Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice.

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.

5-HT inhibition of rat insulin 2 promoter Cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus.

A number of anti-obesity agents have been developed that enhance hypothalamic 5-HT transmission. Various studies have demonstrated that arcuate neurons, which express proopiomelanocortin peptides (POMC neurons), and neuropeptide Y with agouti-related protein (NPY/AgRP) neurons, are components of the hypothalamic circuits responsible for energy homeostasis. An additional arcuate neuron population, rat insulin 2 promoter Cre recombinase transgene (RIPCre) neurons, has recently been implicated in hypothalamic melanocortin circuits involved in energy balance. It is currently unclear how 5-HT modifies neuron excitability in these local arcuate neuronal circuits. We show that 5-HT alters the excitability of the majority of mouse arcuate RIPCre neurons, by either hyperpolarization and inhibition or depolarization and excitation. RIPCre neurons sensitive to 5-HT, predominantly exhibit hyperpolarization and pharmacological studies indicate that inhibition of neuronal firing is likely to be through 5-HT(1F) receptors increasing current through a voltage-dependent potassium conductance. Indeed, 5-HT(1F) receptor immunoreactivity co-localizes with RIPCre green fluorescent protein expression. A minority population of POMC neurons also respond to 5-HT by hyperpolarization, and this appears to be mediated by the same receptor-channel mechanism. As neither POMC nor RIPCre neuronal populations display a common electrical response to 5-HT, this may indicate that sub-divisions of POMC and RIPCre neurons exist, perhaps serving different outputs.

Cortical alveoli of Paramecium: a vast submembranous calcium storage compartment.

The plasma membrane of Paramecium is underlain by a continuous layer of membrane vesicles known as cortical alveoli, whose function was unknown but whose organization had suggested some resemblance with muscle sarcoplasmic reticulum. The occurrence of antimonate precipitates within the alveoli first indicated to us that they may indeed correspond to a vast calcium storage site. To analyze the possible involvement of this compartment in calcium sequestration more directly, we have developed a new fractionation method, involving a Percoll gradient, that allows rapid purification of the surface layer (cortex) of Paramecium in good yield and purity and in which the alveoli retain their in vivo topological orientation. This fraction pumped calcium very actively in a closed membrane compartment, with strict dependence on ATP and Mg2+. The pumping activity was affected by anti-calmodulin drugs but no Triton-soluble calmodulin binding protein could be identified, using gel overlay procedures. The high affinity of the pump for calcium (Km = 0.5 microM) suggests that it plays an important role in the normal physiological environment of the cytosol. This may be related to at least three calcium-regulated processes that take place in the immediate vicinity of alveoli: trichocyst exocytosis, ciliary beating and cytoskeletal elements dynamics during division.

Hepatitis B virus-related insertional mutagenesis implicates SERCA1 gene in the control of apoptosis.

We have used the Hepatitis B Virus DNA genome as a probe to identify genes clonally mutated in vivo, in human liver cancers. In a tumor, HBV-DNA was found to be integrated into the gene encoding Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA), which pumps calcium, an important intracellular messenger for cell viability and growth, from the cytosol to the endoplasmic reticulum. The HBV X gene promoter cis-activates chimeric HBV X/SERCA1 transcripts, with splicing of SERCA1 exon 11, encoding C-terminally truncated SERCA1 proteins. Two chimeric HBV X/SERCA1 proteins accumulate in the tumor and form dimers. In vitro analyses have demonstrated that these proteins localize to the ER, determine its calcium depletion and induce cell death. We have also shown that these biological effects are related to expression of the SERCA, rather than of the viral moiety. This report involves for the first time the expression of mutated SERCA proteins in vivo in a tumor cell proliferation and in vitro in the control of cell viability. Oncogene (2000).

Alterations in human erythrocyte shape and the state of spectrin and phospholipid phosphorylation induced by cholesterol depletion.

Cholesterol depletion of erythrocytes, obtained after incubation with phosphatidylcholine vesicles, induces in most of the experiments: (1) a discocytestomatocyte transformation as observed by scanning electron microscopy; (2) a specific decrease in spectrin phosphorylation of intact erythrocytes; (3) an increase in lipid phosphorylation. It is concluded that the effect of cholesterol on erythrocyte shape is probably mediated through its action on the activity o of membrane-bound enzymes, proteases or kinases.

Alpha-adrenergically mediated changes in membrane lipid fluidity and Ca2/ binding in isolated rat liver plasma membranes.

