Figure 1. Lipocalin signaling pathway
An overview of lipocalin
Lipocalin protein belongs to a large class of adipocytokines, and lipocalin family proteins include LCN2, LCN6, LCN9, LCN11-13, retinol-binding protein, α-microglobulin, α1 acid glycoprotein, insect bile pigment binding protein and β-lactoglobulin, etc. All have a homologous beta barrel structure. More and more studies have shown that the lipocalin protein family is involved in the development of many diseases, such as obesity, insulin resistance, atherosclerosis, etc., and LCN2 is a hot topic in the family. Experiments show that it is involved in the development and progression of inflammation, obesity, insulin resistance, metabolic diseases, and cardiovascular diseases. Increased expression of LCN2 in obesity and metabolic diseases is associated with coronary sclerosing heart disease, severity of atherosclerosis, and mortality in patients with heart failure, suggesting the role of LCN2 as a marker for the diagnosis and prognosis of metabolic-related cardiovascular diseases. Cell and animal experiments suggest that LCN2 may affect cardiovascular function by participating in various ways such as inflammatory reaction, glycolipid metabolism and action on myocardial and vascular endothelium, which may be an important part of the relationship between obesity and cardiovascular disease. Existing studies have contradicted the role of LCN2 in inflammatory responses and insulin resistance, and the interaction of LCN2 with other adipokines and inflammatory factors is still unclear and needs further study.
Studies have shown that the Lipocalin family share some commonalities: the spatial structure of the beta barrel consisting of eight folded sheets; the presence of a pair of conserved disulfide bonds stabilizes the structure of the protein. Studies have found that these lipocalin proteins change spatial structure and kinetics through changes in pH, binding and transporting ligands at high pH and releasing ligands at low pH. In the lipocalin family, the most thoroughly studied is LCN2. LCN2, also known as neutrophil gelatinase-associated lipocalin (NGAL) or tropoprotein, is mainly secreted by fat cells, and other liver cells, activated lymphocytes and various epithelial cells, such as airway epithelium, lung epithelium, gastric mucosal epithelial cells, etc. There are three forms of LCN2 in vivo: monomers, homodimers, and heterodimers that bind to matrix metalloproteinases (MMPs). LCN2 belongs to the family of lipocalins, which are capable of binding to hydrophobic small molecules and certain cell membrane receptors, as well as complexes with soluble macromolecules. LCN2 has the same protein folding pattern as other members of the family, but it has a unique, large and polar binding site. The binding of LCN2 to the common ligands of the lipocalin family such as leukotriene B4 and platelet activating factor is weak, and its high-affinity endogenous ligand has not been found yet. The study found that LCN2 is involved in the innate immunity of bacterial infection by regulating iron metabolism. In addition, LCN2 can induce a variety of apoptosis, suggesting that it is involved in the tumor process.
Lipocalin signaling pathway
Lipocalin signaling pathway cascade
The most important LCN signal pathway cascades is the LCN2 signaling cascade. LCN2 is elevated in the inflammatory response and inflammatory state. Macrophages activate the inflammatory pathway of hepatocytes and induce a 17-fold increase in LCN2 levels. A range of pro-inflammatory factors promote LCN2 secretion, such as lipopolysaccharide, interleukin (IL)-1β, IL-17 and tumor necrosis factor alpha (TNF-α). In primary renal fibroblasts, lipopolysaccharide induces LCN2 expression via the activator protein 1 (AP-1) pathway, and CCAAT/enhancer binding protein δ (C/EBPδ) pathway induces longer-lasting expression of LCN2. Both pathways are dependent. IL-1β induces LCN2 mRNA expression via a nuclear factor κB (NF-κB) pathway. Similarly, in human and rat vascular smooth muscle cells, the increase in LCN2 mRNA and protein expression levels is also dependent on the NF-κB pathway. An in vitro test suggests that LCN2 has an anti-inflammatory effect. LCN2 can counteract the effects of TNF-α on adipocytes and macrophages then reduce the expression of inflammatory factors in lipopolysaccharide-stimulated macrophages. At the same time, LCN2 increases the levels of adiponectin and leptin secretion, increases fatty acid synthesis and lipoprotein lipase in fat cells by up-regulating the levels of peroxisome proliferator-activated receptor gamma (PPAR-γ) and its target genes expression and synthesis. In its subsequent in vivo experiments, LCN2 knocked out the up-regulation of monocyte chemoattractant protein 1 (MCP-1) and TNF-α expression in mouse adipose tissue. However, the results of some other animal experiments were reversed: the inflammatory markers and lipid peroxidation products in the adipose