Deficiency of p62/Sequestosome 1 causes hyperphagia due to leptin resistance in the brain.

The cytoplasmic regulatory protein p62 (Sequestosome 1/A170) is known to modulate various receptor-mediated intracellular signaling pathways. p62 deficiency was shown to result in mature-onset obesity in mice, but the mechanisms underlying this abnormality remained unclear. Here we report that hyperphagia due to central leptin resistance is the cause of obesity in p62(-/-) mice. We found that these mice show hyperphagia. Restriction of food to the amount eaten by wild-type mice prevented excess body weight gain and fat accumulation, suggesting that overfeeding is the primary cause of obesity in p62(-/-) mice. Brain-specific p62 deficiency caused mature-onset obesity to the same extent as in p62(-/-) mice, further supporting a neuronal mechanism as the major cause of obesity in these mice. Immunohistochemical analysis revealed that p62 is highly expressed in hypothalamic neurons, including POMC neurons in the arcuate nucleus. Central leptin resistance was observed even in young preobese p62(-/-) mice. We found a defect in intracellular distribution of the transcription factor Stat3, which is essential for the action of leptin, in p62(-/-) mice. These results indicate that brain p62 plays an important role in bodyweight control by modulating the central leptin-signaling pathway and that lack of p62 in the brain causes leptin resistance, leading to hyperphagia. Thus, p62 could be a clinical target for treating obesity and metabolic syndrome.

Enhanced neointimal hyperplasia and carotid artery remodelling in sequestosome 1 deficient mice.

Deficiency in the signal adaptor protein sequestosome 1 (SQSTM1/A170/p62) in mice is associated with mature-onset obesity, accompanied by insulin and leptin resistance. We previously established that redox sensitive transcription factor Nrf2 up-regulates SQSTM1 expression in response to atherogenic stimuli or laminar shear stress in vascular cells, and here examine the role of SQSTM1 in neointimal hyperplasia and vascular remodelling in vivo following carotid artery ligation. Neointimal hyperplasia was markedly enhanced at ligation sites after 3 weeks in SQSTM1(-/-) compared with wild-type (WT) mice. The intimal area and stenotic ratio were, respectively, 2.1- and 1.7-fold higher in SQSTM1(-/-) mice, indicating enhanced proliferation of vascular smooth muscle cells (SMCs). When aortic SMCs were isolated from WT and SQSTM1(-/-) mice and cultured in vitro, we found that SQSTM1(-/-) SMCs proliferated more rapidly in response to foetal calf serum (FCS) and attained 2-3-fold higher cell densities compared to WT SMCs. Moreover, migration of SQSTM1(-/-) SMCs was enhanced compared to WT SMCs. Early and late phases of p38(MAPK) activation in response to FCS stimulation were also more enhanced in SQSTM1(-/-) SMCs, and inhibitors of p38 and ERK1/2 signalling pathways significantly attenuated SMC proliferation. In summary, SQSTM1(-/-) mice exhibit enhanced neointimal hyperplasia and vascular remodelling following arterial ligation in vivo. The enhanced proliferation of SQSTM1(-/-) aortic SMCs in vitro highlights a novel role for SQSTM1 in suppressing smooth muscle proliferation following vascular injury.

The alpha-glucosidase inhibitor acarbose prevents obesity and simple steatosis in sequestosome 1/A170/p62 deficient mice.

