Resveratrol-Induced AMP-Activated Protein Kinase Activation Is Cell-Type Dependent: Lessons from Basic Research for Clinical Application.

Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol's effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD⁺ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50-100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD⁺ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical setting.

Knockdown of GSK3ß increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells.

High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3ß (GSK3ß). To determine whether GSK3ß affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3ß activity and impaired lysosome acidification. Suppression of GSK3ß in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3ß attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3ß increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3ß inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3ß may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Unraveling the actions of AMP-activated protein kinase in metabolic diseases: Systemic to molecular insights.

AMP-activated protein kinase (AMPK) plays a critical role both in sensing and regulating cellular energy state. In experimental animals, its activation has been shown to reduce the risk of obesity and diabetes-related co-morbidities such as insulin resistance, the metabolic syndrome and atherosclerotic cardiovascular disease. However, in humans, AMPK activation alone often does not completely resolve these conditions. Thus, an improved understanding of AMPK action and regulation in metabolic and other diseases is needed. Herein, we provide a brief description of the enzymatic regulation of AMPK and review its role in maintaining energy homeostasis. We then discuss tissue-specific actions of AMPK that become distorted during such conditions as obesity, type 2 diabetes and certain cancers. Finally, we explore recent findings regarding the interactions of AMPK with mammalian target of rapamycin complex 1 and the lysosome and discuss how changes in these relationships during overnutrition may lead to AMPK dysfunction. A more thorough understanding of AMPK's molecular interactions during diseases of overnutrition may provide key insights for the development of AMPK-based combinatorial treatments for metabolic disease.

Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.

Glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells.

Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.

Nutritional modulation of cataract.

Lens opacification or cataract reduces vision in over 80 million people worldwide and blinds 18 million. These numbers will increase dramatically as both the size of the elderly demographic and the number of those with carbohydrate metabolism-related problems increase. Preventative measures for cataract are critical because the availability of cataract surgery in much of the world is insufficient. Epidemiologic literature suggests that the risk of cataract can be diminished by diets that are optimized for vitamin C, lutein/zeaxanthin, B vitamins, omega-3 fatty acids, multivitamins, and carbohydrates: recommended levels of micronutrients are salutary. The limited data from intervention trials provide some support for observational studies with regard to nuclear - but not other types of - cataracts. Presented here are the beneficial levels of nutrients in diets or blood and the total number of participants surveyed in epidemiologic studies since a previous review in 2007.

Nutritional modulation of age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. It affects 30-50 million individuals and clinical hallmarks of AMD are observed in at least one third of persons over the age of 75 in industrialized countries (Gehrs et al., 2006). Costs associated with AMD are in excess of $340 billion US (American-Health-Assistance-Foundation, 2012). The majority of AMD patients in the United States are not eligible for clinical treatments (Biarnes et al., 2011; Klein et al., 2011). Preventive interventions through dietary modulation are attractive strategies because many studies suggest a benefit of micro- and macronutrients with respect to AMD, as well as other age-related debilities, and with few, if any, adverse effects (Chiu, 2011). Preservation of vision would enhance quality of life for millions of elderly people, and alleviate the personal and public health financial burden of AMD (Frick et al., 2007; Wood et al., 2011). Observational studies indicate that maintaining adequate levels of omega-3 fatty acids (i.e. with 2 servings/week of fish) or a low glycemic index diet may be particularly beneficial for early AMD and that higher levels of carotenoids may be protective, most probably, against neovascular AMD. Intervention trials are needed to better understand the full effect of these nutrients and/or combinations of nutrients on retinal health. Analyses that describe effects of a nutrient on onset and/or progress of AMD are valuable because they indicate the value of a nutrient to arrest AMD at the early stages. This comprehensive summary provides essential information about the value of nutrients with regard to diminishing risk for onset or progress of AMD and can serve as a guide until data from ongoing intervention trials are available.

Glycation-altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics).

