APPL1 is an adaptor protein that binds to both AKT and adiponectin receptors and is hypothesised to mediate the effects of adiponectin in activating downstream effectors such as AMP-activated protein kinase (AMPK). We aimed to establish whether APPL1 plays a physiological role in mediating glycogen accumulation and insulin sensitivity in muscle and the signalling pathways involved. In vivo electrotransfer of cDNA- and shRNA-expressing constructs was used to over-express or silence APPL1 for 1 week in single tibialis cranialis muscles of rats. Resulting changes in glucose and lipid metabolism and signalling pathway activation were investigated under basal conditions and in high-fat diet (HFD)- or chow-fed rats under hyperinsulinaemic–euglycaemic clamp conditions. APPL1 over-expression (OE) caused an increase in glycogen storage and insulin-stimulated glycogen synthesis in muscle, accompanied by a modest increase in glucose uptake. Glycogen synthesis during the clamp was reduced by HFD but normalised by APPL1 OE. These effects are likely explained by APPL1 OE-induced increase in basal and insulin-stimulated phosphorylation of IRS1, AKT, GSK3β and TBC1D4. On the contrary, APPL1 OE, such as HFD, reduced AMPK and acetyl-CoA carboxylase phosphorylation and PPARγ coactivator-1α and uncoupling protein 3 expression. Furthermore, APPL1 silencing caused complementary changes in glycogen storage and phosphorylation of AMPK and PI3-kinase pathway intermediates. Thus, APPL1 may provide a means for crosstalk between adiponectin and insulin signalling pathways, mediating the insulin-sensitising effects of adiponectin on muscle glucose disposal. These effects do not appear to require AMPK. Activation of signalling mediated via APPL1 may be beneficial in overcoming muscle insulin resistance.
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M E Cleasby, Q Lau, E Polkinghorne, S A Patel, S J Leslie, N Turner, G J Cooney, A Xu and E W Kraegen
A Tsuchiya, T Kanno and T Nishizaki
Insulin stimulated translocation of the glucose transporter GLUT4 from the cytosol to the plasma membrane in a concentration (1 nM–1 μM)-dependent manner and increased glucose uptake in 3T3-L1 adipocytes. Insulin-induced GLUT4 translocation to the cell surface was prevented by the phosphoinositide 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase 1 (PDK1) inhibitor BX912 or the Akt1/2 inhibitor MK2206, and by knocking-down PI3K, PDK1 or Akt1/2. Insulin increased phosphorylation of Akt1/2 at Thr308/309 and Ser473/474, to activate Akt1/2, in the adipocytes. Insulin-induced phosphorylation of Akt1/2 was suppressed by wortmannin and knocking-down PI3K, while no significant inhibition of the phosphorylation was obtained with BX912 or knocking-down PDK1. In the cell-free Akt assay, PI3K phosphorylated Akt1 both at Thr308 and Ser473 and Akt2 at Ser474 alone. In contrast, PDK1 phosphorylates Akt1 at Thr308 and Akt2 at Thr309. The results of this study indicate that PI3K activates Akt1, independently of PDK1, and Akt2 by cooperating with PDK1 in the insulin signal transduction pathway linked to GLUT4 translocation.
Bernadette M Trojanowski, Heba H Salem, Heike Neubauer, Eric Simon, Martin Wagner, Rajkumar Dorajoo, Bernhard O Boehm, Leticia Labriola, Thomas Wirth and Bernd Baumann
Maturity-onset diabetes of the young (MODY) is a group of monogenetic forms of diabetes mellitus caused by mutations in genes regulating β-cell development and function. MODY represents a heterogeneous group of non-insulin-dependent diabetes arising in childhood or adult life. Interestingly, clinical heterogeneity in MODY patients like variable disease onset and severity is observed even among individual family members sharing the same mutation, an issue that is not well understood. As high blood glucose levels are a well-known factor promoting β-cell stress and ultimately leading to cell death, we asked whether additional β-cell stress might account for the occurrence of disease heterogeneity in mice carrying a MODY4 mutation. In order to challenge β-cells, we established a MODY4 animal model based on Pdx1 (pancreatic and duodenal homeobox 1) haploinsufficiency, which allows conditional modulation of cell stress by genetic inhibition of the stress-responsive IKK/NF-κB signalling pathway. While Pdx1+/− mice were found glucose intolerant without progressing to diabetes, additional challenge of β-cell function by IKK/NF-κB inhibition promoted rapid diabetes development showing hyperglycaemia, hypoinsulinemia and loss of β-cell mass. Disease pathogenesis was characterized by deregulation of genes controlling β-cell homeostasis and function. Importantly, restoration of normal IKK/NF-κB signalling reverted the diabetic phenotype including normalization of glycaemia and β-cell mass. Our findings implicate that the avoidance of additional β-cell stress can delay a detrimental disease progression in MODY4 diabetes. Remarkably, an already present diabetic phenotype can be reversed when β-cell stress is normalized.
