José G. Soñanez-Organis

Molecular characterization of hypoxia inducible factor-1 (HIF-1) from the white shrimp Litopenaeus vannamei and tissue-specific expression under hypoxia.

Hypoxia inducible factor 1 (HIF-1) is a key transcription factor that regulates a variety of molecular responses to hypoxia. Some marine crustaceans experience changes of oxygen tension in their aquatic environment, but knowledge about the function and expression of HIF-1 is very limited. HIF-1 is a heterodimer composed by alpha and beta subunits. We report the complete cDNA sequences of HIF-1alpha and HIF-1beta from the white shrimp Litopenaeus vannamei. HIF-1alpha (LvHIF-1alpha) is 3672bp and codes for 1050 amino acids, while HIF-1beta is 2135bp (LvHIF-1beta) and 608 amino acids. Both, the alpha and beta subunits have the helix-loop-helix (bHLH) and PAS domains. HIF-1alpha also has the oxygen dependent degradation (ODD) and the C-terminal transactivation domain (C-TAD), important for regulation in normoxia. Phylogenetic analyses of the proteins indicate separation of invertebrates from vertebrates. Large differences of HIF-1alpha and HIF-1beta transcripts abundance were detected in gills, hepatopancreas and muscle under normoxia (6mg/L dissolved oxygen, DO) and hypoxia (2.5 and 1.5mg/L DO). HIF-1alpha was more abundant in gills and HIF-1beta in hepatopancreas. Large changes in response to hypoxia were detected for HIF-1alpha in gills, while HIF-1beta remained fairly constant. Glucose and lactate in hemolymph increased rapidly in hypoxia in all cases and up to 4.7 and 5.0-fold, respectively, in response to 1.5mg/L DO for 1h.

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.

Hexokinase from the white shrimp Litopenaeus vannamei: cDNA sequence, structural protein model and regulation via HIF-1 in response to hypoxia

Hexokinase (HK) catalyzes the phosphorylation of glucose, the first rate-limiting step in glycolysis. HKs are a conserved family of tissue-specific isozymes, from which very little is known in marine crustaceans. This study describes the cloning and characterization of the full-length cDNA sequence for HK from the shrimp Litopenaeus vannamei, the theoretical tridimensional protein structure and response to short-term hypoxia. The full-length cDNA is 1872bp long with an open reading frame of 1452bp coding for a protein of 484 amino acids and predicted molecular mass of 53kDa. Highly conserved amino acid residues are present in the glucose, glucose-6-phosphate, ATP and Mg(+2) binding sites. Phylogenetic analysis shows that the shrimp HK is closer to invertebrates than to vertebrate HKs. Hypoxia induced HK expression in gills with the concomitant increase in the specific enzyme activity. In muscle, hypoxia decreased HK mRNAs but increased the enzyme activity. Silencing of the hypoxia inducible factor 1 (HIF-1) affected the expression of HK differentially. In gills, silencing of α or β subunits blocked the induction of HK expression and the enzymatic activity, but not in muscle. This suggests the existence of tissue-specific HK isozymes and post-transcriptional and post-translational regulation of HK.

Antioxidant capacity develops with maturation in the deep-diving hooded seal

SUMMARY Maturation in hooded seals is characterized by the rapid development of their physiological diving capacity and is accompanied by increases in oxidant production but not oxidative damage. To test the hypothesis that the antioxidant system of hooded seals develops as they transition from a terrestrial to an aquatic environment, we obtained the complete cDNA sequence that encodes the NF-E2-related factor 2 (Nrf2), a central regulator of the antioxidant response, and compared Nrf2 mRNA and protein expression levels in muscle samples from neonate, weaned pups and adult hooded seals, along with glutathione (GSH) levels and the activity/protein content of the antioxidant enzymes catalase, glutathione peroxidase (GPx), peroxyredoxin VI (PrxVI), thioredoxin 1 (Trx1), thioredoxin reductase (TrxR), glutaredoxin 1 (Glrx1), glutathione disulphide reductase, glutathione S-transferase and glutamate-cysteine ligase. The Nrf2 of the hooded seal is 1822 bp long and encodes a protein of 606 amino acids with a leucine zipper domain and Keap1-mediated proteosomal degradation residues, which are key for Nrf2 function and regulation. Although neither Nrf2 mRNA nor Nrf2 nuclear protein content are higher in adults than in pups, GSH levels along with GPx, PrxVI, Trx1, TrxR and Glrx1 activity/protein content increase with maturation, suggesting that the potential for peroxide removal increases with development in hooded seals, and that these enzymes contribute to the regulation of the intracellular redox state and the prevention of oxidative damage in these deep-diving mammals.

