Proteasomes play a key role in living organisms in maintaining optimal protein levels and generating functional intracellular peptides. The catalytic activity of proteasomes is present in archaebacteria, whereas the well-known antigen-generating function of proteasomes emerges only in jawed vertebrates. Therefore, proteasomes may have evolved from a pure degradation function in prokaryotes to a protein-processing function in eukaryotes. Intracellular peptides generated by proteasomes that are not related to antigens have been shown to be functional inside and outside cells. Hemopressin (HP, PVNFKFLSH) was the first functional intracellular peptide shown to act outside cells as an inverse agonist of cannabinoid type 1 receptors (CB1R). N-terminal extended versions of HP, named RVD-HP and VD-HP, were later shown to occur naturally in rodent brains and to have agonist activity at CB1R. Similar to HP, one peptide (DITADDEPLT; corresponding to residues 9–17 of peptidyl-prolyl cis-trans isomerase A) screened in this study with conformational-sensitive antibodies so as to identify novel pharmacologically active peptides, exhibited inverse agonistic activity at CB1R. This original DITADDEPLT sequence was rationally modified to improve CB1R inverse agonist activity, resulting in the peptide sequence DIIADDEPLT (hereafter named “Pep19”). Pep19 displaces [3 H]SR141716A binding to membranes of 3T3-L1 cells expressing CB1R with an IC 50 ~4.9 × 10−12 M. In this study we also examined the effect of Pep19 on signaling pathways of 3T3-L1 differentiated adipocytes and on body-weight and metabolic parameters of diet induced obese (DIO) rats. We find that Pep19 ameliorates several metabolic parameters of DIO rats, without detectable central nervous system (CNS) action. The mechanism of action of Pep19 seems to include induction of uncoupling protein 1 (UCP1) expression in adipocytes.
Oral administration of Pep19 to DIO Wistar rats improved several metabolic parameters, including a reduction in serum glucose, triacylglycerol and blood pressure, without changing heart rate. Pep19 reduced the whole adiposity index and the mass of gonadal and mesenteric adipose tissues. Oral administration of Pep19 significantly increased the expression of UCP1 in specific cells of the inguinal adipose tissue, although this was not seen when the overall expression of UCP1 was evaluated by Western blotting; these results can be explained by the fact that the difference in UCP1 expression is quite small, and this could contribute to the lack in overall change in signal detected by Western blotting. Next, we analyzed the inguinal adipose tissue of DIO rats treated with Pep19 for changes in the number and size of adipocytes. We find an increase in the number of adipocytes with a significant decrease in their size.
Quantitative analyses in inguinal adipose tissue of DIO rats. Panels a–d: Immunohistochemical representative images (400x magnification, scale bar = 50 µm) showing specific UCP1-immunostained cells from the inguinal white adipose tissue of DIO Wistar rats (oral treatments: panel a, saline; panel b, Pep19, 100 μg/Kg; panel c, Pep19, 300 μg/Kg; panel d, Pep19, 600 μg/Kg). Quantitative analyses of UCP1 labeled cells were determined from at least 25 different fields in each slice (n = 8–10), and examined using ImageJ software at 400x magnification; results are expressed as number of UCP1 positive cells per mm 2. Representative UCP1-immunostained cells (fast red dye) obtained from each treatment are indicated by arrows. Panel e: Quantitative analyses suggest that oral administration of Pep19 600 μg/Kg increases the number of UCP1-immunostained cells in the inguinal adipose tissue compared to saline or other treatments (n = 8–10; *p < 0.05, saline vs Pep19 600 μg/Kg). Note that in Pep19 treated animals (panels, c and d) immunostained-UCP1 cells seem to occur in fibrotic-like areas of the adipose tissue. Panel f: Quantitative Western blot analysis suggested similar levels of UCP1 expression in either saline or Pep19 (600 μg/Kg) treated groups of DIO animals; (each line shown on the upper panel f, is representative of one individual animal treated with either saline or Pep19 600 μg/Kg; n = 7). Note that the ratio of UCP1 negative cells to positive cells is fairly large and this could contribute to lack in change in the protein levels seen in the Western Blots (panel f). Panel g: Quantitative analyses of the number of adipocytes (adipocytes/µm 2; 500–800 cells were counted per animal (n = 3), in 14–18 different fields of the H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.05). Panel h: Quantitative analyses of the adipocyte area were performed measuring 15 different adipocytes in each of the 6 fields analyzed per H&E stained slices from each animal (n = 3), using H&E stained slices of adipose tissue from animals treated with either saline or Pep19 (600 μg/Kg; *p < 0.01). All results are expressed as the means ± standard error of the mean (SEM). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test using GraphPad Prism software. Crude Western blot membranes are shown on Supplemental material.
The pharmacological effects of Pep19 on UCP1 protein expression were further investigated in 3T3-L1 adipose cells, to evaluate if Pep19 could be regulating energy metabolism as previously described for endocannabinoids. 3T3-L1 is a well-characterized cell model for studying the adipose tissue metabolism. Rosiglitazone (RSG), used as a positive control for UCP1 activation, is an agonist for peroxisome proliferator-activated receptor-γ (PPAR-γ), a nuclear receptor highly expressed in brown and white adipose tissues that acts as a master transcriptional regulator of brown adipocyte differentiation required for tissue development, function and survival. Pep19 and HP similar to RSG increased the expression of UCP1 after 24 h treatment in 3T3-L1 adipose cells. The CB1R antagonist AM251, but not the agonist WIN55,212–2, prevented UCP1 activation by Pep19. Pep19 was observed to transiently activate pERK1/2 and AKT phosphorylation in 3T3-L1 cells. These data suggest that Pep19 could trigger UCP1 increased expression through activation of AKT and pERK1/2 pathways, which are known to induce the expression of PPARγ coactivator 1α.
