Dysregulation of striatal dopamine signaling by amphetamine inhibits feeding by hungry mice

Amphetamine (AMPH) releases monoamines, transiently stimulates locomotion, and inhibits feeding. Using a genetic approach, we show that mice lacking dopamine (DA-deficient, or DD, mice) are resistant to the hypophagic effects of a moderate dose of AMPH (2 microg/g) but manifest normal AMPH-induced hypophagia after restoration of DA signaling in the caudate putamen by viral gene therapy. By contrast, AMPH-induced hypophagia in response to the same dose of AMPH is not blunted in mice lacking the ability to make norepinephrine and epinephrine (Dbh(-/-)), dopamine D(2) receptors (D2r(-/-)), dopamine D(1) receptors (D1r(-/-)), serotonin 2C receptors (Htr2c(-/Y)), neuropeptide Y (Npy(-/-)), and in mice with compromised melanocortin signaling (A(y)). We suggest that, at this moderate dose of AMPH, dysregulation of striatal DA is the primary cause of AMPH-induced hypophagia and that regulated striatal dopaminergic signaling may be necessary for normal feeding behaviors.     

Multivitamin restriction increases adiposity and disrupts glucose homeostasis in mice

A strong association between obesity and low plasma concentrations of vitamins has been widely reported; however, the causality of this relationship is still not established. Our goal was to evaluate the impact of a multivitamin restriction diet (MRD) on body weight, adiposity and glucose homeostasis in mice. The mice were given a standard diet or a diet containing 50 % of the recommended vitamin intake (MRD) for 12 weeks. At the end of the experiment, total body weight was 6 % higher in MRD animals than in the control group, and the adiposity of the MRD animals more than doubled. The HOMA-IR index of the MRD animals was significantly increased. The adipose tissue of MRD animals had lower expression of mRNA encoding adiponectin and Pnpla2 (47 and 32 %, respectively) and 43 % higher leptin mRNA levels. In the liver, the mRNA levels of Pparα and Pgc1α were reduced (29 and 69 %, respectively) in MRD mice. Finally, the level of β-hydroxybutyrate, a ketonic body reflecting fatty acid oxidation, was decreased by 45 % in MRD mice. Our results suggest that MRD promotes adiposity, possibly by decreasing adipose tissue lipolysis and hepatic β-oxidation. These results could highlight a possible role of vitamin deficiency in the etiology of obesity and associated disorders.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0410-x) contains supplementary material, which is available to authorized users.     

Propentophylline increases striatal dopamine release but dampens methamphetamine-induced dopamine dynamics: A microdialysis study

While there are currently no medications approved for methamphetamine (METH) addiction, it has been shown that propentofylline (PPF), an atypical methylxanthine, can suppress the rewarding effects of methamphetamine (METH) in mice. This experiment studied the interactions of PPF with METH in striatal dopaminergic transmission. Herein, the impact of PPF (10–40 mM, intrastriatally perfused (80 min) on the effect of METH (5 mg/kg, i.p.) on striatal dopamine (DA) release was evaluated using brain microdialysis in Sprague–Dawley adult rats. METH was injected at the 60 min time point of the 80 min PPF perfusion. The extracellular levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were determined using high performance liquid chromatography with electrochemical detection (HPLC-ED). PPF induced a concentration-dependent increase in DA release beginning 30 min after the onset of PPF perfusion. DA peak levels evoked by 40 mM PPF were similar to those induced by 5 mg/kg METH i.p. Only the highest concentration of PPF decreased the METH-induced DA peak (circa 70%). The significant decreases in extracellular levels of DOPAC and HVA evoked by METH were partially blocked by 10 and 20 mM PPF. Although 40 mM of PPF also partially blocked the METH-induced DOPAC decrease, it completely blocked HVA depletion after a transient increase in HVA levels in METH-treated rats. Data indicates for the first time that while PPF increases presynaptic striatal DA dynamics it attenuates METH-induced striatal DA release and metabolism.     