Epidemiologic studies indicate that the risks for major age-related debilities including coronary heart disease, diabetes, and age-related macular degeneration (AMD) are diminished in people who consume lower glycemic index (GI) diets, but lack of a unifying physiobiochemical mechanism that explains the salutary effect is a barrier to implementing dietary practices that capture the benefits of consuming lower GI diets. We established a simple murine model of age-related retinal lesions that precede AMD (hereafter called AMD-like lesions). We found that consuming a higher GI diet promotes these AMD-like lesions. However, mice that consumed the lower vs. higher GI diet had significantly reduced frequency (P < 0.02) and severity (P < 0.05) of hallmark age-related retinal lesions such as basal deposits. Consuming higher GI diets was associated with > 3 fold higher accumulation of advanced glycation end products (AGEs) in retina, lens, liver, and brain in the age-matched mice, suggesting that higher GI diets induce systemic glycative stress that is etiologic for lesions. Data from live cell and cell-free systems show that the ubiquitin-proteasome system (UPS) and lysosome/autophagy pathway [lysosomal proteolytic system (LPS)] are involved in the degradation of AGEs. Glycatively modified substrates were degraded significantly slower than unmodified substrates by the UPS. Compounding the detriments of glycative stress, AGE modification of ubiquitin and ubiquitin-conjugating enzymes impaired UPS activities. Furthermore, ubiquitin conjugates and AGEs accumulate and are found in lysosomes when cells are glycatively stressed or the UPS or LPS/autophagy are inhibited, indicating that the UPS and LPS interact with one another to degrade AGEs. Together, these data explain why AGEs accumulate as glycative stress increases.

Natural history of age-related retinal lesions that precede AMD in mice fed high or low glycemic index diets.

PURPOSE: Epidemiologic data indicate that people who consume low glycemic index (GI) diets are at reduced risk for the onset and progression of age-related macular degeneration (AMD). The authors sought corroboration of this observation in an animal model. METHODS: Five- and 16-month-old C57BL/6 mice were fed high or low GI diets until they were 17 and 23.5 months of age, respectively. Retinal lesions were evaluated by transmission electron microscopy, and advanced glycation end products (AGEs) were evaluated by immunohistochemistry. RESULTS: Retinal lesions including basal laminar deposits, loss of basal infoldings, and vacuoles in the retinal pigment epithelium were more prevalent in the 23.5- than in the 17-month-old mice. Within each age group, consumption of a high GI diet increased the risk for lesions and the risk for photoreceptor abnormalities and accumulation of AGEs. CONCLUSIONS: Consuming high GI diets accelerates the appearance of age-related retinal lesions that precede AMD in mice, perhaps by increasing the deposition of toxic AGEs in the retina. The data support the hypothesis that consuming lower GI diets, or simulation of their effects with nutraceuticals or drugs, may protect against AMD. The high GI-fed C57BL/6 mouse is a new model of age-related retinal lesions that precede AMD and mimic the early stages of disease and may be useful for drug discovery.

Differential protection among fractionated blueberry polyphenolic families against DA-, Abeta(42)- and LPS-induced decrements in Ca(2+) buffering in primary hippocampal cells.

It has been postulated that at least part of the loss of cognitive function in aging may be the result of deficits in Ca(2+) recovery (CAR) and increased oxidative/inflammatory (OX/INF) stress signaling. However, previous research showed that aged animals supplemented with blueberry (BB) extract showed fewer deficits in CAR, as well as motor and cognitive functional deficits. A recent subsequent experiment has shown that DA- or Abeta(42)-induced deficits in CAR in primary hippocampal neuronal cells (HNC) were antagonized by BB extract, and (OX/INF) signaling was reduced. The present experiments assessed the most effective BB polyphenol fraction that could protect against OX/INF-induced deficits in CAR, ROS generation, or viability. HNCs treated with BB extract, BB fractions (e.g., proanthocyanidin, PAC), or control medium were exposed to dopamine (DA, 0.1 mM), amyloid beta (Abeta(42), 25 muM) or lipopolysaccharide (LPS, 1 microg/mL). The results indicated that the degree of protection against deficits in CAR varied as a function of the stressor and was generally greater against Abeta(42) and LPS than DA. The whole BB, anthocyanin (ANTH), and PRE-C18 fractions offered the greatest protection, whereas chlorogenic acid offered the lowest protection. Protective capabilities of the various fractions against ROS depended upon the stressor, where the BB extract and the combined PAC (high and low molecular weight) fraction offered the best protection against LPS and Abeta(42) but were less effective against DA-induced ROS. The high and low molecular weight PACs and the ANTH fractions enhanced ROS production regardless of the stressor used, and this reflected increased activation of stress signals (e.g., P38 MAPK). The viability data indicated that the whole BB and combined PAC fraction showed greater protective effects against the stressors than the more fractionated polyphenolic components. Thus, these results suggest that, except for a few instances, the lesser the polyphenolic fractionation, the greater the effects, especially with respect to prevention of ROS and stress signal generation and viability.