REFERENCE RANGES AND AGE-RELATED AND DIVING EXERCISE EFFECTS ON HEMATOLOGY AND SERUM CHEMISTRY OF FEMALE STELLER SEA LIONS ( EUMETOPIAS JUBATUS).

Decreased health may have lowered the birth and survival rates of Steller sea lions ( Eumetopias jubatus) in the Gulf of Alaska and Aleutian Islands over the past 30 yr. Reference ranges for clinical hematology and serum chemistry parameters needed to assess the health of wild sea lion populations are limited. Here, blood parameters were serially measured in 12 captive female Steller sea lions ranging in age from 3 wk to 16 yr to establish baseline values and investigate age-related changes. Whether diving activity affects hematology parameters in animals swimming in the ocean compared with animals in a traditional aquarium setting was also examined. Almost all blood parameters measured exhibited significant changes with age. Many of the age-related changes reflected developmental life history changes, including a change in diet during weaning, an improvement of diving capacity, and the maturity of the immune system. Mean corpuscular hemoglobin and mean corpuscular volume were also higher in the ocean diving group compared with the aquarium group, likely reflecting responses to increased exercise regimes. These data provide ranges of hematology and serum chemistry values needed to evaluate and compare the health and nutritional status of captive and wild Steller sea lions.

Steller sea lions (Eumetopias jubatus) have greater blood volumes, higher diving metabolic rates and a longer aerobic dive limit when nutritionally stressed.

Marine mammal foraging behaviour inherently depends on diving ability. Declining populations of Steller sea lions may be facing nutritional stress that could affect their diving ability through changes in body composition or metabolism. Our objective was to determine whether nutritional stress (restricted food intake resulting in a 10% decrease in body mass) altered the calculated aerobic dive limit (cADL) of four captive sea lions diving in the open ocean, and how this related to changes in observed dive behaviour. We measured diving metabolic rate (DMR), blood O2 stores, body composition and dive behaviour prior to and while under nutritional restriction. We found that nutritionally stressed sea lions increased the duration of their single long dives, and the proportion of time they spent at the surface during a cycle of four dives. Nutritionally stressed sea lions lost both lipid and lean mass, resulting in potentially lower muscle O2 stores. However, total body O2 stores increased due to rises in blood O2 stores associated with having higher blood volumes. Nutritionally stressed sea lions also had higher mass-specific metabolic rates. The greater rise in O2 stores relative to the increase in mass-specific DMR resulted in the sea lions having a longer cADL when nutritionally stressed. We conclude that there was no negative effect of nutritional stress on the diving ability of sea lions. However, nutritional stress did lower foraging efficiency and require more foraging time to meet energy requirements due to increases in diving metabolic rates and surface recovery times.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

Lack of hemodynamic effects after extended heme synthesis inhibition by succinylacetone in rats.

Hypertyrosinemia (HT) is a life-threatening condition caused in large part by the buildup of tyrosine metabolites and their derivatives. One such metabolite is succinylacetone (SA), a potent irreversible inhibitor of heme biosynthesis. Heme is a key component of numerous enzymes involved in arterial blood pressure (BP) regulation, including nitric-oxide synthase (NOS) and its downstream mediator soluble guanylyl cyclase (sGC). Because NOS and sGC are important regulators of cardiovascular function, we hypothesized that inhibition of heme supply to these enzymes by SA would result in the induction of a measurable hypertensive response. Male Sprague-Dawley rats were treated with SA (80 mg x kg(-1) x day(-1) i.p.) for 14 days, resulting in a marked increase in urinary SA and delta-aminolevulinic acid (P < 0.001 for both parameters) and decreased heme concentrations in kidney, liver, spleen, and vascular tissues (P < 0.05 for all parameters). After SA treatment, systemic nitrite/nitrate excretion was reduced by 72% (P < 0.001), and renal NOS and sGC activities were decreased by 32 (P < 0.05) and 38% (P < 0.01), respectively. SA administration also compromised the ex vivo sensitivity of aorta to endothelium-dependent and -independent vasodilation. Despite these effects, SA treatment failed to induce any changes in BP, as assessed by radiotelemetry. Moreover, BP profiles in the SA-treated animals were less responsive to altered sodium intake. The present results demonstrate that extended inhibition of heme synthesis with SA affects hemoenzyme function, albeit without consequent effects on BP regulation and sodium excretion.

