Novel blockade of protein kinase A-mediated phosphorylation of AMPA receptors.

The phosphorylation state of the glutamate receptor subtype 1 (GluR1) subunit of the AMPA receptor (AMPAR) plays a critical role in synaptic expression of the receptor, channel properties, and synaptic plasticity. Several Gs-coupled receptors that couple to protein kinase A (PKA) readily recruit phosphorylation of GluR1 at S845. Conversely, activation of the ionotropic glutamate NMDA receptor (NMDAR) readily recruits dephosphorylation of the same GluR1 site through Ca2+-mediated recruitment of phosphatase activity. In a physiological setting, receptor activation often overlaps and crosstalk between coactivation of multiple signaling cascades can result in differential regulation of a given substrate. After investigating the effect of coactivation of the NMDAR and the Gs-coupled beta-adrenergic receptor on GluR1 phosphorylation state, we have observed a novel signal that prevents PKA-mediated phosphorylation of GluR1 at serine site 845. This blockade of GluR1 phosphorylation is dependent on cellular depolarization recruited by either NMDAR or AMPAR activation, independent of Ca2+ and independent of calcineurin, protein phosphatase 1, and/or protein phosphatase 2A activity. Thus, in addition to the typical kinase-phosphatase rivalry mediating protein phosphorylation state, we have identified a novel form of phospho-protein regulation that occurs at GluR1 and may also occur at several other PKA substrates.

NMDA and beta1-adrenergic receptors differentially signal phosphorylation of glutamate receptor type 1 in area CA1 of hippocampus.

Glutamatergic synaptic transmission is mediated primarily through the AMPA-type glutamate receptor (AMPAR); the regulation of this receptor underlies many forms of synaptic plasticity. In particular, phosphorylation of GluR1, an AMPAR subunit, by PKA at serine 845 (S845) increases peak open channel probability and is permissive for both the synaptic expression of the receptor and NMDA-receptor (NMDAR)-dependent long-term potentiation (LTP). Robust NMDAR activation activates PKA as well as other signaling enzymes; however, we find that maximal NMDAR activation dephosphorylates GluR1 at the PKA site S845. Coincident inhibition of phosphatases blocks NMDAR-induced dephosphorylation of S845, but surprisingly does not promote PKA phosphorylation at this site. However, we find that phosphorylation of S845 is increased by the activation of a Gs-coupled receptor, the beta1-adrenergic receptor. Interestingly, this divergent signaling occurs despite a more robust coupling of the NMDAR to cAMP generation. In addition, NMDAR activation plays a dominant role in S845 regulation, because activation of beta1AR after NMDAR activation has no detectable effect on S845 phosphorylation. These data (1) demonstrate highly specific coupling between these receptors and this substrate, (2) provide an example of a substrate critical in NMDAR-dependent LTP that is incompletely regulated by the NMDAR, and (3) highlight the importance of identifying the physiological signals that regulate these critical synaptic substrates.

Regulation of cAMP levels in area CA1 of hippocampus by Gi/o-coupled receptors is stimulus dependent in mice.

The cAMP signaling cascade plays a critical role in regulating synaptic efficacy and cellular excitability in hippocampus. Adenylyl cyclase (AC) activation and subsequent generation of cAMP occurs through a number of mechanisms in area CA1 of hippocampus, including Galpha(s)-mediated stimulation upon G-protein coupled receptor (GPCR) activation and Ca2+ -mediated stimulation upon NMDA receptor activation. In addition, activation of Gi/o-coupled receptor subtypes can regulate cAMP levels through modulation of AC activity. Multiple Gi/o-coupled receptor subtypes are expressed in area CA1, where they are commonly thought to dampen synaptic transmission and excitability, in part through inhibition of AC activity and cAMP generation. However, a large family of ACs exists, and in recombinant systems, the cAMP signals generated by these AC isoforms are both inhibited and enhanced by Gi/o-coupled receptors in a manner dependent on the AC isoform and stimulus. Thus, we have assessed the effects of activating several different Gi/o-coupled receptors on the generation of cAMP induced either by NMDA receptor activation or by beta-adrenergic receptor activation within area CA1 of mouse hippocampus. We find that in situ the effect of Gi/o-coupled receptor activation does indeed depend upon the means by which ACs are activated. In addition, the effects are also Gi/o-coupled receptor-specific, suggesting additional complexity. These data indicate that Gi/o-coupled receptors may play roles in "routing" second messenger signaling in area CA1.

Melanocortin-4 receptor signaling is required for weight loss after gastric bypass surgery.

