RNA sequences for glucolipotoxicity-associated disorders

Abstract

The present disclosure relates to the use of an isolated or artificial ribonucleic acid sequence in any condition susceptible of being improved or prevented by modulating glucolipotoxicity, namely diabetes and obesity. The present disclosure further relates to a vector or a construct, a host cell, a pharmaceutical composition and / or a kit comprising said isolated or artificial ribonucleic acid sequence. The present disclosure further relates to an in vitro or ex vivo method for diagnosis or prognosis of diabetes.

Description

D E S C R I P T I O NRNA SEQUENCES FOR GLUCOLIPOTOXICITY-ASSOCIATED DISORDERSTECHNICAL FIELD

[0001] The present disclosure relates to short non-coding ribonucleic acid (RNA) sequences for use as biomarkers and therapeutic targets of glucolipotoxicity, which results from chronic exposure to high levels of glucose and lipids, being implicated in several metabolic disorders, in particularly diabetes and obesity.BACKGROUND

[0002] Type 2 diabetes mellitus (T2DM) is a chronic, progressive and multifactorial disease characterized by uncontrolled elevated glucose levels secondary to -cell dysfunction and / or death1.

[0003] Obesity is a critical risk factor for the development of T2DM and it is associated with elevated concentrations of circulating free fatty acids (FFA)2. Increased plasma concentrations of saturated FFA negatively affect p-cell function and may constitute a link between obesity and diabetes3. In vitro exposure of p-cells to high concentrations of FFA and glucose recapitulates glucolipotoxicity (GLT) - a pathophysiological condition found in vivo in pancreatic islets of obese patients and a well-established model to study the mechanisms governing p-cell dysfunction and / or death4. GLT impinges on p-cell health and function3via several different mechanisms including: oxidative stress and mitochondrial dysfunction, lipid droplet formation, modulation of survival pathways, loss of p-cell identity genes and impairment of P-cell glucose stimulated insulin secretion (GSIS)2.

[0004] Conventional diagnostic tools frequently identify GLT only after substantial tissue and organ damage has occurred.

[0005] The molecular mechanisms driving GLT and its role in the pathogenesis of metabolic disorders remain incompletely elucidated. The lack of validated biomarkers results in non-specific and often indirect methods for monitoring disease progression and therapeutic efficacy, thereby hindering the development of targeted and precise interventions.

[0006] Notably, the risk of developing diabetes is not uniform among individuals with obesity, suggesting the existence of protective mechanisms that mitigate the effects of GLT. This variability highlights the limitations of current diagnostic and therapeutic methods, which fail to account for individual variations in disease progression and treatment response. Consequently, there is a critical need for personalized therapeutic strategies that target specific molecular pathways involved in GLT.

[0007] Existing therapies for metabolic disorders often focus on managing symptoms (e.g., lowering blood glucose or lipid levels), rather than addressing the underlying molecular mechanisms thus limiting the efficacy of therapies and leading to side effects.

[0008] Bariatric Surgery (BS) remains a highly effective strategy for addressing obesity and T2DM, and a high proportion of patients achieve remission in a relatively short period4. Other therapies, such as microRNA (miRNA)-based therapies, present an innovative and promising approach for addressing complex diseases such as diabetes and obesity and are still being investigated.

[0009] Document US2023175066A1 discloses a method for assessing a biological or medical condition, particularly cancer, of a subject by obtaining information of miRNAs and analysing the condition based on that information. Furthermore, it reveals an accurate and efficient method for cancer detection and monitoring.

[0010] However, there remains an unmet need for methods enabling earlier diagnosis of metabolic disorders, particularly diabetes and obesity, as well as improved monitoring of disease progression and treatment efficacy.

[0011] These facts are disclosed to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0012] The present disclosure relates to a short non-coding RNA sequence, namely a miRNA, for modulating GLT in metabolic associated disorders, such as diabetes and obesity.

[0013] Surprisingly, the miRNA sequence of the present disclosure, namely miR-642a-3p, protects p-cells from GLT-induced cell death (GICD) and restores the GLT-induced changes in insulin secretion (hyperinsulinemia) as well as the expression of -cell identity genes (Mafa and Nkx6.1), thus rescuing p-cell function.

[0014] In addition, miR-642a-3p, increased the expression of Glplr and enhanced intracellular lipid droplet (LD) accumulation.

[0015] The miRNA sequence of the present disclosure, namely miR-642a-3p, has as putative targets several genes associated with obesity and / or p-cells proliferation in diabetes, namely Akt2, WdrlS, Prkab2, Cmtm6, Zdhhc7, Dusp4 and Dnajc27. The miRNA sequence of the present disclosure downregulates Akt2 in p-cells transfected with miR-642a-3p and increases AKT phosphorylation at Ser473.

[0016] The expression of the miRNA sequence of the present disclosure is downregulated in human islets isolated from T2DM patients compared to non-diabetic controls, suggesting loss of metabolic control. Higher levels of the miRNA sequence in non-diabetic obese patients eligible for BS might contribute to the preservation of p-cells despite their obesity.

[0017] The miRNA sequence of the present disclosure is a suitable biomarker for early diagnosis of diabetes. The discovery of new biomarkers and therapeutic targets for diabetes solves critical technical problems by enabling earlier diagnosis, providing specific molecular targets for drug development, improving understanding of disease mechanisms, facilitating personalized medicine, and enhancing the monitoring of disease progression and treatment efficacy. These advancements have the potential to transform the management of metabolic disorders, such as diabetes and obesity, and their complications.

[0018] The present disclosure relates to an isolated or artificial ribonucleic acid sequence for use in any condition susceptible of being improved or prevented by modulating GLT comprising a nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46 or SEQ ID NO: 85 to SEQ ID NO: 109; or comprises an isolated double-stranded ribonucleic acid molecule including an antisense strand complementary to CMTM6 mRNA.The double-stranded ribonucleic acid molecule is capable of inducing RNA interference (RNAi)-mediated degradation of CMTM6 mRNA, thereby effecting knockdown of CMTM6 expression. The invention is intended for use in conditions that may be improved or prevented through modulation of GLT and / or reduction of CMTM6 expression.

[0019] As used herein, the term "knockdown of CMTM6" refers to a reduction in the expression of CMTM6 at the mRNA level and / or at the protein level in a cell or subject compared to an appropriate control. Knockdown may comprise a partial reduction or a substantially complete reduction of CMTM6 expression. In preferred embodiments, knockdown comprises a substantially complete reduction, defined as a reduction of at least 95% or a reduction to levels below the limit of detection using standard quantitative methods. Knockdown is preferably achieved by RNA interference-mediated degradation of CMTM6 mRNA. Namely, sequence comprises a nucleotide sequence at least 91 % identical to at least one of the following sequences: SEQ ID NO: 110 to SEQ ID NO: 111; preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical

[0020] In certain embodiments, administration of the ribonucleic acid molecule results in a reduction of CMTM6 expression of at least 50%, preferably at least 90%, and more preferably a substantially complete reduction. Namely, sequence comprises a nucleotide sequence at least 91 % identical to at least one of the following sequences: SEQ ID NO: 110 to SEQ ID NO: 111; preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0021] In an embodiment, the isolated or artificial ribonucleic acid sequence of the present disclosure, wherein the antisense strand comprises a nucleotide sequence of at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to at least one of the following sequences: SEQ ID NQ:110 or SEQ ID NO:111.

[0022] In an embodiment, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 91 % identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO:46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably 92% 93% 94% 95% 96% 97% 98% 99% or 100% identical.

[0023] In an embodiment, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 96% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably 97%, 98%, 99%, or 100% identical.

[0024] In an embodiment, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably 91%, 92%, 93%, 94% or 95% identical.

[0025] In an embodiment, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 96% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably 97%, 98%, 99%, or 100% identical.

[0026] In an embodiment for better results, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 16 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably SEQ ID NO: 16 or SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

[0027] In an embodiment for better results, the isolated or artificial ribonucleic acid sequence comprises a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 46, SEQ ID NO: 110, or SEQ ID NO: 111.

