Lack of obestatin effects on food intake: should obestatin be renamed ghrelin-associated peptide (GAP)?

Obestatin is a newly identified ghrelin-associated peptide (GAP) that is derived from post-translational processing of the prepro-ghrelin gene. Obestatin has been reported initially to be the endogenous ligand for the orphan receptor G protein-coupled receptor 39 (GPR39), and to reduce refeeding- and ghrelin-stimulated food intake and gastric transit in fasted mice, and body weight gain upon chronic peripheral injection. However, recent reports indicate that obestatin is unlikely to be the endogenous ligand for GPR39 based on the lack of specific binding on GRP39 receptor expressing cells and the absence of signal transduction pathway activation. In addition, a number of studies provided convergent evidence that ghrelin injected intracerebroventricularly or peripherally did not influence food intake, body weight gain, gastric transit, gastrointestinal motility, and gastric vagal afferent activity, as well as pituitary hormone secretions, in rats or mice. Similarly, obestatin did not alter ghrelin-induced stimulation of food intake or gastric transit. Therefore, the present state-of-knowledge on obestatin and GPR39 is leaving many unanswered questions that deserve further consideration. Those relate not only to redefining the biological action of obestatin that should be renamed GAP, but also the identification of the native ligand for GPR39.     

Gestational diabetes mellitus epigenetically affects genes predominantly involved in metabolic diseases

Offspring exposed to gestational diabetes mellitus (GDM) have an increased risk for chronic diseases, and one promising mechanism for fetal metabolic programming is epigenetics. Therefore, we postulated that GDM exposure impacts the offspring’s methylome and used an epigenomic approach to explore this hypothesis. Placenta and cord blood samples were obtained from 44 newborns, including 30 exposed to GDM. Women were recruited at first trimester of pregnancy and followed until delivery. GDM was assessed after a 75-g oral glucose tolerance test at 24–28 weeks of pregnancy. DNA methylation was measured at > 485,000 CpG sites (Infinium HumanMethylation450 BeadChips). Ingenuity Pathway Analysis was conducted to identify metabolic pathways epigenetically affected by GDM. Our results showed that 3,271 and 3,758 genes in placenta and cord blood, respectively, were potentially differentially methylated between samples exposed or not to GDM (p-values down to 1 × 10⁻⁰⁶; none reached the genome-wide significance levels), with more than 25% (n = 1,029) being common to both tissues. Mean DNA methylation differences between groups were 5.7 ± 3.2% and 3.4 ± 1.9% for placenta and cord blood, respectively. These genes were likely involved in the metabolic diseases pathway (up to 115 genes (11%), p-values for pathways = 1.9 × 10⁻¹³ < p < 4.0 × 10⁻⁰³; including diabetes mellitus p = 4.3 × 10⁻¹¹). Among the differentially methylated genes, 326 in placenta and 117 in cord blood were also associated with newborn weight. Our results therefore suggest that GDM has epigenetic effects on genes preferentially involved in the metabolic diseases pathway, with consequences on fetal growth and development, and provide supportive evidence that DNA methylation is involved in fetal metabolic programming.     

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague–Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-¹H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), β-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35–47% lower but monounsaturated fatty acids were 40–52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.     

Cycloheximide as a tool to investigate protein import in peroxisomes: a case study of the subcellular localization of isoprenoid biosynthetic enzymes

Cytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes.     

Monitoring by laser-flow-cytometry of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. strain 107 during biotreatment of a contaminated soil

A flow cytometric method (FCM) was used to detect and accurately enumerate a polycyclic aromatic hydrocarbon-degrading bacterial strain, Sphingomonas sp. 107, inoculated into a soil sample artificially contaminated with pyrene. To compare the FCM method with colony forming unit (CFU) assays, a rifampicin-resistant Sphingomonas sp. 107 was obtained which could be distinguished from the indigenous microflora, since there was no organism resistant to rifampicin in the soil that could transform indole to indigo (naphthalene dioxygenase activity). By combining light-scattering profiles (i.e., morphological properties), ethidium bromide influx (i.e., cell wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could enumerate the bacterial population of interest from the indigenous microflora and soil debris during the biotreatment. The FCM technique revealed that the number of inoculated Sphingomonas cells decreased gradually for 15 days of incubation before reaching a steady level of 7 to 12 x 10(5) cells.g-1 of soil. Similar values were obtained with the CFU assay. During this period, pyrene concentration decreased from 632 to 26 mg.kg-1 of dry soil. The FCM detection was improved by adding blocking reagent to the hybridization buffer to minimize the non-specific attachment of the fluorescent probe to soil particles. Combined with the improvements in probe technology, FCM detection was shown to be a good alternative to the conventional culture methods for the analysis of bacterial populations in environmental samples. This technique could be potentially useful for the detection of microorganisms that grow poorly in culture.     

