Abstract Obesity prevalence within the 60+ years-old (yrs) population is drastically increasing. This alarmingly high rate of obesity compels comparative studies to identify common players involved in aging-derived and obesity-associated inflammation. Blood CD4+ T cells from lean older humans show an upregulated Th17 signature accompanied by a defective redox system. Treatment of these cells with the anti-diabetic drug metformin (100μM) promoted a more young-like anti-inflammatory/antioxidant phenotype. Here, we investigate whether the protective effects of metformin observed in CD4+ T cells from lean aging adults persist in obesity. Blood CD4+ T cells from obese older (60+ yrs) donors secrete twice the amount of the Th17 cytokines IL-17F and IL-21 when compared to their younger counterparts (25–35 yrs). Glutamate production, which supports Th17 cytokines secretion, is 1.5-times higher in cells from older compared to younger subjects. Mitochondrial superoxide production in these cells was 25% higher than in younger cells, with no changes in total peroxide. Contrary to findings from cells of lean subjects, metformin failed to reduce Th17 cytokines in cells from obese older subjects, perhaps due to metformin’s inability to reduce glutamate production. However, metformin reduced hydrogen peroxide production 1.75-fold, with no effect on superoxide, in cells from obese older subjects. These results collectively indicate that 1) obesity-associated upregulation of Th17 cytokines is mechanistically linked to glutamine metabolism and mitochondrial superoxide production and 2) metformin’s action targets peroxide production rather than superoxide, which is insufficient for reducing age-associated inflammation in obesity. The Biological mechanisms of Metformin Effects on Aging-Associated Inflammation 1R56AG069685-01
Our work in progress indicates that endothelial dysfunction and reductions in vascular p-eNOS S1177 to total eNOS in db/db mice are less severe in db/db mice supplemented with γ-tocopherol (γ-T). Vasoprotective activities of γ-T could be attributed to antioxidant properties of its metabolite γ-carboxyethyl-hydroxychroman (γ-CEHC) that increased in liver following γ-T supplementation. We hypothesized that γ-CEHC attenuates oxidative stress in endothelial cells treated with high glucose to an extent that preserves eNOS function. Human aortic endothelial cells (HAECs) incubated with 0 or 3 µM γ-CEHC for 24 h prior to treatment with normal (5 mM) or high (25 mM) glucose in the presence or absence of 3 µM γ-CEHC for 48 h. In separate studies HAECs were challenged with insulin to stimulate NO generation. High glucose increased (p<0.05) reactive oxygen species generation (DCF), nitrotyrosine protein expression, and mRNA expression of inflammatory mediators (ICAM-1, VCAM-1, E-selectin, MCP-1, IL-8). High-glucose decreased (p<0.05) eNOS dimer:monomer protein levels and prevented insulin-stimulated increases in p-AktS473 to total Akt, p-eNOSS1177 to total eNOS, and NO metabolite production. Each response evoked by high glucose was normalized when HAECs co-incubated with γ-CEHC and high glucose. Collectively, vascular protection by γ-T in vivo may be mediated through antioxidant and anti-inflammatory activities of γ-CEHC.
and distinct features of aging and obesity, two conditions characterized by chronic, low-grade systemic 55 inflammation and dysfunctional immunity and associated to several clinical problems, including reduced 56 vaccine responses. The cited work favors the hypothesis that obesity is is a state of accelerated immune 57 aging.The original research paper "Altered nutrient uptake causes mitochondrial dysfunction in senescent CD8 + Taken together, this series extends the current understanding of immunometabolism in aging and 78 obesity, while identifying new mechanisms foundational to the interrelationship between these conditions.The editors are optimistic that the assembled series will spark more research into actionable changes in 80 immunometabolism that support the clinical problems triggered by these naturally occurring events.
