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. 2020 Feb 17;10(2):321.
doi: 10.3390/biom10020321.

The Relevance of Thimet Oligopeptidase in the Regulation of Energy Metabolism and Diet-Induced Obesity

Mayara C F Gewehr  1 , Alexandre A S Teixeira  2 , Bruna A C Santos  1 , Luana A Biondo  2 , Fábio C Gozzo  3 , Amanda M Cordibello  3 , Rosangela A S Eichler  1 , Patrícia Reckziegel  4 , Renée N.O. Da Silva  1 , Nilton B Dos Santos  1 , Niels O S Camara  5 , Angela Castoldi  5 , Maria L M Barreto-Chaves  6 , Camila S Dale  6 , Nathalia Senger  6 , Joanna D C C Lima  2 , Marilia C L Seelaender  2 , Aline C Inada  1 , Eliana H Akamine  1 , Leandro M Castro  7 , Alice C Rodrigues  1 , José C Rosa Neto  2 , Emer S Ferro  1
Affiliations

The Relevance of Thimet Oligopeptidase in the Regulation of Energy Metabolism and Diet-Induced Obesity

Mayara C F Gewehr et al. Biomolecules. .

Abstract

Thimet oligopeptidase (EC 3.4.24.15; EP24.15; THOP1) is a potential therapeutic target, as it plays key biological functions in processing biologically functional peptides. The structural conformation of THOP1 provides a unique restriction regarding substrate size, in that it only hydrolyzes peptides (optimally, those ranging from eight to 12 amino acids) and not proteins. The proteasome activity of hydrolyzing proteins releases a large number of intracellular peptides, providing THOP1 substrates within cells. The present study aimed to investigate the possible function of THOP1 in the development of diet-induced obesity (DIO) and insulin resistance by utilizing a murine model of hyperlipidic DIO with both C57BL6 wild-type (WT) and THOP1 null (THOP1-/-) mice. After 24 weeks of being fed a hyperlipidic diet (HD), THOP1-/- and WT mice ingested similar chow and calories; however, the THOP1-/- mice gained 75% less body weight and showed neither insulin resistance nor non-alcoholic fatty liver steatosis when compared to WT mice. THOP1-/- mice had increased adrenergic-stimulated adipose tissue lipolysis as well as a balanced level of expression of genes and microRNAs associated with energy metabolism, adipogenesis, or inflammation. Altogether, these differences converge to a healthy phenotype of THOP1-/- fed a HD. The molecular mechanism that links THOP1 to energy metabolism is suggested herein to involve intracellular peptides, of which the relative levels were identified to change in the adipose tissue of WT and THOP1-/- mice. Intracellular peptides were observed by molecular modeling to interact with both pre-miR-143 and pre-miR-222, suggesting a possible novel regulatory mechanism for gene expression. Therefore, we successfully demonstrated the previously unanticipated relevance of THOP1 in energy metabolism regulation. It was suggested that intracellular peptides were responsible for mediating the phenotypic differences that are described herein by a yet unknown mechanism of action.

