Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Mitochondrial ion transport pathways: Role in metabolic diseases

Mitochondria are the central coordinators of energy metabolism and alterations in their function and number have long been associated with metabolic disorders such as obesity, diabetes and hyperlipidemias. Since oxidative phosphorylation requires an electrochemical gradient across the inner mitochondrial membrane, ion channels in this membrane certainly must play an important role in the regulation of energy metabolism. However, in many experimental settings, the relationship between the activity of mitochondrial ion transport and metabolic disorders is still poorly understood. This review briefly summarizes some aspects of mitochondrial H(+) transport (promoted by uncoupling proteins, UCPs). Ca(2+) and K(+) uniporters which may be determinant in metabolic disorders. (C) 2009 Elsevier B.V. All rights reserved.

Expression of Na+/H+ exchanger mRNA in the gills of the Atlantic hagfish (Myxine glutinosa) in response to metabolic acidosis

Sodium/proton exchangers (NHE) are transmembrane proteins that facilitate the exchange of a Na⁺ ion for a H⁺ ion across cellular membranes. The NHE are present in the gills of fishes and are believed to function in acid-base regulation by driving the extrusion of protons across the branchial epithelium in exchange for Na⁺ in the water. In this study, we have used reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the presence of a branchial NHE in the gills of the Atlantic hagfish, Myxine glutinosa. The subsequent partial cDNA sequence shares homology with other vertebrate and invertebrate NHE isoforms. In addition, using semi-quantitative, multiplex RT-PCR we demonstrate that mRNA expression of hagfish gill NHE is upregulated following an induced metabolic acidosis. Expression was increased to 4.4 times basal levels at 2-h post-infusion and had decreased to 1.6 times basal by 6 h. Expression had returned to basal levels by 24-h post-infusion. The inference from this study is that a gill NHE which is potentially important in acid-base regulation has been present in the vertebrate lineage since before the divergence of the hagfishes from the main vertebrate line.

Mining frequent patterns for AMP-activated protein kinase regulation on skeletal muscle

Background
AMP-activated protein kinase (AMPK) has emerged as a significant signaling intermediary that regulates metabolisms in response to energy demand and supply. An investigation into the degree of activation and deactivation of AMPK subunits under exercise can provide valuable data for understanding AMPK. In particular, the effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. As more AMPK regulation data are accumulated, data mining techniques can play an important role in identifying frequent patterns in the data. Association rule mining, which is commonly used in market basket analysis, can be applied to AMPK regulation.

Results
This paper proposes a framework that can identify the potential correlation, either between the state of isoforms of α, β and γ subunits of AMPK, or between stimulus factors and the state of isoforms. Our approach is to apply item constraints in the closed interpretation to the itemset generation so that a threshold is specified in terms of the amount of results, rather than a fixed threshold value for all itemsets of all sizes. The derived rules from experiments are roughly analyzed. It is found that most of the extracted association rules have biological meaning and some of them were previously unknown. They indicate direction for further research.

Conclusion
Our findings indicate that AMPK has a great impact on most metabolic actions that are related to energy demand and supply. Those actions are adjusted via its subunit isoforms under specific physical training. Thus, there are strong co-relationships between AMPK subunit isoforms and exercises. Furthermore, the subunit isoforms are correlated with each other in some cases. The methods developed here could be used when predicting these essential relationships and enable an understanding of the functions and metabolic pathways regarding AMPK.

Mining frequent itemsets for protein kinase regulation

Protein kinases, a family of enzymes, have been viewed as an important signaling intermediary by living organisms for regulating critical biological processes such as memory, hormone response and cell growth. The
unbalanced kinases are known to cause cancer and other diseases. With the increasing efforts to collect, store and disseminate information about the entire kinase family, it not only leads to valuable data set to understand cell regulation but also poses a big challenge to extract valuable knowledge about metabolic pathway from the data. Data mining techniques that have been widely used to find frequent patterns in large datasets can be extended and adapted to kinase data as well. This paper proposes a framework for mining frequent itemsets from the collected kinase dataset. An experiment using AMPK regulation data demonstrates that our approaches are useful and efficient in analyzing kinase regulation data.

Learning dependency model for AMP-activated protein kinase regulation

The AMP-activated protein kinase (AMPK) acts as a metabolic master switch regulating several intracellular systems. The effect of AMPK on muscle cellular energy status makes this protein a promising pharmacological target for disease treatment. With increasingly available AMPK regulation data, it is critical to develop an efficient way to analyze the data since this assists in further understanding AMPK pathways. Bayesian networks can play an important role in expressing the dependency and causality in the data. This paper aims to analyse the regulation data using B-Course, a powerful analysis tool to exploit several theoretically elaborate results in the fields of Bayesian and causal modelling, and discover a certain type of multivariate probabilistic dependencies. The identified dependency models are easier to understand in comparison with the traditional frequent patterns.