[0028] Another aspect of the present disclosure relates to the isolated or artificial ribonucleic acid sequence for use in the treatment or prevention of GLT-associated disorders.

[0029] Another aspect of the present disclosure relates to a vector or a construct for use in any condition susceptible of being improved or prevented by modulating GLT comprising the isolated or artificial ribonucleic acid sequence of the present disclosure.

[0030] In an embodiment for better results, the vector is selected from the group consisting of viral vectors, plasmids, extracellular vesicles, nanoparticles, dendrimers, or virus-like particles.

[0031] Another aspect of the present disclosure relates to a host cell for use in any condition susceptible of being improved or prevented by modulating GLT comprising the isolated or artificial ribonucleic acid sequence and / or the vector of the present disclosure.

[0032] In an embodiment for better results, the host cell is a beta-cell, or endothelial cell.

[0033] Another aspect of the present disclosure relates to a pharmaceutical composition for use in any condition susceptible of being improved or prevented by modulating GLT using the isolated or artificial ribonucleic acid sequence and / or the vector of the present disclosure.

[0034] In an embodiment for better results, the pharmaceutical composition comprises at least a solvent and at least one pharmaceutically acceptable excipient.

[0035] In an embodiment for better results, the pharmaceutically acceptable excipient is a buffering agent, stabilizer, cryoprotectant, chelating agent, preservative, viscosity modifier, solubilizer, antioxidant, filler, adjuvant, pH adjuster, among others.

[0036] Another aspect of the present disclosure relates to a kit for use in any condition susceptible of being improved or prevented by modulating GLT comprising the isolated or artificial ribonucleic acid sequence and / or the vector and / or the pharmaceutical composition of the present disclosure.

[0037] Another aspect of the present disclosure relates to the use of the isolated or artificial ribonucleic acid sequence of the present disclosure as a biomarker to detect or monitor GLT-associated disorders in a sample; wherein a measured level of the isolated or artificial ribonucleic acid sequence into a sample is compared to a control reference value and a reduction relative to the control is indicative of diagnosis or poor prognosis of diabetes; wherein said sample is a biological sample selected from the list consisting of: interstitial fluid, blood, plasma, serum, or urine.

[0038] Another aspect of the present disclosure relates to the use of the isolated or artificial ribonucleic acid sequence of the present disclosure for the manufacture of a medicament for the treatment of GLT-associated disorders; preferably for the treatment of diabetes and / or obesity.

[0039] Another aspect of the present disclosure relates to a method for treating or preventing GLT-associated disorders, preferably diabetes and / or obesity, in a subject, the method comprising administering the isolated or artificial ribonucleic acid sequence of the present disclosure.

[0040] Another aspect of the present disclosure relates to an in vitro or ex vivo method for diagnosis or prognosis of GLT-associated disorders, comprising the following steps: providing a biological sample of a patient, measuring the content or amount of the isolated or artificial ribonucleic acid sequence described in any of the previous claims, comparing said context or amount to a control reference value wherein the reduction of the context or amount of the sequence relative to the control reference value is indicative of diagnosis of diabetes and wherein the biological sample is selected from the list consisting of interstitial fluid, blood, plasma, serum or urine.BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0042] Figure 1: High-throughput screening assay to identify miRNAs capable of protecting -cells from GLT. (A) Schematic overview of the screening workflow used in the HTS. (B) Metabolic activity versus cell mass (% to BSA) for each individual miRNA tested in the primary screening. miRNAs capable of increasing the metabolic activity are coloured (the ones that increase both the cell mass as well as the metabolic activity are highlighted in blue); (C) Validation of the 7 miRNAs identified in the primary screening as capable of increasing both the metabolic activity as well as the cell mass. The results were normalized to BSA-containing medium condition (n=3 independent experiments); (D) miR-642 location on chromosome 19 and mature sequence for miR-642a-3p and miR-642b-3p, respectively. Bold: seed sequence, underlined: different nucleotide between miR-642a-3p and miR-642b-3p; (E-F) Metabolic activity and cell mass (% to BSA control) and brightfield images, respectively, of INS1E cells transfected with miR-642a-3p and exposed to GLT conditions for 24 h. Statistical analysis was performed by Two-way ANOVA, followed by Tukey's multiple comparisons test. **** p<0.001. Results are presented as mean±SD (n=4 biological replicates, each biological replicate containing three technical replicates). Scale bar is 50 pm.

[0043] Figure 2: Effect of miR-642a-3p in p-cell function. (A) Schematic overview of glucose-stimulated insulin secretion (GSIS) assay in INS1E cells; (B) Quantification of insulin secretion evaluated by ELISA; (C-D) Representative images of immunofluorescence staining for insulin in INS1E cells exposed to control, GLT and miR-642a-3p and their respective quantification; (E) RT-PCR analysis of insulin expression in INS1E cells exposed to GLT or miR-642a-3p and GLT. The results are the average of three independent experiments. Statistics was determined using Two-Way ANOVA followed by Tukey's Multiple Comparison Test. *p<0.0388, **p<.0051, ***p=0.0007, ****p<0.0001. Error bars represent SD. Bar corresponds to 25 pm.

[0044] Figure 3: RNA-sequencing for INS1E cells exposed to GLT and miR-642a-3p. (A) Volcano plot for INS1E cells treated with BSA and GLT showing the magnitude of the gene expression changes (Iog2 foldchange; x-axis) and statistical differences (-loglO q-value; y-axis); (B) Heat maps highlighting the differential expression of genes between BSA vs GLT (412 down-regulated and 210 up-regulated); (C) Gene ontology analysis (biological processes (BP) and KEGG pathways) for genes up-regulated by GLT; (D) GO:BP and KEGG down-regulated by GLT; (E) Volcano plot GLT vs miR-642a-3p showing the magnitude of the gene expression change (Iog2 fold-change; x-axis) and statistical differences of this change (-loglO q-value; y-axis); (F) Heat maps between miR-642a-3p vs GLT performed with 701 genes (115 down-regulated and 586 up-regulated); (G) GO:BP and KEGG up-regulated by miR-642a-3p; (H) GO:BP and KEGG down-regulated by miR-642a-3p; (I) Common target miR642a-3p genes; (J) qRT-PCR quantification of the expression of miR-642a-3p putative targets; (K) Metabolic activity following siRNA-mediated knock-downof Cmtm6, Dusp4, Wrdl3, Dnajc27 and Zdhhc7 followed by exposure to GLT; Statistical analysis was determined using One way ANOVA, followed byTukey's Multiple Comparison test. *p<0.045, **p<0.0066, ***p=0.0002, ****p<0.0001. The results are presented as mean±SD (n=3 biological replicates).

[0045] Figure 4: (A) Predicted sequence conservation between the 3' UTR region of Cmtm6 mRNA across species (human vs. rat and rat vs. mouse), with grey regions indicating perfect nucleotide pairing. Predicted seed regions of human hsa-miR-642a-3p are annotated with triangles and color-coded based on miRanda in silica binding scores; (B) Alignment of predicted hsa-miR-642a-3p binding sites to the 3' UTRs of mouse, rat, and human Cmtm6, considering both Watson-Crick and Wobble base pairing.

[0046] Figure 5: Effect of miR-642a-3p on the expression of genes involved in p-cell identity and on prosurvival signalling pathway. (A-D) FPKM and RT-PCR analysis of the transcription factors known to be involved in p-cell identity (Foxa2, Nkx6.1, Mafa and Pdxl); (E-F) Western-blotting was used to analyse and quantify the expression of proteins known to be involved in cell survival; (G-H) Representative immunofluorescence images of FOXO1 expression in INS1E cells exposed to GLT and miR-642a-3p, and quantification of the cytoplasmatic:nuclear ratio of FOXO1, respectively. Statistical analysis was determined using One-way ANOVA, followed byTukey's multiple comparisons test * p<0.03, **p<0.0086, *** p=0.0007, ****p<0.0001. The results are presented as mean±SD (n=3 biological replicates, each biological replicate containing three technical replicates). Bar corresponds to 25 pm. FPKM: Fragments per kilobase of transcript per million mapped reads.

