PYY3-36 as an anti-obesity drug target.

The neuropeptide Y (NPY)/peptide YY (PYY) system has been implicated in the physiology of obesity for several decades. More recently ignited enormous interest in PYY3-36, an endogenous Y2-receptor agonist, as a promising anti-obesity compound. Despite this interest, there have been remarkably few subsequent reports reproducing or extending the initial findings, while at the same time studies finding no anti-obesity effects have surfaced. Out of 41 different rodent studies conducted (in 16 independent labs worldwide), 33 (83%) were unable to reproduce the reported effects and obtained no change or sometimes increased food intake, despite use of the same experimental conditions (i.e. adaptation protocols, routes of drug administration and doses, rodent strains, diets, drug vendors, light cycles, room temperatures). Among studies by authors in the original study, procedural caveats are reported under which positive effects may be obtained. Currently, data speak against a sustained decrease in food intake, body fat, or body weight gain following PYY3-36 administration and make the previously suggested role of the hypothalamic melanocortin system unlikely as is the existence of PYY deficiency in human obesity. We review the studies that are in the public domain which support or challenge PYY3-36 as a potential anti-obesity target.

Physiology: does gut hormone PYY3-36 decrease food intake in rodents?

Batterham et al. report that the gut peptide hormone PYY3-36 decreases food intake and body-weight gain in rodents, a discovery that has been heralded as potentially offering a new therapy for obesity. However, we have been unable to replicate their results. Although the reasons for this discrepancy remain undetermined, an effective anti-obesity drug ultimately must produce its effects across a range of situations. The fact that the findings of Batterham et al. cannot easily be replicated calls into question the potential value of an anti-obesity approach that is based on administration of PYY3-36.

Presence of a plasma membrane targeting sequence in the amino-terminal region of the rat somatostatin receptor 3.

Although peptide hormone receptors commonly exert their actions at the plasma membrane the cellular mechanisms that route the receptor proteins to the cell surface during biosynthesis are not well characterized. Here we report on the identification of a plasma membrane targeting sequence of rat somatostatin receptor subtype 3. While type 3 somatostatin receptors are present almost exclusively at the cell surface, type 1 receptors localize in addition largely in intracellular vesicular compartments. Chimeric receptors were constructed between rat somatostatin receptors 3 and 1. They were tagged by recombinant DNA techniques with a herpes simplex virus glycoprotein D epitope at the carboxyl-termini to facilitate their detection using fluorescence microscopic methods. Following transfection of the constructs in human embryonic kidney and rat insulinoma cells the chimeric receptors were analyzed by indirect immunofluorescence using anti-epitope monoclonal antibody and confocal laser scanning microscopy. The results demonstrate that the amino-terminal domain of somatostatin receptor 3 suffices to guide chimeric receptors to the cell surface. In marked contrast, chimeric receptors that lack this sequence but contain instead the amino-terminus of somatostatin type 1 receptor localize in an intracellular vesicular compartment.

Agonist-mediated endocytosis of rat somatostatin receptor subtype 3 involves beta-arrestin and clathrin coated vesicles.

Agonist-induced endocytosis of somatostatin receptors determines subsequent cellular responsiveness to peptide agonist and influences somatostatin receptor scintigraphy, a technique to image various tumours. We examined the internalization of sst3HSV, an epitope-tagged type 3 somatostatin receptor, in transfected rat neuroendocrine insulinoma cells. Stimulation of these cells with somatostatin induced trafficking of coexpressed enhanced green fluorescence protein/beta-arrestin1 fusion protein and sst3HSV to colocalize in the same endocytic vesicles. Coexpression of a dominant negative mutant of the arrestin fusion protein with the receptor blocked the internalization of sst3HSV. Stimulation with somatostatin also induced the transient translocation of alpha-adaptin, a component of the adaptor protein complex 2, to the plasma membrane. alpha-adaptin and clathrin colocalized with the receptor. By electron microscopy, we observed internalized sst3 in clathrin coated pits, endosomes and at the limiting membrane of multivesicular bodies, a location typical for receptors being recycled. Concordantly, we observed sst3HSV colocalized with Rab11 in a perinuclear compartment which is likely to correspond to the pericentriolar recycling endosome. Thus, agonist-induced endocytosis of sst3 depends on its interaction with beta-arrestin, involves the adaptor protein complex 2 and proceeds via clathrin coated vesicles to the recycling compartment.

Cellular and subcellular localization of gastrointestinal glutathione peroxidase in normal and malignant human intestinal tissue.

The gastrointestinal glutathione peroxidase (GI-GPx) is believed to prevent absorption of hydroperoxides. GI-GPx is expressed in the intestine together with the other three glutathione peroxidase isoenzymes, raising the question of the physiological role of the different GPx types. We therefore studied the cellular and subcellular distribution of GI-GPx in normal and malignant tissue obtained from patients with colorectal cancer or familial polyposis by immunohistochemistry. In healthy ileum epithelium GI-GPx was preferentially enriched in Paneth cells. In unaffected crypts of colon and rectum, it decreased gradually from the ground to the luminal surface. In crypt ground, GI-GPx was uniformly distributed, whereas in cells at the luminal surface it was concentrated in structures capping the nuclei at the apical pole. In colorectal cancer, GI-GPx expression depended on the stage of malignant transformation. In early stages, GI-GPx was increased and pronouncedly associated with the vesicular structures. In progressed stages of malignancy, structures disintegrated and GI-GPx distribution became more diffuse. These observations support the hypothesis that GI-GPx, apart from being a barrier against hydroperoxide absorption, might be involved in cell growth and differentiation.

Translocation of the interleukin-1 receptor-associated kinase-1 (IRAK-1) into the nucleus.

Interleukin-1 (IL-1) signal transduction involves the recruitment of the IL-1 receptor-associated kinase-1 (IRAK-1). Subsequent signaling finally leads to nuclear translocation of NFkappaB. We here show that the association and autophosphorylation of IRAK-1 was already detectable 30 s after IL-1 stimulation of ECV 304 cells. Significant levels of IRAK-1 accumulated in the nucleus 30 min after IL-1 stimulation shown by Western blot analysis and confocal laser scanning microscopy. Nuclear transfer of IRAK-1 upon IL-1 stimulation was confirmed in the murine T cell line EL-4. This characterizes nuclear localization of IRAK-1 as a possibly essential event in the IL-1 signaling cascade.

Cloning, expression, pharmacology and tissue distribution of the mouse somatostatin receptor subtype 5.

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.