DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans.

The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has α, ß and γ subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory γ subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ∼75% of total γ subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 γ subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK ß subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.

Anthranilate fluorescence marks a calcium-propagated necrotic wave that promotes organismal death in C. elegans.

For cells the passage from life to death can involve a regulated, programmed transition. In contrast to cell death, the mechanisms of systemic collapse underlying organismal death remain poorly understood. Here we present evidence of a cascade of cell death involving the calpain-cathepsin necrosis pathway that can drive organismal death in Caenorhabditis elegans. We report that organismal death is accompanied by a burst of intense blue fluorescence, generated within intestinal cells by the necrotic cell death pathway. Such death fluorescence marks an anterior to posterior wave of intestinal cell death that is accompanied by cytosolic acidosis. This wave is propagated via the innexin INX-16, likely by calcium influx. Notably, inhibition of systemic necrosis can delay stress-induced death. We also identify the source of the blue fluorescence, initially present in intestinal lysosome-related organelles (gut granules), as anthranilic acid glucosyl esters--not, as previously surmised, the damage product lipofuscin. Anthranilic acid is derived from tryptophan by action of the kynurenine pathway. These findings reveal a central mechanism of organismal death in C. elegans that is related to necrotic propagation in mammals--e.g., in excitotoxicity and ischemia-induced neurodegeneration. Endogenous anthranilate fluorescence renders visible the spatio-temporal dynamics of C. elegans organismal death.

Endothelial Protease Activated Receptor 1 (PAR1) Signalling Is Required for Lymphocyte Transmigration across Brain Microvascular Endothelial Cells.

Lymphocyte transendothelial migration (TEM) relies on ICAM-1 engagement on the luminal surface of the endothelial cells (ECs). In blood-brain barrier (BBB) ECs, ICAM-1 triggers TEM signalling, including through JNK MAP kinase and AMP-activated protein kinase (AMPK), which lead to the phosphorylation and internalisation of the adherens junction protein VE-cadherin. In addition to ICAM-1, G protein-coupled receptors (GPCRs) are also required for lymphocytes TEM across BBB ECs. Here, we investigated the role of protease activated GPCRs (PARs) and found a specific role for PAR1 in support of lymphocyte TEM across BBB ECs in vitro. PAR1 requirement for TEM was confirmed using protease inhibitors, specific small molecule and peptide antagonists, function blocking antibodies and siRNA-mediated knockdown. In BBB ECs, PAR1 stimulation led to activation of signalling pathways essential to TEM; notably involving JNK and endothelial nitric oxide synthase (eNOS), with the latter downstream of AMPK. In turn, nitric oxide production through eNOS was essential for TEM by modulating VE-cadherin on Y731. Collectively, our data showed that non-canonical PAR1 activation by a lymphocyte-released serine protease is required for lymphocyte TEM across the BBB in vitro, and that this feeds into previously established ICAM-1-mediated endothelial TEM signalling pathways.

Publisher Correction: Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110α inactivation in adipose tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced ß-adrenergic signalling underlies an age-dependent beneficial metabolic effect of PI3K p110α inactivation in adipose tissue.

The insulin/IGF-1 signalling pathway is a key regulator of metabolism and the rate of ageing. We previously documented that systemic inactivation of phosphoinositide 3-kinase (PI3K) p110α, the principal PI3K isoform that positively regulates insulin signalling, results in a beneficial metabolic effect in aged mice. Here we demonstrate that deletion of p110α specifically in the adipose tissue leads to less fat accumulation over a significant part of adult life and allows the maintenance of normal glucose tolerance despite insulin resistance. This effect of p110α inactivation is due to a potentiating effect on ß-adrenergic signalling, which leads to increased catecholamine-induced energy expenditure in the adipose tissue. Our findings provide a paradigm of how partial inactivation of an essential component of the insulin signalling pathway can have an overall beneficial metabolic effect and suggest that PI3K inhibition could potentiate the effect of ß-adrenergic agonists in the treatment of obesity and its associated comorbidities.

14-3-3 regulates life span by both DAF-16-dependent and -independent mechanisms in Caenorhabditis elegans.

