The ß-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla.

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is ß-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of ß-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

Recurrent hypoglycemia inhibits the counterregulatory response by suppressing adrenal activity.

Hypoglycemia activates the counterregulatory response (CRR), a neural-endocrine reflex that restores euglycemia. Although effective if occasionally activated, repeated induction of the CRR leads to a decline in responsiveness and prolonged exposure to hypoglycemia. The mechanism underlying this impairment is not known. We found that the reduction in epinephrine release that characterizes a suppressed CRR involves a long-lasting form of sympatho-adrenal synaptic plasticity. Using optogenetically evoked catecholamine release, we show that recurrent hypoglycemia reduced the secretory capacity of mouse adrenal chromaffin cells. Single activation of the CRR increased the adrenal levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, but this was prevented by repeated activation. In contrast, the level of neuropeptide Y (NPY), an adrenal cotransmitter, remained elevated after recurrent hypoglycemia. Inhibition of NPY or Y1 signaling, either transgenically or pharmacologically, prevented the attenuation of both TH expression and epinephrine release. These results indicate that impairment of the CRR involves suppressed activity at the adrenal level. Interfering with the peripheral NPY-dependent negative feedback loop may provide a way to avoid the pathophysiological consequences of recurrent hypoglycemia which are common in the diabetic state.

AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.

Fasting evokes a homeostatic response that maintains circulating levels of energy-rich metabolites and increases the drive to eat. Centrally, this reflex activates a small population of hypothalamic neurons that are characterized by the expression of AgRP, a neuropeptide with an extremely restricted distribution. Apart from the hypothalamus, the only other site with substantial expression is the adrenal gland, but there is disagreement about which cells synthesize AgRP. Using immunohistochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, we show AgRP is present in the mouse adrenal medulla and is expressed by neuroendocrine chromaffin cells that also synthesize the catecholamines and neuropeptide Y. Short-term fasting led to an increase in adrenal AgRP expression. Because AgRP can act as an antagonist at MC3/4 receptors, we tested whether melanotan II, an MC3/4 receptor agonist, could regulate pre- and postsynaptic signaling within the adrenal medulla. Melanotan II decreased the paired-pulse ratio of evoked synaptic currents recorded in chromaffin cells; this effect was blocked by exogenous AgRP. In contrast, neither melanotan II nor AgRP altered the optogenetically evoked release of catecholamines from isolated chromaffin cells. These results are consistent with the idea that AgRP regulates the strength of the sympathetic input by modulation of presynaptic MC3/4 receptors located on preganglionic neurons. We conclude that a small population of neuroendocrine cells in the adrenal medulla, and the arcuate nucleus of the hypothalamus, express AgRP and neuropeptide Y and are functionally involved in the systemic response to fasting.

17ß-estradiol attenuates homocysteine-induced oxidative stress and inflammatory response as well as MAPKs cascade via activating PI3-K/Akt signal transduction pathway in Raw 264.7 cells.

Oxidative stress, inflammatory response, and mitogen-activated protein kinases (MAPKs) cascade are significant pathogenic factors of osteoporosis. It has been reported that elevated homocysteine (Hcy) may activate oxidative stress and reduce bone mineral density in post-menopausal osteoporosis. Moreover, hormone replacement therapy has been widely used in clinic to prevent and treat post-menopausal women with osteoporosis and osteoporotic fracture, but the molecular mechanisms and relevant signal transduction pathways underlying the action of Hcy remain unclear. In this study, we investigated the effects of 17ß-estradiol (17ß-E2) on the Hcy-induced oxidative stress, inflammatory response and MAPKs cascade, as well as the underlying signal transduction pathway in murine Raw 264.7 cells. The reactive oxygen species (ROS) was assessed by fluorospectrophotometry. The proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß were analyzed by double-immunofluorescence labeling and reverse transcriptase polymerase chain reaction assay, respectively. Furthermore, phosphorylation levels of MAPKs cascade were measured by western blot analysis. A specific phosphatidylinositol 3-kinase (PI3-K) inhibitor, Wortmannin (1 µM) was employed to determine whether PI3-K/Akt signaling pathway mediated the 17ß-E2's effect on Raw 264.7 cells. 17ß-E2 markedly decreased the ROS production induced by Hcy, the expression of TNF-α and IL-1ß at protein and mRNA levels, and down-regulated the phosphorylation of MAPKs (ERK1/2, JNK and p38). These suppressing effects of 17ß-E2 on Hcy-induced changes were reversed by pretreatment with PI3-K inhibitor Wortmannin. The results indicate that 17ß-estradiol may attenuate Hcy-induced oxidative stress, inflammatory response and up-regulation of MAPKs in Raw 264.7 cells via PI3-K/Akt signal transduction pathway.

