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Mice lacking melanin-concentrating hormone receptor 1 demonstrate increased heart rate associated with altered autonomic activity. Am J Physiol Regul Integr Comp Physiol 287 R749-R758, 2004. First published May 6, 2004; 10.115/ajpregu.00134.2004.--Melanin-concentrating hormone (MCH) plays an important role in energy balance. The current studies were carried out on a new line of mice lacking the rodent MCH receptor (MCHR[1.sup.-/-] mice). These mice confirmed the previously reported lean phenotype characterized by increased energy expenditure and modestly increased caloric intake. Because MCH is expressed in the lateral hypothalamic area, which also has an important role in the regulation of the autonomic nervous system, heart rate and blood pressure were measured by a telemetric method to investigate whether the increased energy expenditure in these mice might be due to altered autonomic nervous system activity. Male MCHR[1.sup.-/-] mice demonstrated a significantly increased heart rate [24-h period: wild type 495 [+ or -] 4 vs. MCHR[1.sup.-/-] 561 [+ or -] 8 beats/min (P < 0.001/; dark phase: wild type 506 [+ or -] 8 vs. MCHR[1.sup.-/-] 582 [1.sup.-/-] 9 beats/min (P < 0.001); light phase: wild type 484 [+ or -] 13 vs. MCHR[1.sup.-/-] 539 [+ or -] 9 beats/min (P < 0.005)] with no significant difference in mean arterial pressure [wild type 110 [+ or -] 0.3 vs. MCHR[1.sup.-/-] 113 [+ or -] 0.4 mmHg (P > 0.05)]. Locomotor activity and cure body temperature were higher in the MCHR[1.sup.-/-] mice during the dark phase only and thus temporally dissociated from heart rate differences. On fasting, wild-type animals rapidly downregulated body temperature and heart rate. MCHR[1.sup.-/-] mice displayed a distinct delay in the onset of this downregulation. To investigate the mechanism underlying these differences, autonomic blockade experiments were carried out. Administration of the adrenergic antagonist metoprolol completely reversed the tachycardia seen in MCHR[1.sup.-/-] mice, suggesting an increased sympathetic tone. melanin-concentrating hormone: sympathetic; fasting

Similar 3'-processing activities of mitochondrially encoded tRNAs by mitochondrial endonucleases were characterized in transcripts of Saccharomyces cerevisiae nuclear tRNA genes. Artificially extended 3'trailer sequences from some tRNA substrates were processed and removed pertly by an endoculease which cleaved near or precisely at the 3'-end of the tRNA coding region.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the [Alpha] (NR1C1) and [Gamma] (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the [Delta] (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPAR[Delta] agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPAR[Delta] agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic ß-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant ß-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical. [ABSTRACT FROM AUTHOR]