[0047] Figure 6: (A) RT-PCR confirming the knock-down of miR-642a-3p target genes following siRNA-mediated silencing; (B) RT-PCR analysis of p-cell identity markers following siRNA-mediated knock-down of Cmtm6.

[0048] Figure 7: Effect of the miR-642a-3p on the expression of genes involved in lipid metabolism (Fasn), involved in oxidative stress (Cat) and PAX6. FPKM and qRT-PCR analysis of Fasn, Cat and Pax6. Statistical analysis was determined using Two-Way Anova, followed byTukey's Multiple Comparison Test. * p<0,03, ** p<0,0051, ****p<0.0001. Error bars represent mean ± SD (n=3 biological replicates, each biological replicate containing two technical replicates). FPKM: Fragments per kilobase of transcript per million mapped reads.

[0049] Figure 8: Effect of miR-642a-3p on Glplr expression. (A) Representative Immunofluorescence images of Glplr; (B) Quantification of the Immunofluorescence for Glplr; (C) FPKM and (D) RT-PCR for Glplr; (E-F) Western-blotting was used to analyse and quantify the expression of Glplr. Statistical analysis was determined using One-way Anova, followed by Tukey's Multiple Comparison Test. * p<0,0268, ** p=0,0062, ****p<0.0001. Error bars represent mean ± SD (n=3 biological replicates, each biological replicate containing three technical replicates).

[0050] Figure 9: Effect of miR-642a-3p on lipid droplet accumulation by INS1E cells. (A-B) Representative brightfield images of INS1E cells exposed to different concentrations of fatty acids (PA, oleate or a combination) in the presence or absence of miR-642a-3p and quantification of liquid droplets (B). Cells were stained with Nile red; (C) FPKM values obtained by RNA-Seq analysis for Plin3 and Plin5. The results of lipid accumulation are the average of two independent experiments. Statistical analysis was determined using One-Way ANOVA, followed by Tukey's Multiple Comparison Test. **p<0.0019, ***p<0.0008. (D) Metabolic activity (% to BSA) of INS1E cells cultured for 24 h of 0.25 mM Oleate, 0.75 mM of PA and the combination of 0.25 mM Oleate with 0.75 mM of PA, in 25 mM glucose, in 1% FBS. Statistical analysis was determined using One-way ANOVA, followed by Tukey's multiple comparisons test. **p<0.0019, ***p<0.0008, ****p<0.0001. Error bars represent mean±SD (at least two independent biological replicates were performed, each biological replicate containing three technical replicates).

[0051] Figure 10: miR-642a-3p protects INS1E cells against GICD. (A) Representative images of polarized mitochondria after TMRM staining. The following morphometric parameters were measured: TMRM fluorescence intensity (i), number of particles per cell (ii), circularity (iii), TMRM area per cell (iv), aspect ratio (v) and Feret's diameter. (B) Total DNA was extracted and amplified by real-time RT-PCR. Mitochondrial DNA copy number was normalized to B2m levels. (C) The Seahorse XFe96 Extracellular Flux Analyzer was used to measure Oxygen Consumption Rate (OCR). Several OCR parameters were evaluated: basal cell respiration (i), ATP production-linked OCR (ii) and maximal cell respiration (iii). (D and E) MitoSOX and Lysotracker red fluorescence intensities were measured by epifluorescence microscopy. Fluorescence intensity was normalized by cell number. (F) Caspase-3 / 7-like activities were measured using the Caspase-Gio 3 / 7 assay kit, following the manufacturer's instructions. Data are expressed as mean ± SEM of 3-4 different experiments. Statistical analysis was determined using One-way ANOVA, followed by Tukey's multiple comparisons test *p<0.05, **p<0.001, ***p<0.0005, ****p<0.0001 to BSA vs GLT or miR-642-3p+GLT, #p<0.05, ##p<0.001, ### p<0.0005, ####p<0.0001 to GLT vs miR-642-3p+GLT.

[0052] Figure 11: Cmtm6 knockdown protects INS1E cells against GICD in a mitochondrial-independent manner. (A) Representative images of polarized mitochondria after TMRM staining. Several mitochondria morphometric parameters were measured: TMRM fluorescence intensity (i), number of particles per cell (ii), circularity (iii), TMRM area per cell (iv), aspect ratio (v) and Feret's diameter (vi). (B) Total DNA was extracted and amplified by real-time RT-PCR. Mitochondrial DNA copy number was normalized to B2m levels. (C) The Seahorse XFe96 Extracellular Flux Analyzer was used to measure Oxygen Consumption Rate (OCR). Several OCR parameters were evaluated: basal cell respiration (i), ATP production-linked OCR (ii) and maximal cell respiration (iii). (D and E) MitoSOX and Lysotracker red fluorescence intensities were measured by epifluorescence microscopy. The fluorescence intensity was normalized by cell number. (F) Caspase-3 / 7-like activities were measured using the Caspase-Gio 3 / 7 assay kit, following the manufacturer's instructions. Data are expressed as mean ± SEM of 3-4 different experiments. Statisticalanalysis was determined using One-way ANOVA, followed by Tukey's multiple comparisons test *p<0.05, **p<0.001, ***p<0.0005, ****p<0.0001 to BSA vs GLT or GLT+Cmtme7#p<0.05, ##p<0.001, ###p<0.0005, ####p<0.0001 to GLT vs GET+Cmtme7'.

[0053] Figure 12: Expression of GLT-protective miRNAs in the pancreas and liver of mice fed with a control or hypercaloric diet. (A) Experimental graphical abstract of a mouse model of diabetes induced by a hypercaloric diet; (B) pancreatic and (C) hepatic expression of miR-20a-5p, miR17-5p, miR20b-5p and miR-432-5; (D) expression analysis of miR-642a-3p in human pancreatic islets from control (ND) and T2DM donors (T2D). Statistical analysis was determined using unpaired t-test. *p=0.0324. Error bars represent SD.

[0054] Figure 13: miRNA signature from plasma-derived EVs of obese patients selected for BS. (A) Schematic overview of sample collection; (B) Patient BMI and HbAicbefore and after BS; (C) copies of miR-642a-3p, miR-20a-5p, miR-17-5p, miR-20b-5p and miR-432-5p per EV particle in plasma. Statistical analyses were performed using t-test. **p=0.0041. Results are presented as mean ± SEM. BMI: Body Mass Index; HbAlC: Glycated haemoglobin; pnd: people non-diabetic; pwd: people with diabetes.

[0055] Figure 14: miR-372 modulates mitochondrial bioenergetics and function. (A) Representative images of polarized mitochondria after TMRM staining. Several mitochondria morphometric parameters were measured: TMRM fluorescence intensity (i), number of particles per cell (ii), circularity (iii), TMRM area per cell (iv), aspect ratio (v) and Feret's diameter (vi). (B) Total DNA was extracted and amplified by real-time RT-PCR. Mitochondrial DNA copy number was normalized to B2m levels. (C) The Seahorse XFe96 Extracellular Flux Analyzer was used to measure Oxygen Consumption Rate (OCR). Several OCR parameters were evaluated: basal cell respiration (i), ATP production-linked OCR (ii) and maximal cell respiration (iii). (D and E) MitoSOX and Lysotracker red fluorescence intensities were measured by epifluorescence microscopy. The fluorescence intensity was normalized by cell number. (F) Caspase-3 / 7-like activities were measured using the Caspase-Gio 3 / 7 assay kit, following the manufacturer's instructions. Data are expressed as mean ± SEM of 3-4 different experiments. Statistical analysis was determined using One-way ANOVA, followed by Tukey's multiple comparisons test *p<0.05, **p<0.001, ***p<0.0005, ****p<0.0001 to BSA vs GLT or GLT+miR-372, #p<0.05, ##p<0.001, ###p<0.0005, ####p<0.0001 to GLT vs GLT+miR-372.