Noradrenaline (0.1-5 microM, in the presence of 5 microM propranolol to block beta-receptors), ATP (100 microM) and angiotensin II (0.1 microM), which are thought to increase cytosolic Ca2+ concentration by mobilizing Ca2+ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the alpha-antagonists phenoxybenzamine (50 microM) and phentolamine (1 microM). The response to a maximal dose of noradrenaline (5 microM) and that to ATP (100 microM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 microM) along with the foreign amphiphile Na+-oleate (1-30 microM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg2+, reducing free Ca2+ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca2+ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 microM) and angiotensin II (0.1 microM) also promoted a small release of 45Ca2+ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the alpha-antagonist phentolamine (5 microM). It is proposed that noradrenaline, via alpha-adrenergic receptors and other Ca2+ -mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca2+ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+-K+ pump in erythrocytes.

cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.

Asymmetric distribution of phosphoinositides and phosphatidic acid in the human erythrocyte membrane.

The distribution of phosphoinositides and phosphatidic acid (PA) between the outer and inner layers of the human erythrocyte membrane was investigated by using two complementary methodologies: hydrolysis by phospholipase A2 (PLA2) and immunofluorescence detection with monoclonal antibodies against polyphosphoinositides. The contents of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP) and PA were decreased by 15-20% after 60 min incubation with PLA2, while that of phosphatidylinositol (PI) was increased. Studies with 32P-labelled cells revealed that PLA2 treatment led to indirect effects on the metabolism of these phospholipids. Therefore, the asymmetric distribution of phosphoinositides and PA was inferred from the data obtained in ATP-depleted erythrocytes. In these cells with arrested phosphoinositide metabolism, the asymmetric distribution of the major phospholipids was maintained: PLA2 hydrolyzed approx. 20% of PI, PIP2 and PA (but no PIP) indicating their localization in the outer layer of the membrane. This finding was confirmed by immunofluorescence studies with antibodies specific to each phosphoinositide. External addition of anti-PIP2 but not anti-PIP gave a positive reaction both in control and in ATP-depleted erythrocytes. A pretreatment of cells with PLA2 led to a decrease in the intensity of anti-PIP2 staining. These results demonstrate that significant fractions of PIP2, PI and PA are localized on the outer surface of the erythrocyte membrane.

Characterization of two forms of inositol 1,4,5-trisphosphate receptor in rat liver.

Two binding sites for [32P]myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) were detected in a crude particulate fraction prepared from rat liver homogenate and in permeabilized hepatocytes. The same high- and low-affinity sites with KDs of 1.8-2.6 nM and 35-71 nM, respectively, were detected in subcellular fractions enriched in plasma membranes, mitochondria and microsomes, with relative proportions close to those found in the crude membrane fraction. The order of potency of three inositol phosphates in inhibiting [32P]Ins(1,4,5)P3 binding to the two sites, i.e. Ins(1,4,5)P3 greater than Ins(2,4,5)P3] greater than Ins(1,3,4,5)P4, and the inhibition by heparin, strongly suggest that neither of the binding sites reflected components due to the 3-kinase or the 5-phosphatase. A close correlation was observed between the dose-response curves for Ca2+ release by Ins(1,4,5)P3 and Ins(2,4,5)P3 and the occupancy of the low-affinity binding site by these agonists. These results support the view that the two [32P]Ins(1,4,5)P3 binding sites are two forms of the same receptor.

Cellular distribution of polyphosphoinositides in rat hepatocytes.

The distribution of total phospholipids, phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was studied in isolated rat hepatocytes: (i) by mass assay and isotopic labelling in the fractions of plasma membranes, microsomes, mitochondria and nuclei prepared from isolated hepatocytes and (ii) by immunolocalization of PIP2 with a specific antibody (kt3g) in whole hepatocytes and isolated nuclei. Mass measurement and isotopic labelling showed that PIP was distributed in all four fractions. PIP2 was present in the plasma membrane and the nuclei. In whole cells, PIP2 was also detected in the plasma membrane by immunolocalization with the anti-PIP2 antibody kt3g. In unpolarized single hepatocytes, PIP2 distributed evenly throughout the plasma membrane. However, in polarized cell couplets, PIP2 was the most often undetectable in the lateral domain between the cells, and distributed preferentially in the sinusoidal domain of the plasma membrane. These results suggest that hepatocytes segregate PIP2 in particular domains of their plasma membrane. In purified fractions of nuclei, immunolocalization experiments showed that PIP2 was present uniquely in the nuclear envelope.

Are third-trimester adipokines associated with higher metabolic risk among women with gestational diabetes?