The regulation of AMP-activated protein kinase by upstream kinases.

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a major role in maintaining energy homoeostasis. Within individual cells, AMPK is activated by a rise in the AMP/ATP ratio that occurs following a fall in ATP levels. AMPK is also regulated by the adipokines, adiponectin and leptin, hormones that are secreted from adipocytes. AMPK regulates a wide range of metabolic pathways, including fatty acid oxidation, fatty acid synthesis, glycolysis and gluconeogenesis. In peripheral tissues, activation of AMPK leads to responses that are beneficial in counteracting the deleterious effects that arise in the metabolic syndrome. Recent studies have demonstrated that modulation of AMPK activity in the hypothalamus plays a role in feeding. A decrease in hypothalamic AMPK activity is associated with decreased feeding, whereas activation of AMPK leads to increased food intake. Furthermore, signalling pathways occurring in the hypothalamus lead to changes in AMPK activity in peripheral tissues, such as skeletal muscle, via the sympathetic nervous system. AMPK, therefore, provides a mechanism for monitoring changes in energy metabolism within individual cells and at the level of the whole body. Activation of AMPK requires phosphorylation of threonine 172 (Thr-172) within the catalytic subunit. Recent studies have shown that both LKB1 and Ca(2+)/calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) play important roles in phosphorylating and activating AMPK. In addition, there is evidence that AMPK can be activated by other upstream kinases, although the physiological significance of this is not clear at present. This review focuses on the role of LKB1 and CaMKKbeta in the regulation of AMPK.

Aspartate aminotransferase-to-platelet ratio index increases significantly 3 years prior to liver-related death in HIV-hepatitis-coinfected men.

: The utility of longitudinal AST-to-platelet ratio index (APRI), a surrogate for hepatic fibrosis, is unknown. We compared APRI up to 9 years before liver-related death among 57 cases of viral hepatitis-infected men (91% HIV+) to matched controls. APRI was stable among controls but, among cases, increased 4.6%/year from 9 to 3 years predeath (P = 0.10) and 30%/year during the 3 years predeath (P < 0.001). Thus, rapid APRI increase may predict impending liver-related death in HIV-viral hepatitis coinfection.

Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia.

BACKGROUND: The role of protein phosphorylation in the Pasteur effect--the phenomenon whereby anaerobic conditions stimulate glycolysis--has not been addressed. The AMP-activated protein kinase (AMPK) is activated when the oxygen supply is restricted. AMPK acts as an energy-state sensor and inhibits key biosynthetic pathways, thus conserving ATP. Here, we studied whether AMPK is involved in the Pasteur effect in the heart by phosphorylating and activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. RESULTS: Heart PFK-2 was phosphorylated on Ser466 and activated by AMPK in vitro. In perfused rat hearts, anaerobic conditions or inhibitors of oxidative phosphorylation (oligomycin and antimycin) induced AMPK activation, which correlated with PFK-2 activation and with an increase in fructose 2,6-bisphosphate concentration. Moreover, in cultured cells transfected with heart PFK-2, oligomycin treatment resulted in a parallel activation of endogenous AMPK and PFK-2. In these cells, the activation of PFK-2 was due to the phosphorylation of Ser466. A dominant-negative construct of AMPK abolished the activation of endogenous and cotransfected AMPK, and prevented both the activation and phosphorylation of transfected PFK-2 by oligomycin. CONCLUSIONS: AMPK phosphorylates and activates heart PFK-2 in vitro and in intact cells. AMPK-mediated PFK-2 activation is likely to be involved in the stimulation of heart glycolysis during ischaemia.

Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase.

In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism, e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP-activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (alpha1(312)) acts as a constitutively active kinase, while the other (alpha1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of alpha1(312) increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of alpha1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucose-activated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

Physiological constraints and energetic costs of diving behaviour in marine mammals: a review of studies using trained Steller sea lions diving in the open ocean.