AIM: Sequestosome 1 (SQSTM1)/A170/p62 plays an important role in membrane-receptor mediated signal transduction and autophagic protein degradation. Although the mechanism involved is not clear, sqstm1 gene knockout (KO) mice develop mature-onset obesity and insulin resistance, leading to type II diabetes. KO mice show accumulation of fat in white adipose tissue and the liver when fed a standard diet. Acarbose is an alpha-glucosidase inhibitor that improves insulin sensitivity and decreases postprandial hyperglycemia, and it is used to treat type 2 diabetes. We examined whether or not dietary acarbose prevented obesity and simple steatosis in KO mice. METHODS: Wild-type (WT) and KO mice were fed a standard diet with or without acarbose (0.8% w/w) from 15-25 weeks of age. The body weight and the fat content of adipose tissue and the liver were measured, and changes of lipid metabolism in these tissues were assessed from gene expression. RESULTS: Acarbose treatment suppressed weight gain and the development of hepatic steatosis in KO mice. Acarbose treatment up-regulated hepatic expression of the pparalpha, ucp-2, and abca1 genes, as well as srebp1c, pparalpha, and ppargamma in adipose tissue. In WT mice, however, acarbose treatment had little influence on weight gain and gene expression. CONCLUSIONS: The results of this study suggest that long-term administration of acarbose is effective for prevention of obesity and simple steatosis in SQSTM1-KO mice.

Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice.

Inactivation of constitutive autophagy results in formation of cytoplasmic protein inclusions and leads to liver injury and neurodegeneration, but the details of abnormalities related to impaired autophagy are largely unknown. Here we used mouse genetic analyses to define the roles of autophagy in the aforementioned events. We report that the ubiquitin- and LC3-binding protein "p62" regulates the formation of protein aggregates and is removed by autophagy. Thus, genetic ablation of p62 suppressed the appearance of ubiquitin-positive protein aggregates in hepatocytes and neurons, indicating that p62 plays an important role in inclusion body formation. Moreover, loss of p62 markedly attenuated liver injury caused by autophagy deficiency, whereas it had little effect on neuronal degeneration. Our findings highlight the unexpected role of homeostatic level of p62, which is regulated by autophagy, in controlling intracellular inclusion body formation, and indicate that the pathologic process associated with autophagic deficiency is cell-type specific.

Tissue Prx I in the protection against Fe-NTA and the reduction of nitroxyl radicals.

Peroxiredoxin I (Prx I) is a key cytoplasmic peroxidase that reduces intracellular hydroperoxides in concert with thioredoxin. To study the role of tissue Prx I in protection from oxidative stress, we generated Prx I-/- mice by gene trapping. We then evaluated the acute-phase tissue damage caused by ferric-nitrilotriacetate (Fe-NTA). Increases in serum aspartate aminotransferase and alanine aminotransferase levels were significantly greater in Prx I-/- than wild-type mice, 4 and 12 h after the injection of Fe-NTA. Using real-time EPR imaging, we examined the reduction of the stable paramagnetic nitroxyl radical 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl in vivo, and found that the half-life of this spin probe in the liver and kidney was significantly prolonged in the Prx I-/- mice. These results demonstrate that Prx I-/- mice have less reducing activity and are more susceptible to the damage mediated by reactive oxygen species in vivo than wild-type mice.

Nrf2 deficiency causes tooth decolourization due to iron transport disorder in enamel organ.

Rodents have brownish-yellow incisors whose colour represents their iron content. Iron is deposited into the mature enamel by ameloblasts that outline enamel surface of the teeth. Nrf2 is a basic region-leucine zipper type transcription factor that regulates expression of a range of cytoprotective genes in response to oxidative and xenobiotic stresses. We found that genetically engineered Nrf2-deficient mice show decolourization of the incisors. While incisors of wild-type mice were brownish yellow, incisors of Nrf2-deficient mice were greyish white in colour. Micro X-ray imaging analysis revealed that the iron content in Nrf2-deficient mouse incisors were significantly decreased compared to that of wild-type mice. We found that iron was aberrantly deposited in the papillary layer cells of enamel organ in Nrf2-deficient mouse, suggesting that the iron transport from blood vessels to ameloblasts was disturbed. We also found that ameloblasts of Nrf2-null mouse show degenerative atrophy at the late maturation stage, which gives rise to the loss of iron deposition to the surface of mature enamel. Our results thus demonstrate that the enamel organ of Nrf2-deficient mouse has a reduced iron transport capacity, which results in both the enamel cell degeneration and disturbance of iron deposition on to the enamel surface.