José Pablo Vázquez-Medina

Prolonged fasting does not increase oxidative damage or inflammation in postweaned northern elephant seal pups

SUMMARY Elephant seals are naturally adapted to survive up to three months of absolute food and water deprivation (fasting). Prolonged food deprivation in terrestrial mammals increases reactive oxygen species (ROS) production, oxidative damage and inflammation that can be induced by an increase in the renin–angiotensin system (RAS). To test the hypothesis that prolonged fasting in elephant seals is not associated with increased oxidative stress or inflammation, blood samples and muscle biopsies were collected from early (2–3 weeks post-weaning) and late (7–8 weeks post-weaning) fasted seals. Plasma levels of oxidative damage, inflammatory markers and plasma renin activity (PRA), along with muscle levels of lipid and protein oxidation, were compared between early and late fasting periods. Protein expression of angiotensin receptor 1 (AT1), pro-oxidant (Nox4) and antioxidant enzymes (CuZn- and Mn-superoxide dismutases, glutathione peroxidase and catalase) was analyzed in muscle. Fasting induced a 2.5-fold increase in PRA, a 50% increase in AT1, a twofold increase in Nox4 and a 70% increase in NADPH oxidase activity. By contrast, neither tissue nor systemic indices of oxidative damage or inflammation increased with fasting. Furthermore, muscle antioxidant enzymes increased 40–60% with fasting in parallel with an increase in muscle and red blood cell antioxidant enzyme activities. These data suggest that, despite the observed increases in RAS and Nox4, an increase in antioxidant enzymes appears to be sufficient to suppress systemic and tissue indices of oxidative damage and inflammation in seals that have fasted for a prolonged period. The present study highlights the importance of antioxidant capacity in mammals during chronic periods of stress to help avoid deleterious systemic consequences.

Glut4 is Upregulated Despite Decreased Insulin Signaling during Prolonged Fasting in Northern Elephant Seal Pups

Postprandial cellular glucose uptake is dependent on an insulin-signaling cascade in muscle and adipose tissue, resulting in the translocation of the insulin-dependent glucose transporter 4 (Glut4) into the plasma membrane. Additionally, extended food deprivation is characterized by suppressed insulin signaling and decreased Glut4 expression. Northern elephant seals are adapted to prolonged fasts characterized by high levels of plasma glucose. To address the hypothesis that the fasting-induced decrease in insulin is associated with reduced insulin signaling in prolonged fasted seals, we compared the adipose protein levels of the cellular insulin-signaling pathway, Glut4 and plasma glucose, insulin, cortisol, and adiponectin concentrations between Early (n = 9; 2-3 wks postweaning) and Late (n = 8; 6-8 wks postweaning) fasted seals. Plasma adiponectin (230 ± 13 vs. 177 ± 11 ng/ml), insulin (2.7 ± 0.4 vs. 1.0 ± 0.1 μU/ml), and glucose (9.8 ± 0.5 vs. 8.0 ± 0.3 mM) decreased, while cortisol (124 ± 6 vs. 257 ± 30 nM) doubled with fasting. Glut4 increased (31%) with fasting despite the significant decreases in the cellular content of phosphatidylinositol 3-kinase as well as phosphorylated insulin receptor, insulin receptor substrate-1, and Akt2. Increased Glut4 may have contributed to the decrease in plasma glucose, but the decrease in insulin and insulin signaling suggests that Glut4 is not insulin-dependent in adipose tissue during prolonged fasting in elephant seals. The reduction of plasma glucose independent of insulin may make these animals an ideal model for the study of insulin resistance.

Coping with physiological oxidative stress: a review of antioxidant strategies in seals

While diving, seals are exposed to apnea-induced hypoxemia and repetitive cycles of ischemia/reperfusion. While on land, seals experience sleep apnea, as well as prolonged periods of food and water deprivation. Prolonged fasting, sleep apnea, hypoxemia and ischemia/reperfusion increase oxidant production and oxidative stress in terrestrial mammals. In seals, however, neither prolonged fasting nor apnea-induced hypoxemia or ischemia/reperfusion increase systemic or local oxidative damage. The strategies seals evolved to cope with increased oxidant production are reviewed in the present manuscript. Among these strategies, high antioxidant capacity and the oxidant-mediated activation of hormetic responses against hypoxia and oxidative stress are discussed. In addition to expanding our knowledge of the evolution of antioxidant defenses and adaptive responses to oxidative stress, understanding the mechanisms that naturally allow mammals to avoid oxidative damage has the potential to advance our knowledge of oxidative stress-induced pathologies and to enhance the translative value of biomedical therapies in the long term.

