Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, ß-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

Effect of ageing and hypertension on the expression and activity of PEPT2 in normal and hypertrophic hearts.

Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether the expression and activity of the cardiac-type PEPT2 cotransporter could be affected by ageing and/or hypertension. Sarcolemmal vesicles (SV) were isolated from the hearts of all rat groups using a standard procedure to investigate the transport activity and protein abundance by fluorescence spectroscopy and Western blot, respectively. SLC15A2 "PEPT2" gene expression was relatively quantified by RT-qPCR. In the Wistar rat groups, the protein and gene expression of PEPT2 were upregulated with ageing. These changes were accompanied by corresponding increases in the competitive inhibition and the transport rate (Vmax) of ß-Ala-Lys (AMCA) into SV isolated from middle-aged hearts. Although, the transport rate of ß-Ala-Lys (AMCA) into SV isolated from old hearts was significantly the lowest compared to middle-aged and young adult hearts, the inhibition percentage of ß-Ala-Lys (AMCA) transport by Gly-Gln was the highest. In the WKY and SHR rat groups, Y-SHR hypertrophied hearts showed an increase in PEPT2 gene expression accompanied by a significant decrease in protein expression and activity. With advanced age, however, M-SHR hypertrophied hearts revealed significantly lower gene expression, but higher protein expression and activity than Y-SHR hearts. These findings suggest that increased expression of PEPT2 cotransporter in all types of middle-aged hearts could be exploited to facilitate di-and tripeptide transport by PEPT2 in these hearts, which subsequently could result in improved myocardial protection in these populations.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.

A new use of ß-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla.

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide ß-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH6.6 and different concentrations of ß-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the ß-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations >100µM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100µM ß-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of ß-Ala-Lys (AMCA) at 20min showed that the Km and Vmax were 783.7±115.7µM and 2191.2±133.9ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km=93.6±21.9µM and Vmax=935.8±50.2ΔF/min/mg. These findings demonstrate the applicability of ß-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 µM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

Homocysteine Exposure Impairs Myocardial Resistance to Ischaemia Reperfusion and Oxidative Stress.

BACKGROUND/AIMS: Hyperhomocysteinaemia is recognised as a strong independent risk factor for developing cardiovascular disease. This study investigated how an acute homocysteine dose affected cardiac performance during ischaemia reperfusion and cardiomyocyte contractility and morphology under normal conditions and during oxidative stress. METHODS: Cardiac function was measured in isolated and perfused rat hearts before and after 40 minutes' global normothermic ischaemia. Where used, 0.1 mM L-homocysteine was present prior to, and throughout ischaemia, before wash out after 10 minutes' reperfusion. Calcium transients under normal conditions and changes in contractile synchronicity during oxidative stress (exposure to 0.2 mM H2O2) were measured in freshly isolated rat cardiomyocytes incubated for 60 minutes ± 0.1 mM L-homocysteine. RESULTS: During ischaemia reperfusion 0.1 mM L-homocysteine significantly reduced the rate pressure product during reperfusion (10,038 ± 749 vs. 5955 ± 567 mmHg bpm, p < 0.001), but did not affect time to ischaemic contracture. Incubation of freshly isolated cardiomyocytes with 0.1 mM L-homocysteine significantly decreased the amplitude of the calcium transient and slowed the time to half relaxation. CONCLUSIONS: These findings suggest that homocysteine exposure affected myocardial recovery from ischaemia and contractile homeostasis although the exact mechanisms for these changes remain to be determined.

L-Glycyl-L-glutamine provides the isolated and perfused young and middle-aged rat heart protection against ischaemia-reperfusion injury.

The amino acids glycine and glutamine have been implicated in myocardial protection of the much studied young adult heart. This study aimed to determine whether such protection could be enhanced using the dipeptide, L-glycyl-L-glutamine (gly-gln) in both young hearts and in middle-aged hearts representative of a more clinically relevant age group. Hearts from 8-week-old and 36-week-old rats were perfused in the Langendorff mode for 20 min, before 40 min global normothermic ischaemia and 30 min reperfusion. Where present, 0.5, 2, or 5 mM gly-gln was added 10 min into baseline perfusion, was present throughout ischaemia and was washed out after 10 min reperfusion. Reperfusion damage was assessed from the release of lactate dehydrogenase. Metabolic fitness was assessed from the time to ischaemic contracture and the accumulation of lactate and thiobarbituric acid reactive substances during ischaemia. The presence of 5 mM gly-gln significantly improved the post-ischaemic rate pressure product (RPP) and decreased reperfusion damage in both the 8 (RPP in control on reperfusion 5527 ± 957 vs. 10,320 ± 795 mmHg beat min(-1) in 5 mM gly-gln, n = 6 ± SE, p < 0.05) and 36-week-old (RPP in control on reperfusion 1964.33 ± 876.3 vs. 4008 ± 675 mmHg beat min(-1), n = 6 ± SE, p < 0.01) hearts. Five mM gly-gln also increased the time to ischaemic contracture and was able to protect against the rise in lactate that occurred in the controls during ischaemia. These results suggest that gly-gln has good potential as a combatant against ischaemia-reperfusion injury in both the young adult and middle-aged populations.

