ERRgamma directs and maintains the transition to oxidative metabolism in the postnatal heart

At birth, the heart undergoes a critical metabolic switch from a predominant dependence on carbohydrates during fetal life to a greater dependence on postnatal oxidative metabolism. This remains the principle metabolic state throughout life, although pathologic conditions such as heart failure and cardiac hypertrophy reactivate components of the fetal genetic program to increase carbohydrate utilization. Disruption of the ERRgamma gene (Esrrg), which is expressed at high levels in the fetal and postnatal mouse heart, blocks this switch, resulting in lactatemia, electrocardiographic abnormalities, and death during the first week of life. Genomic ChIP-on-chip and expression analysis identifies ERRgamma as both a direct and an indirect regulator of a nuclear-encoded mitochondrial genetic network that coordinates the postnatal metabolic transition. These findings reveal an unexpected and essential molecular genetic component of the oxidative metabolic gene program in the heart and highlight ERRgamma in the study of cardiac hypertrophy and failure.     

JNK1 is required to preserve cardiac function in the early response to pressure overload

Cardiac stress consistently activates c-Jun NH(2)-terminal kinase (JNK) pathways, however the role of different members of the JNK family is unclear. In this study, we applied pressure overload (TAC) in mice with selective deletion of the three JNK genes (Jnk1(-/-), Jnk2(-/-), and Jnk3(-/-)). Following TAC, all three JNK knockout mouse lines developed cardiac hypertrophy similar to wild-type mice (WT), but only JNK1(-/-) mice displayed a significant reduction in fractional shortening after 3 and 7 days of pressure overload, associated with a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining and marked inflammatory infiltrate. After the acute deterioration stage, JNK1(-/-) mice underwent a slow recovery followed by a steady progression of cardiac dysfunction, becoming indistinguishable from WT after 12 weeks of TAC. These data suggest that JNK1 plays a protective role in response to pressure overload, preventing the early deterioration in cardiac function following an acute increase in afterload.     

Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production

Insulin action in the central nervous system regulates energy homeostasis and glucose metabolism. To define the insulin-responsive neurons that mediate these effects, we generated mice with selective inactivation of the insulin receptor (IR) in either pro-opiomelanocortin (POMC)- or agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus. While neither POMC- nor AgRP-restricted IR knockout mice exhibited altered energy homeostasis, insulin failed to normally suppress hepatic glucose production during euglycemic-hyperinsulinemic clamps in AgRP-IR knockout (IR^ΔAgRP) mice. These mice also exhibited reduced insulin-stimulated hepatic interleukin-6 expression and increased hepatic expression of glucose-6-phosphatase. These results directly demonstrate that insulin action in POMC and AgRP cells is not required for steady-state regulation of food intake and body weight. However, insulin action specifically in AgRP-expressing neurons does play a critical role in controlling hepatic glucose production and may provide a target for the treatment of insulin resistance in type 2 diabetes.     

PERK EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required for postnatal glucose homeostasis

Mutations in PERK (EIF2AK3) result in permanent neonatal diabetes as well as several other anomalies that underlie the human Wolcott-Rallison syndrome, and these anomalies are mirrored in Perk knockout mice. To identify the cause of diabetes in PERK-deficient mice, we generated a series of tissue- and cell-specific knockouts of the Perk gene and performed a developmental analysis of the progression to overt diabetes. We discovered that PERK is specifically required in the insulin-secreting β cells during the fetal and early neonatal period as a prerequisite for postnatal glucose homeostasis. However, PERK expression in β cells is not required at the adult stage to maintain β cell functions and glucose homeostasis. We show that PERK-deficient mice exhibit severe defects in fetal/neonatal β cell proliferation and differentiation, resulting in low β cell mass, defects in proinsulin trafficking, and abrogation of insulin secretion that culminate in permanent neonatal diabetes.     