Caenorhabditis elegans life span, stress resistance and metabolism are regulated by the Insulin/IGF-1/DAF-2/DAF-16 pathway. DAF-16, a member of FOXO/Forkhead transcription factor family, can be targeted by 14-3-3 proteins to promote stress resistance. We have identified a 14-3-3 C. elegans homolog which promotes life span by both DAF-2-dependent and -independent mechanisms and by an unexpected DAF-16-independent mechanism. Our results demonstrate that C. elegans 14-3-3 proteins modulate stress-responsive genes throughout adulthood. In conclusion, 14-3-3 can be considered as an acute stress-responsive regulator as well as a sustained modulator of the Insulin/IGF-1/DAF-2/DAF-16 regulatory pathway in promoting life expectancy of growing old worms.

Involvement of oxidative stress and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the fructose-fed rat.

Growing evidences suggest a role of oxidative stress in hypertension and cardiac hypertrophy. The fructose (60%)-fed rat represents a model of metabolic syndrome, associating insulin resistance and high blood pressure. In this model, hypertension, cardiac and vessels hypertrophy and markers of oxidative stress were determined. In addition, the production of reactive oxygen species (ROS) was evaluated at different times after the initiation of fructose-enriched diet in aorta, heart and polymorphonuclear cells. High fructose feeding was associated with an early (1-week) increase in ROS production by aorta, heart and circulatory polymorphonuclear cells, in association with enhanced markers of oxidative stress. Vascular and cardiac hypertrophy was also rapidly observed, while the rise in blood pressure was significant only after 3 weeks. In summary, our study suggests that the production of reactive oxygen species can be a key-event in the initiation and development of cardiovascular complications associated with insulin resistance.

Extracts enriched in different polyphenolic families normalize increased cardiac NADPH oxidase expression while having differential effects on insulin resistance, hypertension, and cardiac hypertrophy in high-fructose-fed rats.

Insulin resistance and oxidative stress act synergistically in the development of cardiovascular complications. The present study compared the efficacy of three polyphenolic extracts in their capacity to prevent hypertension, cardiac hypertrophy, increased production of reactive oxygen species (ROS) by the aorta or the heart, and increased expression of cardiac NAD(P)H oxidase in a model of insulin resistance. Rats were fed a 60%-enriched fructose food and were treated once a day (gavage) for 6 weeks with 10 mL/kg of water only (F group) or the same amount of solution containing a red grape skin polyphenolic extract enriched in anthocyanins (ANT), a grape seed extract enriched in procyanidins and rich in galloylated procyanidins (PRO), or the commercial preparation Vitaflavan (VIT), rich in catechin oligomers. All treatments were administered at the same dose of 21 mg/kg of polyphenols. Our data indicate that (a) the ANT treatment prevented hypertension, cardiac hypertrophy, and production of ROS, (b) the PRO treatment prevented insulin resistance, hypertriglyceridemia, and overproduction of ROS but had only minor effects on hypertension or hypertrophy, while (c) Vitaflavan prevented hypertension, cardiac hypertrophy, and overproduction of ROS. All polyphenolic treatments prevented the increased expression of the p91phox NADPH oxidase subunit. In summary, our study suggest that (a) the pathogeny of cardiac hypertrophy in the fructose-fed rat disease involves both hypertension and hyperproduction of ROS, (b) polyphenolic extracts enriched in different types of polyphenols possess differential effects on insulin resistance, hypertension, and cardiac hypertrophy, and (c) polyphenols modulate the expression of NAD(P)H oxidase.

Red wine polyphenols alone or in association with ethanol prevent hypertension, cardiac hypertrophy, and production of reactive oxygen species in the insulin-resistant fructose-fed rat.