Effects of ageing on metabolite and oxidant concentrations in different regions of rat kidney under normal and stress conditions.

Metabolic and oxidative stresses have been implicated in ageing and the pathogenesis of chronic kidney disease. In this study, we investigated the glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and lactate concentrations in different kidney regions under control conditions and after exposure to oxidative stress invoked by 0.2 mM H2O2. Slices of superficial cortex, outer or inner medulla were dissected from kidneys of male Wistar rats of 5-, 12-, 36- and 60-week old. Samples were incubated for 30 min ± 0.2 mM H2O2 prior to homogenisation and centrifugation. The concentrations of GSH, TBARS and lactate were measured by colorimetry. Each metabolite showed a distinctive pattern. For GSH, this was 12 weeks > 36 weeks > 60 weeks and 5 weeks with the highest concentration measured in the superficial cortex at 12 weeks. For TBARS and lactate, the pattern was for the lowest concentration at 12 weeks and the highest at 60 and 5 weeks. The highest lactate and TBARS concentrations were measured under oxidative stress conditions, particularly at 5 and 60 weeks. These results suggest that GSH in different kidney regions peaks at maturity and then reduces with increasing age.

Effect of ageing and oxidative stress on antioxidant enzyme activity in different regions of the rat kidney.

Oxidative stress has been implicated in ageing and the pathogenesis of chronic kidney disease. We examined levels of antioxidant enzymes glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and superoxide dismutase as modulated by age and oxidative stress in different regions of the kidney. Antioxidant enzymes were examined in different regions of the kidney in male Wistar rats. Kidneys from rats of different ages (5, 12, 36 and 60 weeks) were dissected into cortex, outer medulla and inner medulla. Tissues were incubated for 30 min with or without 0.2 mM H2O2 to induce oxidative stress. Antioxidant enzyme activities progressively decreased with age under both control and stress conditions (P < 0.05) after peaking at 12 weeks. Antioxidant enzyme activities were greater in the cortex (P < 0.05) by comparison with the outer and inner medulla, respectively.

Oxygenation response to positive end-expiratory pressure predicts mortality in acute respiratory distress syndrome. A secondary analysis of the LOVS and ExPress trials.

RATIONALE: Previous trials of higher positive end-expiratory pressure (PEEP) for acute respiratory distress syndrome (ARDS) failed to demonstrate mortality benefit, possibly because of differences in lung recruitability among patients with ARDS. OBJECTIVES: To determine whether the physiological response to increased PEEP is associated with mortality. METHODS: In a secondary analysis of the Lung Open Ventilation Study (LOVS, n = 983), we examined the relationship between the initial response to changes in PEEP after randomization and mortality. We sought to corroborate our findings using data from a different trial of higher PEEP (ExPress, n = 749). MEASUREMENTS AND MAIN RESULTS: The oxygenation response (change in ratio of arterial partial pressure of oxygen to fraction of inspired oxygen: P/F) after the initial change in PEEP after randomization varied widely (median, 9.5 mm Hg; interquartile range, -16 to 47) and was only weakly related to baseline P/F or the magnitude of PEEP change. Among patients in whom PEEP was increased after randomization, an increase in P/F was associated with reduced mortality (multivariable logistic regression; adjusted odds ratio, 0.80 [95% confidence interval, 0.72-0.89] per 25-mm Hg increase in P/F), particularly in patients with severe disease (baseline P/F [less-than-or-equal-to] 150 mm Hg). Changes in compliance and dead space were not associated with mortality. These findings were confirmed by a similar analysis of data from the ExPress trial. CONCLUSIONS: Patients with ARDS who respond to increased PEEP by improved oxygenation have a lower risk of death. The oxygenation response to PEEP might be used to predict whether patients will benefit from higher versus lower PEEP.

Increase in skeletal-muscle glycogenolysis and perceived exertion with progressive dehydration during cycling in hydrated men.