The ERRalpha orphan nuclear receptor controls morphogenetic movements during zebrafish gastrulation

Gastrulation is a process involving cellular commitment and movements whereby the three fundamental germ layers are established in vertebrates embryos. Estrogen Receptor-Related (ERR) alpha is a nuclear receptor displaying high sequence identity to the Estrogen Receptors (ERs). However, ERRalpha is unable to bind and to be regulated by estrogens or any natural ligand to date. Whereas recent studies have suggested roles for ERRalpha in bone and adipose tissue metabolism in the mouse, little is known about its roles during embryonic development. In zebrafish embryos, ERRalpha is expressed from the beginning of gastrulation at the margin of the blastoderm that represents the presumptive mesendoderm. Using loss of function (morpholinos or a dominant-negative version of the protein) and gain of function (mRNA injection) strategies, we show here that ERRalpha is involved in epiboly and convergent-extension (CE) processes in the zebrafish. Altogether, these results propose ERRalpha as a new regulator of morphogenetic movement during gastrulation, independently of cell fate determination.     

AMPK and metabolic adaptation by the heart to pressure overload

Accelerated glycolysis in hypertrophied hearts may be a compensatory response to reduced energy production from long-chain fatty acid oxidation with 5'-AMP-activated protein kinase (AMPK) functioning as a cellular signal. Therefore, we tested the hypothesis that enhanced fatty acid oxidation improves energy status and normalizes AMPK activity and glycolysis in hypertrophied hearts. Glycolysis, fatty acid oxidation, AMPK activity, and energy status were measured in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. Hearts from halothane (3-4%)-anesthetized rats were perfused with KH solution containing either palmitate, a long-chain fatty acid, or palmitate plus octanoate, a medium-chain fatty acid whose oxidation is not impaired in hypertrophied hearts. Compared with control, fatty acid oxidation was lower in hypertrophied hearts perfused with palmitate, whereas it increased to similar values in both groups with octanoate plus palmitate. Glycolysis was accelerated in palmitate-perfused hypertrophied hearts and was normalized in hypertrophied hearts by the addition of octanoate. AMPK activity was increased three- to sixfold with palmitate alone and was reduced to control values by octanoate plus palmitate. Myocardial energy status improved with the addition of octanoate but did not differ between groups. Our findings, particularly the correspondence between glycolysis and AMPK activity, provide support for the view that activation of AMPK is responsible, in part, for the acceleration of glycolysis in cardiac hypertrophy. Additionally, they indicate myocardial AMPK is activated by energy state-independent mechanisms in response to pressure overload, demonstrating AMPK is more than a sensor of the heart's energy status.     

The nuclear orphan receptor COUP-TFII is required for limb and skeletal muscle development

The nuclear orphan receptor COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development, suggesting that COUP-TFII is involved in multiple aspects of embryogenesis. Because of the early embryonic lethality of COUP-TFII knockout mice, the role of COUP-TFII during limb development has not been determined. COUP-TFII is expressed in lateral plate mesoderm of the early embryo prior to limb bud formation. In addition, COUP-TFII is also expressed in the somites and skeletal muscle precursors of the limbs. Therefore, in order to study the potential role of COUP-TFII in limb and skeletal muscle development, we bypassed the early embryonic lethality of the COUP-TFII mutant by using two methods. First, embryonic chimera analysis has revealed an obligatory role for COUP-TFII in limb bud outgrowth since mutant cells are unable to contribute to the distally growing limb mesenchyme. Second, we used a conditional-knockout approach to ablate COUP-TFII specifically in the limbs. Loss of COUP-TFII in the limbs leads to hypoplastic skeletal muscle development, as well as shorter limbs. Taken together, our results demonstrate that COUP-TFII plays an early role in limb bud outgrowth but not limb bud initiation. Also, COUP-TFII is required for appropriate development of the skeletal musculature of developing limbs.     

