Acarbose improves health and lifespan in aging HET3 mice

To follow‐up on our previous report that acarbose (ACA), a drug that blocks postprandial glucose spikes, increases mouse lifespan, we studied ACA at three doses: 400, 1,000 (the original dose), and 2,500 ppm, using genetically heterogeneous mice at three sites. Each dose led to a significant change (by log‐rank test) in both sexes, with larger effects in males, consistent with the original report. There were no significant differences among the three doses. The two higher doses produced 16% or 17% increases in median longevity of males, but only 4% or 5% increases in females. Age at the 90th percentile was increased significantly (8%–11%) in males at each dose, but was significantly increased (3%) in females only at 1,000 ppm. The sex effect on longevity is not explained simply by weight or fat mass, which were reduced by ACA more in females than in males. ACA at 1,000 ppm reduced lung tumors in males, diminished liver degeneration in both sexes and glomerulosclerosis in females, reduced blood glucose responses to refeeding in males, and improved rotarod performance in aging females, but not males. Three other interventions were also tested: ursolic acid, 2‐(2‐hydroxyphenyl) benzothiazole (HBX), and INT‐767; none of these affected lifespan at the doses tested. The acarbose results confirm and extend our original report, prompt further attention to the effects of transient periods of high blood glucose on aging and the diseases of aging, including cancer, and should motivate studies of acarbose and other glucose‐control drugs in humans.     

Longer lifespan in male mice treated with a weakly estrogenic agonist,an antioxidant,an α‐glucosidase inhibitor or a Nrf2‐inducer

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin – the latter with and without rapamycin, and two drugs previously examined: 17‐α‐estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17‐α‐estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male‐specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α‐glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.     

Cap‐independent translation: A shared mechanism for lifespan extension by rapamycin,acarbose, and 17α‐estradiol

We hypothesized that rapamycin (Rapa), acarbose (ACA), which both increase mouse lifespan, and 17α‐estradiol, which increases lifespan in males (17aE2) all share common intracellular signaling pathways with long‐lived Snell dwarf, PAPPA‐KO, and Ghr−/− mice. The long‐lived mutant mice exhibit reduction in mTORC1 activity, declines in cap‐dependent mRNA translation, and increases in cap‐independent translation (CIT). Here, we report that Rapa and ACA prevent age‐related declines in CIT target proteins in both sexes, while 17aE2 has the same effect only in males, suggesting increases in CIT. mTORC1 activity showed the reciprocal pattern, with age‐related increases blocked by Rapa, ACA, and 17aE2 (in males only). METTL3, required for addition of 6‐methyl‐adenosine to mRNA and thus a trigger for CIT, also showed an age‐dependent increase blunted by Rapa, ACA, and 17aE2 (in males). Diminution of mTORC1 activity and increases in CIT‐dependent proteins may represent a shared pathway for both long‐lived‐mutant mice and drug‐induced lifespan extension in mice.     

17‐a‐estradiol late in life extends lifespan in aging UM‐HET3 male mice; nicotinamide riboside and three other drugs do not affect lifespan in either sex

In genetically heterogeneous mice produced by the CByB6F1 x C3D2F1 cross, the “non‐feminizing” estrogen, 17‐α‐estradiol (17aE2), extended median male lifespan by 19% (p < 0.0001, log‐rank test) and 11% (p = 0.007) when fed at 14.4 ppm starting at 16 and 20 months, respectively. 90th percentile lifespans were extended 7% (p = 0.004, Wang–Allison test) and 5% (p = 0.17). Body weights were reduced about 20% after starting the 17aE2 diets. Four other interventions were tested in males and females: nicotinamide riboside, candesartan cilexetil, geranylgeranylacetone, and MIF098. Despite some data suggesting that nicotinamide riboside would be effective, neither it nor the other three increased lifespans significantly at the doses tested. The 17aE2 results confirm and extend our original reports, with very similar results when started at 16 months compared with mice started at 10 months of age in a prior study. The consistently large lifespan benefit in males, even when treatment is started late in life, may provide information on sex‐specific aspects of aging.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

17‐β estradiol attenuates the pro‐oxidant activity of corticotropin‐releasing hormone in macroendothelial cells