Impaired electrical signaling disrupts gamma frequency oscillations in connexin 36-deficient mice

Neural processing occurs in parallel in distant cortical areas even for simple perceptual tasks. Associated cognitive binding is believed to occur through the interareal synchronization of rhythmic activity in the gamma (30-80 Hz) range. Such oscillations arise as an emergent property of the neuronal network and require conventional chemical neurotransmission. To test the potential role of gap junction-mediated electrical signaling in this network property, we generated mice lacking connexin 36, the major neuronal connexin. Here we show that the loss of this protein disrupts gamma frequency network oscillations in vitro but leaves high frequency (150 Hz) rhythms, which may involve gap junctions between principal cells (Schmitz et al., 2001), unaffected. Thus, specific connexins differentially deployed throughout cortical networks are likely to regulate different functional aspects of neuronal information processing in the mature brain.     

Chronic estradiol treatment increases ovariectomized rat striatal D-1 dopamine receptors

Striatal D-1 dopamine (DA) receptors were investigated following chronic 17 beta-estradiol (10 micrograms, b.i.d., s.c., for two weeks) to ovariectomized (OVX) female rats. This treatment initiated the day after ovariectomy has revealed that the maximal density in homogenates of striatal D-1 DA receptors (Bmax) labelled with [3H] SCH 23390 was increased (44% without and 28% with 120 mM NaCl in the assay buffer). Estradiol treatments initiated 2 or 4 weeks after ovariectomy did not induce D-1 DA receptor binding modifications. The affinity (Kd) of the ligand for the receptor remains unchanged by the steroid treatment while NaCl increased both the density and the affinity of [3H] SCH 23390 binding to striatal D-1 DA receptors. By autoradiography, the increase of striatal [3H] SCH 23390 binding to D-1 DA receptors after chronic estradiol treatment was found to be homogenously distributed in this brain region. Thus, chronic treatment with estradiol of ovariectomized rats leads to an increased density of striatal D-1 DA receptors but, this hormonal modulation of D-1 DA receptors is lost when treatment is started 2 weeks after ovariectomy or later.     

Leptin receptor signaling in midbrain dopamine neurons regulates feeding

The leptin hormone is critical for normal food intake and metabolism. While leptin receptor (Lepr) function has been well studied in the hypothalamus, the functional relevance of Lepr expression in the ventral tegmental area (VTA) has not been investigated. The VTA contains dopamine neurons that are important in modulating motivated behavior, addiction, and reward. Here, we show that VTA dopamine neurons express Lepr mRNA and respond to leptin with activation of an intracellular JAK-STAT pathway and a reduction in firing rate. Direct administration of leptin to the VTA caused decreased food intake while long-term RNAi-mediated knockdown of Lepr in the VTA led to increased food intake, locomotor activity, and sensitivity to highly palatable food. These data support a critical role for VTA Lepr in regulating feeding behavior and provide functional evidence for direct action of a peripheral metabolic signal on VTA dopamine neurons.     

Sham feeding corn oil increases accumbens dopamine in the rat

Both real and sham feeding of sucrose increase dopamine (DA) overflow in the nucleus accumbens (NAc). Fat is another constituent of foods that is inherently preferred by humans and rodents. We examined the affect of sham feeding corn oil in rats that were food and water deprived overnight. Rats were implanted with guide cannulas aimed at the NAc, as well as gastric fistulas. On alternate days, they were trained to sham lick 100% corn oil or distilled water (dH2O) for 20 min in the morning. Twenty-minute microdialysis samples were taken before, during, and after sham licking. DA and monoamines were analyzed by reverse-phase HPLC with coulometric detection. The results show that DA release in the NAc was significantly increased during sham licking of corn oil compared with the prior baseline (157.5+/-18.8%, n=12). During sham licking of dH2O, DA release in the NAc was not changed (93.0+/-4.0%, n=15). This experiment demonstrates that sham feeding of corn oil releases accumbens DA in a manner similar to ingestion of sucrose. Although both stimuli may have an olfactory component, sucrose is a gustatory, and 100% corn oil appears to be a trigeminal stimulus. Thus these data support the hypothesis that different sensory modalities produce reward using the same or closely related substrates in the forebrain.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.     

Activation of Wnt signaling by chemically induced dimerization of LRP5 disrupts cellular homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.     