CONTEXT: Roux-en-Y gastric bypass (RYGB) is one of the most effective long-term therapies for the treatment of severe obesity. Recent evidence indicates that RYGB effects weight loss through multiple physiological mechanisms, including changes in energy expenditure, food intake, food preference, and reward pathways. OBJECTIVE: Because central melanocortin signaling plays an important role in the regulation of energy homeostasis, we investigated whether genetic disruption of the melanocortin-4 receptor (MC4R) in rodents and humans affects weight loss after RYGB. METHODS AND RESULTS: Here we report that MC4R(-/-) mice lost substantially less weight after surgery than wild-type animals, indicating that MC4R signaling is necessary for the weight loss effects of RYGB in this model. Mice heterozygous for MC4R remain fully responsive to gastric bypass. To determine whether mutations affect surgically induced weight loss in humans, we sequenced the MC4R gene in 972 patients undergoing RYGB. Patients heterozygous for MC4R mutations exhibited the same magnitude and distribution of postoperative weight loss as patients without such mutations, suggesting that although two normal copies of the MC4R gene are necessary for normal weight regulation, a single normal copy of the MC4R gene is sufficient to mediate the weight loss effects of RYGB. CONCLUSIONS: MC4R is the first gene identified that is required for the sustained effects of bariatric surgery. The need for MC4R signaling for the weight loss effects of RYGB in mice underscores the physiological mechanisms of action of this procedure and demonstrates that RYGB both influences and is dependent on the normal pathways that regulate energy balance.

Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose inflammation: comparison to high-fat diet.

Obesity has reached epidemic proportions worldwide and reports estimate that American children consume up to 25% of calories from snacks. Several animal models of obesity exist, but studies are lacking that compare high-fat diets (HFD) traditionally used in rodent models of diet-induced obesity (DIO) to diets consisting of food regularly consumed by humans, including high-salt, high-fat, low-fiber, energy dense foods such as cookies, chips, and processed meats. To investigate the obesogenic and inflammatory consequences of a cafeteria diet (CAF) compared to a lard-based 45% HFD in rodent models, male Wistar rats were fed HFD, CAF or chow control diets for 15 weeks. Body weight increased dramatically and remained significantly elevated in CAF-fed rats compared to all other diets. Glucose- and insulin-tolerance tests revealed that hyperinsulinemia, hyperglycemia, and glucose intolerance were exaggerated in the CAF-fed rats compared to controls and HFD-fed rats. It is well-established that macrophages infiltrate metabolic tissues at the onset of weight gain and directly contribute to inflammation, insulin resistance, and obesity. Although both high fat diets resulted in increased adiposity and hepatosteatosis, CAF-fed rats displayed remarkable inflammation in white fat, brown fat and liver compared to HFD and controls. In sum, the CAF provided a robust model of human metabolic syndrome compared to traditional lard-based HFD, creating a phenotype of exaggerated obesity with glucose intolerance and inflammation. This model provides a unique platform to study the biochemical, genomic and physiological mechanisms of obesity and obesity-related disease states that are pandemic in western civilization today.

MafA and MafB regulate Pdx1 transcription through the Area II control region in pancreatic beta cells.

Pancreatic-duodenal homeobox factor-1 (Pdx1) is highly enriched in islet beta cells and integral to proper cell development and adult function. Of the four conserved 5'-flanking sequence blocks that contribute to transcription in vivo, Area II (mouse base pairs -2153/-1923) represents the only mammalian specific control domain. Here we demonstrate that regulation of beta-cell-enriched Pdx1 expression by the MafA and MafB transcription factors is exclusively through Area II. Thus, these factors were found to specifically activate through Area II in cell line transfection-based assays, and MafA, which is uniquely expressed in adult islet beta cells was only bound to this region in quantitative chromatin immunoprecipitation studies. MafA and MafB are produced in beta cells during development and were both bound to Area II at embryonic day 18.5. Expression of a transgene driven by Pdx1 Areas I and II was also severely compromised during insulin+ cell formation in MafB(-/-) mice, consistent with the importance of this large Maf in beta-cell production and Pdx1 expression. These findings illustrate the significance of large Maf proteins to Pdx1 expression in beta cells, and in particular MafB during pancreatic development.

ERK activation by G-protein-coupled receptors in mouse brain is receptor identity-specific.

In transfected cells and non-neuronal tissues many G-protein-coupled receptors activate p44/42 MAP kinase (ERK), a kinase involved in both hippocampal synaptic plasticity and learning and memory. However, it is not clear to what degree these receptors couple to ERK in brain. G(s)-coupled beta-adrenergic receptor activation of ERK in neurons is critical in the regulation of synaptic plasticity in area CA1 of the hippocampus. In addition, alpha(1)- and alpha(2)-adrenergic receptors, present in CA1, could potentially activate ERK. We find that, like the beta-adrenergic receptor, the G(q)-coupled alpha(1)AR activates ERK in adult mouse CA1. However, activation of the G(i/o)-coupled alpha(2)AR does not activate ERK, nor does activation of a homologous G(i/o)-coupled receptor enriched in adult mouse CA1, the 5HT(1A) receptor. In contrast, the nonhomologous G(i/o)-coupled gamma-aminobutyric acid type B receptor does activate ERK in adult mouse CA1. Surprisingly, activation of alpha(2)ARs in CA1 from immature animals where basal phospho-ERK is low induces ERK phosphorylation. These data suggest that although most G-protein-coupled receptor subtypes activate ERK in non-neuronal cells, the coupling of G(i/o) to ERK is tightly regulated in brain.