Influence of preexercise muscle glycogen content on transcrptional activity of metabolic and myogenic genes in well-trained humans

To determine whether preexercise muscle glycogen content influences the transcription of several early-response genes involved in the regulation of muscle growth, seven male strength-trained subjects performed one-legged cycling exercise to exhaustion to lower muscle glycogen levels (Low) in one leg compared with the leg with normal muscle glycogen (Norm) and then the following day completed a unilateral bout of resistance training (RT). Muscle biopsies from both legs were taken at rest, immediately after RT, and after 3 h of recovery. Resting glycogen content was higher in the control leg (Norm leg) than in the Low leg (435 ± 87 vs. 193 ± 29 mmol/kg dry wt; P < 0.01). RT decreased glycogen content in both legs (P < 0.05), but postexercise values remained significantly higher in the Norm than the Low leg (312 ± 129 vs. 102 ± 34 mmol/kg dry wt; P < 0.01). GLUT4 (3-fold; P < 0.01) and glycogenin mRNA abundance (2.5-fold; not significant) were elevated at rest in the Norm leg, but such differences were abolished after exercise. Preexercise mRNA abundance of atrogenes was also higher in the Norm compared with the Low leg [atrogin: 14-fold, P < 0.01; RING (really interesting novel gene) finger: 3-fold, P < 0.05] but decreased for atrogin in Norm following RT (P < 0.05). There were no differences in the mRNA abundance of myogenic regulatory factors and IGF-I in the Norm compared with the Low leg. Our results demonstrate that 1) low muscle glycogen content has variable effects on the basal transcription of select metabolic and myogenic genes at rest, and 2) any differences in basal transcription are completely abolished after a single bout of heavy resistance training. We conclude that commencing resistance exercise with low muscle glycogen does not enhance the activity of genes implicated in promoting hypertrophy.

Epidemiology of metabolic syndrome

Metabolic syndrome (MetS), also previously known by a variety of other names, including insulin resistance syndrome and the deadly quartet, is characterized by clustering of abdominal (visceral and retroperitoneal) obesity and other cardiovascular risk factors, including impaired glucose regulation, raised triglycerides, decreased high-density lipoprotein cholesterol (HDL-C), elevated blood pressure (BP).

Associated with increased risk of both type 2 diabetes and cardiovascular disease (CVD), MetS is believed to be a contributor to the modern-day epidemics of diabetes and CVD and has become a major public health challenge around the world [I]. Currently, there are five different sets of criteria which have been developed to characterize the syndrome. These definitions differ in the components included and the cut-points used for each component. The prevalence of MetS in the westernized world is significant (10-50%) and believed to be increasing over time. The pathophysiology of the syndrome is unclear, but it is thought that obesity and/or insulin resistance are key underlying components. Genetics, lifestyle and environment factors are also important causes of MetS.

This chapter provides:

• a historical overview of the evolution of MetS;
• a summary of the value of the different definitions used to characterize the syndrome;
• a summary of the underlying pathophysiology, the causes and other important risk factors of MetS;
• a summary of the evidence describing the association of MetS with CVD and diabetes;
• a summary of the prevalence of MetS using the various definitions in different countries.

AMPK and transcriptional regulation

The AMP-activated protein kinase (AMPK) is an energy sensing enzyme that once activated, promotes energy production and limits energy utilisation to ensure cellular survival. In addition to targeting numerous metabolic enzymes for this purpose, it is becoming apparent that AMPK can also regulate a number of transcriptional processes. These processes ensure cell survival through the inhibition of cell cycle and growth mechanisms, and also prepare the cell for future perturbations in energy balance by increasing the capacity of the cell to produce ATP. While these adaptations might be inextricably linked through regulation of the proliferation-differentiation process, recent studies have identified a number of transcriptional regulators as AMPK substrates that give insights into the regulation of transcription by AMPK in a number of metabolically active tissues.

Histone modifications and skeletal muscle metabolic gene expression

1.      Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases.

2.      Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression.

3.      Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response.

4.      The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified.

5.      Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases.

6.      Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

Novel hypothalamic genes involved in body weight regulation in Psammomys obesus

Focuses on the discovery, characterisation and validation of a gene in the brain, named FIT, that regulates body weight. When suppressed in rats, food intake is inhibited and body weight is reduced. FIT regulates both appetite and metabolic rate, and is therefore a new and exciting target for obesity therapy.