Light activation of vindoline biosynthesis does not require cytomorphogenesis in Catharanthus roseus seedlings

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC) and desacetoxyvindoline 4-hydroxylase (D4H), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. The expression of TDC was restricted to the upper epidermis of cotyledons, whereas that of D4H was confined to laticifer cells. Light exposure of etiolated seedlings significantly induced D4H enzyme activity without changing the steady-state levels of D4H immunoreactive protein or modifying the cellular distribution of D4H expression in dark-grown seedlings. These results suggest that the early and late stages of vindoline biosynthesis occupy different cellular compartments, even in the early phases of etiolated seedling development. The role of light in activating the late stages of vindoline biosynthesis does not, therefore, seem to be related to the formation of the laticifer and idioblast cell types. It is concluded that light is not required for formation of these cell types, whereas regulatory factors, restricted to idioblasts and laticifers, may respond to light to activate localised expression of the late stages of vindoline biosynthesis.     

Nimodipine inhibits the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb) in the rat

Impaired nitric oxide (NO) activity is associated with an increase in blood pressure in rats. Voltage-regulated calcium channels are believed to participate in this hemodynamic event. To further test this hypothesis, we examined the effect of nimodipine and verapamil (calcium antagonists) on the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb), a well-known NO scavenger, in anesthetized rats. Nimodipine, the most potent of the two calcium antagonists used, was also tested against phenylephrine (alpha1-adrenoceptor agonist). The pressor effect of DCLHb was reduced markedly by nimodipine and verapamil, whereas that elicited by phenylephrine, particularly the tonic phase of its pressor response, was resistant to blockade by nimodipine. The bradycardia and tachycardia associated with the pressor effects of DCLHb and phenylephrine, respectively, were not affected by nimodipine. The pressor effect elicited by DCLHb and its alteration by nimodipine were also examined in rats pretreated with 100% O2. This treatment was found to potentiate the pressor effect of DCLHb. However, this synergism did not impair the inhibitory action of nimodipine towards the pressor activity of DCLHb. Altogether these results suggest that the pressor activity of DCLHb in our animal model might involve the participation of voltage-regulated calcium channels.     

Characterization of the histamine releasing effect of neurotensin in the rat heart

Bolus injections of neurotensin (NT) in the rat perfused heart elicited a transient, dose-dependent histamine release. The histamine releasing effect of NT appears to be independent of the heart rate and coronary perfusion pressure and it was not influenced by atropine, propanolol, prazosin, methysergide, ketanserin, indomethacin, morphine, lidocaine or by removal of the atria. However, it was potentiated by adenosine, inhibited by sub-stimulatory concentrations of NT and the mast cell membrane stabilizing drug cromoglycate but was unaltered by the calcium antagonist verapamil. The absence of calcium in the heart perfusate suppressed the histamine releasing effect of NT. These results suggest that the histamine releasing effect of NT in the rat heart results from a direct effect on ventricular mast cells and is calcium-dependent.     

Synthesis and trafficking of alkaloid biosynthetic enzymes

The biosynthesis of plant natural products involves a large number of enzymes that create and elaborate a bewildering array of chemical structures, which are generally involved in ecophysiological interactions. Alkaloids are one of the largest groups of natural products and are generally produced through an assortment of intricate pathways. The application of molecular biochemical approaches to investigate the cell biology of alkaloid pathways has revealed a paradigm for the complex, yet highly ordered, organization of biosynthetic enzymes at both the cellular and subcellular levels. Many different cell types have been implicated in alkaloid formation and storage, in one case suggesting the intercellular transport of enzymes. The localization of enzymes to numerous cellular compartments shows the importance of protein targeting in the assembly of alkaloid pathways. Recent studies have also pointed to the possible interaction of biosynthetic enzymes in multi-enzyme complexes. These processes must be considered to be integral components of the mechanisms that regulate alkaloid biosynthesis and perhaps other natural product pathways.