N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes.

Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes.

GPR55 promotes migration and adhesion of colon cancer cells indicating a role in metastasis.

BACKGROUND AND PURPOSE: Tumour cell migration and adhesion constitute essential features of metastasis. G-protein coupled receptor 55 (GPR55), a lysophospholipid receptor, has been shown to play an important role in carcinogenesis. Here, we investigated the involvement of GPR55 in migration and metastasis of colon cancer cells. EXPERIMENTAL APPROACH: Adhesion and migration assays using the highly metastatic colon cancer cell line HCT116 and an in vivo assay of liver metastasis were performed. The GPR55 antagonist CID16020046, cannabidiol, a putative GPR55 antagonist and GPR55 siRNA were used to block GPR55 activity in HCT116 colon cancer cells. KEY RESULTS: HCT116 cells showed a significant decrease in adhesion to endothelial cells and in migration after blockade with CID16020046 or cannabidiol. The inhibitory effects of CID16020046 or cannabidiol were averted by GPR55 siRNA knock down in cancer cells. The integrity of endothelial cell monolayers was increased after pretreatment of HCT116 cells with the antagonists or after GPR55 siRNA knockdown while pretreatment with lysophosphatidylinositol (LPI), the endogenous ligand of GPR55, decreased integrity of the monolayers. LPI also induced migration in GPR55 overexpressing HCT116 cells that was blocked by GPR55 antagonists. In a mouse model of metastasis, the arrest of HCT116 cancer cells in the liver was reduced after treatment with CID16020046 or cannabidiol. Increased levels of LPI (18:0) were found in colon cancer patients when compared with healthy individuals. CONCLUSIONS AND IMPLICATIONS: GPR55 is involved in the migratory behaviour of colon carcinoma cells and may serve as a pharmacological target for the prevention of metastasis. © 2015 The British Pharmacological Society.

Clinical applicability of dOFM devices for dermal sampling.

BACKGROUND: Sampling the dermal interstitial fluid (ISF) allows the pharmacokinetics and pharmacodynamics of dermatological drugs to be studied directly at their site of action. Dermal open-flow microperfusion (dOFM) is a recently developed technique that can provide minimally invasive, continuous, membrane-free (thus unfiltered) access to the dermal ISF. Herein, we evaluate the clinical applicability and reliability of novel wearable dOFM devices in a clinical setting. METHODS: Physicians inserted 141 membrane-free dOFM probes into the dermis of 17 healthy and psoriatic volunteers and sampled dermal ISF for 25 h by using wearable push-pull pumps. The tolerability, applicability, reproducibility, and reliability of multiple insertions and 25 h continuous sampling was assessed by pain scoring, physician feedback, ultrasound probe depth measurements, and 25 h-drift and variability of the sodium relative recovery. RESULTS: Insertion pain was moderate and decreased with each additional probe. Probe insertion was precise, although slightly deeper in lesional skin. The wearable push-pull pump enabled uninterrupted ISF sampling over 25 h with low variability. The relative recovery was drift-free and highly reproducible. CONCLUSION: dOFM sampling devices are tolerable and reliable for prolonged continuous dermal sampling in a multiprobe clinical setting. These devices should enable the study of a wide range of drugs and their biomarkers in the skin.

The propeptide of yeast cathepsin D inhibits programmed necrosis.

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.

Insulin glulisine, insulin lispro and regular human insulin show comparable end-organ metabolic effects: an exploratory study.

AIMS: To compare the end-organ metabolic effects of insulin glulisine (glulisine), insulin lispro (lispro) and regular human insulin (RHI) in patients with type 1 diabetes mellitus. METHODS: Eighteen patients with type 1 diabetes mellitus (mean age 36.9 +/- 8.6 years, BMI 23.6 +/- 2.8 kg/m(2), haemoglobin A(1c) 7.4 +/- 0.9%) were randomized in this single-centre, double-blind, three-period cross-over, standard Latin-square, euglycaemic glucose clamp trial. Patients received sequential, primed stepwise intravenous infusions of glulisine, lispro or RHI (infusion rates were increased in a stepwise manner from an initial rate of 0.33 [180 min] to 0.66 [180 min] and 1.00 [180 min] mU/kg/min). The primary variables were the suppression of endogenous glucose production (S(EGP)) and glucose uptake (GU). RESULTS: Mean basal endogenous glucose production (EGP) was 1.88, 2.12 and 2.12 mg/kg/min for glulisine, lispro and RHI respectively. Mean (+/-s.e.) maximum absolute S(EGP) (adjusted for basal EGP) was -1.64 +/- 0.06, -1.72 +/- 0.05 and -1.56 +/- 0.05 mg/kg/min respectively. Mean (+/-s.e.) maximum absolute increase in GU (adjusted for basal GU) was 6.46 +/- 0.26, 6.23 +/- 0.24 and 6.72 +/- 0.24 mg/kg/min respectively. There were no clinically relevant differences between the three insulin treatments with respect to serum insulin, free fatty acid (FFA), glycerol or lactate levels. No serious adverse events and no episodes of severe hypoglycaemia were reported. CONCLUSIONS: This study shows that glulisine, lispro and RHI have similar effects on S(EGP), GU, FFA, glycerol and lactate levels, providing evidence for similar end-organ metabolic effects.

Proportional dose-response relationship and lower within-patient variability of insulin detemir and NPH insulin in subjects with type 1 diabetes mellitus.