Water deprivation induces appetite and alters metabolic strategy in Notomys alexis : unique mechanisms for water production in the desert

Like many desert animals, the spinifex hopping mouse, Notomys alexis, can maintain water balance without drinking water. The role of the kidney in producing a small volume of highly concentrated urine has been well-documented, but little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. In Notomys, we found that water deprivation (WD) induced a sustained high food intake that exceeded the pre-deprivation level, which was driven by parallel changes in plasma leptin and ghrelin and the expression of orexigenic and anorectic neuropeptide genes in the hypothalamus; these changed in a direction that would stimulate appetite. As the period of WD was prolonged, body fat disappeared but body mass increased gradually, which was attributed to hepatic glycogen storage. Switching metabolic strategy from lipids to carbohydrates would enhance metabolic water production per oxygen molecule, thus providing a mechanism to minimize respiratory water loss. The changes observed in appetite control and metabolic strategy in Notomys were absent or less prominent in laboratory mice. This study reveals novel mechanisms for appetite regulation and energy metabolism that could be essential for desert rodents to survive in xeric environments.

New gene targets of PGC-1α and ERRα co-regulation in C2C12 myotubes

As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P < 0.001) were identified. From these, further genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P < 0.001) and of Mtfp1, Mrm1, Oxnad1 and Cluh (P < 0.05). Screening of the promoter and intron 1 regions also revealed all genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P < 0.05) except for Akr1b10. These findings provide preliminary evidence for the novel regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.

ATGL-mediated triglyceride turnover and the regulation of mitochondrial capacity in skeletal muscle

Emerging evidence indicates that skeletal muscle lipid droplets are an important control point for intracellular lipid homeostasis and that regulating fatty acid fluxes from lipid droplets might influence mitochondrial capacity. We used pharmacological blockers of the major triglyceride lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase, to show that a large proportion of the fatty acids that are transported into myotubes are trafficked through the intramyocellular triglyceride pool. We next tested whether increasing lipolysis from intramyocellular lipid droplets could activate transcriptional responses to enhance mitochondrial and fatty acid oxidative capacity. ATGL was overexpressed by adenoviral and adenoassociated viral infection in C2C12 myotubes and the tibialis anterior muscle of C57Bl/6 mice, respectively. ATGL overexpression in C2C12 myotubes increased lipolysis, which was associated with increased peroxisome proliferator-activated receptor (PPAR)-∂ activity, transcriptional upregulation of some PPAR∂ target genes, and enhanced mitochondrial capacity. The transcriptional responses were specific to ATGL actions and not a generalized increase in fatty acid flux in the myotubes. Marked ATGL overexpression (20-fold) induced modest molecular changes in the skeletal muscle of mice, but these effects were not sufficient to alter fatty acid oxidation. Together, these data demonstrate the importance of lipid droplets for myocellular fatty acid trafficking and the capacity to modulate mitochondrial capacity by enhancing lipid droplet lipolysis in vitro; however, this adaptive program is of minor importance when superimposing the normal metabolic stresses encountered in free-moving animals.

Regulation of breathing and body temperature of a burrowing rodent during hypoxic-hypercapnia

Burrowing mammals usually have low respiratory sensitivity to hypoxia and hypercapnia. However, the interaction between ventilation (V), metabolism and body temperature (Tb) during hypoxic-hypercapnia has never been addressed. We tested the hypothesis that Clyomys bishopi, a burrowing rodent of the Brazilian cerrado, shows a small ventilatory response to hypoxic-hypercapnia, accompanied by a marked drop in Tb and metabolism. V, Tb and O-2 consumption (VO2) of C. bishopi were measured during exposure to air, hypoxia (10% and 7% O-2), hypercapnia (3% and 5% CO2) and hypoxic-hypercapnia (10% O-2 + 3% CO2). Hypoxia of 7% but not 10%, caused a significant increase in V, and a significant drop in Tb. Both hypoxic levels decreased VO2 and 7% O-2 significantly increased V/VO2. Hypercapnia of 5%, but not 3%, elicited a significant increase in V, although no significant change in Tb, VO2 or V/VO2 was detected. A combination of 10% O-2 and 3% CO2 had minor effects on V and Tb, while VO2 decreased and V/VO2 tended to increase. We conclude that C. bishopi has a low sensitivity not only to hypoxia and hypercapnia, but also to hypoxic-hypercapnia, manifested by a biphasic ventilatory response, a drop in metabolism and a tendency to increase V/VO2. The effect of hypoxic-hypercapnia was the summation of the hypoxia and hypercapnia effects, with respiratory responses tending to have hypercapnic patterns while metabolic responses, hypoxic patterns. (C) 2004 Elsevier B.V. All rights reserved.