This study investigated the effects of progressive mild dehydration during cycling on whole-body substrate oxidation and skeletal-muscle metabolism in recreationally active men. Subjects (N = 9) cycled for 120 min at ~65% peak oxygen uptake (VO2peak 22.7 °C, 32% relative humidity) with water to replace sweat losses (HYD) or without fluid (DEH). Blood samples were taken at rest and every 20 min, and muscle biopsies were taken at rest and at 40, 80, and 120 min of exercise. Subjects lost 0.8%, 1.8%, and 2.7% body mass (BM) after 40, 80, and 120 min of cycling in the DEH trial while sweat loss was not significantly different between trials. Heart rate was greater in the DEH trial from 60 to 120 min, and core temperature was greater from 75 to 120 min. Rating of perceived exertion was higher in the DEH trial from 30 to 120 min. There were no differences in VO2, respiratory-exchange ratio, total carbohydrate (CHO) oxidation (HYD 312 ± 9 vs. DEH 307 ± 10 g), or sweat rate between trials. Blood lactate was significantly greater in the DEH trial from 20 to 120 min with no difference in plasma free fatty acids or epinephrine. Glycogenolysis was significantly greater (24%) over the entire DEH vs. HYD trial (433 ± 44 vs. 349 ± 27 mmol · kg-1 · dm-1). In conclusion, dehydration of <2% BM elevated physiological parameters and perceived exertion, as well as muscle glycogenolysis, during exercise without affecting whole-body CHO oxidation.

Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2.

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). Additional strategies to lower levels of ataxin-2 could be beneficial. Here, we perform a genome-wide arrayed small interfering RNA (siRNA) screen in human cells and identify RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a potent modifier of ataxin-2 levels. RTN4R knockdown, or treatment with a peptide inhibitor, is sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro, and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. We provide evidence that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

The Diversity of Volatile Compounds in Australia's Semi-Desert Genus Eremophila (Scrophulariaceae).

Australia's endemic desert shrubs are commonly aromatic, with chemically diverse terpenes and phenylpropanoids in their headspace profiles. Species from the genus Eremophila (Scrophulariaceae ex. Myoporaceae) are the most common, with 215 recognised taxa and many more that have not yet been described, widely spread across the arid parts of the Australian continent. Over the years, our research team has collected multiple specimens as part of a survey to investigate the chemical diversity of the genus and create leads for further scientific enquiry. In the current study, the diversity of volatile compounds is studied using hydrodistilled essential oils and leaf solvent extracts from 30 taxa. Several rare terpenes and iridoids were detected in chemical profiles widely across the genus, and three previously undescribed sesquiterpenes were isolated and are assigned by 2D NMR-E-11(12)-dehydroisodendrolasin, Z-11-hydroxyisodendrolasin and 10-hydroxydihydro-α-humulene acetate. Multiple sampling from Eremophila longifolia, Eremophila arbuscular, Eremophila latrobei, Eremophila deserti, Eremophila sturtii, Eremophila oppositifolia and Eremophila alternifolia coneys that species in Eremophila are highly chemovariable. However, taxa are generally grouped according to the expression of (1) furanosesquiterpenes, (2) iridoids or oxides, (3) mixtures of 1 and 2, (4) phenylpropanoids, (5) non-furanoid terpenes, (6) mixtures of 4 and 5, and less commonly (7) mixtures of 1 and 5. Furthermore, GC-MS analysis of solvent-extracted leaves taken from cultivated specimens conveys that many heavier 'volatiles' with lower vapour pressure are not detected in hydrodistilled essential oils and have therefore been neglected in past chemical studies. Hence, our data reiterate that chemical studies of the genus Eremophila will continue to describe new metabolites and that taxon determination has limited predictive value for the chemical composition.

Hypothalamic POMC deficiency increases circulating adiponectin despite obesity.

OBJECTIVE: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc-/-) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc-/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels. METHODS: Circulating adiponectin was measured in obese ArcPomc-/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc-/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc-/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc-/- mice. Finally, we generated compound Adiopoq-/-:ArcPomc-/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls. RESULTS: Obese ArcPomc-/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc-/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc-/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc-/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc-/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc-/- mice did not exacerbate their metabolic abnormalities. CONCLUSION: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc-/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Expression of a hypomorphic Pomc allele alters leptin dynamics during late pregnancy.

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

Prostanthera (Lamiaceae) as a 'Cradle of Incense': Chemophenetics of Rare Essential Oils from Both New and Forgotten Australian 'Mint Bush' Species.