Apnea stimulates the adaptive response to oxidative stress in elephant seal pups.

SUMMARY Extended breath-hold (apnea) bouts are routine during diving and sleeping in seals. These apneas result in oxygen store depletion and blood flow redistribution towards obligatory oxygen-dependent tissues, exposing seals to critical levels of ischemia and hypoxemia. The subsequent reperfusion/reoxygenation has the potential to increase oxidant production and thus oxidative stress. The contributions of extended apnea to oxidative stress in adapted mammals are not well defined. To address the hypothesis that apnea in seals is not associated with increased oxidative damage, blood samples were collected from northern elephant seal pups (N=6) during eupnea, rest- and voluntary submersion-associated apneas, and post-apnea (recovery). Plasma 4-hydroxynonenal (HNE), 8-isoprostanes (8-isoPGF2α), nitrotyrosine (NT), protein carbonyls, xanthine and hypoxanthine (HX) levels, along with xanthine oxidase (XO) activity, were measured. Protein content of XO, superoxide dismutase 1 (Cu,ZnSOD), catalase and myoglobin (Mb), as well as the nuclear content of hypoxia inducible factor 1α (HIF-1α) and NF-E2-related factor 2 (Nrf2), were measured in muscle biopsies collected before and after the breath-hold trials. HNE, 8-iso PGF2α, NT and protein carbonyl levels did not change among eupnea, apnea or recovery. XO activity and HX and xanthine concentrations were increased at the end of the apneas and during recovery. Muscle protein content of XO, CuZnSOD, catalase, Mb, HIF-1α and Nrf2 increased 25–70% after apnea. Results suggest that rather than inducing the damaging effects of hypoxemia and ischemia/reperfusion that have been reported in non-diving mammals, apnea in seals stimulates the oxidative stress and hypoxic hormetic responses, allowing these mammals to cope with the potentially detrimental effects associated with this condition.

Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals

SUMMARY Northern elephant seals experience prolonged periods of absolute food and water deprivation (fasting) while breeding, molting or weaning. The postweaning fast in elephant seals is characterized by increases in the renin–angiotensin system, expression of the oxidant-producing protein Nox4, and NADPH oxidase activity; however, these increases are not correlated with increased oxidative damage or inflammation. Glutathione (GSH) is a potent reductant and a cofactor for glutathione peroxidases (GPx), glutathione-S transferases (GST) and 1-cys peroxiredoxin (PrxVI) and thus contributes to the removal of hydroperoxides, preventing oxidative damage. The effects of prolonged food deprivation on the GSH system are not well described in mammals. To test our hypothesis that GSH biosynthesis increases with fasting in postweaned elephant seals, we measured circulating and muscle GSH content at the early and late phases of the postweaning fast in elephant seals along with the activity/protein content of glutamate-cysteine ligase [GCL; catalytic (GCLc) and modulatory (GCLm) subunits], γ-glutamyl transpeptidase (GGT), glutathione disulphide reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), GST and PrxVI, as well as plasma changes in γ-glutamyl amino acids, glutamate and glutamine. GSH increased two- to four-fold with fasting along with a 40–50% increase in the content of GCLm and GCLc, a 75% increase in GGT activity, a two- to 2.5-fold increase in GR, G6PDH and GST activities and a 30% increase in PrxVI content. Plasma γ-glutamyl glutamine, γ-glutamyl isoleucine and γ-glutamyl methionine also increased with fasting whereas glutamate and glutamine decreased. Results indicate that GSH biosynthesis increases with fasting and that GSH contributes to counteracting hydroperoxide production, preventing oxidative damage in fasting seals.

Angiotensin receptor-mediated oxidative stress is associated with impaired cardiac redox signaling and mitochondrial function in insulin-resistant rats

