Bioinformatic analysis
To uncover plausible molecular mechanisms responsible for the observed
impact of GLP-1R agonists treatment on urinary peptides in T2DM
patients, the proteases potentially responsible for cleavage of the 70
statistically significant peptides were investigated using Proteasix. In
total, 10 endopeptidases were retrieved as a result of the default
search with the “Observed Prediction tool” of Proteasix, putatively
responsible for cleaving 38 urinary peptides (36 downregulated and 2
upregulated) out of the 70 urinary peptides. The results are provided in
Table S2. Most of the predicted endopeptidases belonged to the matrix
metalloproteinase (MMP) family of proteases (7 out of 10 proteases),
responsible for cleaving peptides at both the N’ and C’ terminals.
Further proteases predicted as potentially responsible for cleaving the
N’ terminal belonged to the cathepsin family (CTSL and CTSD) while that
cleaving at the C’ terminus was A disintegrin and metalloproteinase with
thrombospondin motifs 5 (ADAMTS5). Notably, the proteases MMP2, MMP9 and
MMP13 were mapped to at least 6 cleavage sites each.
The protein-protein interactome was constructed using 26 parental
proteins identified from 70 urinary GLP-1R agonist-associated peptides,
using the STRING database. The network consisted of 27 nodes and 113
edges, as depicted in Figure 2I. The protein-protein interaction
enrichment yielded a significant p-value <
1.0e–16. While, most of the collagen proteins can be
observed to interact with all the other collagen proteins, interestingly
none of these interacted with the non-collagen proteins. Furthermore, a
protein-protein interaction network within the non-collagen proteins
could also be observed, indicating their involvement in the
pathophysiology of T2DM. Within this network, a total of 9 KEGG pathways
were predicted to be significantly enriched, including pathways related
to protein digestion and absorption, ECM-receptor interaction, AGE-RAGE
signaling pathway in diabetic complications, amoebiasis, relaxin
signaling, focal adhesion, human papillomavirus infection, PI3K-Akt
signaling pathway, small cell lung cancer and platelet activation,
detailed results are provided in Table S3.
Discussion
In the last decade, GLP-1R agonists have been the recommended and
preferred second line treatment for T2DM patients. Despite the various
advantages of GLP-1R agonists over other anti-hyperglycemic drugs, the
underlying molecular mechanisms of treatment with GLP-1R agonists have
not been studied in-depth. Aiming to understand the effect of GLP-1R
agonist treatment on T2DM patients, the urinary peptidome of thirty-two
T2DM patients were analyzed for the first time in this study with CE-MS.
The untargeted peptidomic analysis coupled with statistical tools
identified 70 statistically significant (adjusted for multiple testing)
urinary peptide fragments in abundance between the pre- and
post-treatment samples. These urinary peptides generated from 26
parental proteins. Uniform distribution of intensity of the 70 peptides
(red spots in Figure 2A), emphasized that their observed significant
change with GLP-1R agonist treatment was not a function of abundance in
the urine samples. For most of these 70 peptides, a comparative analysis
further revealed a combined downregulation with GLP-1R agonist treatment
(66/70 peptides).
In total, 59 out of the 70 statistically significant urinary peptides,
majorly generated from three prominent collagen proteins COL3A1
(n =16), COL1A1 (n =15) and COL1A2 (n =10). Recently,
He et al. ,[41] reported about
the high abundance of collagen peptides observed in urine samples, as a
result of proline hydroxylation which plausibly inhibits its
reabsorption in the kidney. 55 out of the 59 collagen peptides were
observed to be significantly downregulated with GLP-1R agonists
treatment in the T2DM urinary proteome, while 4 peptides (COL3A1;n =3 and COL1A2; n =1) showed a significant upregulation on
treatment which could plausibly be attributed to the variation in the
post-translational modification by hydroxylation of proline residues in
the peptides and varied proteolytic cleavage. In-line with the extensive
recent report by Mavrogeorgis et
al. ,[42] all the urinary collagen
peptides identified in this study were devoid of the signal peptide,
N-terminal pro-peptide and C-terminal pro-peptide; corresponding only to
the mature protein region (Figure 2G and 2H). The observed
downregulation of the collagen peptides in our study could potentially
represent rather an attenuated degradation of the mature collagen
protein, instead of resulting from protein synthesis or protein
assembling processes. Rossing et
al.[43] and Genovese et
al. ,[44] have earlier speculated
that the decrease of urinary collagen proteins could attribute to
decreased proteolysis of collagen molecules, resulting from an increased
resistance to proteolytic cleavage or an increased expression of
protease inhibition.
To further corroborate the above speculation, endopeptidases responsible
for putatively cleaving 38 statistically significant collagen peptides
were majorly accounted to the MMP family of proteases (89% of the
cleavage sites, i.e., 34 out of the 38 urinary peptides) by MMP2
(21.1%), MMP9 (21.1%) and MMP13 (15.5%). The suggested downregulation
in the activity of MMP peptidases as observed by decrease in intensity
of collagen peptides in this study, is in-line with literature.
Down-regulation in expression of MMP9 was observed on treatment with
Liraglutide in a study that included induced-DM rabbit
models[45]. In an another study, 45%
and 60% reduction in activity of MMP2 and MMP9, respectively, in
addition to 60% reduced COL1A1 levels, was observed in a male C57BL/6
mice on treatment with Semaglutide (GLP-1R agonist
type)[46]. Research groups exploring
the effect of Exenatide (GLP-1R agonist type) treatment on tumor
necrosis factor-α human coronary artery smooth muscle
cells[47] and human retinal pigment
epithelium cells[48], reported the
downregulated expression of MMP2 and MMP9, respectively on treatment.
