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.

The threshold for hippocampal-dependent synaptic plasticity and memory storage is thought to be determined by the balance between protein phosphorylation and dephosphorylation mediated by the kinase PKA and the phosphatase calcineurin. To establish whether endogenous calcineurin acts as an inhibitory constraint in this balance, we examined the effect of genetically inhibiting calcineurin on plasticity and memory. Using the doxycycline-dependent rtTA system to express a calcineurin inhibitor reversibly in the mouse brain, we find that the transient reduction of calcineurin activity facilitates LTP in vitro and in vivo. This facilitation is PKA dependent and persists over several days in vivo. It is accompanied by enhanced learning and strengthened short- and long-term memory in several hippocampal-dependent spatial and nonspatial tasks. The LTP and memory improvements are reversed fully by suppression of transgene expression. These results demonstrate that endogenous calcineurin constrains LTP and memory.

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.

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.

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 ␤-adrenergic receptor activation of ERK in neurons is critical in the regulation of synaptic plasticity in area CA1 of the hippocampus. In addition, ␣ 1 -and ␣ 2 -adrenergic receptors, present in CA1, could potentially activate ERK. We find that, like the ␤-adrenergic receptor, the G q -coupled ␣ 1 AR activates ERK in adult mouse CA1. However, activation of the G i/o -coupled ␣ 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 ␥-aminobutyric acid type B receptor does activate ERK in adult mouse CA1. Surprisingly, activation of ␣ 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.Protein phosphorylation plays a critical role in synaptic plasticity and learning and memory in vertebrates. A growing body of evidence suggests that the p44/42 MAP 1 kinase (ERK) cascade in particular plays important roles in the modulation of long-term potentiation in area CA1 of the hippocampus and is required for several forms of learning and memory (1). Given the roles of this kinase cascade in transcriptionally regulated processes, initial studies focused on its roles in long term forms of both long-term potentiation of synaptic transmission in the hippocampus and hippocampus-dependent long term memory formation (2-7). More recent studies, however, implicate a role for this kinase in more moment-to-moment cellular excitability (8, 9). Thus, the ERK signaling cascade regulates several aspects of synaptic transmission.Because of the multiple roles ERK may play in neuronal function it is critical to understand how the activation of this kinase is regulated in neurons. A number of receptor signaling pathways critical to synaptic plasticity recruit ERK activation in the hippocampus. As in many cell types, ligands for receptor tyrosine kinases, such as neurotrophins, recruit ERK activation in neurons, as does N-methyl D-aspartate receptor activation (10, 11). In addition, neuromodulators such as norepinephrine (NE) that activate GPCRs play critical roles in learning and memory and synaptic plasticity, at least in part through the regulation of ERK activity. For example, the activation of one adrenergic receptor, the G s -coupled ␤-adrenergic receptor, results in increased ERK activity and facilitates ERK-dependent forms of long-term potentiation in CA1 (8, 9, 12, 13). However, this receptor is not likely to be activated alone in vivo but rather in concert with other NE receptors, including the G qcoupled ␣ 1 -adre...

Pancreatic-duodenal homeobox factor-1 (Pdx1) is highly enriched in islet ␤ 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 ␤-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 ␤ cells was only bound to this region in quantitative chromatin immunoprecipitation studies. MafA and MafB are produced in ␤ 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 ␤-cell production and Pdx1 expression. These findings illustrate the significance of large Maf proteins to Pdx1 expression in ␤ cells, and in particular MafB during pancreatic development.Much effort is currently being directed to define the biochemical pathways essential to islet ␤-cell formation, with the hope that such insight will aid in the development of treatment strategies aimed at reducing ␤-cell dysfunction in diabetic patients. A transcription factor that is critical to both ␤-cell development and function is pancreatic-duodenal homeobox-1 (Pdx1, 5 IPF-1 in humans), the first pancreas-enriched gene product expressed in budding epithelium at embryonic day 8.5 (E8.5) (1, 2). Pdx1 is produced in early pancreatic endocrine, exocrine, and ductal progenitors, and complete loss in humans and mice results in an apancreatic phenotype (3-5). Later in development, islet ␤ cells can be distinguished from pancreatic exocrine and ductal cells by their high Pdx1 levels (1, 2). In mature pancreas, Pdx1 is principally localized to ␤ cells (1), with specific removal leading to a severe diabetic phenotype due to ␤-cell dysfunction in mice (6, 7). Pdx1 is also one of few genes associated with an autosomal dominant form of diabetes in humans (5) and is viewed as a master regulator of ␤-cell formation and function (8,9).Endogenous Pdx1 expression is predominately controlled by four conserved 5Ј-flanking sequence domains, referred to as Area I (mouse AI, base pairs (bp) Ϫ2761/Ϫ2457), AII (bp Ϫ2153/Ϫ1923), AIII (bp Ϫ1879/Ϫ1600), and AIV (bp Ϫ6529/ Ϫ6047) (10 -12). AI-III mediates pancreas-specific expression, as early targeted removal of these regions from the endogenous gene profoundly reduces pancreas formation in vivo, while leaving Pdx1 expression in the stomach and duodenum intact (13). Several transgenic reporter lines encompassing AI, AII, and AIII have been developed to more precisely define their roles in pancreatic expression. Interactions between AIII and AI/II have been shown to be necessary in the early...

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 G s -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 Ca 2ϩ -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 G s -coupled ␤-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 Ca 2ϩ 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 phosphoprotein regulation that occurs at GluR1 and may also occur at several other PKA substrates.

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