Activation of angiotensin receptor type 1 (AT1) contributes to NADPH oxidase (Nox)-derived oxidative stress during metabolic syndrome. However, the specific role of AT1 in modulating redox signaling, mitochondrial function, and oxidative stress in the heart remains more elusive. To test the hypothesis that AT1 activation increases oxidative stress while impairing redox signaling and mitochondrial function in the heart during diet-induced insulin resistance in obese animals, Otsuka Long Evans Tokushima Fatty (OLETF) rats (n = 8/group) were treated with the AT1 blocker (ARB) olmesartan for 6 wk. Cardiac Nox2 protein expression increased 40% in OLETF compared with age-matched, lean, strain-control Long Evans Tokushima Otsuka (LETO) rats, while mRNA and protein expression of the H₂O₂-producing Nox4 increased 40-100%. ARB treatment prevented the increase in Nox2 without altering Nox4. ARB treatment also normalized the increased levels of protein and lipid oxidation (nitrotyrosine, 4-hydroxynonenal) and increased the redox-sensitive transcription factor Nrf2 by 30% and the activity of antioxidant enzymes (SOD, catalase, GPx) by 50-70%. Citrate synthase (CS) and succinate dehydrogenase (SDH) activities decreased 60-70%, whereas cardiac succinate levels decreased 35% in OLETF compared with LETO, suggesting that mitochondrial function in the heart is impaired during obesity-induced insulin resistance. ARB treatment normalized CS and SDH activities, as well as succinate levels, while increasing AMPK and normalizing Akt, suggesting that AT1 activation also impairs cellular metabolism in the diabetic heart. These data suggest that the cardiovascular complications associated with metabolic syndrome may result from AT1 receptor-mediated Nox2 activation leading to impaired redox signaling, mitochondrial activity, and dysregulation of cellular metabolism in the heart.

Decreased expression of adipose CD36 and FATP1 are associated with increased plasma non-esterified fatty acids during prolonged fasting in northern elephant seal pups (Mirounga angustirostris)

SUMMARY The northern elephant seal pup (Mirounga angustirostris) undergoes a 2–3 month post-weaning fast, during which it depends primarily on the oxidation of fatty acids to meet its energetic demands. The concentration of non-esterified fatty acids (NEFAs) increases and is associated with the development of insulin resistance in late-fasted pups. Furthermore, plasma NEFA concentrations respond differentially to an intravenous glucose tolerance test (ivGTT) depending on fasting duration, suggesting that the effects of glucose on lipid metabolism are altered. However, elucidation of the lipolytic mechanisms including lipase activity during prolonged fasting in mammals is scarce. To assess the impact of fasting and glucose on the regulation of lipid metabolism, adipose tissue and plasma samples were collected before and after ivGTTs performed on early (2 weeks, N=5) and late (6–8 weeks; N=8) fasted pups. Glucose administration increased plasma triglycerides and NEFA concentrations in late-fasted seals, but not plasma glycerol. Fasting decreased basal adipose lipase activity by 50%. Fasting also increased plasma lipase activity twofold and decreased the expressions of CD36, FAS, FATP1 and PEPCK-C by 22–43% in adipose tissue. Plasma acylcarnitine profiling indicated that late-fasted seals display higher incomplete LCFA β-oxidation. Results suggest that long-term fasting induces shifts in the regulation of lipolysis and lipid metabolism associated with the onset of insulin resistance in northern elephant seal pups. Delineation of the mechanisms responsible for this shift in regulation during fasting can contribute to a more thorough understanding of the changes in lipid metabolism associated with dyslipidemia and insulin resistance in mammals.

Oxidative stress is a potential cost of breeding in male and female northern elephant seals

The trade-off between current reproductive effort and survival is a key concept of life history theory. A variety of studies support the existence of this trade-off but the underlying physiological mechanisms are not well-understood. Oxidative stress has been proposed as a potential mechanism underlying the observed inverse relationship between reproductive investment and lifespan. Prolonged fasting is associated with oxidative stress including increases in the production of reactive oxygen species, oxidative damage and inflammation.Northern elephant seals (NES) undergo prolonged fasts while maintaining high metabolic rates during breeding. We investigated NES of both sexes to assess oxidative stress associated with extended breeding fasts. We measured changes in the plasma activity or concentrations of markers for oxidative stress in 30 adult male and 33 adult female northern elephant seals across their 1-3 month breeding fasts. Markers assessed included a pro-oxidant enzyme, several antioxidant enzymes, markers for oxidative damage to lipids, proteins and DNA, and markers for systemic inflammation.Plasma xanthine oxidase (XO), a pro-oxidant enzyme that increases production of oxidative radicals, and several protective antioxidant enzymes increased over breeding in both sexes. Males showed increased oxidative damage to lipids and DNA and increased systemic inflammation, while oxidative damage to proteins declined across breeding. In contrast, females showed no oxidative damage to lipids or DNA or changes in inflammation, but showed increases in oxidative damage to proteins. XO activity, antioxidant enzymes, oxidative damage markers, and inflammatory markers were strongly correlated in males but these relationships were weaker or non-existent in females.NES provide evidence for oxidative stress as a physiological cost of reproduction in a capital breeding mammal. Both sexes strongly up-regulated antioxidant defenses during breeding. Despite this response, and in contrast to similar duration non-breeding fasts in previous studies on conspecifics, there was evidence of oxidative damage to tissues. These data demonstrate the utility of using plasma markers to examine oxidative stress but also suggest the necessity of measuring a broad suite of plasma markers to assess systemic oxidative stress.