[0056] Figure 15: miR-17-5p modulates mitochondrial bioenergetics and function. (A) Representative images of polarized mitochondria after TMRM staining. Several mitochondria morphometric parameters were measured: TMRM fluorescence intensity (i), number of particles per cell (ii), circularity (iii), TMRM area per cell (iv), aspect ratio (v) and Feret's diameter (vi). (B) Total DNA was extracted and amplified by real-time RT-PCR. Mitochondrial DNA copy number was normalized to B2m levels. (C) The Seahorse XFe96 Extracellular Flux Analyzer was used to measure Oxygen Consumption Rate (OCR). Several OCR parameters were evaluated: basal cell respiration (i), ATP production-linked OCR (ii) and maximal cellrespiration (iii). (D and E) MitoSOX and Lysotracker red fluorescence intensities were measured by epifluorescence microscopy. The fluorescence intensity was normalized by cell number. (F) Caspase-3 / 7-like activities were measured using the Caspase-Gio 3 / 7 assay kit, following the manufacturer's instructions. Data are expressed as mean ± SEM of 3-4 different experiments. Statistical analysis was determined using One-way ANOVA, followed by Tukey's multiple comparisons test *p<0.05, **p<0.001, ***p<0.0005, ****p<0.0001 to BSA vs GLT or GLT+miR-17-5p, #p<0.05, ##p<0.001, ###p<0.0005, ####p<0.0001 to GLT vs GLT+miR-17-5p.DETAILED DESCRIPTION

[0057] The present disclosure relates to the use of an isolated or artificial ribonucleic acid sequence in any condition susceptible of being improved or prevented by modulating GLT, namely diabetes and obesity. The present disclosure further relates to a vector or a construct, a host cell, a pharmaceutical composition and / or a kit comprising said isolated or artificial ribonucleic acid sequence. The present disclosure further relates to an in vitro or ex vivo method for diagnosis or prognosis of diabetes.

[0058] The present disclosure relates to short non-coding RNA sequences (miRNAs) capable of increasing the metabolicactivity and / or cell mass more than three times the mean of the GLT condition. In particular, the present disclosure relates to miRNAs, wherein the miRNAs sequences are SEQ ID NO: 1 to SEQ ID NO: 46, disclosed in Table 2.

[0059] It was surprisingly found that the 46 miRNAs (SEQ ID NO: 1 to SEQ ID NO: 46), disclosed in Table 2, increase the metabolic activity and / or cell mass more than three times the mean of the GLT condition, wherein 17 miRNAs (SEQ ID NO: 1 to SEQ ID NO: 17) increased the metabolic activity and 28 miRNAs (SEQ ID NO: 28 to SEQ ID NO: 45) increased the cell mass.

[0060] Furthermore, 7 miRNAs (miR-432-5p - SEQ ID NO: 12, miR-370, SEQ ID NO: 6, miR-377-3p, SEQ ID NO: 8, miR-17-5p, SEQ ID NO: 1, miR-642b-3p, SEQ ID NO: 16, miR-372, SEQ ID NO: 7 and miR-20a-5p, SEQ ID NO: 2), , increase both metabolic activity and cell mass and, therefore, are able to protect p-cells from GICD.

[0061] In an embodiment, miR-642a-3p (SEQ ID NO: 46) effectively protects p-cells from GICD in a 1.57-fold increase in metabolic activity and 1.97-fold increase in cell mass, compared to GLT condition.

[0062] The present disclosure shows that genes upregulated by miR-642a-3p (SEQ ID NO: 46) are associated with chromatin remodelling, cell migration, mitotic cytokinesis and axon guidance, focal adhesion, MAPK signalling pathway and phosphatidylinositol 3-kinase / protein kinase B (PI3K-Akt) signalling pathway whereas down-regulated genes are associated with intrinsic apoptotic signalling pathway in response to endoplasmic reticulum stress, negative regulation of fat cell differentiation,positive regulation of apoptotic process and dopaminergic synapse, relaxin signalling pathway and insulin signalling pathway.Materials and MethodsCell culture

[0063] INS1E cell line was cultured in RPMI 1640, supplemented with 10% (v / v) foetal bovine serum (FBS) (#10270106, Gibco), 50 U / ml of penicillin, 50 pg / mL of streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 pM p-mercaptoethanol, and maintained in 5% CO2 at 37 °C. Cells were used between passage 61-72 and were tested regularly for mycoplasma infection, using Mycoalert mycoplasma detection kit (#LT07-418), Lonza, according to the manufacturer's instructions.Palmitate in bovine serum albumin (BSA)-containing medium

[0064] Sodium palmitate (# P9767, Sigma-Aldrich, St. Louis, MO, USA) was prepared at 50 mM stock concentration in 90% (v / v) ethanol, heated at 60 °C until dissolved and diluted to a final concentration of 0.75% (v / v) fatty acid-free bovine serum albumin (BSA)-containing medium (#3117057001, Roche), in 11 mM or 25 mM of Glucose. Control for equal amounts of ethanol were tested.Glucolipotoxicity (GLT) assay

[0065] Palmitate stock was diluted in 0.75% (v / v) fatty acid-free BSA-containing RPMI 1640 media (#3117057001, Roche), with either 11 or 25 mM of glucose, as before.

[0066] Next, 4xl04INS1E cells were seeded in a flat-bottom 96-well plate (#734-1662, Corning) in RPMI 1640, supplemented with 10% (v / v) FBS (#10270106, Gibco), 50 U / ml of penicillin, 50 pg / mL of streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 pM p-mercaptoethanol, for 24 h before adding increasing concentrations of PA, for another 24 h.Cell Metabolic activity assay

[0067] After being subject to different treatments, cell metabolic activity was evaluated through resazurin reduction assay (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions. Briefly, the medium was replaced with RPMI supplemented with 10% (v / v) FBS, 50 U / ml of penicillin, 50 pg / mL of streptomycin and 10 pg / mL resazurin. Cells were incubated for 1 to 2 h at 37 °C and 5% of CO2 upon which fluorescence was measured (540 nm excitation and 590 nm emission) using the microplate reader Synergy™ Hl (Biotek Instruments, Winooski, VT, USA).Cell mass (sulforhodamine B, SRB) assay

[0068] The sulforhodamine B (SRB) assay is based on the measurement of cellular protein content. In brief, after applying different treatments, cells were fixed with 1% (v / v) acetic acid in methanol for 18 h at -20 °C upon which the fixation solution was removed, and the plates dried at 37 °C for 1-2 h. Next, 150 pL of 0.05% (v / v) SRB solution was added to the wells and incubated at 37 °C for 1 h. Upon which the wellswere washed with 1% (v / v) acetic acid in water and dried. Following, 10 mM of Tris (pH 10) was added and the plates were stirred for 15 min and optical density was measured (540 nm), using the microplate reader Synergy™ Hl (Biotek Instruments, Winooski, VT, USA).High-throughput screening (HTS) assay

[0069] INS1E (4xl04cells) were reverse transfected in a 96-well plate with a library of miRNAmimics (Dharmacon miRIDIAN miRNA Library- Human mimic miRBase version 19.0; 2080 miR mimics), according to the manufacturer's instructions. Per well, complexes of miRNA: Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, # 13778) were prepared 50 nM miRNA and 0.3 pL Lipofectamine™ RNAiMAX in OptiMEM medium (Gibco, # 51985-026), in a final volume of 100 pL. Complexes were allowed to form during 30 min at room temperature. Lipofectamine™ RNAiMAX-treated cells were used as a control. After the incubation, INS1E cells were added to the well and transfection was allowed to proceed for 48 h, upon which the GLT stimulus (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v), was added for further 24 h. After 24 h, the metabolic activity and cell mass were evaluated and a bright field image was acquired for each individually transfected miRNA using the InCell Analyzer 2200 automated high-content imager (GE Healthcare) at 40x magnification.Glucose-stimulated hormone secretion (GSIS) and Insulin levels by ELISA