tissue of LCN2 knockout mice were significantly reduced. After increasing age or giving a high-fat diet, 12-lipoxygenase expression was increased in mouse adipose tissue, whereas in LCN2-extracted mice, 12-lipoxygenase expression was decreased and TNF-α production was decreased. Further studies have shown that IL-6 expression is decreased in adipose tissue of female LCN2 knockout mice. The number of infiltrating granulocytes after reperfusion after heart transplantation was also reduced in LCN2 knockout mice, suggesting that LCN2 may have an initial role in inflammatory responses. Cell and animal experiments have shown that LCN2 plays an important role in the development of insulin resistance. In the 3T3-L1 adipocyte cell line, the decrease of LCN2 expression level by RNA interference can enhance glucose uptake under insulin stimulation, while the administration of exogenous recombinant LCN2 can increase hepatocyte glucose production. These phenomena suggest that LCN2 may play a role in the formation of insulin resistance and hyperglycemia. In obese animal models, circulating LCN2 levels are elevated and LCN2 mRNA expression is increased in liver tissue. In the case of aging, high-fat-induced obesity, and obesity caused by gene defects, fasting blood glucose and circulating insulin levels in LCN2 knockout mice were significantly lower than that in wild-type mice, and insulin sensitivity was elevated. The protective effect of LCN2 knockout on insulin sensitivity is related to the regulation of 12-lipoxygenase and TNF-α in adipose tissue. However, in two other animal experiments given a high-fat diet, LCN2 knockout mice had different glycemic phenotypes. One of the studies found that after LCN2 was removed, the glucose tolerance of male mice given a high-fat diet is improved, but the insulin sensitivity of both female and male LCN2 knockout mice did not increase. Another study found that LCN2 was excluded. It exacerbates the harmful effects of high-fat diet such as dyslipidemia, fatty liver, insulin resistance and decreased mitochondrial oxidative capacity. The researchers believe that the results of the study may be related to two points. First, the resection range of the lcn2 exon is different in different experiments, which may be the main cause of different phenotypes. Secondly, the components of the high-fat diet given by different experiments, together with the temperature and cleanliness of the feeding environment, are different, causing differences in the level of lipopolysaccharide in the circulation, which may be one of the reasons for the contradictory results. In conclusion, although LCN2 is directly linked to insulin resistance, the specific mechanism of action remains to be further studied. Previous studies have shown that post-translational modifications may be involved in the regulation of LCN2 secretion and influence its role in metabolic stress. LCN2 is a selective regulator of PPAR-γ activation. Rosiglitazone improved the sensitivity of insulin in wild-type and LCN2 knockout mice. After treatment with rosiglitazone for 25 days, fasting blood glucose, fasting non-esterified fatty acids, and fasting triglycerides were all decreased in LCN2 knockout mice, but the glyceride-induced fat deposition and regulation of fatty acid homeostasis, such as increased body mass, promotion of subcutaneous fat tissue and liver fat accumulation, and fat synthesis, were significantly decreased after LCN2 rejection. Ketones are involved in the promotion of fat synthesis and in the induction of the uptake and oxidation of non-esterified fatty acids in brown adipose tissue. The results of this study indicate that LCN2 is involved in the regulation of energy consumption and homeostasis of lipid metabolism during obesity. Apolipoprotein E knockout and low-density lipoprotein receptor knockout mice spontaneously produce atherosclerosis, during which LCN2 expression is significantly elevated in atherosclerotic plaques. In the rat carotid injury model, the carotid intima of rats after angioplasty highly expresses LCN2 by activating NF-κB pathway. The increase in LCN2 expression in cardiomyocytes is associated with stimulation of the innate immune system such as Toll-like receptors 2 and 4, IL-1β, X-ray and H2O2. IL-1β also stimulates vascular smooth muscle cells to secrete LCN2. The elevation of local LCN2 levels is involved in the development of atherosclerosis. LCN2 coexists with matrix metalloproteinases-9 (MMP-9) in rat atherosclerotic plaques and damaged intima. Immunohistochemistry of human carotid endarterectomy specimens demonstrated that macrophages coexisted with elevated LCN2 and MMP-9. In atherosclerotic plaques, especially in plaques with internal hemorrhage and central necrosis, the expression of LCN2 and MMP-9 is up-regulated in neutrophils. MMP-9 is an enzyme involved in the degradation of extracellular matrix, and its degradation of extracellular matrix exists in a variety of physiological