TLR4: Contributing to Metabolic Syndrome in Multiple Tissues: R&D Systems

Obesity has increased dramatically in the United States with serious health consequences.¹ The metabolic consequences of obesity, often termed metabolic syndrome, include lipotoxicity, inflammation, and insulin resistance, which increase the risk of type II diabetes, atherosclerosis, hypertension, and cardiovascular disease. Obesity-related lipotoxicity is thought to result from overloading the body's mechanisms for dealing with dietary lipids. Under normal physiological conditions, adipocytes efficiently convert dietary fatty acids to triglycerides, which can be safely stored in fat droplets. Excess lipids in the form of diacylglycerides and ceramides create stress within the cell, while excess free fatty acids enter the circulation. Adipose tissue hypoxia likely increases oxidative stress on the endoplasmic reticulum, contributing to the unfolded protein response.¹ Kinases including IKK (IkB Kinase) and JNK (c-Jun N terminal Kinase) are activated, leading to the secretion of inflammatory cytokines and inhibitory phosphorylation of IRS-1 (Insulin Receptor Substrate 1), which promotes insulin resistance.¹ During the investigation of these signaling pathways, it has become increasingly apparent that the toll-like receptors (TLRs), and in particular TLR4, may be involved in their initiation.^1,²

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TLR4 Activation by Fatty Acids Triggers a Cellular Response that Promotes Insulin Resistance. In adipose, muscle, brain, and liver tissue, activation of TLR4 by long-chain saturated fatty acids (FA) in either macrophage-lineage cells or resident cells initiates signaling that influences cellular metabolism. An increase in PKC activity leads to the phosphorylation and activation of IKK. IKK phosphorylates IkB, resulting in the nuclear translocation of NFkB, which promotes the transcription of inflammatory cytokines. Formation of diacylglycerols (DAG) and ceramides from internalized fatty acids leads to oxidative stress and the unfolded protein response (UPR). In addition, TLR4 activation stimulates the phosphorylation and activation of JNK, resulting in inhibitory phosphorylation of IRS-1 and insulin resistance.

TLRs are mainstays of the innate immune system. Mammalian TLRs include intracellular and extracellular receptors that recognize microbial proteins, nucleic acids, carbohydrates, and lipids to activate host defense mechanisms. TLR4 is a cell surface, transmembrane protein that primarily recognizes bacterial lipopolysaccharides. At sufficiently high concentrations, endogenous lipids can also be recognized by TLR4.² TLR4, and to a lesser extent TLR2, are candidates for activating the immune system in response to a diet rich in long-chain saturated fatty acids. In 2007, a pivotal study demonstrated that mice with a loss-of-function mutation in TLR4 resist becoming obese on a high-fat diet.³ This places TLR4 among several other proteins whose inactivation can protect against high fat diet-induced insulin resistance.

TLR4 is expressed on macrophages, which are recruited by inflammatory cytokines in tissues such as obese adipose tissue. In addition, it is also expressed on resident, non-immune cells, such as adipocytes and muscle myocytes.^4,⁵ Muscle accounts for most insulin-stimulated glucose use and is therefore a key insulin target tissue. Treatment of isolated muscle with long-chain saturated fatty acids such as palmitate activates both JNK- and IKK/NF kappa B-mediated pathways, promotes insulin resistance, and enhances cellular output of inflammatory cytokines such as IL-6, CCL2/JE/MCP-1, and TNF-alpha.^3,⁶ In contrast, muscle deficient in TLR4 activity lacks these effects.^3,⁶ In humans, recent studies show that TLR4 expression is upregulated in muscle or adipose tissue from obese or type II diabetic subjects, and this increase correlates with insulin resistance.^7,⁸ In addition, there is an increase in TLR4-mediated NF kappa B activation. Interestingly, monounsaturated fatty acids such as olein, which is enriched in olive oil, appear to neutralize the effects of palmitate and thus have a positive rather than a negative effect.⁹ Exercise also has a positive effect, downregulating the expression of TLR4 and decreasing palmitate accumulation in human muscle.¹⁰

New data also links activation of TLR4, and possibly TLR2, on macrophages or their tissue-specific relatives, microglia and Kupffer cells, to obesity-related events in their respective tissues. In the brain, TLR engagement increases cytokine expression, stress in the endoplasmic reticulum, and leptin resistance. These effects stimulate systemic inflammation, which promotes atherosclerosis in blood vessels and contributes to non-alcoholic fatty liver disease.^1,^11,^12,¹³ While far from being the only cell surface molecule involved, TLR4 does seem to link many known signaling pathways involved in the metabolic consequences of obesity.