The pancreatic beta cell is a key site for mediating the effects of leptin on glucose homeostasis

The hormone leptin plays a crucial role in maintenance of body weight and glucose homeostasis. This occurs through central and peripheral pathways, including regulation of insulin secretion by pancreatic beta cells. To study this further in mice, we disrupted the signaling domain of the leptin receptor gene in beta cells and hypothalamus. These mice develop obesity, fasting hyperinsulinemia, impaired glucose-stimulated insulin release, and glucose intolerance, similar to leptin receptor null mice. However, whereas complete loss of leptin function causes increased food intake, this tissue-specific attenuation of leptin signaling does not alter food intake or satiety responses to leptin. Moreover, unlike other obese models, these mice have reduced fasting blood glucose. These results indicate that leptin regulation of glucose homeostasis extends beyond insulin sensitivity to influence beta cell function, independent of pathways controlling food intake. These data suggest that defects in this adipoinsular axis could contribute to diabetes associated with obesity.     

Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.     

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C. elegans somatic gonad. Specifically, nhr-6 is required for the development of the hermaphrodite spermatheca, a somatic gonad organ that serves as the site of sperm storage and oocyte fertilization. Using a variety of spermatheca cell markers, we have determined that loss of nhr-6 function causes severe morphological defects in the spermatheca and associated spermathecal valves. This appears to be due to specific requirements for nhr-6 in regulating cell proliferation and cell differentiation during development of these structures. The improper development of these structures in nhr-6(lg6001) mutants leads to defects in ovulation and significantly reduced fecundity of C. elegans hermaphrodites. The phenotypes of nhr-6(lg6001) mutants are consistent with a role for nhr-6 in organogenesis, similar to the functions of its mammalian homologs.     

The nuclear receptor unfulfilled is required for free-running clocks in Drosophila pacemaker neurons

An intricate neural circuit composed of multiple classes of clock neurons controls circadian locomotor rhythms in Drosophila. Evidence indicates that the small ventral lateral neurons (s-LNvs, M cells) are the dominant pacemaker neurons that synchronize the clocks throughout the circuit and drive free-running locomotor rhythms. Little is known, however, about the molecular underpinning of this unique function of the s-LNvs. Here, we show that the nuclear receptor gene unfulfilled (unf; DHR51) is required for the function of the s-LNvs. UNFULFILLED (UNF) is rhythmically expressed in the s-LNvs, and unf mutant flies are behaviorally arrhythmic. Knockdown of unf in developing LNvs irreversibly destroys the ability of adult s-LNvs to generate free-running rhythms, whereas depletion of UNF from adult LNvs dampens the rhythms of the s-LNvs only in constant darkness. These temporally controlled LNv-targeted unf knockdowns desynchronize circuit-wide molecular rhythms and disrupt behavioral rhythms. Therefore, UNF is a prerequisite for free-running clocks in the s-LNvs and for the function of the entire circadian circuit.     

The foot structure from the type 1 ryanodine receptor is required for functional coupling to store-operated channels

In the present study we have explored structural determinants of the functional interaction between skeletal muscle ryanodine receptor (RyR1) and transient receptor potential channel 1 (TRPC1) channels expressed in Chinese hamster ovary cells. We have illustrated a functional interaction between TRPC1 channels and RyR1 for the regulation of store-operated calcium entry (SOCE) initiated after releasing calcium from a caffeine-sensitive intracellular calcium pool. RNA interference experiments directed to reduce the amount of TRPC1 protein indicate that RyR1 associates to at least two different types of store-operated channels (SOCs), one dependent and one independent of TRPC1. In contrast, bradykinin-induced SOCE is completely dependent on the presence of TRPC1 protein, as we have previously illustrated. Removing the foot structure from RyR1 results in normal caffeine-induced release of calcium from internal stores but abolishes the activation of SOCE, indicating that this structure is require for functional coupling to SOCs. The footless RyR1 protein shows a different cellular localization when compared with wild type RyR1. The later protein shows a higher percentage of colocalization with FM-464, a marker of plasma membrane. The implications of the foot structure for the functional and physical coupling to TRPC and SOCs is discussed.     