Prolonged fasting increases purine recycling in post-weaned northern elephant seals

SUMMARY Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of absolute food and water deprivation (fasting). In terrestrial mammals, food deprivation stimulates ATP degradation and decreases ATP synthesis, resulting in the accumulation of purines (ATP degradation byproducts). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) salvages ATP by recycling the purine degradation products derived from xanthine oxidase (XO) metabolism, which also promotes oxidant production. The contributions of HGPRT to purine recycling during prolonged food deprivation in marine mammals are not well defined. In the present study we cloned and characterized the complete and partial cDNA sequences that encode for HGPRT and xanthine oxidoreductase (XOR) in northern elephant seals. We also measured XO protein expression and circulating activity, along with xanthine and hypoxanthine plasma content in fasting northern elephant seal pups. Blood, adipose and muscle tissue samples were collected from animals after 1, 3, 5 and 7 weeks of their natural post-weaning fast. The complete HGPRT and partial XOR cDNA sequences are 771 and 345 bp long and encode proteins of 218 and 115 amino acids, respectively, with conserved domains important for their function and regulation. XOR mRNA and XO protein expression increased 3-fold and 1.7-fold with fasting, respectively, whereas HGPRT mRNA (4-fold) and protein (2-fold) expression increased after 7 weeks in adipose tissue and muscle. Plasma xanthine (3-fold) and hypoxanthine (2.5-fold) levels, and XO (1.7- to 20-fold) and HGPRT (1.5- to 1.7-fold) activities increased during the last 2 weeks of fasting. Results suggest that prolonged fasting in elephant seal pups is associated with increased capacity to recycle purines, which may contribute to ameliorating oxidant production and enhancing the supply of ATP, both of which would be beneficial during prolonged food deprivation and appear to be adaptive in this species.

Prolonged fasting activates hypoxia inducible factors-1α, -2α and -3α in a tissue-specific manner in northern elephant seal pups

Hypoxia inducible factors (HIFs) are important regulators of energy homeostasis and cellular adaptation to low oxygen conditions. Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of food deprivation (fasting) which result in metabolic changes that may activate HIF-1. However, the effects of prolonged fasting on HIFs are not well defined. We obtained the full-length cDNAs of HIF-1α and HIF-2α, and partial cDNA of HIF-3α in northern elephant seal pups. We also measured mRNA and nuclear protein content of HIF-1α, -2α, -3α in muscle and adipose during prolonged fasting (1, 3, 5 & 7 weeks), along with mRNA expression of HIF-mediated genes, LDH and VEGF. HIF-1α, -2α and -3α are 2595, 2852 and 1842 bp and encode proteins of 823, 864 and 586 amino acid residues with conserved domains needed for their function (bHLH and PAS) and regulation (ODD and TAD). HIF-1α and -2α mRNA expression increased 3- to 5-fold after 7 weeks of fasting in adipose and muscle, whereas HIF-3α increased 5-fold after 7 weeks of fasting in adipose. HIF-2α protein expression was detected in nuclear fractions from adipose and muscle, increasing approximately 2-fold, respectively with fasting. Expression of VEGF increased 3-fold after 7 weeks in adipose and muscle, whereas LDH mRNA expression increased 12-fold after 7 weeks in adipose. While the 3 HIFα genes are expressed in muscle and adipose, only HIF-2α protein was detectable in the nucleus suggesting that HIF-2α may contribute more significantly in the up-regulation of genes involved in the metabolic adaptation during fasting in the elephant seal.

Prolonged fasting activates Nrf2 in post-weaned elephant seals

SUMMARY Elephant seals naturally experience prolonged periods of absolute food and water deprivation (fasting). In humans, rats and mice, prolonged food deprivation activates the renin–angiotensin system (RAS) and increases oxidative damage. In elephant seals, prolonged fasting activates RAS without increasing oxidative damage likely due to an increase in antioxidant defenses. The mechanism leading to the upregulation of antioxidant defenses during prolonged fasting remains elusive. Therefore, we investigated whether prolonged fasting activates the redox-sensitive transcription factor Nrf2, which controls the expression of antioxidant genes, and if such activation is potentially mediated by systemic increases in RAS. Blood and skeletal muscle samples were collected from seals fasting for 1, 3, 5 and 7 weeks. Nrf2 activity and nuclear content increased by 76% and 167% at week 7. Plasma angiotensin II (Ang II) and transforming growth factor β (TGF-β) were 5000% and 250% higher at week 7 than at week 1. Phosphorylation of Smad2, an effector of Ang II and TGF signaling, increased by 120% at week 7 and by 84% in response to intravenously infused Ang II. NADPH oxidase 4 (Nox4) mRNA expression, which is controlled by smad proteins, increased 430% at week 7, while Nox4 protein expression, which can activate Nrf2, was 170% higher at week 7 than at week 1. These results demonstrate that prolonged fasting activates Nrf2 in elephant seals and that RAS stimulation can potentially result in increased Nox4 through Smad phosphorylation. The results also suggest that Nox4 is essential to sustain the hormetic adaptive response to oxidative stress in fasting seals.

Glucose delays the insulin-induced increase in thyroid hormone-mediated signaling in adipose of prolong-fasted elephant seal pups

Prolonged food deprivation in mammals typically reduces glucose, insulin, and thyroid hormone (TH) concentrations, as well as tissue deiodinase (DI) content and activity, which, collectively, suppress metabolism. However, in elephant seal pups, prolonged fasting does not suppress TH levels; it is associated with upregulation of adipose TH-mediated cellular mechanisms and adipose-specific insulin resistance. The functional relevance of this apparent paradox and the effects of glucose and insulin on TH-mediated signaling in an insulin-resistant tissue are not well defined. To address our hypothesis that insulin increases adipose TH signaling in pups during extended fasting, we assessed the changes in TH-associated genes in response to an insulin infusion in early- and late-fasted pups. In late fasting, insulin increased DI1, DI2, and THrβ-1 mRNA expression by 566%, 44%, and 267% at 60 min postinfusion, respectively, with levels decreasing by 120 min. Additionally, we performed a glucose challenge in late-fasted pups to differentiate between insulin- and glucose-mediated effects on TH signaling. In contrast to the insulin-induced effects, glucose infusion did not increase the expressions of DI1, DI2, and THrβ-1 until 120 min, suggesting that glucose delays the onset of the insulin-induced effects. The data also suggest that fasting duration increases the sensitivity of adipose TH-mediated mechanisms to insulin, some of which may be mediated by increased glucose. These responses appear to be unique among mammals and to have evolved in elephant seals to facilitate their adaptation to tolerate an extreme physiological condition.

Activation of systemic, but not local, renin–angiotensin system is associated with upregulation of TNF-α during prolonged fasting in northern elephant seal pups

SUMMARY Northern elephant seal pups naturally endure a 2–3 month post-weaning fast that is associated with activation of systemic renin–angiotensin system (RAS), a decrease in plasma adiponectin (Acrp30), and insulin resistance (IR)-like conditions. Angiotensin II (Ang II) and tumor necrosis factor-alpha (TNF-α) are potential causal factors of IR, while Acrp30 may improve insulin signaling. However, the effects of fasting-induced activation of RAS on IR-like conditions in seals are not well described. To assess the effects of prolonged food deprivation on systemic and local RAS, and their potential contribution to TNF-α as they relate to an IR condition, the mRNA expressions of adipose and muscle RAS components and immuno-relevant molecules were measured along with plasma RAS components. Mean plasma renin activity and Ang II concentrations increased by 89 and 1658%, respectively, while plasma angiotensinogen (AGT) decreased by 49% over the fast, indicative of systemic RAS activation. Prolonged fasting was associated with decreases in adipose and muscle AGT mRNA expressions of 69 and 68%, respectively, corresponding with decreases in tissue protein content, suggesting suppression of local AGT production. Muscle TNF-α mRNA and protein increased by 239 and 314%, whereas those of adipose Acrp30 decreased by 32 and 98%, respectively. Collectively, this study suggests that prolonged fasting activates a systemic RAS, which contributes to an increase in muscle TNF-α and suppression of adipose Acrp30. This targeted and tissue-specific regulation of TNF-α and Acrp30 is likely coordinated to synergistically contribute to the development of an IR-like condition, independent of local RAS activity. These data enhance our understanding of the adaptive mechanisms evolved by elephant seals to tolerate potentially detrimental conditions.

HIF-1α and PPARγ during physiological cardiac hypertrophy induced by pregnancy: Transcriptional activities and effects on target genes.

Hypoxia inducible factor 1-α (HIF-1α) and peroxisome proliferator-activated receptor γ (PPARγ) are transcription factors that activate genes involved in cellular metabolism. Physiological cardiac hypertrophy induced by pregnancy initiates compensatory changes in metabolism. However, the contributions of HIF-1α and PPARγ to this physiological status and to its reversible, metabolic process (postpartum) in the heart are not well-defined. Therefore, the aim of the present study was to evaluate the transcriptional activities of HIF-1α and PPARγ in the left ventricle of rats before, during, and after pregnancy. Furthermore, the effects of pregnancy on target genes of glycolysis and glycerol-lipid biosynthesis, key regulatory enzymes, and metabolic intermediates were evaluated. The activities of HIF-1α and PPARγ increased 1.2- and 1.6-fold, respectively, during pregnancy, and decreased to basal levels during postpartum. Expressions of mRNA for glucose transport 1 (GLUT1), enzymes of glycolysis (HK2, PFKM, and GAPDH) and glycerol-lipid biosynthesis (GPAT and GPD1) increased 1.6- to 14-fold during pregnancy and returned to basal levels postpartum. The increase in GPD1 expression translated to an increase in its activity, but such was not the case for GAPDH suggesting that post-translational regulation of these proteins is differential during pregnancy. Glycolytic (glucose, lactate, and DHAP) and glycerol-lipid biosynthesis (G3P and FFA) intermediates increased with pregnancy and were maintained postpartum. The results demonstrate that pregnancy-induced, physiological cardiac hypertrophy activates the expression of genes involved in glycolytic and glycerol-lipid biosynthesis suggesting that the shift in cardiac metabolism is mediated by the activation of HIF-1α and PPARγ.