AIMS: This study was conducted to evaluate the dose ratio of insulin detemir and neutral protamine Hagedorn (NPH) insulin over a range of therapeutically relevant subcutaneous doses. METHODS: The study was a randomized, double-blind, crossover 24-h-iso-glycemic clamp trial in 12 C-peptide-negative type 1 diabetic patients. Each subject received, by an incomplete block design selection, two of three possible doses of insulin detemir (0.15, 0.3, 0.6 U/kg) and NPH insulin (0.15, 0.3, 0.6 IU/kg), respectively. A detailed assessment of endogenous glucose production (EGP) and glucose uptake was performed, by use of stable isotopic labeled glucose tracer (D-[6,6- (2)H (2)] glucose). RESULTS: Dose proportionality was observed within the tested dose range. Regarding unit dose ratio, 0.68 U insulin detemir equals 1 IU NPH insulin (95% CI [0.35; 1.30]). There was no statistically significant difference in effect on the area under the curve (AUC) of glucose infusion rate (GIR) (AUC (GIR)) and the maximal GIR (GIR (max)) values, when comparing U (insulin detemir) to IU (NPH insulin). The pharmacodynamic within-subject profile was lower with insulin detemir in regard to AUC (GIR 0-24 h), GIR (max) and duration of action ( P<0.05). There was a tendency for a greater reduction of EGP with insulin detemir than with NPH insulin in regard to the area over the curve (AOC) of EGP in 24 hours (AOC (EGP 0-24 h)) ( P=0.07) and minimal EPG (EGP (min)) ( P=0.02). CONCLUSIONS: These data show that insulin detemir is dose-proportional to NPH insulin in type 1 diabetic patients at clinically relevant doses. The data indicate that insulin detemir has a lower degree of within-subject variability.

In vivo deleterious effects of a right shift of the HbO2 curve during hypoxemia.

We investigated the influence of a right shift of the oxyhemoglobin dissociation curve on tissue oxygenation in two groups of anesthetized rabbits subjected to short periods of graded hypoxia: Group 1 (n = 5) with elevated P50 due to increased RBC 2,3-diphosphoglycerate and adenosine triphosphate and Group 2 (n = 5) with normal P50. Hemoglobin fell progressively in all animals due to blood letting for necessary measurements. During 16% inspired O2 (FIO2), both groups remained stable. During 13% FIO2, arterial pO2 was the same in both groups, but only in Group I did it fall below the crossover point (C.O.P.), which was raised by the high P50. Arterial pH and arterial-venous O2 content difference remained within the normal range in both groups throughout the experiment. During 13% FIO2, animals with high P50 showed a fall in cardiac output and oxygen consumption while animals with normal P50 remained stable. We postulate that when systemic O2 content is sufficiently reduced and tissue O2 extraction is maximal, the O2 needs of the myocardium perfused with a pO2 below the C.O.P. cannot be met: under these conditions cardiac output and systemic O2 consumption fall, presumably due to a reduction in coronary blood flow.

Oxygen affinity of hemoglobin and peripheral nerve degeneration in experimental diabetes.

Peripheral neuropathy remains a major complication of diabetes. Numerous etiological theories of metabolic and/or vascular disturbances have been suggested including decreased endoneurial oxygen tension with presumed tissue hypoxia. Increases in the affinity of hemoglobin for oxygen (Hb-O2 affinity) may also produce tissue hypoxia and such Hb-O2 affinity changes have been implicated in the pathogenesis of diabetic microangiopathy. In order to test whether affinity hypoxia might contribute to the development of diabetic peripheral neuropathy, we have utilized a rat model of high and normal Hb-O2 affinity produced by backcrossing animals with increased and decreased levels of 2,3-diphosphoglycerate (DPG). Diabetes was induced in ten high and ten low DPG animals with a tail vein injection of 55 mg/kg streptozotocin (STZ). Five animals in each group were treated with 2.4 U protamine zinc insulin (PZI)/day while the remaining animals were untreated. All rats were killed after 30 days, sections of tibial and sural nerve were rapidly removed and processed for teased fiber analysis. A minimum of 125 axons were assessed per nerve for E degeneration (myelin ovoids) using the classification developed by Dyck et al. Untreated animals, regardless of DPG levels, demonstrated 0% neuropathy. In contrast, all insulin-treated animals showed degeneration (0.4-17%) that inversely correlated with the DPG level (r = -0.59, P less than 0.04). The results of this study suggest that the level of RBC DPG (and presumably the Hb-O2 affinity) with its attendant effect on tissue oxygen release may play a role in the development of peripheral neuropathy in STZ-induced diabetic rats treated with insulin.

Spatial relationships between fibroblasts during the growth of rat-tail tendon.

Sections of tendons from the base of the tail of rats were taken at eight time intervals from 18 days in utero until 244 days after birth and were examined in the electron microscope. For each time period, measurements were made of the relative area of fibroblasts, collagen and interstitial material, of the number of fibroblasts per unit area of tendon and of the average area of individual fibroblasts. The spatial arrangement of fibroblasts in the tendon sections was described quantitatively using the "nearest neighbor" method. Initially there was a rapid increase in the area of collagen accompanied by a decrease in the area occupied by fibroblasts but after 104 days of age these values changed very little. The numbers of fibroblasts per unit area decreased steadily from the embryo until 104 days whereas the average size of each cell increased to reach a maximum area at 40 days of age and then declined. At all time intervals cells were arranged in a regular, dispersed pattern across the tendon fascicles. Growth in width of the rat tail appears to involve the secretion of collagen and other intercellular material symmetrically around each fibroblast, so as to gradually separate the cells until a stage is reached at which cells are sufficiently far apart that there is little contact between adjacent cell processes. This may interfere with the integration of metabolic activity in the tissue. As a consequence, there is shrinkage of the cell bodies and a reduction in secretory activity so that, between 55 and 104 days of age, the tendon enters a period of terminal senescence.