[0070] INS1E (4xl04cells) were reverse transfected in a 96-well plate with 50 nM of miR-642a-3p for 48 h and exposed to GLT conditions (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v)) for 24 h. Afterwards, supernatant was used to quantify the degree of glucose-stimulated hormone secretion (GSIS). Briefly, to enhance the sensitivity of INS1E cells to a high-glucose challenge, cells were washed in krebs-ringer solution (KRB) (HEPES-buffered (#J67795.AP, Alfa Aesar) supplemented with 120 mM NaCI, 5 mM KCI, 1 mM MgCL, 5.5 mM HEPES, 2 mM CaCL, 25 mM NaHCOs; 1 mM Glucose pH 7.2 supplemented with 0.5% (v / v) BSA. Subsequently, cells were incubated in KRB low glucose (1.67 mM) for 1 h, washed, another 1 h in KRB high glucose (16.7 mM), washed 3 times and finally 1 h in KRB low glucose (1.67 mM). Samples were collected between each treatment, centrifuged at 300 x g for 3 min and the supernatant was stored at -20 °C for subsequent analysis. Secretion of insulin was measured using the High Range Rat Insulin Enzyme-linked Immunosorbent Assay (ELISA) (# 10-1145-01, Mercodia, Uppsala, Sweden), according to the manufacturer's recommendations. Briefly, 10 pL of each of the Calibrators, controls and samples were pipetted into a mouse monoclonal anti-insulin coated plate 50 pL of enzyme conjugate IX solution was added and incubated on a plate shaker (700-900 rpm) for 2 h at room temperature (18-25 °C). Afterwards, the wells were washed 6 times with 350 pL wash buffer IX solution, 200 pLTMB substrate was added per well and incubated for 15 min on the bench at room temperature (18 - 25 °C). Finally, 50 pL Stop Solution was added to each well, the plate was placed on the shaker for approximately 5 sec to ensure mixing and the optical density was read at 450 nm. The amount of insulin secreted was normalized for the total protein content.siRNA-mediated knockdown

[0071] INS1E (4xl04cells) were reverse transfected in a 96-well plate with siRNA pre-designed to target specific genes (IDT, Coralville, USA). Per well, complexes of siRNA: Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, # 13778) were prepared using 50 nM siRNA and 0.3 pL Lipofectamine™ RNAiMAX in OptiMEM medium (Gibco, # 51985-026), in a final volume of 100 pL. Complexes were allowed to form during 30 min at room temperature. Lipofectamine™ RNAiMAX-treated cells were used as a control. After the incubation, INS1E cells were added to the well and transfection was allowed to proceed for 48 h, upon which the GLT stimulus (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v), was added for further 24 h. After 24 h, the metabolic activity and beta-cell identity markers were evaluated by RT-PCR.Immunofluorescence microscopy

[0072] INS1E (4xl04cells) were reverse transfected with 50 nM of miR-642a-3p for 48 h and exposed to GLT conditions (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v)), for 24 h. Afterwards, cells were washed with 100 pL of PBS and fixed with 50 pL of 4% (v / v) paraformaldehyde (PFA) for 10-20 min at room temperature. Next, cells were permeabilized using 0.2% (v / v) Triton X-100 for 10-20 min at room temperature. To block non-specific interactions, cells were incubated in 1% (v / v) BSA, with 5% (v / v) normal goat serum (NGS) and 0.3% (v / v) Triton X-100 in PBS, for 45 min. The following primary antibodies were used insulin (1:50; FLEX Polyclonal Guinea Pig Anti Insulin (#IR00261-2, Agilent), FOXO1 (1:100 (C29H4) Rabbit mAb (#1672880S, Cell Signaling Technology)) and Rabbit Polyclonal GLP-1R Antibody (1:250; NBP1-97308SS, Novus Biologicals). All the antibodies were diluted in PBS with 1% (v / v) BSA and 0.3% (v / v) Triton X-100 and incubated for 1 h at room temperature. Subsequently, cells were washed and incubated with secondary Alexa 568 anti-guinea pig (1:1000) or Alexa 488 anti-rabbit (1:1000), diluted in a PBS solution containing 1% (v / v) BSA and 0.3% (v / v) Triton X-100 for 1 h at room temperature. Finally, the nuclei were stained with DAPI solution (1 pg / mL) for 10 min at room temperature. Randomly, 8 fields per well were selected for image acquisition using the InCell Analyzer 2200 automated high-content imager (GE Healthcare) using 40x magnification. InCell Investigator software (GE Healthcare) was used for quantification. Cytosolic and nuclear FOXO1 were resolved by following an image-based segmentation strategy, using InCell Investigator software (GE Healthcare).Western-Blotting

[0073] INS1E (4xl04cells) were reverse transfected with 50 nM of miR-642a-3p for 48 h and exposed to GLT conditions (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v)), for 24 h. Afterwards, cells were gently washed with PBS, lysed in RIPA Lysis Buffer containing lOOx protease / phosphatase inhibitor cocktail (#5872, Cell Signaling), vortexed, and kept on ice for 30 min. The supernatant was collected after centrifugation at 13,000 rpm for 15 min. Protein was quantified by the microBCA method using BSA as a standard (#23235; ThermoFisher). Protein samples (20-50 pg) were separated by electrophoresis using 12% (v / v) SDS-polyacrylamide gels (SDS-PAGE). After electrophoresis, the proteins were transferred ontoPVDF membranes (Millipore, Billerica, MA). The membranes were blocked in 5% (v / v) BSA (Roche, Basel, Switzerland) in Tris-Buffered Saline - Tween (TBS-T) at room temperature for 60 min. The following antibodies were incubated overnight at 4 °C: Rabbit Polyclonal p-AKT Antibody (1:500; #4060s, Cell signaling), Rabbit polyclonal AKT Antibody (1:500; #4691S, Cell signaling), Rabbit Polyclonal Phospho-p44 / 42 MAPK Antibody (Erkl / 2) (1:500; #4376S, Cell signaling), Rabbit Polyclonal p44 / 42 MAPK (Erkl / 2) Antibody (1:500; 4695S, Cell signaling), Mouse polyclonal P-tubulin Antibody (1:5000; #T8328, Sigma Aldrich), Rabbit Polyclonal GLP-1R Antibody (1:250; NBP1-97308SS, Novus Biologicals). Next, the membranes were incubated at room temperature for 60 min with anti-rabbit-HRP conjugated (1:5000; Cell Signaling Technology) or anti-goat-HRP conjugated secondary antibody (1:5000; Cell Signaling). Blots were imaged using a Biospectrum— Multispectral imaging system (UVP; LLC Upland, CA; Cambridge, UK) and the densities of each band were calculated with Quantity one software (Biorad).Nile red

[0074] Lipid accumulation was assessed by Nile red staining assay, as previously described. Briefly, a 1:200 dilution from Nile Red was prepared from the stock (0.5 mg / mL in acetone) in medium without FBS and 100 pL of this solution was added to the cells for 1 h in the dark at 37 °C. Subsequently, the lipid content was measured fluorimetrically (excitation: 520 nm; emission: 620 nm) using the microplate reader SynergyTM Hl (Biotek Instruments, Winooski, VT, USA). The percentage of lipid accumulation was normalized to the nuclear counts. Images of the stained cells were acquired using InCell Analyzer 2200 automated high-content imager (GE Healthcare) at 40x magnification.RT-PCR

[0075] INS1E (4xl04cells) were reverse transfected with 50 nM of miR-642a-3p for 48 h and exposed to GLT conditions (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v)), for 24 h. Afterwards, cells were lysed and RNA was isolated using a RNeasy Micro Kit (# 50974034, Qiagen, Hilden, Germany) according to the manufacturer's instructions and quantified on a spectrophotometer. Next, 500 ng of RNA was reverse transcribed, using a qScript SuperMix kit (#95048; Quantabio, Massachusetts, USA) and the obtained cDNA was used to quantify the m RNA expression of target genes using quantitative real-time PCR analysis. For this, PCR products were amplified using the NZYSpeedy qPCR Green Master Mix kit (#MB22403, NZYtech, Lisbon, Portugal) according to the manufacturer's recommendations. The housekeeping gene Actb was used for normalization and fold changes were determined using the 2-AAClmethod7. Primer sequences (SEQ ID NO: 47 to SEQ ID NO: 84) are available in Table 1 and in Table 3.Table 1: List of Rat primer sequencesAnalysis of mitochondrial DNA (mtDNA) copy number