Marine mammals are characterized as having physiological specializations that maximize the use of oxygen stores to prolong time spent under water. However, it has been difficult to undertake the requisite controlled studies to determine the physiological limitations and trade-offs that marine mammals face while diving in the wild under varying environmental and nutritional conditions. For the past decade, Steller sea lions (Eumetopias jubatus) trained to swim and dive in the open ocean away from the physical confines of pools participated in studies that investigated the interactions between diving behaviour, energetic costs, physiological constraints, and prey availability. Many of these studies measured the cost of diving to understand how it varies with behaviour and environmental and physiological conditions. Collectively, these studies show that the type of diving (dive bouts or single dives), the level of underwater activity, the depth and duration of dives, and the nutritional status and physical condition of the animal affect the cost of diving and foraging. They show that dive depth, dive and surface duration, and the type of dive result in physiological adjustments (heart rate, gas exchange) that may be independent of energy expenditure. They also demonstrate that changes in prey abundance and nutritional status cause sea lions to alter the balance between time spent at the surface acquiring oxygen (and offloading CO2 and other metabolic by-products) and time spent at depth acquiring prey. These new insights into the physiological basis of diving behaviour further our understanding of the potential scope for behavioural responses of marine mammals to environmental changes, the energetic significance of these adjustments, and the consequences of approaching physiological limits.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

The substrate and sequence specificity of the AMP-activated protein kinase. Phosphorylation of glycogen synthase and phosphorylase kinase.

In addition to acetyl-CoA carboxylase and HMG-CoA reductase, the AMP-activated protein kinase phosphorylates glycogen synthase, phosphorylase kinase, hormone-sensitive lipase and casein. A number of other substrates for the cyclic AMP-dependent protein kinase, e.g., L-pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, are not phosphorylated at significant rates. Examination of the sites phosphorylated on acetyl-CoA carboxylase, hormone-sensitive lipase, glycogen synthase and phosphorylase kinase suggests a consensus recognition sequence in which the serine residue phosphorylated by the AMP-activated protein kinase has a hydrophobic residue on the N-terminal side (i.e., at -1) and at least one arginine residue at -2, -3 or -4. Substrates for cyclic AMP-dependent protein kinase which lack the hydrophobic residue at -1 are not substrates for the AMP-activated protein kinase.

Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase.

Glucose transport in skeletal muscle is stimulated by two distinct stimuli, insulin and exercise. The mechanism by which exercise stimulates glucose transport is not known, although it is distinct from the insulin-mediated pathway. Recently, it has been shown that AMP-activated protein kinase (AMPK) is activated by exercise in skeletal muscle, whereas pharmacological activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) leads to increased glucose transport. It has been postulated, therefore, that AMPK may be the link between exercise and glucose transport. To address this, we have examined the signaling pathway involved in the stimulation of glucose uptake after activation of AMPK. Here we show that activation of AMPK by AICAR in rat muscle and mouse H-2Kb muscle cells activates glucose transport approximately twofold. AMPK in H-2Kb cells is also activated by hyperosmotic stress and the mitochondrial uncoupling agent, dinitrophenol, both of which lead to increased glucose transport. In contrast, insulin, which activates glucose transport two- to-threefold in both rat muscle and H-2Kb cells, has no effect on AMPK activity. A previous study has shown that AMPK phosphorylates and activates endothelial nitric oxide synthase (NOS). We show here that NOS activity in H-2Kb cells is activated after stimulation of AMPK by AICAR. Treatment of H-2Kb cells or rat muscle with NOS inhibitors completely blocks the increase in glucose transport after activation of AMPK. In addition, an inhibitor of guanylate cyclase also blocks activation of glucose transport by AICAR in H-2Kb cells. These results indicate that activation of AMPK in muscle cells stimulates glucose transport by activation of NOS coupled to downstream signaling components, including cyclic GMP.

A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis.

A highly purified rat liver protein kinase phosphorylates and inactivates acetyl-CoA carboxylase, and causes rapid inactivation of microsomal HMG-CoA reductase in the presence of MgATP. Both effects are stimulated in an identical manner by AMP, and are greatly reduced by prior treatment of the kinase with purified protein phosphatase. The dephosphorylated kinase can be reactivated in the presence of MgATP, apparently due to a distinct kinase kinase, and this reactivation is stimulated by nanomolar concentrations of palmitoyl-CoA. These results show that a common, bicyclic protein kinase cascade can potently inactivate the regulatory enzymes of both fatty acid and cholesterol biosynthesis.

Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases.