processes, such as embryonic development, reproductive physiology and tissue remodeling. At the same time, MMP-9 is also considered to be a key enzyme in chronic inflammation and atherosclerosis. In human atherosclerotic plaques, elevated levels of LCN2 and LCN2/MMP-9 complexes are paralleled by a series of changes, including elevated IL-6, IL-8, increased lipid content and macrophages. The zymography also demonstrated that the regions rich in LCN2 and MMP-9 in the plaques showed high proteolytic activity. LCN2 binds to MMP-9 to form a stable heterodimer, thereby inhibiting the inactivation of MMP-9, resulting in the hydrolysis of MMP-9 and the enhancement of collagen degradation. LCN2 and MMP-9 complexes regulate extracellular matrix reorganization and may also mediate myocardial fibrosis and vascular damage. The systolic blood pressure of wild mice increased after administration of a high-fat diet, whereas LCN2 knockout mice did not change this, and the mechanism was associated with improved endothelial dysfunction after LCN2 knockout, which was shown to give insulin stimulation after LCN2 knockout mice had a high-fat diet. Injecting exogenous LCN2 into mice can lead to uncoupling of endothelial nitric oxide synthase and increased expression of cyclooxygenase in arterial blood vessels, and the improvement is neutralized. Endothelial dysfunction caused by LCN2 may be involved in the regulation of cytochrome C450 activity. A recent study found that LCN2 is expressed in endothelial cells of the cerebral blood vessels and is partially expressed by cyclooxygenase 2 after lipopolysaccharide stimulation, and the expression of cyclooxygenase 2 mRNA is decreased in female LCN2 knockout mice. LCN2 and myocardial injury studies have found that LCN2 directly induces apoptosis in rat cardiomyocytes by increasing intracellular iron content and increasing the translocation of proapoptotic protein Bax. After 14 days of injection of recombinant LCN2 into mice, the apoptosis of cardiomyocytes increased and caused an acute inflammatory reaction, which further led to changes in compensatory cardiac function parameters. In rat cardiomyocytes with post-infarction heart failure, the expression level of LCN2 also increased, and the expression of LCN2 in non-ischemic myocardium increased with the time after infarction, suggesting that LCN2 is involved in the development of chronic heart failure. In cardiomyocytes, overexpression of mineralocorticoid receptor strongly induced LCN2 gene expression in mouse myocardium. Injection of aldosterone into mice also increased LCN2 expression, suggesting that LCN2 may become a marker of mineralocorticoid-dependent cardiovascular damage.
The transcription of the lipocalin gene is regulated by a variety of nuclear factors. Interleukin-1 (IL-1) activates nuclear factor (NF)-κB, while the latter binds to the lipocalin-2 gene. At this point, the promoter rapidly induces gene transcription of peritoneal macrophages. In atherosclerosis, IL-1 can induce the expression of this gene through the above pathways, and it is also found that vascular smooth muscle cells also induce the expression of mRNA by activating NF-κB pathway under injury. Yan et al. demonstrated that tumor necrosis factor alpha (TNF-α) and dexamethasone (DEX) can induce the expression of lipocalin-2 gene in adipocytes by using microarray experiments. The former can increase the lipocalin-2 expression level by 30-fold. The latter can increase lipocalin-2 level by 80 times, while the insulin sensitizer rosiglitazone has the opposite effect.
Relationship with disease
The experimental results suggest that serum lipocalin-2 levels are elevated in patients with increased body fat and may be associated with metabolic abnormalities associated with obesity. However, the exact source of elevated lipocalin-2 still requires more animal and clinical trials.
Both in vitro and clinical studies suggest that lipocalin-2 may have an insulin-promoting effect, may be an independent risk factor for elevated fasting blood glucose and insulin resistance. However, the molecular mechanism of insulin resistance induced by lipocalin-2 is still unclear and needs further study.
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Xing C, Wang X, Cheng C, et al. Neuronal production of lipocalin-2 as a help-me signal for glial activation. Stroke. 2014, 45(7):2085-2092.
Guo H, Jin D, Chen X. Lipocalin 2 is a regulator of macrophage polarization and NF-κB/STAT3 pathway activation. Molecular Endocrinology. 2014, 28(10):1616-28.
Wei N. Role of Lipocalin-2 in Brain Injury after Intracerebral Hemorrhage. Journal of Cerebral Blood Flow & Metabolism Official Journal of the International Society of Cerebral Blood Flow & Metabolism. 2015, 35(9):1454.
Mesquita S D, Ferreira A C, Falcao A M, et al. Lipocalin 2 modulates the cellular response to amyloid beta. Cell Death & Differentiation. 2014, 21(10):1588-99.
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