Hyo Youl Moon, Parkyong Song, Cheol Soo Choi, Sung Ho Ryu and Pann-Ghill Suh
Physical inactivity can lead to obesity and fat accumulation in various tissues. Critical complications of obesity include type II diabetes and nonalcoholic fatty liver disease (NAFLD). Exercise has been reported to have ameliorating effects on obesity and NAFLD. However, the underlying mechanism is not fully understood. We showed that liver expression of macrophage migration inhibitory factor (MIF) was increased after 4 weeks of treadmill exercise. Phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase in human hepatocyte cell lines was enhanced after MIF treatment. These responses were accompanied by increases in lipid oxidation. Moreover, inhibition of either AMPK or cluster of differentiation 74 resulted in inhibition of MIF-induced lipid oxidation. Furthermore, the administration of MIF to a human hepatocyte cell line and mice liver reduced liver X receptor agonist-induced lipid accumulation. Taken together, these results indicate that MIF is highly expressed in the liver during physical exercise and may prevent hepatic steatosis by activating the AMPK pathway.
Nami Kim, Jung Ok Lee, Hye Jeong Lee, Yong Woo Lee, Hyung Ip Kim, Su Jin Kim, Sun Hwa Park, Chul Su Lee, Sun Woo Ryoo, Geum-Sook Hwang and Hyeon Soo Kim
Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle.
Jin-Ran Chen, Oxana P Lazarenko, Haijun Zhao, Alexander W Alund and Kartik Shankar
Intrauterine or early postnatal high-fat diet (HFD) has substantial influences on adult offspring health; however, studies of HFD-induced maternal obesity on regulation of adult offspring bone formation are sparse. Here, we investigated the effects of HFD-induced maternal obesity on both fetal and adult offspring skeletal development. We found that HFD-induced maternal obesity significantly decreased fetal skeletal development, but enhanced fetal osteoblastic cell senescence signaling and significantly increased the expression of inflammatory factors of the senescence-associated secretory phenotype (SASP) in osteo-progenitors. It was found that p300/CBP activation led to H3K27 acetylation to increase the expression of senescence-related genes and PPARγ in embryonic mouse osteogenic calvarial cells from HFD obese dams. These results were recapitulated in human umbilical cord mesenchymal stem cells (UC MSCs) isolated from offspring of pregnant obese and lean mothers following delivery. Regardless of postnatal HFD challenge, adult offspring from HFD obese dams showed significantly suppressed bone formation. Such early involution of bone formation of adult offspring from HFD obese dams may at least in part due to histone acetylation, i.e., epigenetic regulation of genes involved in cell senescence signaling in pre-osteoblasts from prenatal development. These findings indicate fetal pre-osteoblastic cell senescence signaling is epigenetically regulated by maternal obesity to repress bone formation in adult offspring in rodents and suggest that at least some of these effects may also manifest in humans.