We have reported previously that cyclic AMP-dependent protein kinase phosphorylates two sites on acetyl-CoA carboxylase (site 1: Arg-Met-Ser(P)-Phe, and site 2: Ser-Ser(P)-Met-Ser-Gly-Leu), while the AMP-activated protein kinase also phosphorylates site 1, plus site 3 (Ser-Ser-Met-Ser(P)-Gly-Leu), the latter being two residues C-terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP-dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in Vmax produced by the AMP-activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP-activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP-dependent protein kinase.

The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro.

The AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). Two isoforms of the catalytic subunit (alpha1 and alpha2) have been identified. We show here that the alpha1- and alpha2-containing complexes contribute approximately equally to total AMPK activity in rat liver. Furthermore, expression of alpha1 or alpha2 with beta and gamma in mammalian cells demonstrates that both complexes have equal specific activity measured with the SAMS peptide. Using variant peptides, however, we show that alpha1 and alpha2 exhibit slightly different substrate preferences, which suggest that the two isoforms could play different physiological roles within the cell.

Evidence that the AMP-activated protein kinase stimulates rat liver carnitine palmitoyltransferase I by phosphorylating cytoskeletal components.

The activity of hepatic carnitine palmitoyltransferase I (CPT-I) may be modulated by interactions with cytoskeletal components [Velasco et al. (1998) J. Biol. Chem. 273, 21497-21504]. We have studied whether the AMP-activated protein kinase (AMPK) is involved in this process. AMPK stimulated CPT-I in permeabilized hepatocytes but not in isolated liver mitochondria. In addition, AMPK abrogated the inhibition of CPT-I of isolated mitochondria induced by a cytoskeletal fraction. These two effects of AMPK were not evident when the kinase was inactivated by pretreatment with protein phosphatase 2C. Cytokeratins 8 and 18 were phosphorylated by AMPK in vitro and by incubation of intact hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside, a cell-permeable activator of AMPK. These results provide the first evidence that AMPK stimulates CPT-I by direct phosphorylation of cytoskeletal components.

Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase.

In vivo, hormone-sensitive lipase (HSL) is known to be phosphorylated on two sites termed the regulatory and basal sites. However, the intracellular role of the basal site or the identity of the protein kinase phosphorylating this site has not been established. We show that 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR) markedly activates cellular AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. As expected for an agent that activates AMPK intracellularly, AICAR had no effect on the basal activity of HSL. However, preincubation of adipocytes with AICAR led to a reduced response of these cells to the lipolytic agent isoprenaline. AICAR was also shown to profoundly inhibit lipogenesis through increased phosphorylation of acetyl-CoA carboxylase (ACC). Thus it appears that in addition to regulating lipogenesis, AMPK also plays an important antilipolytic role by regulating HSL in rat adipocytes.

The SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1.

Mig1p is a zinc finger protein required for repression of glucose-regulated genes in budding yeast. On removal of medium glucose, gene repression is relieved via a mechanism that requires the SNF1 protein kinase complex. We show that Mig1p expressed as a glutathione-S-transferase fusion in bacteria is readily phosphorylated by the SNF1 kinase in vitro. Four phosphorylation sites were identified, i.e. Ser-222, Ser-278, Ser-311 and Ser-381. The latter three are exact matches to the recognition motif we previously defined for SNF1 and lie within regions shown to be required for SNF1-dependent derepression and nuclear-to-cytoplasmic translocation.

Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase.

Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity. One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments. AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic [32P]phosphopeptide containing exclusively phospho-Ser621. AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.

Molecular cloning, expression and chromosomal localisation of human AMP-activated protein kinase.

A cDNA encoding rat liver AMP-activated protein kinase (AMPK) was used to isolate human skeletal muscle AMPK cDNA clones. Human AMPK cDNA is more than 90% homologous to the rat sequence and predicts a protein of molecular mass 62.3 kDa, which closely agrees with the mass observed in Western blots of human tissues. AMPK antibodies were also shown to immunoprecipitate AMPK from human liver extracts. A cDNA probe was used to identify a 9.5kb transcript in several human tissues and to isolate human genomic clones. PCR mapping of rodent/human hybrid cell lines localised the human AMPK gene to chromosome 1, and fluorescent in situ hybridisation with a human genomic clone was used to sub-localise the human AMPK gene to 1p31.