[0076] For the mtDNA copy number, total DNA was extracted from cell pellets using the QIAamp DNA mini kit (Qiagen, Dusseldorf, Germany), following manufacturer's instructions, sonicated in a water bath for 2 min. and quantified using a Nanodrop2000 (ThermoScientific, Waltham, MA, USA). qRT-PCR was performed using the NZYSpeedy qPCR Green Master Mix (2x) (NZYtech; ref. MB22403) in a CFX96 real time-PCR system (Bio-Rad, Hercules, CA, USA), according to the manufacturer's specifications and using the primers described in Table 1, at 4 pM. mtDNA copy number was determined in each sample using the ratio between the amount of a fragment of the mitochondrial cytochrome B (mt CytB) and the amount of the beta-2-microglobulin (B2m) nuclear gene.RNA Sequencing and Data Analysis

[0077] INS1E (4xl04cells) were reverse transfected with 50 nM of miR-642a-3p for 48 h and exposed to GLT conditions (0.75 mM of PA, 25 mM of glucose, 1% FBS (v / v)), for 24 h (n=2). Afterwards, cells were lysed and RNA was isolated using a RNeasy Micro Kit (# 50974034, Qiagen, Hilden, Germany) according to the manufacturer's instructions and quantified on a spectrophotometer. Two biological replicates wereperformed. The mRNA Library construction was done with different steps: (i) the Oligo dT Selection (mRNA enrichment) was done using oligo dT beads to enrich mRNA with poly A tail; (ii) the fragment the RNA and first-strand cDNA was generated using random N6-primed reverse transcription, followed by second-strand cDNA synthesis with dUTP instead of dTTP; (iii) the synthesized cDNA was subjected to endrepair, 3' adenylated and adaptors were ligated to the ends of these 3' adenylated cDNA fragments; (iv) before PCR amplification, the dUTP-marked strand was selectively degraded by Uracil-DNA-Glycosylase (UDG) and the remaining strand was amplified to generate a cDNA library suitable for sequencing. Many rounds of PCR amplification were performed to enrich the purified cDNA template using PCR primer; the PCR product was heat denatured, the single strand DNA was cyclized by splint oligo and DNA ligase followed by a DNA nanoball synthesis and Sequencing on DNBSEQ. (DNBSEQ Technology) platform. Subsequent analysis was performed after the filtered clean reads were aligned to the reference sequence. The sequencing data filtering was done using the software SOAPnuke. Hierarchical Indexing for Spliced Alinment of Transcripts (HISAT) was the software used for mapping RNA-seq reads9and significantly up and downregulated genes (differentially expressed) were selected based on criteria described below. Filtering criteria:

[0078] Compared to the control (lipofectamine-treated cells), transfection of p-cells with 50 nM miR-642a-3p for 48 h resulted in a total of 7495 DEG (3538 up and 3921 down). This list was further filtered based on genes with a log2FC> 111 and FPKM>1 for each of the replicas of one of the conditions, ending up with 1381 genes (903 downregulated and 478 upregulated).

[0079] To analyse the transcriptional changes induced by GLT on p-cells, Volcano plots were constructed with the applied criteria log2FC> 111 , resulting in 3950 genes. From this list, genes with a log2FC> 121 and FPKM>1 for each of the replicas of one of the conditions were selected, ending up with 622 genes (412 downregulated and 210 upregulated) to build the heat map.

[0080] To analyse the transcriptional changes induced by miR-642a-3p on p-cells, volcano plots were constructed with selected genes with a log2FC> 111 , resulting in 4069 genes and from this list, genes with a log2FC> 121 and FPKM>1 for each of the replicas of one of the conditions were selected, ending up with 701 genes: 586 upregulated and 115 downregulated to build the heat map.

[0081] Targets of the miR-642a-3p: the analysis was focused on genes downregulated by miR-642a-3p (903 genes). To that end, genes with a FPKM>1 for each of the replicas of one of the conditions and a log2FC> 11.51 were selected, giving a list of 238 genes.miR-642 binding conservation

[0082] Genome FASTA and GTF files were retrieved from the Ensembl database for human (Homo sapiens GRCh38, Ensembl GTF version 113), mouse (Mus musculus GRCm39, Ensembl GTF version 113), and rat (Rattus norvegicus mRatBN7.2, Ensembl GTF version 113). Mature miRNA sequences for hsa-miR-642a-3p in FASTA format were downloaded from miRbase (release 22.1). For each species, the chromosome position and strand orientation of the Cmtm63'UTR were extracted from the GTF file, and the output was saved into a BED file. A FASTA file with the full length 3'UTR sequences was made by using bedtools (version 2.30.0) getfasta with the option -s to force the strandedness. miRNA binding scores were computed using the miRanda software, with the miRNA FASTA and 3'UTR FASTAs as input. The settings applied were: score threshold of 140, energy threshold of 1 kcal / mol, scaling parameter of 4, gapopen penalty of -4, and gap-extend penalty of -9. Sequence conservation between the human, mouse, and rat 3'UTRs was performed in python, using needle pairwise sequence alignment (emboss version 6.6.0.0; psa version 1.0.1). Sequence conservation, miRNA binding scores, and binding position for each species were visualized using pygenomeviz (version 1.3.0). Code is available at https: / / github.com / tjj-de-winter / miRNA_conservation.GLT-protective miRNA selection

[0083] The GLT-protective miRNA selection was based on literature mining of the transcriptome and proteome of palmitate-treated INS1E cells and / or human islets preparations. Seventeen miRNAs were identified using HTS and selected five (miR-642a-3p, miR20a-5p, miR-17-5p, miR20b-5p, and miR-432-5p) based on their targeting of a higher number of deregulated genes. Other miRNAs were disregarded due to their low expression in plasma-extracellular vesicles (pEVs) of patients undergoing bariatric surgery (BS).Cellular oxygen consumption rate (OCR) measurements

[0084] Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at 379C using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies, Germany). The test performed were the Seahorse XF Cell Mito Stress and Seahorse XF Cell GlicoStress (Agilent Technologies, Santa Clara, CA). INS1E cell line was seeded under the same conditions described above at a density of 75,000 cells / well. A XFe96 sensor cartridge for each cell plate was placed in a 96-well calibration plate containing 200 pL / well calibration buffer and left to hydrate overnight at 379C. The cell culture medium from the plates was replaced the following day with 175 pL / well of pre-warmed, low-buffered, serum-free minimal DMEM (102,353, Bioscience) medium, with the pH adjusted to 7.4 and incubated at 379C for 1 h to allow the temperature and pH of the medium to reach equilibrium before the first-rate measurement. Oligomycin, BAM-15, rotenone, and antimycin A were prepared in DMSO. Two pM oligomycin, 2 pM BAM-15, and 1 pM rotenone plus 1 pM antimycin A were injected into reagent delivery port A, B, and C, respectively. Next, 25 pL of compounds were pre-loaded into the ports, respectively, of each well in the XFe96 sensor cartridge. Three baseline rate measurements of OCR were made using a 3 min mix, 5 min measure cycle. The compounds were then pneumatically injected by the XFe96 Analyzer into each well, mixed, and measurements of OCR made using a 3 min mix, 5 min measure cycle. In the end of the experiment, cells were fixed by adding 50 pL of 60% trichloroacetic acid and stored for at leastone day at 49C. Sulforhodamine B assay was used to evaluate cell mass as described above to normalize results. Results were analyzed by using Software Wave Desktop Version 2.2.Vital epifluorescence microscopy

[0085] Vital epifluorescence microscopy was used to assess alterations in mitochondrial membrane potential and network distribution, as well as to evaluate reactive oxygen species production and lysosomal content in the INS-1E cell line following GLT exposure. Mitochondrial membrane potential (AQjmt) was measured using the potential-sensitive dye TMRM, oxidative stress was assessed using MitoSOX, lysosomal content was evaluated using Lysotracker Red, and nuclear DNA was stained with Hoechst 33,342. After GLT exposure, the fluorescent dyes TMRM (100 nM), MitoSOX (3 pM), Lysotracker Red (75 nM) and Hoescht 33,324 (1 mg / mL) were added to each well in RPMI medium without FBS. Cells were incubated for 15-30 min at 379C with a 5% CO2 atmosphere. Images were obtained using the 40x objective of the In Cell Analyzer 2200 (GE, Healthcare) microscope and were analyzed in the InCell Developer Toolbox program (GE Healthcare).Mitochondria morphogenic parameters

