Alliin, a Garlic (Allium sativum) Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes
Abstract (Garlic Extract)
Garlic (Allium sativum L.) has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide), a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS-) stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 μmol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile.
In conclusion, the results presented here demonstrate the possible mechanism by which alliin, a garlic compound, controls the inflammatory state of adipocytes by decreasing IL-6 and MCP-1 expressions (both at mRNA and protein levels), as well as diminishing ERK1/2 phosphorylation in LPS-stimulated 3T3-L1 adipocytes and generating an anti-inflammatory gene expression profile in adipocytes and modifying their metabolic profile.
Additionally, we confirmed that alliin modifies the mRNA expression of genes involved in phospholipid and organophosphate metabolic processes, in positive regulation of the related immune process (expressing immunoglobulin (Ig) and in T-cell receptor-related genes), some enzymes for metabolic and energy process, and genes involved in cancer. All of these processes can be somehow involved in adipocyte protection against a proinflammatory stimulus; thus, they constitute interesting groups of genes to be further explored as involved in adipocyte physiology derived from the alliin action. A deeper understanding of the mechanisms that regulate anti-inflammatory signaling in adipocytes by alliin action may contribute to unraveling possible treatments for obesity-induced inflammation and insulin resistance.
Obesity has been traditionally linked to metabolic dysfunction, led by adipocyte proliferation and hypertrophy . However, some other systemic alterations are being studied as related to obesity, for example, inflammation and immune dysfunction . Adipose tissue is now widely considered as an endocrine tissue capable of producing chronic inflammatory responses [3–5]. Obesity has been shown to cause an increase in plasma concentrations of a number of proinflammatory markers (e.g., IL-6, TNF-α) that are expressed and released by adipocytes . Diet-induced obesity increases local and systemic inflammatory adipocytokines in humans and in rodents; these factors contribute to adverse health outcomes . The homeostatic balance between pro- and anti-inflammatory cytokines and adipokines defines the profile and magnitude of inflammation and its effects on insulin sensitivity and glucose homeostasis . Therefore, therapies able to modulate the inflammatory state of adipose tissue are being considered for the treatment of obesity . However, factors that might mitigate or act against this inflammatory response have remained elusive.
Garlic (Allium sativum) is one of the oldest medicinal plants used by different cultures . Allium vegetables comprise one natural source of organic sulfur-containing compounds and have been widely investigated regarding their therapeutic applications [11, 12], mainly due to its cardioprotective effect and to its anticancerogenic properties [10, 13]. However, its anti-inflammatory effects have received less attention. For example, garlic compounds can exert an anti-inflammatory effect by inhibiting the oxidative stress-induced activation of nuclear factor-kappa B (NF-κB) , which is implicated in the expression of proinflammatory enzymes such as inducible nitric oxide synthase (NOS) and cyclooxygenase-II (COX-II). In particular, allicin and ajoene, two garlic compounds, significantly suppressed nitric oxide production by lipopolysaccharide (LPS) stimulation, accompanied by suppression in inducible nitric oxide (iNOS) expression and iNOS activity . Although it appears promising that garlic and its derivatives possess antidiabetic potential, an understanding of the antidiabetic effects of garlic is still in its early stages. Furthermore, the active compounds in garlic, and doses thereof, which can effectively provide antidiabetic effects (i.e., glycemic control and amelioration of diabetic complications) remain to be established.
Alliin (S-allyl cysteine sulfoxide), first identified by Stoll and Seebeck in 1947, is considered the main specific principle of garlic since that time . Alliin can be found in intact garlic and its reduced form; S-allyl-cysteine is the major component of aged garlic extract (AGE), together with other derived organosulfur compounds [12, 13]. It is absorbed in the intestine via the amino acid transported for cysteine , exhibits an hypoglycemic effect, and also increases blood insulin concentrations , and its antioxidant activity has been widely studied . Nonetheless, to our knowledge, its anti-inflammatory potential has not yet been explored.