The highly aromatic Australian mint bushes from the genus Prostanthera Labill. produce a high yield of essential oil on hydrodistillation. Together with its rich history, horticultural potential, iconic flowers, and aromatic leaves, it achieves high ornamental and culinary value. Species in the genus express highly diverse and chemically unique essential oils that demonstrate intra- and inter-specific patterns that have inspired taxonomic reinterpretation for over a hundred years. Previous studies have conveyed that phenoplastic expression of volatiles creates chemotypes within taxa, adding complexity to chemophenetic exploration. The current study chemically characterised essential oils from 64 highly aromatic specimens, representative of 25 taxa, giving yields as high as >2% g/g. The chemical profiles of essential oils are diverse, but generally include 1,8-cineole and signatory compounds such as sesquiterpene oxides, caryophyllene oxide, kessane and cis-dihydroagarofuran; sesquiterpene alcohols, globulol, epiglobulol, maaliol, prostantherol, spathulenol and ledol; and monoterpene derivatives of common scaffolds, borneol, bornyl acetate, bornanone, linalool and linalyl acetate. As in previous studies, analysis of chemical data confirms that the chemistry strongly agrees with taxonomic classifications. Importantly, as in classical taxonomy, the current chemical study complemented morphological analysis but conveys chemovariation, obscuring the taxonomic agreement. Nevertheless, variation within taxa may be due to environmental factors, meaning that cultivation of species in gardens will create different chemical profiles as compared to those published here.

Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network.

Hypothalamic POMC deficiency leads to obesity and metabolic deficiencies, largely due to the loss of melanocortin peptides. However, POMC neurons in the arcuate nucleus (ARC) are comprised of glutamatergic and GABAergic subpopulations. The developmental program, relative proportion and function of these two subpopulations are unresolved. To test whether glutamatergic POMC neurons serve a distinct role in maintaining energy homeostasis, we activated Pomc expression Cre- dependently in Vglut2-expressing neurons of mice with conditionally silenced Pomc alleles. The Vglut2-Pomc restored mice had normal ARC Pomc mRNA levels, POMC immunoreactivity, as well as body weight and body composition at age 12 weeks. Unexpectedly, the cumulative total of Vglut2+ glutamatergic- and Gad67+ GABAergic-Pomc neurons detected by in situ hybridization (ISH) exceeded 100% in both Vglut2- Pomc restored and control mice, indicating that a subpopulation of Pomc neurons must express both neuronal markers. Consistent with this hypothesis, triple ISH of C57BL/6J hypothalami revealed that 35% of ARC Pomc neurons were selectively Gad67+, 21% were selectively Vglut2+, and 38% expressed both Gad67 and Vglut2. The single Gad67+ and Vglut2+Pomc neurons were most prevalent in the rostral ARC, while the Vglut2/Gad67+ dual-phenotype cells predominated in the caudal ARC. A lineage trace using Ai9-tdTomato reporter mice to label fluorescently all Vglut2-expressing neurons showed equal numbers of tdTomato+ and tdTomato- POMC immunoreactive neurons. Together, these data suggest that POMC neurons exhibit developmental plasticity in their expression of glutamatergic and GABAergic markers, enabling re-establishment of normal energy homeostasis in the Vglut2-Pomc restored mice.

Hypothalamic POMC or MC4R deficiency impairs counterregulatory responses to hypoglycemia in mice.

OBJECTIVE: Life-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation. METHODS: To test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes. RESULTS: Stimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice. CONCLUSION: In conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes.

Keeping it simple: lessons from the golden era of antibiotic discovery.

Bacteria are becoming increasingly resistant to currently used antibiotics. At the same time, little progress has been made in discovering new antibacterial drugs to combat resistant organisms. History teaches us that 'high tech' target-based complex methods are not synonymous with success and a return to simple, systematic screening of natural products against bacteria from traditional and novel resources holds our greatest hope of success.

Reprogramming the body weight set point by a reciprocal interaction of hypothalamic leptin sensitivity and Pomc gene expression reverts extreme obesity.

OBJECTIVE: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice. METHODS: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc (-/-)) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc (-/-) mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc (-/-) mice to mimic the super-elevated leptin levels of obese mice. RESULTS: ArcPomc (-/-) mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc (-/-) mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc (-/-) mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc (-/-) mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc (-/-) mice. CONCLUSIONS: Pomc reactivation in previously obese, calorie-restricted ArcPomc (-/-) mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc (-/-) mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc (-/-) mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc (-/-) mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight.