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The prolonged, postweaning fast of northern elephant seal (Mirounga angustirostris) pups is characterized by a reliance on lipid metabolism and reversible, fasting-induced insulin resistance, providing a unique model to examine the effects of insulin on lipid metabolism. We have previously shown that acute insulin infusion induced a shift in fatty acid metabolism dependent on fasting duration. This study complements the previous study by examining the effects of fasting duration and insulin infusion on circulating levels of oxylipins, bioactive metabolites derived from the oxygenation of polyunsaturated fatty acids. Northern elephant seal pups were studied at two postweaning periods (n = 5/period): early fasting (1--2wk postweaning; 127 ± 1 kg) and late fasting (6--7wk postweaning; 93 ± 4 kg). Different cohorts of pups were weighed, sedated, and infused with 65 mU/kg of insulin. Plasma was collected prior to infusion (T0) and at 10, 30, 60, and 120min postinfusion. A profile of ~80 oxylipins was analyzed by UPLC-ESI-MS/MS. Nine oxylipins changed between early and late fasting and eight were altered in response to insulin infusion. Fasting decreased prostaglandin F2a (PGF2a) and increased 14,15-dihydroxyicosatrienoic acid (14,15-DiHETrE), 20-hydroxyeicosatetraenoic acid (20-HETE), and 4-hydroxy-docosahexaenoic acid (4- HDoHE) (P < 0.03) in T0 samples, whereas insulin infusion resulted in an inverse change in area-under-the-curve (AUC) levels in these same metabolites (P < 0.05). In addition, 12-12-hydroperoxyeicosatetraenoic acid (HpETE) and 12-HETE decreased with fasting and insulin infusion, respectively (P < 0.04). The oxylipins altered during fasting and in response to insulin infusion may contribute to the manifestation of insulin resistance and participate in the metabolic regulation of associated cellular processes. [ABSTRACT FROM AUTHOR]

The postweaning fast of northern elephant seal pups is characterized by a lipid-dependent metabolism and associated with a decrease in plasma glucagon-like peptide-1 (GLP-1), insulin, and glucose and increased gluconeogenesis (GNG) and ketogenesis. We have also demonstrated that exogenous GLP-1 infusion increased plasma insulin despite simultaneous increases in cortisol and glucagon, which collectively present contradictory regulatory stimuli of GNG, ketogenesis, and glycolysis. To assess the effects of GLP-1 on metabolism using primary carbon metabolite profiles in late-fasted seal pups, we dose-dependently infused late-fasted seals with low (LDG; 10 pM/kg; n = 3) or high (HDG; 100 pM/kg; n = 4) GLP-1 immediately following a glucose bolus (0.5 g/kg), using glucose without GLP-1 as control (n = 5). Infusions were performed in similarly aged animals 6-8wk into their postweaning fast. The plasma metabolome was measured from samples collected at five time points just prior to and during the infusions, and network maps constructed to robustly evaluate the effects of GLP-1 on primary carbon metabolism. HDG increased key tricarboxylic acid (TCA) cycle metabolites, and decreased phosphoenolpyruvate and acetoacetate (P < 0.05) suggesting that elevated levels of GLP-1 promote glycolysis and suppress GNG and ketogenesis, which collectively increase glucose clearance. These GLP-1-mediated effects on cellular metabolism help to explain why plasma GLP-1 concentrations decrease naturally in fasting pups as an evolved mechanism to help conserve glucose during the late-fasting period. [ABSTRACT FROM AUTHOR]

Hepatosteatosis, which is frequently associated with development of metabolic syndrome and insulin resistance, manifests when triglyceride (TG) input in the liver is greater than TG output, resulting in the excess accumulation of TG. Dysregulation of lipogenesis therefore has the potential to increase lipid accumulation in the liver, leading to insulin resistance and type 2 diabetes. Recently, efforts have been made to examine the epigenetic regulation of metabolism by histone-modifying enzymes that alter chromatin accessibility for activation or repression of transcription. For regulation of lipogenic gene transcription, various known lipogenic transcription factors, such as USF1, ChREBP, and LXR, interact with and recruit specific histone modifiers, directing specificity toward lipogenesis. Alteration or impairment of the functions of these histone modifiers can lead to dysregulation of lipogenesis and thus hepatosteatosis leading to insulin resistance and type 2 diabetes. [ABSTRACT FROM AUTHOR]

De novo lipogenesis is precisely regulated by nutritional and hormonal conditions. The genes encoding various enzymes involved in this process, such as fatty acid synthase (FASN), are transcriptionally activated in response to insulin. We showed that USF1, a key transcription factor for FASN activation, directly interacted with the Mediator subunit MED17 at the FASN promoter. This interaction recruited Mediator, which can bring POL II and other general transcription machinery to the complex. Moreover, we showed that MED17 was phosphorylated at Ser53 by casein kinase 2 (CK2) in the livers of fed mice or insulin-stimulated hepatocytes, but not in the livers of fasted mice or untreated hepatocytes. Furthermore, activation of the FASN promoter in response to insulin required this CK2-mediated phosphorylation event, which occurred only in the absence of p38 MAPK-mediated phosphorylation at Thr570. Overexpression of a nonphosphorylatable S53A MED17 mutant or knockdown of MED17, as well as CK2 knockdown or inhibition, impaired hepatic de novo fatty acid synthesis and decreased triglyceride content in mice. These results demonstrate that CK2-mediated phosphorylation of Ser53 in MED17 is required for the transcriptional activation of lipogenic genes in response to insulin. [ABSTRACT FROM AUTHOR]

The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissuespecific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance. [ABSTRACT FROM AUTHOR]

Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrß-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrß-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism. [ABSTRACT FROM AUTHOR]

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-a) 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-a 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-a 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-a and suppression of adipose Acrp30. This targeted and tissue-specific regulation of TNF-a 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (6-8weeks; 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 (&bgr;-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Brown adipose tissue harbors UCP1 to dissipate chemical energy as heat. However, the transcriptional network that governs the thermogenic gene program is incompletely understood. Zc3h10, a CCCH-type zinc finger protein, has recently been reported to bind RNA. However, we report here that Zc3h10 functions as a transcription factor to activate UCP1 not through the enhancer region, but by binding to a far upstream region of the UCP1 promoter. Upon sympathetic stimulation, Zc3h10 is phosphorylated at S126 by p38 mitogen-activated protein kinase (MAPK) to increase binding to the distal region of the UCP1 promoter. Zc3h10, as well as mutant Zc3h10, which cannot bind RNA, enhances thermogenic capacity and energy expenditure, protecting mice from diet-induced obesity. Conversely, Zc3h10 ablation in UCP1+ cells in mice impairs thermogenic capacity and lowers oxygen consumption, leading to weight gain. Hence, Zc3h10 plays a critical role in the thermogenic gene program and may present future targets for obesity therapeutics. • Zc3h10 directly binds and activates the UCP1 and other BAT gene promoter regions • Zc3h10 promotes BAT gene expression and protects mice from diet-induced obesity • p38 MAPK phosphorylates Zc3h10 to allow binding to target sites for transcription Zc3h10 is a RNA-binding protein. Here, Yi et al. report Zc3h10 is a transcription factor that activates UCP1 and other BAT genes. Cold/β 3 stimulation causes phosphorylation of Zc3h10 at S126 by p38 MAPK to increase its binding to targets genes and, thus, promotes thermogenic capacity and energy expenditure. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (FGF)-21 is secreted from the liver, pancreas, and adipose in response to prolonged fasting/starvation to facilitate lipid and glucose metabolism. Northern elephant seals naturally fast for several months, maintaining a relatively elevated metabolic rate to satisfy their energetic requirements. Thus, to better understand the impact of prolonged food deprivation on FGF21-associated changes, we analyzed the expression of FGF21, FGF receptor-1 (FGFR1), β-klotho (KLB; a co-activator of FGFR) in adipose, and plasma FGF21, glucose and 3-hydroxybutyrate in fasted elephant seal pups. Expression of FGFR1 and KLB mRNA decreased 98% and 43%, respectively, with fasting duration. While the 80% decrease in mean adipose FGF21 mRNA expression with fasting did not reach statistical significance, it paralleled the 39% decrease in plasma FGF21 concentrations suggesting that FGF21 is suppressed with fasting in elephant seals. Data demonstrate an atypical response of FGF21 to prolonged fasting in a mammal suggesting that FGF21-mediated mechanisms have evolved differentially in elephant seals. Furthermore, the typical fasting-induced, FGF21-mediated actions such as the inhibition of lipolysis in adipose may not be required in elephant seals as part of a naturally adapted mechanism to support their unique metabolic demands during prolonged fasting. [ABSTRACT FROM AUTHOR]

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or β-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance. • Aifm2 is expressed specifically in brown adipose tissue (BAT) • Aifm2 regenerates NAD to support robust glycolysis for thermogenesis • Glucose oxidation is critical for thermogenesis in a fed state • Aifm2 translocated from LD to mitochondria supports ETC for thermogenesis Aifm2, a brown-adipose-tissue-specific, lipid-droplet-associated NADH oxidase, associates with the mitochondrial inner membrane to regenerate cytosolic NAD for robust glycolysis and to support ETC for thermogenesis. [ABSTRACT FROM AUTHOR]

The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

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.

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 pM/kg) or high (100 pM/kg) dosages of glucagon-like peptide-1 (GLP-1) immediately following a glucose bolus (0.5g/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.

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.

Northern elephant seals endure a 2- to 3-month fast characterized by sustained hyperglycemia, hypoinsulinemia, and increased plasma cortisol and free fatty acids, conditions often seen in insulin-resistant humans. We had previously shown that adipose Glut4 expression and 5'AMP-activated protein kinase (AMPK) activity increase and plasma glucose decreases in fasting seals suggesting that AMPK activity contributes to glucose regulation during insulin-resistant conditions. To address the hypothesis that AMPK activity increases during fasting-induced insulin resistance, we performed glucose tolerance tests (GTT) on early (n=5) and late (n=8)-fasted seal pups and compared adipose tissue expression of insulin signaling proteins, peroxisome proliferator-activated receptor γ (PPARγ), and AMPK, in addition to plasma adiponectin, leptin, cortisol, insulin, and non-esterified fatty acid (NEFA) levels. Fasting was associated with decreased glucose clearance, plasma insulin and adiponectin, and intracellular insulin signaling, as well as increased plasma cortisol and NEFAs, supporting the suggestion that seals develop insulin resistance late in the fast. The expression of Glut4 and VAMP2 increased (52 and 63% respectively) with fasting but did not change significantly during the GTT. PPARγ and phosphorylated AMPK did not change in the early fasted seals, but increased significantly (73 and 50% respectively) in the late-fasted seals during the GTT. Increased AMPK activity along with the reduction in the activity of insulin-signaling proteins supports our hypothesis that AMPK activity is increased following the onset of insulin resistance. The association between increased AMPK activity and Glut4 expression suggests that AMPK plays a greater role in regulating glucose metabolism in mammals adapted to prolonged fasting than in non-fasting mammals.