Mouse preadipocyte 3T3-L1 cells are the most commonly studied and available adipogenic cell line. This cell line has been quite useful in identifying key molecular markers, transcription factors, and various interactions that are required for preadipocyte differentiation . Additionally, 3T3-L1 adipocytes are able to respond to LPS by means of a fully intact innate immunity pathway, through the production and secretion of immunomodulatory (mainly proinflammatory) molecules such as IL-6, TNF-α, and TLR-2 , and through a TLR-ligand-induced activation, which triggers a proinflammatory and -diabetic transformation of adipocytes . Interestingly, this immune response activation is resembled by human adipose tissue ; consequently, once differentiated, 3T3-L1 appears to be a suitable model to test the anti-inflammatory response that affects the metabolic regulation of adipocytes elicited by chemical compounds and nutraceutics under controlled conditions [24–27]. Thus, to determine the anti-inflammatory effect of alliin, in the present work we used the LPS-stimulated 3T3-L1 cell line as an inflammatory model in vitro. Our findings indicate that alliin prevents LPS-induced inflammation by inhibiting ERK1/2 in 3T3-L1 adipocytes.
3.1. Alliin Pretreatment Significantly Reduces the mRNA Expression and Protein Levels of Proinflammatory Molecules IL-6 and MCP-1 after LPS Exposure in 3T3-L1 Adipocytes
Previously, we determined the alliin concentration that exerts an effect on the expression of the tested genes; the concentrations probed were 0.1, 0.3, 0.6, and 1.0 mM (data not shown). From this we selected 0.1 mM as the minimum concentration able to elicit a clear effect.
Cytokine IL-6 is correlated with insulin resistance in subjects with obesity and is inducible through TLR-4 receptor activation . After the alliin pretreatment, mRNA levels for IL-6 were significantly reduced (Figure 1(a)). In contrast, the level of TNF-α mRNA was apparently not significantly affected, although a slight tendency toward its decrease in alliin pretreated cells was also noted.
Additionally, we checked for MCP-1 expression because it is produced by a variety of cells, including adipocytes, in response to inflammatory stimuli . As expected, we found a significant increase in MCP-1 expression in LPS-treated adipocytes. Interestingly, we again observed a significant reduction in MCP-1 mRNA levels when LPS-stimulated adipocytes were pretreated with alliin (Figure 1(c)).
Furthermore, we verified the expression of Egr-1, which is described as induced by cytokines and hormones through activation of the MAPK pathway and which are related with insulin resistance . Once again, the mRNA expression level was significantly reduced by alliin pretreatment even after the LPS proinflammatory stimulus (Figure 1(d)).
To corroborate these results, we evaluated the secreted protein levels of these cytokines and determined their release into the culture media by ELISA. Protein levels detected after the LPS stimulus, which are significantly reduced by alliin pretreatment, are shown in the case of IL-6 (Figure 2(a)) and Mcp-1 (Figure 2(d)). Moreover, we observed a reduction in TNF-α levels (Figure 2(b)), although this was small and did not reach statistical significance. Additionally, we tested for adiponectin levels (Figure 2(c)) because this represents an important union between obesity and insulin resistance and is considered as an anti-inflammatory protein . The control group of adipocytes secretes a large amount of adiponectin (Figure 2(c)), which is clearly reduced by LPS stimuli. In the group pretreated with alliin, a slight increase can be observed in the production of this protein; however, it cannot overcome the severe reduction elicited by LPS.
3.2. Alliin Exerts Its Anti-Inflammatory Effect at Least through Diminishing the Phosphorylation of ERK1/2
Since LPS induces inflammation in adipocytes through ERK1/2  and IL-6 and Egr-1 intracellular signaling mechanisms converge in this pathway, we next examined whether alliin pretreatment affects ERK1/2 phosphorylation. LPS stimulus is able to increase the protein levels of phosphorylated ERK1/2, and alliin pretreatment overwhelms this effect by significantly reducing this level, to nearly reach control levels
3.3. Gene Expression Profile of Alliin Pretreated 3T3-L1 Cells after LPS Stimulus Is Consistent with a Shift in Cell Response to Inflammatory Stimulus and Reveals Alliin Action on Adipocyte Physiology
Saray Quintero-Fabián,1,2 Daniel Ortuño-Sahagún ,1 Manuel Vázquez-Carrera,3 and Rocío Ivette López-Roa
Research Article | Open Access
Volume 2013 |Article ID 381815 |