FoxO proteins are major targets of insulin action. To better define the role of FoxO1 in mediating insulin effects in the liver, we generated liver-specific insulin receptor knockout (LIRKO) and IR/FoxO1 double knockout (LIRFKO) mice. Here we show that LIRKO mice are severely insulin resistant based on glucose, insulin and C-peptide levels, and glucose and insulin tolerance tests, and genetic deletion of hepatic FoxO1 reverses these effects. 13C-glucose and insulin clamp studies indicate that regulation of both hepatic glucose production (HGP) and glucose utilization is impaired in LIRKO mice, and these defects are also restored in LIRFKO mice corresponding to changes in gene expression. We conclude that (1) inhibition of FoxO1 is critical for both direct (hepatic) and indirect effects of insulin on HGP and utilization, and (2) extrahepatic effects of insulin are sufficient to maintain normal whole-body and hepatic glucose metabolism when liver FoxO1 activity is disrupted.

Background: FoxO1 regulates expression of lipogenic genes including srebp1. Results: FoxO1 inhibits transcription of SREBP-1c via coordinated effects on key regulatory factors including Sp1 and SREBP-1c itself. Conclusion: FoxO1 acts at multiple levels to prevent assembly of the transcriptional complex on the srebp1 gene. Significance: FoxO1 effectively inhibits SREBP-1c gene expression, a major regulator of hepatic lipogenesis.

Aims/hypothesis The aim of this study was to determine the impact of the common food additive carrageenan (E407) on glucose tolerance, insulin sensitivity and insulin signalling in a mouse model and human hepatic cells, since carrageenan is known to cause inflammation through interaction with toll-like receptor (TLR)4, which is associated with inflammation in diabetes. Methods Male C57BL/6J mice were given carrageenan (10 mg/l) in their drinking water, and underwent a glucose tolerance test (GTT), an insulin tolerance test (ITT) and an ante-mortem intraperitoneal insulin injection. HepG2 cells were exposed to carrageenan (1 mg/l×24 h) and insulin. Levels of phospho(Ser473)-protein kinase B (Akt), phospho (Ser307)-IRS1, phosphoinositide 3-kinase (PI3K) activity and phospho(Ser32)-inhibitor of κB (IκBα) were determined by western blotting and ELISA. Results Glucose tolerance was significantly impaired in carrageenan-treated 12-week-old mice compared with untreated controls at all time points (n=12; p<0.0001). Baseline insulin and insulin levels at 30 min after taking glucose during the GTT were significantly higher following carrageenan treatment. During the ITT, glucose levels declined by more than 80% in controls, but not in carrageenan-treated mice. Carrageenan exposure completely inhibited insulininduced increases in phospho-(Ser473)-Akt and PI3K activity in vivo in mouse liver and in human HepG2 cells. Carrageenan increased phospho(Ser307)-IRS1 levels, and this was blocked when carrageenan-induced inflammation was inhibited. Conclusion This is the first report of the impact of carrageenan on glucose tolerance and indicates that carrageenan impairs glucose tolerance, increases insulin resistance and inhibits insulin signalling in vivo in mouse liver and human HepG2 cells. These effects may result from carrageenan-induced inflammation. The results demonstrate extra-colonic manifestations of ingested carrageenan and suggest that carrageenan in the human diet may contribute to the development of diabetes.

Discogenic low back pain (DLBP) is extremely common and costly. Effective treatments are lacking due to DLBP's unknown pathogenesis. Currently, there are no in vivo mouse models of DLBP, which restricts research in this field. The aim of this study was to establish a reliable DLBP model in mouse that captures the pathological changes in the disc and allows longitudinal pain testing. The model was generated by puncturing the mouse lumbar discs (L4/5, L5/6, and L6/S1) and removing the nucleus pulposus using a microscalpel under the microscope. Histology, molecular pathways, and pain-related behaviors were examined. Over 12 weeks post-surgery, animals displayed the mechanical, heat, and cold hyperalgesia along with decreased burrowing and rearing. Histology showed progressive disc degeneration with loss of disc height, nucleus pulposus reduction, proteoglycan depletion, and annular fibrotic disorganization. Immunohistochemistry revealed a substantial increase in inflammatory mediators at 2 and 4 weeks. Nerve growth factor was upregulated from 2 weeks to the end of the experiment. Nerve fiber ingrowth was induced in the injured discs after 4 weeks. Disc-puncture also produced an upregulation of neuropeptides in dorsal root ganglia neurons and an activation of glial cells in the spinal cord dorsal horn. These findings indicate that the cellular and structural changes in discs, as well as peripheral and central nervous system plasticity, paralleled persistent, and robust behavioral pain responses. Therefore, this mouse DLBP model could be used to investigate mechanisms underlying discogenic pain, thereby facilitating effective drug screening and development of treatments for DLBP.

Summary FoxO proteins are major targets of insulin action. Adipose triacylglycerol (TAG) lipase mediates the first step in TAG hydrolysis and FoxO1 stimulates ATGL expression in adipose tissue. Here, we report that FoxO1 also stimulates ATGL and suppresses expression of its inhibitor, the G0/G1 switch gene protein 2 (G0S2), in the liver. Studies in FoxO transgenic and knockout mice and hepatocytes show that FoxO proteins mediate effects of insulin on ATGL and G0S2 expression. Further, ATGL-dependent lipolysis mediates effects of FoxOs on glycolytic, lipogenic and gluconeogenic gene expression and metabolism, including fatty acid oxidation, which is required for effects of FoxO1 on glucose homeostasis. These results reveal that ATGL-dependent lipolysis and fatty acid oxidation play a critical role in integrating the regulation of glucose and lipid metabolism downstream from FoxO proteins in the liver, and suggest that targeting ATGL-dependent lipolysis and its downstream effectors may provide an effective strategy for improving the treatment of patients with diabetes and hepatic insulin resistance.

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Liver-specific disruption of the type 2 deiodinase gene (Alb-D2KO) results in resistance to both diet-induced obesity and liver steatosis in mice. Here, we report that this is explained by an ∼60% reduction in liver zinc-finger protein-125 (Zfp125) expression. Zfp125 is a Foxo1-inducible transcriptional repressor that causes lipid accumulation in the AML12 mouse hepatic cell line and liver steatosis in mice by reducing liver secretion of triglycerides and hepatocyte efflux of cholesterol. Zfp125 acts by repressing 18 genes involved in lipoprotein structure, lipid binding, and transport. The ApoE promoter contains a functional Zfp125-binding element that is also present in 17 other lipid-related genes repressed by Zfp125. While liver-specific knockdown of Zfp125 causes an "Alb-D2KO-like" metabolic phenotype, liver-specific normalization of Zfp125 expression in Alb-D2KO mice rescues the phenotype, restoring normal susceptibility to diet-induced obesity, liver steatosis, and hypercholesterolemia.

Background: Insulin/IGF-1 stimulates thyroid hormone action via type 2 deiodinase (D2). Results: Insulin/IGF-1-induced activation of the PI3K-mTORC2-Akt pathway transcriptionally up-regulates D2. Conclusion: FOXO1 represses DIO2 during fasting, and derepression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway. Significance: This mechanism explains how T 3 production, serum T 3 levels, and T 3 -dependent cellular metabolic rate are kept at a level proportionate to the availability of energy substrates.