Gulizar Issa Ameen and Silvia Mora
Obesity leads to adipose tissue dysfunction, insulin resistance and diabetes. Adipose tissue produces adipokines that contribute to regulate insulin sensitivity. In turn, insulin stimulates the production and release of some adipokines. Casitas-b-lymphoma proteins (c-Cbl, Cbl-b and Cbl3) are intracellular adaptor signalling proteins that are rapidly phosphorylated by activation of tyrosine kinase receptors. c-Cbl is rapidly phosphorylated by insulin in adipocytes. Here, we tested the hypothesis that Cbl signalling regulates adipokine expression in adipose tissue. We determined the adipokine profile of WAT of Cbl−/− and Cbl+/+ mice in the C57BL6 background. Female Cbl−/− mice exhibited altered expression of adiponectin, leptin and RBP4 in visceral adipose tissue, while no significant changes were seen in male mice. TNFα and IL6 levels were unaffected by Cbl depletion. RBP4 expression was unchanged in liver. Adipose tissue of Cbl−/− animals showed increased basal activation of extracellular regulated kinases (ERK1/2) compared to Cbl+/+. c-Cbl knockdown in 3T3L1 adipocytes also increased basal ERK phosphorylation and RBP4 expression. Inhibition of ERK1/2 phosphorylation in Cbl-depleted 3T3L1 adipocytes or in adipose tissue explants of Cbl−/− mice reduced RBP4 mRNA. 17β-Estradiol increased RBP4 mRNA in adipocytes. Cbl depletion did not change ER expression but increased phosphorylation of ERα at S118, a target site for ERK1/2. ERK1/2 inhibition reduced phosphoER and RBP4 levels. These findings suggest that Cbl contributes to regulate RBP4 expression in adipose of female mice through ERK1/2-mediated activation of ERα. Since Cbl signalling is compromised in diabetes, these data highlight a novel mechanism that upregulates RBP4 locally.
Md Nurul Islam, Yuichiro Mita, Keisuke Maruyama, Ryota Tanida, Weidong Zhang, Hideyuki Sakoda and Masamitsu Nakazato
Ghrelin, a stomach-derived peptide, promotes feeding and growth hormone (GH) secretion. A recent study identified liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous inhibitor of ghrelin-induced GH secretion, but the effect of LEAP2 in the brain remained unknown. In this study, we showed that intracerebroventricular (i.c.v.) administration of LEAP2 to rats suppressed central ghrelin functions including Fos expression in the hypothalamic nuclei, promotion of food intake, blood glucose elevation, and body temperature reduction. LEAP2 did not inhibit neuropeptide Y (NPY)-induced food intake or des-acyl ghrelin-induced reduction in body temperature, indicating that the inhibitory effects of LEAP2 were specific for GHSR. Plasma LEAP2 levels varied according to feeding status and seemed to be dependent on the hepatic Leap2 expression. Furthermore, ghrelin suppressed the expression of hepatic Leap2 via AMPK activation. Together, these results reveal that LEAP2 inhibits central ghrelin functions and crosstalk between liver and stomach.
T Mracek, D Gao, T Tzanavari, Y Bao, X Xiao, C Stocker, P Trayhurn and C Bing
Zinc-α2-glycoprotein (ZAG, also listed as AZGP1 in the MGI Database), a lipid-mobilising factor, has recently been suggested as a potential candidate in the modulation of body weight. We investigated the effect of increased adiposity on ZAG expression in adipose tissue and the liver and on plasma levels in obese (ob/ob) mice compared with lean siblings. The study also examined the effect of the pro-inflammatory cytokine tumour necrosis factor-α (TNFα) on ZAG expression in adipocytes. Zag mRNA levels were significantly reduced in subcutaneous (fourfold) and epididymal (eightfold) fat of ob/ob mice. Consistently, ZAG protein content was decreased in both fat depots of ob/ob mice. In the liver of obese animals, steatosis was accompanied by the fall of both Zag mRNA (twofold) and ZAG protein content (2.5-fold). Plasma ZAG levels were also decreased in obese mice. In addition, Zag mRNA was reduced in epididymal (fivefold) and retroperitoneal (fivefold) adipose tissue of obese (fa/fa) Zucker rats. In contrast to Zag expression, Tnfα mRNA levels were elevated in adipose tissue (twofold) and the liver (2.5-fold) of ob/ob mice. Treatment with TNFα reduced Zag gene expression in differentiated adipocytes, and this inhibition was chronic, occurring at 24 and 48 h following TNFα treatment. It is concluded that ZAG synthesis in adipose tissue and the liver is downregulated, as are its circulating levels, in ob/ob mice. The reduced ZAG production may advance the susceptibility to lipid accumulation in these tissues in obesity, and this could be at least in part attributable to the inhibitory effect of TNFα.
T V Novoselova, R Larder, D Rimmington, C Lelliott, E H Wynn, R J Gorrigan, P H Tate, L Guasti, The Sanger Mouse Genetics Project, S O’Rahilly, A J L Clark, D W Logan, A P Coll and L F Chan
Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2 tm1a/tm1a) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2 tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2 tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.