[0086] Images of TMRM-stained INS1E cells were acquired using IN Cell Analyzer. Mitochondrial morphology was analyzed using Fiji (ImageJ) software, defining regions of interest (ROIs) in the TMRM channel by outlining individual cells. Several mitochondrial parameters were evaluated: number, surface area (mitochondrial size), circularity ([4nxsurface area / perimeter2], Feret's diameter (representing the longest distance (um) between any two points within a given mitochondria), and aspect ratio ([major axis - minor axis], indicating the elliptical shape of mitochondria).Caspase-3 and -7 activities

[0087] Caspase-3 and -7 activities were measured using the Caspase-Gio 3 / 7 assay (Promega, G8090), following the manufacturer's instructions. Briefly, after GLT exposure, the 96 well plates were removed from the incubator and equilibrate at room temperature for 5 min. Then, the cell culture medium was removed and freshly 50 pL of RPMI medium without FBS was added, following with plus 50 pL of Caspase 3 / 7 reagent to each well. After a gently mix use a plate shaker at 300 rpm for 30 sec. and incubate for 30 min. at room temperature. Luminescence was measured in a Biotek Cytation 3 spectrophotometer (BioTek Instruments Inc., USA).Bariatric CohortStudy Population

[0088] All samples were obtained from patients from the surgery department of the Centro Hospitalar e Universitdrio de Santo Antonio (CHUdSA) after receiving an informed consent. This study was approved by the Ethics Commission of the CHUdSA with the reference number 2020-075 (060-DEFI. 061-CE). Withsupport from our National Health Service facilities and adherence to the established project protocol, no costs were incurred by the patient.Sampling

[0089] To obtain plasma, blood samples were collected from each patient in a labelled EDTA tube. Centrifugation was performed at 15000 x g for 10 minutes at 4 °C and the supernatant was stored at -80 °C until use.Animal study

[0090] Animal experiments were conducted in compliant with the Animal Care National and European Directives. The project received approval (#3 / 2022) from the local animal welfare body (ORBEA). The animal study was performed in a mouse model of diabetes induced by a hypercaloric diet, using 13, 6 weeks old male mice C57BL / 6J strain, purchased from Charles-River and maintained in the Coimbra Institute for Clinical and Biomedical Research (iCBR) animal facility at Faculty of Medicine of the University of Coimbra (FMUC). After 2 weeks of quarantine, the animals were randomly divided into the following groups, in a 20-week protocol: control group (n=6) - mice maintained ad libitum on standard chow; diabetic group (n=7) - mice maintained ad libitum with hypercaloric diet (60% carbohydrates, Mucedola, Ref. PF4215). Throughout the study, the weekly evolution of their weight was monitored. In vivo, glucose tolerance tests (GTT) and insulin sensitivity tests (ITT) were performed at weeks 19 and 20 of the study. The sample size was determined with an alpha level set at 0.05 and a power level at 0.95. The variation in the group sizes stems from the loss of an animal in the control group during an in vivo experimental procedure (ITT test). Measures were considered to follow the 3Rs principles, such as, a small number of mice (n=7 per group). The sample size was calculated using G*power software). In terms of refinement, the team members that carried out the animal procedures are very experienced in in vivo tests (including GTT and ITT) and in ex vivo determinations, an appropriate species and strain was used, which allows conclusions to be drawn, a robust model was chosen, excellent conditions in terms of animal facility were available, and monitoring of animal welfare was thorough. Critical limits, such as food / water intake, weight loss weight loss of more than 15% (fluid therapy was used if necessary), and faeces consistency, were monitored, and animals were humanely euthanized under deep anaesthesia. Animal behaviour and body appearance were also closely observed throughout the study. The animals were sacrificed with an anaesthetic overdose and blood was collected for separation into serum. Liver and pancreas were collected for miRNA profile and immediately stored at -80 °C until use.Metabolic characterization

[0091] On the day of sacrifice, after a fasting period of 6 h, glucose levels were determined. A drop of blood was collected from the jugular vein through venipuncture and measured using an Accu-Chek® Aviva glucometer (Roche, Mannheim, Germany).

[0092] Glucose tolerance test (GTT): On week 19, mice were administered intraperitoneally with a glucose bolus of 2 g / kg BW following a 6-hour fasting period. Blood glucose levels were quantified through the tail vein before the injection and 15, 30, 45 and 60 min after, using the portable device Accu-Chek® Aviva glucometer (Roche, Mannheim, Germany). The area under the curve (AUC) for the GTT was calculated by using the trapezoidal method, as previously described.

[0093] Insulin tolerance test (ITT): On week 20, 0.75 U / kg BW of insulin was administrated intraperitoneally (Actrapid Novo Nordisk) following a 6-hour fasting period. A drop of blood was collected from the tail vein before the bolus and blood glucose levels were measured using the portable device Accu-Chek® Aviva glucometer (Roche, Mannheim, Germany) 30, 60 and 120 min after. The area under the curve (AUC) for the ITT was calculated by using the trapezoidal method.

[0094] Serum levels of total cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were assessed by colorimetric methods using an automated analyzer (Hitachi 717, Roche Diagnostics, Mannheim, Germany).Pancreas and liver miRNA profile by RT-PCR

[0095] Total RNA was extracted using the Trizol® reagent (Life Technologies, Carlsband, CA, USA) according to the manufacturer's protocol. RNA concentration and purity were determined using NanoDrop® (Thermo Fisher Scientific, Waltham, MA, USA). Next, RNA was reverse transcribed using 5x miRCURY RT reaction buffer, and lOx miRCURY RT Enzyme Mix, and the obtained cDNA was used to quantify miRNA expression by quantitative real-time PCR analysis. For this, PCR products were amplified using the NZYSpeedy qPCR Green Master Mix kit (#MB22403, NZYtech) according to the manufacturer's instructions. Normalization was done to U6 and fold changes were determined using the 2'AACtmethod.Plasma EVs isolation

[0096] Isolation of plasma EVs (pEVs) was performed as previously described with small modifications. Briefly, samples were centrifuged at 3000 x g for 15 min to remove cells and cell debris. The supernatant was mixed with 5 U / mL of human Thrombin (#T7009, Sigma Aldrich) for 5 min and centrifuged at 10000 x g for 5 min to remove fibrinogen. Next, the corresponding amount of ExoQuick™ (#EXOTC10A-1, System BioSciences) was added according to the manufacturer's instructions (ratio 5:1 (v / v)), mixed and incubated for 45 min at 4 °C. Samples were centrifuged at 1,500 x g for 30 min, resuspended in a final volume of 150 pL, subjected to 2 pg / mL RNase (# R4875, Sigma Aldrich) treatment for 30 min at room temperature, and re-purified again via ExoQuick™ . Finally, pEVs were ressuspended in 150 pL of PBS and stored at -80 °C.EV characterizationNanoparticle tracking analysis (NTA)

[0097] The size and concentration of the pEVs were calculated using a NanoSight NS300 (Malvern Instruments, Malvern, U.K.). To obtain a reliable reading, pEVs were diluted in PBS until a value of 15 - 45 particles / frame was obtained. The PBS used was confirmed to be pure (<5 particles per frame). For each sample, 5 videos of 30 sec were recorded with the camera level set at 13. All the videos were processed with NTA 3.0 analytical software.Zeta potential

[0098] Surface charge of pEVs was measured using the NanoBrook ZetaPALS Potential Analyzer (Brookhaven Instruments Corporation, Long Island, USA). Per sample, 5 pL of EVs were diluted in 1500 pL of biological grade ultrapure water (Fisher Scientific, New Hampshire, USA), the EVs were placed in contact with the zeta potential electrode and allowed to stabilize for 10 min. Five runs (using the Smoluchowski module) were performed for each sample at room temperature.Protein quantification