Angiotensin Receptor Blockade Recovers Hepatic UCP2 Expression and Aconitase and SDH Activities and Ameliorates Hepatic Oxidative Damage in Insulin Resistant Rats

Metabolic syndrome (MetS) is commonly associated with elevated renin-angiotensin system, oxidative stress, and steatohepatitis with down-regulation of uncoupling proteins (UCPs). However, the mechanisms linking renin-angiotensin system, steatosis, and UCP2 to hepatic oxidative damage during insulin resistance are not described. To test the hypothesis that angiotensin receptor activation contributes to decreased hepatic UCP2 expression and aconitase activity and to increased oxidative damage after increased glucose intake in a model of MetS, lean and obese Long Evans rats (n = 10/group) were randomly assigned to the following groups: 1) untreated Long Evans Tokushima Otsuka (lean, strain control), 2) untreated Otsuka Long Evans Tokushima Fatty (OLETF) (MetS model), 3) OLETF + angiotensin receptor blocker (ARB) (10 mg olmesartan/kg·d × 6 wk), 4) OLETF + high glucose (HG) (5% in drinking water × 6 wk), and 5) OLETF + ARB + HG (ARB/HG × 6 wk). HG increased body mass (37%), plasma triglycerides (TGs) (35%), plasma glycerol (87%), plasma free fatty acids (28%), and hepatic nitrotyrosine (74%). ARB treatment in HG decreased body mass (12%), plasma TG (15%), plasma glycerol (23%), plasma free fatty acids (14%), and hepatic TG content (42%), suggesting that angiotensin receptor type 1 (AT1) activation and increased adiposity contribute to the development of obesity-related dyslipidemia. ARB in HG also decreased hepatic nitrotyrosine and increased hepatic UCP2 expression (59%) and aconitase activity (40%), as well as antioxidant enzyme activities (50-120%), suggesting that AT1 activation also contributes to protein oxidation, impaired lipid metabolism, and antioxidant metabolism in the liver. Thus, in addition to promoting obesity-related hypertension, AT1 activation may also impair lipid metabolism and antioxidant capacity, resulting in steatosis via decreased UCP2 and tricarboxylic acid cycle activity.

Insulin and GLP-1 infusions demonstrate the onset of adipose-specific insulin resistance in a large fasting mammal: potential glucogenic role for GLP-1

Prolonged food deprivation increases lipid oxidation and utilization, which may contribute to the onset of the insulin resistance associated with fasting. Because insulin resistance promotes the preservation of glucose and oxidation of fat, it has been suggested to be an adaptive response to food deprivation. However, fasting mammals exhibit hypoinsulinemia, suggesting that the insulin resistance‐like conditions they experience may actually result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. To determine whether fasting results in insulin resistance or in pancreatic dysfunction, we infused early‐ and late‐fasted seals (naturally adapted to prolonged fasting) with insulin (0.065 U/kg), and a separate group of late‐fasted seals with low (10 pmol/L per kg) or high (100 pmol/L per kg) dosages of glucagon‐like peptide‐1 (GLP‐1) immediately following a glucose bolus (0.5 g/kg), and measured the systemic and cellular responses. Because GLP‐1 facilitates glucose‐stimulated insulin secretion, these infusions provide a method to assess pancreatic insulin‐secreting capacity. Insulin infusions increased the phosphorylation of insulin receptor and Akt in adipose and muscle of early‐ and late‐fasted seals; however, the timing of the signaling response was blunted in adipose of late‐fasted seals. Despite the dose‐dependent increases in insulin and increased glucose clearance (high dose), both GLP‐1 dosages produced increases in plasma cortisol and glucagon, which may have contributed to the glucogenic role of GLP‐1. Results suggest that fasting induces adipose‐specific insulin resistance in elephant seal pups, while maintaining skeletal muscle insulin sensitivity, and therefore suggests that the onset of insulin resistance in fasting mammals is an evolved response to cope with prolonged food deprivation.