Corelease of dopamine and serotonin from striatal dopamine terminals

The striatum receives rich dopaminergic and more moderate serotonergic innervation. After vesicular release, dopamine and serotonin (5-hydroxytryptamine, 5-HT) signaling is controlled by transporter-mediated reuptake. Dopamine is taken up by dopamine transporters (DATs), which are expressed at the highest density in the striatum. Although DATs also display a low affinity for 5-HT, that neurotransmitter is normally efficiently taken up by the 5-HT transporters. We found that when extracellular 5-HT is elevated by exogenous application or by using antidepressants (e.g., fluoxetine) to inhibit the 5-HT transporters, the extremely dense striatal DATs uptake 5-HT into dopamine terminals. Immunohistochemical results and measurements using fast cyclic voltammetry showed that elevated 5-HT is taken up by DATs into striatal dopamine terminals that subsequently release 5-HT and dopamine together. These results suggest that antidepressants that block serotonin transporters or other factors that elevate extracellular 5-HT alter the temporal and spatial relationship between dopamine and 5-HT signaling in the striatum.     

Lateral hypothalamic lesions and striatal dopamine levels

Rats with bilateral lateral hypothalamic lesions were killed on the third day after surgery and their brains were assayed for tel-diencephalic norepinephrine and striatal dopamine. Lesion-induced weight loss was highly correlated with depletion of striatal dopamine but not with tel-diencephalic norepinephrine. In rats with severe dopamine depletions, the degree of weight loss was related more to the striatum with the highest remaining level of dopamine suggesting that a critical level of dopamine in one striatum may be essential for lateral hypothalamic recovery.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Although TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (Mtor^A/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promotes mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 coimmunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal cross talk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.     

Activation of pregnane X receptor disrupts glucocorticoid and mineralocorticoid homeostasis

The pregnane X receptor (PXR) was isolated as a xenobiotic receptor that regulates responses to various xenobiotic agents. In this study, we show that PXR plays an important endobiotic role in adrenal steroid homeostasis. Activation of PXR by genetic (transgene) or pharmacological (ligand, such as rifampicin) markedly increased plasma concentrations of corticosterone and aldosterone, the respective primary glucocorticoid and mineralocorticoid in rodents. The increased levels of corticosterone and aldosterone were associated with activation of adrenal steroidogenic enzymes, including cytochrome P450 (CYP)11a1, CYP11b1, CYP11b2, and 3beta-hydroxysteroid dehydrogenase. The PXR-activating transgenic mice also exhibited hypertrophy of the adrenal cortex, loss of glucocorticoid circadian rhythm, and lack of glucocorticoid responses to psychogenic stress. Interestingly, the transgenic mice had normal pituitary secretion of ACTH and the corticosterone-suppressing effect of dexamethasone was intact, suggesting a functional hypothalamus-pituitary-adrenal axis despite a severe disruption of adrenal steroid homeostasis. The ACTH-independent hypercortisolism in the PXR-activating transgenic mice is reminiscent of the pseudo-Cushing's syndrome in patients. The glucocorticoid effect appears to be PXR specific, as the activation of constitutive androstane receptor in transgenic mice had little effect. We propose that PXR is a potential endocrine disrupting factor that may have broad implications in steroid homeostasis and drug-hormone interactions.     

Trisomy 21 increases microtubules and disrupts centriolar satellite localization

Trisomy 21, the source of Down syndrome, causes a 0.5-fold protein increase of the chromosome 21-resident gene Pericentrin (PCNT) and reduces primary cilia formation and signaling. We investigate how PCNT imbalances disrupt cilia. Using isogenic RPE-1 cells with increased chromosome 21 dosage, we find PCNT accumulates around the centrosome as a cluster of enlarged cytoplasmic puncta that localize along microtubules (MTs) and at MT ends. Cytoplasmic PCNT puncta impact the density, stability, and localization of the MT trafficking network required for primary cilia. The PCNT puncta appear to sequester cargo peripheral to centrosomes in what we call pericentrosomal crowding. The centriolar satellite proteins PCM1, CEP131, and CEP290, important for ciliogenesis, accumulate at enlarged PCNT puncta in trisomy 21 cells. Reducing PCNT when chromosome 21 ploidy is elevated is sufficient to decrease PCNT puncta and pericentrosomal crowding, reestablish a normal density of MTs around the centrosome, and restore ciliogenesis to wild-type levels. A transient reduction in MTs also decreases pericentrosomal crowding and partially rescues ciliogenesis in trisomy 21 cells, indicating that increased PCNT leads to defects in the MT network deleterious to normal centriolar satellite distribution. We propose that chromosome 21 aneuploidy disrupts MT-dependent intracellular trafficking required for primary cilia.     