The effects of a red wine polyphenolic extract (RWPE), ethanol, or both combined were evaluated in insulin resistant rats. Rats were fed for 6 weeks with fructose (60%)-enriched food and force-fed with (a) water only (F group), (b) aqueous solution of RWPE (100 mg/kg, FP group), (c) 10% (v/v) mixture of ethanol and water (FE group), or (d) solution containing the same amount of the RWPE and ethanol (FPE group). Animals fed a standard chow (C group) were used for comparison purpose. After 6 weeks, blood pressure was higher in F (130.0 x b1 1.7 mm Hg) than in C animals (109.6 x b1 0.9 mm Hg) and similar to the C group in all other fructose-fed treatment groups. Relative heart weight was higher in F (3.10 x b1 0.05) than in C (2.78 x b1 0.07) and significantly lower in FP (2.92 x b1 0.04) and FPE (2.87 x b1 0.08 mg/g) than in F animals. Left ventricle and aorta productions of reactive oxygen species (O2*-) were higher in F than in C groups and lowered by the RWPE but not by the ethanol treatment. Ethanol but not the RWPE treatment reduced the degree of insulin resistance in the fructose-fed rats. In summary, our study showed that polyphenols are able to prevent cardiac hypertrophy and production of reactive oxygen species in the insulin resistant fructose-fed rat.

Increased life span from overexpression of superoxide dismutase in Caenorhabditis elegans is not caused by decreased oxidative damage.

The superoxide free radical (O(2)(•-)) has been viewed as a likely major contributor to aging. If this is correct, then superoxide dismutase (SOD), which removes O(2)(•-), should contribute to longevity assurance. In Caenorhabditis elegans, overexpression (OE) of the major cytosolic Cu/Zn-SOD, sod-1, increases life span. But is this increase caused by enhanced antioxidant defense? sod-1 OE did not reduce measures of lipid oxidation or glycation and actually increased levels of protein oxidation. The effect of sod-1 OE on life span was dependent on the DAF-16/FoxO transcription factor (TF) and, partially, on the heat shock TF HSF-1. Similarly, overexpression of sod-2 (major mitochondrial Mn-SOD) resulted in life-span extension that was daf-16 dependent. sod-1 OE increased steady-state hydrogen peroxide (H(2)O(2)) levels in vivo. However, co-overexpression of catalase did not suppress the life-span extension, arguing against H(2)O(2) as a cause of longevity. sod-1 OE increased hsp-4 expression, suggesting increased endoplasmic reticulum (ER) stress. Moreover, longevity was partially suppressed by inactivation of ire-1 and xbp-1, mediators of the ER stress response. This suggests that high levels of SOD-1 protein may challenge protein-folding homeostasis, triggering a daf-16- and hsf-1-dependent stress response that extends life span. These findings imply that SOD overexpression increases C. elegans life span, not by removal of O(2)(•-), but instead by activating longevity-promoting transcription factors.

Klotho interferes with a novel FGF-signalling pathway and insulin/Igf-like signalling to improve longevity and stress resistance in Caenorhabditis elegans.

Klotho exerts anti-aging properties in mammals in two different ways. While membrane-bound Klotho, which is primarily expressed in the kidney, acts as an obligate co-receptor of FGF23 to regulate phosphate homeostasis, secreted Klotho, resulting from the shedding of the KL1-KL2 ectodomain into the bloodstream, inhibits Insulin/IGF1 signalling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the biological role of Klotho in Caenorhabditis elegans. Two redundant homologues of the klotho gene exist in C. elegans and encode predicted proteins homologous to the  glucosidase-like KL1 domain of mammalian Klotho. We have used a genetic approach to investigate the functional activity of Klotho in C. elegans. Here, we report that whereas Klotho requires EGL-15 (FGFR) and EGL-17 to promote longevity and oxidative stress resistance, it is not involved in the regulation of fluid homeostasis, controlled by LET-756. Besides revealing a new post-developmental role for EGL-17, our data suggest that the KL1 form of Klotho is involved in FGF23-independent FGF signalling. We also report a genetic interaction between Klotho and the DAF-2 (Ins/IGF1R)/DAF-16 (FOXO) pathway. While the regulation of longevity requires functional DAF-2/DAF-16 signalling, the control of oxidative stress resistance involves a DAF-2- independent, DAF-16-dependent pathway, suggesting that Klotho may target either DAF-2 or DAF-16, depending of environmental conditions. Thus, the predictive KL1 form of Klotho appears to crosstalk with both FGF and Insulin/IGF1/FOXO pathways to exert anti-aging properties in C. elegans.