The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces.

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3 (-), the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.

Identification of a novel gene, CIA6, required for normal pyrenoid formation in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii possesses a CO(2)-concentrating mechanism (CCM) that allows the alga to grow at low CO(2) concentrations. One common feature seen in photosynthetic organisms possessing a CCM is the tight packaging of Rubisco within the cell. In many eukaryotic algae, Rubisco is localized to the pyrenoid, an electron-dense structure within the chloroplast. In order to identify genes required for a functional CCM, insertional Bleomycin resistance (Ble(R)) mutants were generated and screened for growth on minimal medium under high CO(2) conditions (5% CO(2) in air) but only slow or no growth under very low CO(2) conditions (0.01% CO(2) in air). One mutant identified from this screen was named cia6. Physiological studies established that cia6 grows poorly on low levels of CO(2) and has an impaired ability to accumulate inorganic carbon. The inserted Ble(R) disrupted a gene encoding a protein with sequence similarity to proteins containing SET domain methyltransferase, although experiments using overexpressed CIA6 failed to demonstrate the methyltransferase activity. Electron microscopy revealed that the pyrenoid of cia6 mutant cells is highly disorganized. Complementation of the mutant restored the pyrenoid, the ability to grow under low-CO(2) conditions, and the ability to concentrate inorganic carbon. Quantitative reverse transcription-polymerase chain reaction data from a low-CO(2) induction time-course experiment demonstrated that the up-regulation of several CCM components is slower in cia6 compared with the wild type. This slow induction was further confirmed at the protein level using western blots. These results indicated that CIA6 is required for the formation of the pyrenoid and further supported the notion that the pyrenoid is required for a functional CCM in C. reinhardtii.

The carbonic anhydrase isoforms of Chlamydomonas reinhardtii: intracellular location, expression, and physiological roles.

Aquatic photosynthetic organisms, such as the green alga Chlamydomonas reinhardtii, respond to low CO(2) conditions by inducing a CO(2) concentrating mechanism (CCM). Carbonic anhydrases (CAs) are important components of the CCM. CAs are zinc-containing metalloenzymes that catalyze the reversible interconversion of CO(2) and HCO(3)(-). In C. reinhardtii, there are at least 12 genes that encode CA isoforms, including three alpha, six beta, and three gamma or gamma-like CAs. The expression of the three alpha and six beta genes has been measured from cells grown on elevated CO(2) (having no active CCM) versus cells growing on low levels of CO(2) (with an active CCM) using northern blots, differential hybridization to DNA chips and quantitative RT-PCR. Recent RNA-seq profiles add to our knowledge of the expression of all of the CA genes. In addition, protein content for some of the CA isoforms was estimated using antibodies corresponding to the specific CA isoforms: CAH1/2, CAH3, CAH4/5, CAH6, and CAH7. The intracellular location of each of the CA isoforms was elucidated using immunolocalization and cell fractionation techniques. Combining these results with previous studies using CA mutant strains, we will discuss possible physiological roles of the CA isoforms concentrating on how these CAs might contribute to the acquisition and retention of CO(2) in C. reinhardtii.