Hepatocyte-specific c-Met deletion disrupts redox homeostasis and sensitizes to Fas-mediated apoptosis

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-kappaB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-kappaBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH precursor, significantly reduced Jo2-induced cell death. Conversely, the GSH-depleting agent buthionine sulfoximine completely abolished the protective effects of N-acetylcysteine in Met-KO hepatocytes. In conclusion, genetic inactivation of c-Met in mouse hepatocytes caused defects in redox regulation, which may account for the increased sensitivity to Fas-induced apoptosis and adaptive up-regulation of NF-kappaB survival signaling. These data provide evidence that intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous reactive oxygen species.     

CXCR7 expression disrupts endothelial cell homeostasis and causes ligand-dependent invasion

The homeostatic function of endothelial cells (EC) is critical for a number of physiological processes including vascular integrity, immunity, and wound healing. Indeed, vascular abnormalities resulting from EC dysfunction contribute to the development and spread of malignancies. The alternative SDF-1/CXCL12 receptor CXCR7 is frequently and specifically highly expressed in tumor-associated vessels. In this study, we investigate whether CXCR7 contributes to vascular dysfunction by specifically examining the effect of CXCR7 expression on EC barrier function and motility. We demonstrate that CXCR7 expression in EC results in redistribution of CD31/PECAM-1 and loss of contact inhibition. Moreover, CXCR7+ EC are deficient in barrier formation. We show that CXCR7-mediated motility has no influence on angiogenesis but contributes to another motile process, the invasion of CXCR7+ EC into ligand-rich niches. These results identify CXCR7 as a novel manipulator of EC barrier function via alteration of PECAM-1 homophilic junctions. As such, aberrant expression of CXCR7 in the vasculature has the potential to disrupt vascular homeostasis and could contribute to vascular dysfunction in cancer systems.     

Long-term high intake of 9-PAHPA or 9-OAHPA increases basal metabolism and insulin sensitivity but disrupts liver homeostasis in healthy mice

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a new family of endogenous lipids recently discovered. Several studies reported that some FAHFAs have antidiabetic and anti-inflammatory effects. The objective of this study was to explore the impact of two FAHFAs, 9-PAHPA or 9-OAHPA, on the metabolism of mice. C57Bl/6J male mice, 6 weeks old, were divided into 3 groups of 10 mice each. One group received a control diet and the two others groups received the control diet supplemented with 9-PAHPA or 9-OAHPA for 12 weeks. Mouse weight and body composition were monitored throughout the study. Some days before euthanasia, energy expenditure, glucose tolerance and insulin sensitivity were also determined. After sacrifice, blood and organs were collected for relevant molecular, biochemical and histological analyses. Although high intake of 9-PAHPA or 9-OAHPA increased basal metabolism, it had no direct effect on body weight. Interestingly, the 9-PAHPA or 9-OAHPA intake increased insulin sensitivity but without modifying glucose tolerance. Nevertheless, 9-PAHPA intake induced a loss of glucose-stimulated insulin secretion. Surprisingly, both studied FAHFAs induced hepatic steatosis and fibrosis in some mice, which were more marked with 9-PAHPA. Finally, a slight remodeling of white adipose tissue was also observed with 9-PAHPA intake. In conclusion, the long-term high intake of 9-PAHPA or 9-OAHPA increased basal metabolism and insulin sensitivity in healthy mice. However, this effect, highly likely beneficial in a diabetic state, was accompanied by manifest liver damage in certain mice that should deserve special attention in both healthy and pathological studies.