Another study analyzing atherosclerosis associated biomarkers in T2DM
female subjects, reported decrease in MMP2 and MMP9 levels, with an
increase in GLP-1 and GLP-1R
levels[49]. Interestingly, treatment
of Human SW1353 with Dulaglutide (GLP-1R agonist
type)[50] and Fibroblast-like
synoviocytes cell lines with
Exenatide[51], also resulted in the
downregulation of MMP13 and ADAMTS5 proteases.
On the other hand, collectively, all the non-collagen peptides (11 out
of 70) showed lower abundance after GLP-1R agonist treatment and each
generated from a different protein namely, SERPINA1, APOC3, CD99, CPSF6,
CRNN, SERPINA6, HBA2, MB, VGF, PIGR and TTR. In studies analyzing the
effect of Liraglutide (GLP-1R agonist type) on T2DM patients, Rafiullahet al. ,[52] reported the
downregulation of the urinary protein SERPINA1 and Adiels et
al. ,[53] reported decreased
secretion of APOC3, respectively with treatment. No literature was found
reporting regulation of the other non-collagen peptides by GLP-1R
agonist treatment. However, impact of diabetes and/or obesity on these
molecules has been reported in the literature. CD99 transcripts have
been stated to up-regulate in T2DM
profiles[54] and Pasello et
al. ,[55] reported the proteins
involvement in biological processes such as cell death and inflammation.
CPSF6 indirectly modulates glucose homeostasis and insulin
secretion[56]; while, HBA2, a
commonly known marker for anemia and β-thalassaemia, interferes with
glycemic markers of T2DM
patients[57].
Increased levels of TTR have been associated with glucose intolerance,
obesity and decreased pancreatic β-cells percentage in
T2DM[58,
59]. Along the same lines, increased
levels of SERPINA6 have been identified in obese patients and is
reported to play a crucial role in glucose homeostasis, along with
reducing insulin resistance and
inflammation[60,
61]. Benchoula et
al. ,[62] in their extensive review
reported that VGF is expected to induce obesity, while also playing a
role in lipolysis and insulin secretion, hence, acting as a potential
target in T2DM therapy. CRNN is reportedly associated with the immune
system and acts as an inflammation marker in chronic
diseases[63,
64]. In addition, elevated levels of
MB, a known cardiac marker, were reported in T2DM
patients[65] and has been associated
with insulin resistance, dyslipidemia and abnormal glucose metabolism
with elevated levels acting as a biomarker for diabetic kidney
disease[66]. Similarly, inflammatory
mediators have been reported to increase PIGR protein levels in the
renal tubular cells, linking its role in renal
injury[67]. As a result of this
exploratory study and literature search, we report the plausible
molecular mechanisms affected by the treatment of GLP-1R agonists on the
pathophysiology of T2DM, as hypothesized from the functions of the
down-regulated non-collagen proteins in Figure 3. The results in this
study may therefore indicate towards the beneficial effect of GLP-1R
agonists in the context of management of T2DM and prevention or delaying
the progression of its associated diseases.
Regardless of the novel findings, this study comes with its own
limitations. Firstly, the large difference in time points between the
pre-treatment and administration of GLP-1R agonists of 4.4 ± 4.11 may
have resulted in unidentified variations of clinical parameters as well
as the composition of urinary peptides, which were not accounted in this
study. Secondly, the administering of multiple anti-hypertensives and
GLP-1R agonist drugs at varied dosages and different combinations to the
T2DM patients may have produced different effects of the treatment,
which were also not analyzed in this study. Thirdly and surprisingly,
within the follow-up we did not observe significant changes in Hb1Ac and
BMI. The study was not powered to detect such changes, which would
require about 10 times the number of subjects to be included, however,
this does help in eliminating the argument that the reported changes in
this study could be a result of weight loss, and instead support the
proteins’ role in T2DM pathophysiological mechanisms. Fourthly, since
only one urinary peptide was identified per non-collagen protein, the
reported effects of GLP-1R agonist treatment on these proteins cannot be
definitive and require further experimental studies with increased
power. However, to overcome this short-coming, we additionally,
performed a paired Wilcoxon test on a cohort of thirty-two T2DM patients
administering only the anti-hypertensive drugs and no GLP-1R agonists
drugs from the same PROVALID study, that were matched to the GLP-1R
agonist treated cohort by age, sex, BMI, SBP, DBP and eGFR.
Interestingly, we did not identify any statistically significant urinary
peptides between the paired urine samples collected at a similar time
difference as in this study. Finally, we could identify 15 urinary
peptides downregulated by GLP-1R agonist treatment in our study, whose
elevated levels have been previously reported as markers of heart
failure[68]. This observation may
further indicate the positive effect of GLP-1R agonist treatment.
To conclude, this untargeted peptidomic analysis to identify the effect
of GLP-1R agonists treatment on the urinary peptidome of T2DM patients,
indicated as a prominent finding the downregulation of MMP proteases, as
identified by the downregulation of urinary collagen peptides on GLP-1R
agonists treatment. Treatment with GLP-1R agonists also resulted in the
decrease of SERPINA1, APOC3, CD99, CPSF6, CRNN, SERPINA6, HBA2, MB, VGF,
PIGR and TTR peptides; indicating a potential benefit as many of these
proteins express increased levels in T2DM patients. The results also
merit the possibility of larger cohort studies to further understand the
impact on underlying molecular mechanisms such as insulin resistance and
inflammation, behind the findings.
Associated data
Data will be made available upon request directed to the corresponding
author. Proposals will be reviewed and approved by the investigators and
collaborators based on scientific merit. After approval of a proposal,
data will be shared through a secure online platform.