Impaired intracellular autophagy has been implicated as a cause of vascular dysfunction in aging human arteries. Earlier we reported that shear‐stress (180‐min × 20 dyne•cm 2 ) induces autophagy that is concurrent with phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at serine (S) 1177 (p‐eNOS S1177 ) and NO generation in bovine aortic endothelial cells (ECs). Genetic inhibition of autophagy via Atg3 or Atg5 siRNA suppresses shear‐induced p‐eNOS S1177 and NO generation, increases reactive oxygen species generation, and unleashes inflammatory/adhesion responses, suggesting strongly that autophagy is critical for maintaining EC function. Here we report one mechanism through which autophagy regulates shear‐induced p‐eNOS S1177 and NO production in ECs. Tumor suppressor and metabolism regulator, p53, is strongly upregulated by Atg3 siRNA, causing reduced shear‐induced Glut‐1 expression, glucose transport, glycolysis, and ATP production in ECs. As expected, AMPK is activated by the energy deficit, but does not restore p‐eNOS S1177 and NO generation in this setting. However, shear‐evoked p‐eNOS S1177 and NO generation can be restored in autophagy‐impaired ECs by (i) adding extracellular ADP or by (ii) overexpressing Glut‐1. These findings prompted a further investigation of purinergic signaling to eNOS in the context of suppressed EC autophagy. As predicted, P2Y 1 receptor blockade (MRS2179) and P2Y 1 siRNA can recapitulate the Atg3 phenotype i.e., shear‐induced p‐eNOS S1177 and NO generation are prevented. Furthermore, shear‐induced ATP/ADP release increases p‐PKCδ Thr505 , a downstream effector of P2Y 1 and activator of p‐eNOS S1177 . As would be anticipated by these findings, shear‐induced p‐eNOS S1177 and NO generation can be restored in autophagy‐impaired ECs by (i) the PKCδ activator bryostatin or by (ii) PKCδ overexpression, whereas shear‐induced p‐eNOS S1177 and NO generation are prevented when autophagy‐intact ECs are transfected with PKCδ siRNA. Ongoing experiments are determining the extent to which this mechanism is responsible for impaired endothelial function that we have observed in arteries from old mice, that is concurrent with lower Atg3 protein expression, compromised autophagy, and attenuated p‐eNOS S1177 : total eNOS, vs. arteries from adult mice. Collectively, targeted activation of purinergic signaling and/or PKCδ may present a new strategy for treating arterial dysfunction that exists with impaired EC autophagy in the context of healthy aging. Support or Funding Information UU Diabetes and Metabolism Center; UU Center on Aging; UU College of Health
Dysregulation of autophagy is an important underlying cause in the onset and progression of many metabolic diseases, including diabetes. Studies in animal models and humans show that impairment in the removal and the recycling of organelles, in particular, contributes to cellular damage, functional failure, and the onset of metabolic diseases. Interestingly, in certain contexts, inhibition of autophagy can be protective. While the inability to upregulate autophagy can play a critical role in the development of diseases, excessive autophagy can also be detrimental, making autophagy an intricately regulated process, the altering of which can adversely affect organismal health. Autophagy is indispensable for maintaining normal cardiac and vascular structure and function. Patients with diabetes are at a higher risk of developing and dying from vascular complications. Autophagy dysregulation is associated with the development of heart failure, many forms of cardiomyopathy, atherosclerosis, myocardial infarction, and microvascular complications in diabetic patients. Here, we review the recent findings on selective autophagy in hyperglycemia and diabetes-associated microvascular and macrovascular complications.
Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.
Striking age-related changes occur in the human immune system, beginning in the sixth decade of life. Age is a non-modifiable, universal risk factor that results in the dysregulation of many cellular homeostatic processes. The decline in immune cell macroautophagy/autophagy and the increased generation of proinflammatory cytokines during agingfuels the development of diseases in the elderly. We reported that higher Th17 inflammation during aging was secondary to dysregulation in T cell autophagy. However, the mechanism underlying lower anti-CD3 and anti-CD28 activation-induced T cell autophagy during aging remain unknown. Our data fuel the speculation that dysregulation of the glutathione (GSH) system might cause the decline in T cell autophagy in aging, additionally provoked by reactive oxygen species signaling emanating from the mitochondria.