The inositol 1,4,5-trisphosphate receptor is required for maintenance of olfactory adaptation in Drosophila antennae

A role for inositol 1,4,5-trisphosphate (IP(3)) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP(3) signaling pathway, an unequivocal demonstration of IP(3) function in olfaction has not been possible. Here we have investigated the role of a well-established cellular target of IP(3)-the IP(3) receptor (IP(3)R)-in olfactory transduction in Drosophila. For this purpose we tested existing viable combinations of IP(3)R mutant alleles, as well as a newly generated set of viable itpr alleles, for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP(3)R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP(3)R.     

Hepatic insulin resistance is sufficient to produce dyslipidemia and susceptibility to atherosclerosis

Insulin resistance plays a central role in the development of the metabolic syndrome, but how it relates to cardiovascular disease remains controversial. Liver insulin receptor knockout (LIRKO) mice have pure hepatic insulin resistance. On a standard chow diet, LIRKO mice have a proatherogenic lipoprotein profile with reduced high-density lipoprotein (HDL) cholesterol and very low-density lipoprotein (VLDL) particles that are markedly enriched in cholesterol. This is due to increased secretion and decreased clearance of apolipoprotein B-containing lipoproteins, coupled with decreased triglyceride secretion secondary to increased expression of Pgc-1 beta (Ppargc-1b), which promotes VLDL secretion, but decreased expression of Srebp-1c (Srebf1), Srebp-2 (Srebf2), and their targets, the lipogenic enzymes and the LDL receptor. Within 12 weeks on an atherogenic diet, LIRKO mice show marked hypercholesterolemia, and 100% of LIRKO mice, but 0% of controls, develop severe atherosclerosis. Thus, insulin resistance at the level of the liver is sufficient to produce the dyslipidemia and increased risk of atherosclerosis associated with the metabolic syndrome.     

The interaction between thymine DNA glycosylase and nuclear receptor coactivator 3 is required for the transcriptional activation of nuclear hormone receptors

The cellular counterpart of the “soluble” guanylyl cyclase found in tissue homogenates over 30 years ago is now recognized as the physiological receptor for nitric oxide (NO). The ligand-binding site is a prosthetic haem group that, when occupied by NO, induces a conformational change in the protein that propagates to the catalytic site, triggering conversion of GTP into cGMP. This review focuses on recent research that takes this basic information forward to the beginnings of a quantitative depiction of NO signal transduction, analogous to that achieved for other major transmitters. At its foundation is an explicit enzyme-linked receptor mechanism for NO-activated guanylyl cyclase that replicates all its main properties. In cells, NO signal transduction is subject to additional, activity-dependent modifications, notably through receptor desensitization and changes in the activity of cGMP-hydrolyzing phosphodiesterases. The measurement of these parameters under varying conditions in rat platelets has made it possible to formulate a cellular model of NO-cGMP signaling. The model helps explain cellular responses to NO and their modification by therapeutic agents acting on the guanylyl cyclase or phosphodiesterase limbs of the pathway.     

The ciliary neurotrophic factor receptor alpha component induces the secretion of and is required for functional responses to cardiotrophin-like cytokine

Ciliary neurotrophic factor (CNTF) is involved in the survival of a number of different neural cell types, including motor neurons. CNTF functional responses are mediated through a tripartite membrane receptor composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor (LIFR), associated with a non-signalling CNTF binding receptor alpha component (CNTFR). CNTFR-deficient mice show profound neuronal deficits at birth, leading to a lethal phenotype. In contrast, inactivation of the CNTF gene leads only to a slight muscle weakness, mainly during adulthood, suggesting that CNTFR binds to a second ligand that is important for development. Modelling studies of the interleukin-6 family member cardiotrophin-like cytokine (CLC) revealed structural similarities with CNTF, including the conservation of a site I domain involved in binding to CNTFR. Co-expression of CLC and CNTFR in mammalian cells generates a secreted composite cytokine, displaying activities on cells expressing the gp130-LIFR complex on their surface. Correspondingly, CLC-CNTFR activates gp130, LIFR and STAT3 signalling components, and enhances motor neuron survival. Together, these observations demonstrate that CNTFR induces the secretion of CLC, as well as mediating the functional responses of CLC.