Keywords: diet-induced obesity; insulin resistance; mass spectrometry; obesity; peptidases; peptidome; proteases; proteasome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Weight and adipose tissue gain of animals during the 24 weeks of the diets. (A) females; (B–D) males. (A,B) show that wild-type (WT) animals fed a hyperlipidic diet (HD) began to weigh more than THOP1−/− mice after 18 (females) or 12 (males) weeks, depending on gender. After 24 weeks, the adipose tissue (fat) content was observed by X-ray density images (C,D). Results are expressed as mean ± standard error of the mean (SEM). Statistical analyses were conducted using two-way ANOVA followed by Tukey’s test (A,B) or Student’s unpaired t-test (D). One letter, p ≤ 0.05; two letters, p ≤ 0.01; three letters p ≤ 0.001. a, WT standard diet (SD) vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 5–9).
Figure 2
Figure 2
Blood glucose levels. Pre-prandial glucose levels of female (A) and male (B) mice, WT or THOP1−/−, fed either an SD or a HD, were evaluated every two weeks across the 24 weeks. Note that only WT male mice fed a HD showed an increase in the pre-prandial blood glucose levels across the 24 weeks. Results are expressed as mean ± SEM. Statistical analyses were conducted using Two-way ANOVA followed by Tukey’s test or Student’s unpaired t-test (n = 6–9).
Figure 3
Figure 3
Glucose and insulin tolerance tests. (A,C,E) females and (B,D,F) males. (A–D) show the glucose tolerance test (GTT). (E,F) show the insulin tolerance test (ITT). (C,D) show the area under the curve (AUC) for groups A (C) or B (D). Note that only WT male animals fed a HD for 24 weeks were insulin-resistant (F). Data are presented as mean ± SEM. Statistical analyses were conducted using Two-way ANOVA followed by Tukey’s test (A,B,E,F) or Student’s unpaired t-test (C,D). One letter, p ≤ 0.05; two letters, p ≤ 0.01; three letters p ≤ 0.001. a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 6–9).
Figure 4
Figure 4
Pyruvate tolerance test of 24-week-old WT or THOP1−/− mice fed an SD to evaluate their liver gluconeogenesis. (A,B) females; (C,D) males; (B,D) respectively, A and C area under the curve (AUC) for blood glucose curves. Colored curves (A,C) show blood glucose levels after pyruvate injection (2 g/kg sodium pyruvate after 16 h of fasting) at the indicated time point (x-axis). Blood glucose was measured using a glucose meter as detailed in the Methods section. Statistical analyses were performed using Student’s unpaired t-test. aa, p ≤ 0.01 (n = 6).
Figure 5
Figure 5
Resting energy metabolism of WT (black bars) or THOP1−/− (green bars) male mice across 24 h. (A–E) 24 h period (6 a.m.–6 a.m.); (F–J) 5–10 p.m. period; (A,F) spontaneous locomotor activity; (B,G) heat production; (C,H) VCO2 (mL/kg/h) production; (D,I) VO2 (mL/kg/h) consumption; (E,J) respiratory exchange ratios (RERs). The results are shown for mice previously fed with either an SD or a HD for 24 weeks. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s unpaired t-test. One letter, p ≤ 0.05; two letters, p ≤ 0.01; three letters p ≤ 0.001. a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 4). y-axis: bars represent the area under the curve (AUC).
Figure 6
Figure 6
Plasma corticosterone levels of WT and THOP1−/− mice. (A) plasma corticosterone levels (µg/dL) at 7 a.m.; (B) plasma corticosterone levels (µg/dL) at 7 p.m. Results are expressed as mean ± SEM. Statistical analyses were performed using Student’s unpaired t-test. a, p ≤ 0.05 between WT and THOP1−/− mice (n = 4–5).
Figure 7
Figure 7
Food restriction experiments. Twelve-week-old WT and THOP1−/− animals were fed with 1.5 g/day of standard diet (SD; corresponding to 40% of the regular fed for these animals). During four days, once a day, these animals received by gavage either saline or propranolol (10 mg∙kg−1/day). (A) blood glucose variation; (B) area under curve (AUC) of blood glucose variation; (C) animal weight variation; and, (D) AUC of animal weight variation. Results are expressed as mean ± SEM. Statistical analyses were performed using Student’s unpaired t-test. One letter p ≤ 0.05; between: a, WT vs. THOP1−/− WT/HD; b, THOP1−/− vs. THOP1−/− + propranolol (n = 7–10).
Figure 8
Figure 8
Isoproterenol-stimulated lipolytic activity and mRNA expression levels of β-adrenergic receptors 1, 2, or 3 (β1AR, β2AR, and β3AR, respectively). (A) Inguinal adipose tissue was removed, weighed, divided in two pieces, and placed in Krebs buffer, pH 7.2, at 37 °C for 10 min., in the absence (Basal) or presence of isoproterenol (0.10 ng/g; ISO). Lipolytic activity was measured as the amount of glycerol produced, using the “free glycerol reagent” kit (Sigma, MO, USA). Glycerol content (µg/mg) was normalized by tissue weight. Results are expressed as mean ± SEM. (B) mRNA expression levels of adrenergic receptors β1AR, β2AR, and β3AR. Analyses of gene expression were performed by Quantitative Real-Time PCR (qRT-PCR) in female or male retroperitoneal adipose tissue. Data are presented as mean ± standard deviation. Statistical analyses were performed using Student’s unpaired t-test. One letter, p ≤ 0.05; two letters, p ≤ 0.01; three letters p ≤ 0.001. a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 4–6).
Figure 9
Figure 9
Progressive treadmill exercise test. The cardiorespiratory fitness of the animals fed with a standard diet (SD) was evaluated using a graded treadmill. (A,C,E,G) running distance (m); (B,D,F,H) running time (min.). Both running distance and time were evaluated in the high- and low-intensity tests. (A–D) females; (E–F) males. Data are presented as mean ± SEM. Statistical analyses were performed while using Student’s unpaired t-test. One letter, p ≤ 0.05; three letters p ≤ 0.001 (n = 7–9).
Figure 10
Figure 10