Corticotropin‐releasing hormone, which is the predominant regulator of neuroendocrine responses to stress, attenuates inflammation through stimulation of glucocorticoid release. Enhanced corticotropin‐releasing hormone expression has been detected in inflammatory cells of the vascular endothelium, where it acts as a local regulator of endothelial redox homeostasis. Estrogens have beneficial effects on endothelial integrity and function, though the mechanism underlying their antioxidative effect remains as yet largely unknown. We therefore investigated the effect of 17β‐estradiol on pro‐oxidant action of corticotropin‐releasing hormone in vitro in macroendothelial cells, and, more specifically, the role of 17β‐estradiol on corticotropin‐releasing hormone‐induced activities/release of the antioxidant enzymes namely, endothelial nitric oxide synthase, superoxide dismutase, catalase, and glutathione. We observed that 17β‐estradiol abolished the stimulatory effect of corticotropin‐releasing hormone on intracellular reactive oxygen species levels and counteracted its inhibitory effect on endothelial nitric oxide synthase activity and nitric oxide release. In addition, 17β‐estradiol significantly induced superoxide dismutase and catalase activity, an effect that was not significantly influenced by corticotropin‐releasing hormone. Finally, 17β‐estradiol significantly increased glutathione levels and the glutathione/glutathione + glutathione disulfide ratio, an action that was partially blocked by corticotropin‐releasing hormone. Our results reveal that 17β‐estradiol counterbalances corticotropin‐releasing hormone‐mediated pro‐inflammatory action and thereby maintains the physiological threshold of the endothelial cell redox environment. These observations may be of importance, considering the protective role of estrogen in the development of atherosclerosis.     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

Interaction of antidiabetic α‐glucosidase inhibitors and gut bacteria α‐glucosidase

Carbohydrate hydrolyzing α‐glucosidases are commonly found in microorganisms present in the human intestine microbiome. We have previously reported crystal structures of an α‐glucosidase from the human gut bacterium Blaubia (Ruminococcus) obeum (Ro‐αG1) and its substrate preference/specificity switch. This novel member of the GH31 family is a structural homolog of human intestinal maltase‐glucoamylase (MGAM) and sucrase–isomaltase (SI) with a highly conserved active site that is predicted to be common in Ro‐αG1 homologs among other species that colonize the human gut. In this report, we present structures of Ro‐αG1 in complex with the antidiabetic α‐glucosidase inhibitors voglibose, miglitol, and acarbose and supporting binding data. The in vitro binding of these antidiabetic drugs to Ro‐αG1 suggests the potential for unintended in vivo crossreaction of the α‐glucosidase inhibitors to bacterial α‐glucosidases that are present in gut microorganism communities. Moreover, analysis of these drug‐bound enzyme structures could benefit further antidiabetic drug development.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Ultra‐high α‐linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2

For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α‐linolenic acid (ALA) accumulation. RNA‐sequencing analysis in the defective embryo suggested that brassinosteroid synthesis, FA synthesis and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum stress and oxidative stress were activated. Lipidomics analysis showed that ultra‐accumulated ALA is released from phosphatidylcholine as a free FA in cells, inducing severe endoplasmic reticulum and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects and generates strategies to produce higher levels of UFAs.     

17‐β‐Estradiol increases macrophage activity through activation of the G‐protein‐coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17‐β‐estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G‐protein‐coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.     

γ‐glutamyl transpeptidase 1 specifically suppresses green‐light avoidance via GABAA receptors in Drosophila

Drosophila larvae innately show light avoidance behavior. Compared with robust blue‐light avoidance, larvae exhibit relatively weaker green‐light responses. In our previous screening for genes involved in larval light avoidance, compared with control w¹¹¹⁸ larvae, larvae with γ‐glutamyl transpeptidase 1 (Ggt‐1) knockdown or Ggt‐1 mutation were found to exhibit higher percentage of green‐light avoidance which was mediated by Rhodopsin6 (Rh6) photoreceptors. However, their responses to blue light did not change significantly. By adjusting the expression level of Ggt‐1 in different tissues, we found that Ggt‐1 in malpighian tubules was both necessary and sufficient for green‐light avoidance. Our results showed that glutamate levels were lower in Ggt‐1 null mutants compared with controls. Feeding Ggt‐1 null mutants glutamate can normalize green‐light avoidance, indicating that high glutamate concentrations suppressed larval green‐light avoidance. However, rather than directly, glutamate affected green‐light avoidance indirectly through GABA, the level of which was also lower in Ggt‐1 mutants compared with controls. Mutants in glutamate decarboxylase 1, which encodes GABA synthase, and knockdown lines of the GABA_A receptor, both exhibit elevated levels of green‐light avoidance. Thus, our results elucidate the neurobiological mechanisms mediating green‐light avoidance, which was inhibited in wild‐type larvae.