AIM: This study aimed to determine whether third-trimester adipokines during gestational diabetes (GDM) are associated with higher metabolic risk. METHODS: A total of 221 women with GDM (according to IADPSG criteria) were enrolled between 2011/11 and 2013/6 into a prospective observational study (IMAGE), and categorized as having elevated fasting blood glucose (FBG) or impaired fasting glucose (IFG, n = 36) if levels were ≥ 92 mg/dL during a 75-g oral glucose tolerance test (OGTT), impaired glucose tolerance (IGT, n = 116) if FBG was < 92 mg/dL but with elevated 1-h or 2-h OGTT values, or impaired fasting and stimulated blood glucose (IFSG, n = 69) if both FBG was ≥ 92 mg/dL and 1-h or 2-h OGTT values were elevated. RESULTS: Pre-gestational body mass index (BMI) was higher in women with IFG or IFSG compared with IGT (P < 0.001), as were leptin levels in women with IFG vs IGT [34.7 (10.5-119.7) vs 26.6 (3.56-79.4) ng/L; P = 0.008]. HOMA2-IR scores were higher in women with IFG or IFSG vs IGT (1.87 ± 1.2 or 1.72 ± 0.9 vs 1.18 ± 0.8, respectively; P < 0.001). Also, those with IFSG vs those with IGT had significantly lower HOMA2-B scores (111.4 ± 41.3 vs 127.1 ± 61.6, respectively; P < 0.05) and adiponectin levels [5.00 (1.11-11.3) vs 6.19 (2.11-17.7) µg/mL; P < 0.001], and higher levels of IL-6 [1.14 (0.33-20.0) vs 0.90 (0.31-19.0); P = 0.012] and TNF-α [0.99 (0.50-10.5) vs 0.84 (0.45-11.5) pg/mL; P = 0.003]. After adjusting for age, parity, and pre-gestational and gestational BMI, the difference in adiponectin levels remained significant. CONCLUSION: Diagnosing GDM by IADSPG criteria results in a wide range of heterogeneity. Our study has indicated that adipokine levels in addition to FBG may help to select women at high metabolic risk for appropriate monitoring and post-delivery interventions (ClinicalTrials.gov number NCP02133729).

Calcium control on InsP3-induced discharge of calcium from permeabilised hepatocyte pools.

The control exerted by intralumenal and cytosolic Ca2+ on InsP3-induced release of Ca2+ from intracellular Ca2+ pools in suspensions of saponin-permeabilised rat hepatocytes was investigated by combined Quin-2 and 45Ca2+ measurements at 20 degrees C. We failed to detect a major effect of intralumenal Ca2+ in regulating this release, as various manipulations in which the load of the Ca2+ pools was varied by a factor of two did not significantly affect the apparent relative efficiency of InsP3 in releasing Ca2+; these manipulations included loading the Ca2+ pools up to various steady state levels by preliminary equilibration at various external free Ca2+ concentrations, as well as emptying them progressively through the blockade of pump-mediated Ca2+ uptake. As regards Ca2+ on the cytosolic side, in contrast with recent results obtained with other systems, we found that, at maximal doses, InsP3-induced Ca2+ release was not stimulated by raising Ca2+ from very low to submicromolar or micromolar concentrations, and that only relatively high concentrations of free Ca2+ inhibited this release (half-maximal inhibition was between 3 and 15 microM). Such elevated Ca2+ concentrations reduced the size of the InsP3-sensitive Ca2+ pool. We also noted that the apparent cooperativity of InsP3 activation of release at pCa 5 was noticeably less than that observed at pCa 7. As a result, at low InsP3 concentrations, a rise in cytosolic Ca2+ from pCa 7 to pCa 5 stimulated InsP3-mediated Ca2+ release. These results are discussed in the context of the current speculations about tissue specificity, heterogeneity, quantal release, oscillations, and the several different mechanisms that may control InsP3-induced Ca2+ release.

Characterization of the co-agonist effects of strontium and calcium on myo-inositol trisphosphate-dependent ion fluxes in cerebellar microsomes.

Using sheep cerebellum microsomes previously loaded with 45Ca2+ or 90Sr2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP3)-induced efflux of these ions on Ca2+ or Sr2+ on the cytosolic side. At a low InsP3 concentration, Ca2+ in the submicromolar range only poorly activated 45Ca2+ or 90Sr2+ efflux, and higher Ca2+ concentrations were inhibitory. In contrast, Sr2+ in the micromolar range activated release efficiently, while only very high Sr2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP3 concentration, which allowed increasing free Ca2+ to micromolar concentrations without inducing complete inhibition of the InsP3-dependent efflux. Under these conditions, micromolar Ca2+ was found to activate efflux to a large extent, similar to that previously found with Sr2+. Optimal activation by Ca2+ of the InsP3-dependent channel occurs at micromolar rather than submicromolar free Ca2+ concentrations, but at too low an InsP3 concentration, Ca(2+)-induced activation is counteracted by Ca(2+)-induced inactivation. Separate measurements of [3H]-InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]-InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptor's affinity for InsP3. A similar relationship also holds in the case of the activating effect of nucleotides.

Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes.

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.

Do submaximal InsP3 concentrations only induce the partial discharge of permeabilized hepatocyte calcium pools because of the concomitant reduction of intraluminal Ca2+ concentration?

In several types of cells whose cytoplasmic Ca2+ is regulated by inositol phosphate derivatives, low concentrations of InsP3 added to permeabilized cell suspensions induce the rapid discharge of part of the InsPs-sensitive Ca2+ pool instead of slow monophasic release of Ca2+ from the entire pool. As a tentative explanation for this puzzling observation, sometimes called 'quantal release', it was suggested that the reduced intraluminal Ca2+ concentration remaining in the Ca2+ pool after a certain amount of Ca2+ had been released might allosterically reduce the channels' affinity for InsP3 and the corresponding InsP3-dependent Ca2+ efflux, and thus result in partial pool discharge (Irvine, R.F. (1990) FEBS Lett. 263, 5-9). We have tested this hypothesis by manipulating the Ca2+ pool contents with ionophore, and found that the rate of InsP3-dependent Ca2+ efflux after ionophore-induced partial discharge of the Ca2+ pools was much faster than what was predicted on the basis of this hypothesis. Heterogeneity of the Ca2+ pools appears to be a more likely reason for the 'quantal release' behavior.

Effect of the bile acid taurolithocholate on cell calcium in saponin-treated rat hepatocytes.

Neomycin was used to assess the involvement of Ins (1,4,5)P3 in the Ca2+ release from the endoplasmic reticulum induced by the bile acid taurolithocholate. In saponin-permeabilized rat hepatocytes, neomycin via its ability to bind Ins (1,4,5)P3 abolished the release of Ca2+ induced by added Ins (1,4,5)P3. In contrast, it did not alter the Ca2+ release initiated by the bile acid. In intact cells, neomycin had no effect on the [Ca2+]i rises promoted by taurolithocholate and vasopressin. It is suggested that the effect of taurolithocholate in liver is not mediated by Ins (1,4,5)P3 but results from a primary action on endoplasmic reticulum.

Resistance to apamin of the Ca2+-activated K+ permeability in pancreatic B-cells.

The bee venom neurotoxin apamin failed to affect 86Rb outflow and insulin release from rat pancreatic islets stimulated by D-glucose or the Ca2+-ionophore A23187. Apamin, in contrast to quinine or A23187, also failed to affect bioelectrical activity in mouse islet cells. These findings suggest that, like in erythrocytes, and at variance with the situation found in smooth muscle, liver or neuroblastoma cells, the Ca2+-activated K+ permeability in the pancreatic B-cell is resistant to apamin.

Cytosolic free Ca2+ in isolated rat hepatocytes as measured by quin2. Effects of noradrenaline and vasopressin.

Cytosolic [Ca2+] has been measured by using the Ca2+-sensitive indicator quin2 in rat liver cells. Optimal loading and hydrolysis have been obtained by equilibrating the cells with 50 microM quin2 acetoxymethyl ester for 150 s. The increase in [Ca2+]i initiated by noradrenaline and vasopressin was reduced but not abolished by removing external Ca2+.

Norepinephrine-induced loss of phosphatidylinositol from isolated rat liver plasma membrane. Effects of divalent cations.

Norepinephrine at 5 microM induces a rapid (60 s) and specific loss of phosphatidylinositol (PtdIns) when added to isolated rat liver plasma membranes. The hormone action is inhibited by the alpha-adrenergic antagonist phentolamine (20 microM). Depletion of Mg2+ and Ca2+ singly or in combination from the incubation buffer mimicks the hormone effect on PtdIns breakdown. No further effect on PtdIns degradation could be measured when norepinephrine was added to the cation-depleted buffers. Addition of the Ca2+ ionophore A23187 to the isolated membranes has no effect. It is concluded that PtdIns degradation can be provoked in isolated rat liver plasma membrane through alpha-adrenergic receptor activation and that this effect is dependent on divalent cations in the sense that loss of cations from the membrane allows degradation to commence.