparagine, substantial quantities of branched-chain amino acids are ingested and metabolized in the colon. Branched-chain amino acids (BCAA) were metabolized in jejunal mucosal cells, exhibiting significant cytosolic branched-chain amino acid transaminase (BCAT) activity, with around 30% of the BCAA-derived branched-chain α-keto acids undergoing decarboxylation by branched-chain α-keto acid dehydrogenase (BCKD). Numerous studies have demonstrated that BCAA influences the expression of intestinal amino acid transporters. Administering 1.4 g L-leucine/kg body weight to breast-fed infants enhances intestinal growth and elevates the expression of neutral amino acid transporters (ATB^0,+, B0AT1, and b^0,+AT). Our laboratory study established that fulfilling BCAA requirements (supplementing 0.1% L-leucine, 0.34% L-Val, and 0.19% L-isoleucine in a 17% crude protein diet) is essential for preserving intestinal health and the expression of amino acid transporters, potentially via the PI3K/Akt/mTOR and ERK signaling pathways. Notably, we discovered that leucine enhanced the expression of neutral amino acid transporters (ASCT2, rBAT, and 4F2hc) as well as cationic amino acid transporters (CAT1), underscoring the significance of branched-chain amino acids in intestinal nutritional absorption.

Optimal dietary isoleucine enhanced growth performance, immunological barrier function, and physical barrier function of the gut in hybrid catfish. Furthermore, dietary Ile enhanced intestinal β-defensin production in hybrid catfish through the activation of the Sirt1/ERK1/2/p90rsk signaling pathway. The dietary isoleucine reduces cell apoptosis through the AKT/FOXO3a signaling pathway. The results offered limited theoretical support for the enhancement of intestinal immunological and physical barrier functions by isoleucine in fish. Finally, the quadratic regression analysis of PWG calculated the dietary Ile need for hybrid catfish to be 12.43 g/kg of the diet, equating to 32.05 g/kg of dietary protein.

(3) Regulate metabolic function of the body

The energy consumption varies based on the makeup of amino acids in mixed solutions. The identification of amino acids that mitigate hypothermia in a mixed amino acid solution was achieved by eliminating certain amino acids from the mixture. Consequently, researchers discovered that the elimination of BCAAs led to a decrease of hypothermic effects; hence, valine, leucine, and isoleucine were deemed essential for these thermal effects. Leucine is recognized for its role in stimulating protein synthesis through the activation of mTOR. Conversely, isoleucine could promote thermogenesis through glucose metabolism. Leucine and isoleucine promote glucose absorption independently of insulin. This activity is more pronounced for isoleucine compared to leucine, and the reaction occurs via phosphoinositol 3-kinase, excluding mTOR involvement. Moreover, isoleucine is documented to suppress gluconeogenesis and promote the oxidative use of systemic glucose. Isoleucine enhances glucose uptake and oxidative utilization in skeletal muscle while inhibiting gluconeogenesis in the liver.

(4) Isoleucine increases muscle mass

A total of thirty-two male C57BL/6J mice were randomly assigned to one of two groups and subjected to a high-fat diet (HFD) with or without 25 g/L of isoleucine (Ile). Muscle and fat mass were both dramatically boosted by Ile, which also caused insulin resistance and upregulated levels of important adipogenic and myogenic proteins, according to the data. Moreover, Ile disrupted mitochondrial activity through tibialis anterior (TA) and gastrocnemius (GAS) cristae fracture, swelling, and vacuolation, all while lowering expression of genes associated with mitochondrial function. In addition, myogenesis was enhanced and myotubes accumulated more lipid droplets when Ile was present. Several proteins were found to be upregulated in the Ile group when compared to the control group. These proteins include myosin heavy chain (MyHC), myoblast determination protein 1 (MyoD), myogenin (MyoG), PPARg, and FAS. On the other hand, adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL) had their protein levels downregulated. The combined effects of Ile on myogenesis and intramyocellular lipid deposition resulted in an increase in muscle mass. The results give light on a new way to modulate myopathies in obese animals and on how to increase intramyocellular lipid accumulation, two factors that contribute to the lean juiciness of farmed animals.

Isoleucine (Ile) increases muscle mass. (Liu S., et al., 2021)

FAQ

Is Isoleucine basic or acidic?

Isoleucine is an amino acid that is not acidic or basic; it is neutral. The hydrophobic side chain of isoleucine is composed of a branched alkyl group (-CH(CH₃)₂) and is nonpolar. The molecule's acidity or basicity would not be affected by the presence of any ionizable groups on this side chain. Because the amine group's positive charge and the carboxyl group's negative charge cancel each other out, isoleucine takes on a zwitterionic (neutral) form at physiological pH, meaning its net charge is zero.

Is isoleucine polar or nonpolar?

Isoleucine possesses a hydrophobic side chain characterized by a branched alkyl group (–CH(CH3)2). Alkyl groups are often nonpolar due to their composition of carbon and hydrogen atoms, which share electrons almost equally, exhibiting little electronegativity disparities. The hydrophobic side chain of isoleucine renders it nonpolar, leading to its tendency to evade interactions with water molecules. This feature suggests a propensity for localization into the inner of proteins or in hydrophobic environments. Isoleucine is categorized as a nonpolar amino acid.

References

1. Bhagavan N V. Chapter 2-Amino Acids, Medical Biochemistry, 2002: 17-33.
2. Kiani F., et al., Determination of acidic dissociation constants of glutamine and isoleucine in water using ab initio methods, Turkish Journal of Biochemistry/Turk Biyokimya Dergisi, 2014, 39(4).
3. Giuseppe A., et al., Preliminary study for the application of Raman spectroscopy for the identification of Leishmania infected dogs, Scientific Reports, 2022, 12(1): 7489.
4. Zhang S., et al., Novel metabolic and physiological functions of branched chain amino acids: a review, Journal of animal science and biotechnology, 2017, 8: 1-12.
5. Górska-Warsewicz H., et al., Food products as sources of protein and amino acids—The case of Poland, Nutrients, 2018, 10(12): 1977.
6. Gu C., et al., Isoleucine plays an important role for maintaining immune function, Current Protein and Peptide Science, 2019, 20(7): 644-651.
7. Zhao Y., et al., Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli× Leiocassis longirostris, Fish & Shellfish Immunology, 2021, 109: 20-33.
8. Liu S., et al., Isoleucine increases muscle mass through promoting myogenesis and intramyocellular fat deposition, Food & function, 2021, 12(1): 144-153.