Liver slices from WT and THOP1−/− mice stained with periodic acid–Schiff (PAS). WT or THOP1−/− male mice were fed for 24 weeks with either an SD (WT/SD, THOP1−/−/SD) or a HD (WT/HD, THOP1−/−/HD). Note the higher intensity of PAS reaction in THOP1−/−/SD when compared to the other groups; this is an indicative of higher glycogen content in liver of THOP1−/− mice fed an SD. Panels from top to bottom present increased magnifications of 40- (top), 100- (middle), or 200-fold (bottom).
Figure 11
Figure 11
Liver slices from WT and THOP1−/− mice stained with hematoxylin and eosin (HE). WT or THOP1−/− male mice were fed for 24 weeks with either an SD (WT/SD, Table 1. or a HD (WT/HD, THOP1−/−/HD). Note the large number of lipid droplets (white spots) in the liver slices from WT mice fed a HD (WT/HD panels), suggesting the presence of NAFLS. Panels present increased magnifications from 40- (top), 100- (middle) or 200-fold (bottom). Bars, 100 μm.
Figure 12
Figure 12
Dicer mRNA and microRNA expression levels in male liver tissue. (A) dicer mRNA expression levels. (B) Effect of phenotype on the expression levels of the indicated mature microRNAs; * p ≤ 0.05 comparing WT and THOP1−/− mice. (C) Effect of diet and phenotype on the expression levels of the indicated mature microRNAs. (D) Effect of diet and phenotype on the expression level of pri-miR-222. Results are expressed as mean ± SEM. Statistical analyses were performed using Student’s unpaired t-test. One letter, p ≤ 0.05; a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 4–6).
Figure 13
Figure 13
Dicer mRNA and microRNA expression levels in male retroperitoneal adipose tissue. (A) Dicer mRNA expression levels. (B) Effect of phenotype on the expression levels of the indicated microRNAs; * p ≤ 0.05 when comparing WT and THOP1−/− mice. (C) Effect of diet and phenotype on the expression levels of the indicated microRNAs. (D) The effect of diet and phenotype on the expression level of pri-miR-222. Results are expressed as mean ± SEM. Statistical analyses were performed using Student’s unpaired t-test. One letter, p ≤ 0.05; a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 4–6).
Figure 14
Figure 14
Gene expression of peptidases and proteasome beta5-subunit (Protβ5) in adipose tissue from WT and THOP1−/− mice. A–F, female; G–L, male. Analyses of gene expression were conducted by qRT-PCR in female and male retroperitoneal adipose tissue for specific peptidases, dipeptidyl peptidase 4 (DPP4), neprilysin (NEP), insulin-degrading enzyme (IDE), angiotensin converting enzyme 1 (ACE1), prolyl-oligopeptidase (POP), and Protβ5. Data are presented as mean ± standard deviation. Statistical analyses were performed using Student’s unpaired t-test. One letter, p ≤ 0.05; two letters, p ≤ 0.01; three letters p ≤ 0.001. a, WT/SD vs. WT/HD; b, THOP1−/−/SD vs. THOP1−/−/HD; c, WT/SD vs. THOP1−/−/SD; d, WT/HD vs. THOP1−/−/HD (n = 4–6).
Figure 15
Figure 15
Structural modeling of murine May peptides interacting with either miR-143 or miR-222. Each panel contains seven small Figures that represent, from left to right, pre-miR-143 (A) or pre-miR-222 (B) interacting with the indicated intracellular peptide (May1–May7). The top left histogram panel from each small Figure represents the different densities/theoretical affinities (shown on the y-axis) of the top 10 structures predicted for each indicated May peptide. Shown on the x-axis of the histograms are the nucleotide positions from each microRNA that interacted with the predicted structures of May peptides. Note that intracellular peptides were predicted to interact with different regions and with different affinities along the nucleotide sequences of the respective pre-microRNAs. The top right panel on each small Figure shows all of the top ten predicted peptide structures interacting with their respective pre-microRNAs. The lower larger panel shows only one predicted structure of the corresponding May peptide, which was predicted to interact with the highest affinity to the indicated region of the respective pre-microRNA. In these large panels, inside the small blue boxes, are the nucleotides from the mature microRNAs predicted to interact with the peptide of the highest affinity. Note that intracellular peptides May1–May7 frequently interacted with a large portion (both at 5p and 3p) of the mature region of these miRNAs.
Figure 15
Figure 15
Structural modeling of murine May peptides interacting with either miR-143 or miR-222. Each panel contains seven small Figures that represent, from left to right, pre-miR-143 (A) or pre-miR-222 (B) interacting with the indicated intracellular peptide (May1–May7). The top left histogram panel from each small Figure represents the different densities/theoretical affinities (shown on the y-axis) of the top 10 structures predicted for each indicated May peptide. Shown on the x-axis of the histograms are the nucleotide positions from each microRNA that interacted with the predicted structures of May peptides. Note that intracellular peptides were predicted to interact with different regions and with different affinities along the nucleotide sequences of the respective pre-microRNAs. The top right panel on each small Figure shows all of the top ten predicted peptide structures interacting with their respective pre-microRNAs. The lower larger panel shows only one predicted structure of the corresponding May peptide, which was predicted to interact with the highest affinity to the indicated region of the respective pre-microRNA. In these large panels, inside the small blue boxes, are the nucleotides from the mature microRNAs predicted to interact with the peptide of the highest affinity. Note that intracellular peptides May1–May7 frequently interacted with a large portion (both at 5p and 3p) of the mature region of these miRNAs.
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