Aging is associated with suppressed endothelial cell (EC) autophagy and EC nitric oxide (NO) bioavailability. A link might exist among autophagy, mitophagy, and stimulated NO production in ECs. ECs treated with scrambled siRNA exposed to shear stress (3 h x 20 dyn/cm 2 ) exhibit: ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation (indices of increased autophagy); ↓VDAC, ↓m‐aconitase, ↑TOM20:LAMP1 colocalization, ↓ mitochondrial pH indicating lysosomal compartmentalization (indices of increased mitochondrial turnover), ↑ROS generation, ↑endothelial NO synthase (eNOS) phosphorylation, and ↑NO production (p<0.05 for all). ECs transfected with A u T opha G y‐related protein 3 (Atg3) siRNA did not exhibit shear‐induced autophagy, mitochondrial turnover, eNOS phosphorylation, or NO production, but ROS accumulation and cytokine production (ICAM‐1, MCP‐1, IL‐8, E‐selectin) were exaggerated (all p<0.05). Shear‐induced increases in oxygen consumption rate, extracellular acidification rate, and ATP production (all p<0.05) in ECs were prevented by autophagy suppression, indicating impaired mitochondrial function. Dysfunctional mitochondria might be the source of ROS when autophagy is compromised because: (i) shear‐induced increases in NO generation were restored, and ROS production and inflammation were normalized, when ECs +Atg3 siRNA were treated with a mitochondrial‐targeted O 2 •‐ scavenger; and (ii) the EC phenotype +Atg3 siRNA could be recapitulated using approaches that limit mitophagy per se . A crosstalk exists among autophagy, mitophagy, and NO production in ECs.
Obesity promotes the onset and progression of metabolic and inflammatory diseases such as type 2 diabetes. The chronic low-grade inflammation that occurs during obesity triggers multiple signaling mechanisms that negatively affect organismal health. One such mechanism is the persistent activation and mitochondrial translocation of STAT3, which is implicated in inflammatory pathologies and many types of cancers. STAT3 in the mitochondria (mitoSTAT3) alters electron transport chain activity, thereby influencing nutrient metabolism and immune response. PBMCs and CD4+ T cells from obese but normal glucose-tolerant (NGT) middle-aged subjects had higher phosphorylation of STAT3 on residue serine 727 and more mitochondrial accumulation of STAT3 than cells from lean subjects. To evaluate if circulating lipid overabundance in obesity is responsible for age- and sex-matched mitoSTAT3, cells from lean subjects were challenged with physiologically relevant doses of the saturated and monounsaturated fatty acids, palmitate and oleate, respectively. Fatty acid treatment caused robust accumulation of mitoSTAT3 in all cell types, which was independent of palmitate-induced impairments in autophagy. Co-treatment of cells with fatty acid and trehalose prevented STAT3 phosphorylation and mitochondrial accumulation in an autophagy-independent but cellular peroxide-dependent mechanism. Pharmacological blockade of mitoSTAT3 either by a mitochondria-targeted STAT3 inhibitor or ROS scavenging prevented obesity and fatty acid-induced production of proinflammatory cytokines IL-17A and IL-6, thus establishing a mechanistic link between mitoSTAT3 and inflammatory cytokine production.
Circulating fatty acids (FAs) increase with obesity and can drive mitochondrial damage and inflammation. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that positively regulates nicotinamide adenine dinucleotide phosphate (NADPH), a key mediator of energy transduction and redox homeostasis. The role that NNT-regulated bioenergetics play in the inflammatory response of immune cells in obesity is untested. Our objective was to determine how free fatty acids (FFAs) regulate inflammation through impacts on mitochondria and redox homeostasis of peripheral blood mononuclear cells (PBMCs). PBMCs from lean subjects were activated with a T cell-specific stimulus in the presence or absence of generally pro-inflammatory palmitate and/or non-inflammatory oleate. Palmitate decreased immune cell expression of NNT, NADPH, and anti-oxidant glutathione, but increased reactive oxygen and proinflammatory Th17 cytokines. Oleate had no effect on these outcomes. Genetic inhibition of NNT recapitulated the effects of palmitate. PBMCs from obese (BMI >30) compared to lean subjects had lower NNT and glutathione expression, and higher Th17 cytokine expression, none of which were changed by exogenous palmitate. Our data identify NNT as a palmitate-regulated rheostat of redox balance that regulates immune cell function in obesity and suggest that dietary or therapeutic strategies aimed at increasing NNT expression may restore redox balance to ameliorate obesity-associated inflammation.