[0099] Quantification of the total protein of pEVs was performed using the microBCA™ protein assay kit (Thermo Fisher Scientific, Massachusetts, USA), following the manufacturer's recommendations. In short, a 10-point standard curve was obtained with BSA. For disrupting the EV membrane, samples were diluted in 2% (v / v) sodium dodecyl sulphate (SDS) at room temperature. Then, 50 pLof the latest mixwas pipetted in duplicate in a 96-well Corning® Costar® cell culture plates (Corning Inc., New York, USA), the reaction solution was added and incubated for 2 h at 37 °C. Finally, the plate was equilibrated at room temperature for 15 min and the absorbance at 562 nm was read in the microplate reader SynergyTM Hl (Biotek, Vermont, USA).EV-miRNA signature by RT PCR

[0100] RNA from plasma-derived EVs was isolated by miRNeasy Micro Kit (# 217084, Qiagen, Hilden, Germany) according to the manufacturer's instructions and quantified on a spectrophotometer. Next, RNA was reverse transcribed using a 5x miRCURY RT reaction buffer, and lOx miRCURY RT Enzyme Mix. The resulting cDNA was used to quantify miRNA expression by quantitative real-time PCR. For this, PCR products were amplified using the NZYSpeedy qPCR Green Master Mix kit (#MB22403, NZYtech) according to the manufacturer's instructions. Normalization was done to 5S. miRNA-EV signature was represented as copies of each miRNA per particle.Statistics

[0101] GraphPad Prism (version 9) software was used to perform statistical analysis. All experiments were performed in at least two independent experiments. Results were expressed as the mean ± SD. For statistical comparison, analysis of variance (ANOVA) or t-tests were used as indicated.ResultsIdentification of miRNAs capable of protecting p-cells from GLT-induced cell death

[0102] In an embodiment, INS1E cells, insulin producing p-cells known to be susceptible to PA-induced apoptosis were selected for the high-throughput screening (HTS) assay. INS1E cells were exposed to increasing concentrations of PA for 24 h in media containing 1% (v / v) FBS and either 11 or 25 mM glucose. Metabolic activity and cell mass were quantified as indirect measurements of metabolism, viability and proliferation, respectively. The glucolipotoxicity (GLT) condition selected for the HTS assay (0.75 mM PA and 25 mM glucose in media with 1% (v / v) FBS) induced a 62% and a 71% decrease in metabolic activity and cell mass, respectively. A library containing 2080 human miRNA mimics was screened (Fig. 1 A). A non-targeting miRNA (miR-SRC) and a Lipofectamine (LP) control were included in the HTS assay. miRNAs capable of increasing the metabolic activity and / or cell mass more than three times the mean of the GLT condition were considered a hit. On this basis, 45 hits (SEQ ID NO: 1 to SEQ ID NO: 45) were identified: 17 miRNAs (SEQ ID NO: 1 to SEQ ID NO: 17) increased the metabolic activity and 28 (SEQ ID NO: 18 to SEQ ID NO: 45) the cell mass (Fig. 1 B). Notably, 7 miRNAs [miR-432-5p (SEQ ID NO: 12), miR-370 (SEQ ID NO: 6), miR-377-3p (SEQ ID NO: 8), miR-17-5p (SEQ ID NO: 1), miR-642b-3p (SEQ ID NO: 16), miR-372 (SEQ ID NO: 7) and miR-20a-5p (SEQ ID NO: 2)], increased both metabolic activity and cell mass (blue dots; Fig. 1 B).Using the same experimental conditions of the primary screening and three technical replicates, these 7 miRNAs were able to protect p-cells from GICD (Fig. 1 C).miR-642a-3p protects p-cells from GLT-induced cell death and loss of function

[0103] In an embodiment, one of the hits, miR-642b-3p (SEQ ID NO: 16), belongs to the same family as miR-642a-3p (SEQ ID NO: 46), a miRNA recently identified as capable of enhancing the survival of endothelial cells (ECs) exposed to ischemia- mimicking conditions26. These two miRNAs differ by one single nucleotide and, more importantly, share the same seed sequence (Fig. 1 D).

[0104] In an embodiment, given the potential impact of a single miRNA on two phenotypes highly relevant to diabetes (protection against GLT and survival of ECs), miR-642a-3p was selected for mechanistic studies.

[0105] In an embodiment, it was confirmed that miR-642a-3p effectively protected p-cells from GICD resulting in a 1.57-fold increase in metabolic activity and 1.97-fold increase in cell mass, compared to GLT condition (Fig. 1 E-F).

[0106] In another embodiment, the impact of GLT and miR-642a-3p was determined in p-cell function. The results showed that p-cells increased insulin secretion in response to glucose stimulation, reverting to baseline levels upon exposure to low glucose concentration. Compared to the control, exposure of p-cells to GLT led to a significant increase in insulin secretion, regardless of the glucose concentration. Notably, transfection of p-cells with miR-642a-3p protected p-cells from the changes elicited by GLT (Fig.2 A-B). These results were further confirmed using immunofluorescence and qRT-PCR (Fig. 2 C-E).miR-642a-3p opposes GLT-induced transcriptional changes in p-cells

[0107] In an embodiment, an RNA-Seq analysis showed that, compared to the control, exposure of p-cells to GLT resulted in a total of 4666 differentially expressed genes (DEG) (4221 upregulated and 445 downregulated). Volcano plots and hierarchical clustering were generated to compare gene expression between GLT and bovine serum albumin (BSA)-treated p-cells (Fig. 3 A-B). These analyses showed that genes upregulated by GLT were associated with positive regulation of apoptotic processes, intrinsic apoptotic signalling pathways in response to endoplasmic reticulum stress, cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MARK) signalling pathway and apoptosis whereas down-regulated genes were associated with cell division, chromosome segregation, mitotic cytokinesis, cell cycle and homologous recombination (Fig.3 C-D). Overall, these results suggest that GLT treatment down-regulates cell division pathways and upregulates those involved in inflammation and apoptosis.

[0108] In an embodiment, compared to GLT-treated p-cells, transfection with miR-642a-3p followed by GLT exposure resulted in a total of 5039 DEG (2969 upregulated and 2070 downregulated). Volcano plots and hierarchical clustering were generated to compare gene expression between miR-642a-3p and GLT-treated p-cells (Fig. 3 E-F). These analyses showed that genes upregulated by miR-642a-3p were associated with chromatin remodelling, cell migration, mitotic cytokinesis and axon guidance, focal adhesion, MARK signalling pathway and phosphatidylinositol 3-kinase / protein kinase B (PI3K-Akt) signalling pathway whereas down-regulated genes were associated with intrinsic apoptotic signalling pathway in response to endoplasmic reticulum stress, negative regulation of fat cell differentiation, positive regulation of apoptotic process and dopaminergic synapse, relaxin signalling pathway and insulin signalling pathway (Fig. 3 G-H).

[0109] In an embodiment, given the fact that the putative targets of miR-642a-3p can be found among the downregulated genes, p-cells were transfected with miR-642a-3p for 48 h and, compared to the control. The results showed a total of 7495 DEG (3538 up and 3921 down). Using the filtering criteria described in the methods, functional annotation and gene ontology (GO) enrichment analysis revealed that the upregulated genes were involved in synapse organization, neuron differentiation and regulation of inflammatory responses whereas the downregulated genes were involved in synapse assembly and miRNA loading onto the RNA-induced silencing complex (RISC). From the list of downregulated genes, and after applying the filtering criteria, 238 genes were selected. To narrow the search, this list was intersected with the list of putative targets retrieved from TargetScan and miRWalk, resulting in a final list of 72 genes (set A). Finally, this list of 72 putative targets was intersected with the list of genes downregulated upon transfection of p-cells with miR-642a-3p followed by exposure to GLT (115 genes) (set B), resulting in 7 common genes: Wdrl3, Zdhhc7, PrkabZ, Cmtm6, Akt2, Dusp4 and Dnajc27 (Fig. 3 I). To validate these targets, an independent experiment was performed, and RT-PCR was used to analyse the expression of the selected genes. Compared to the control, the results showed that exposure of p-cells to GLT did notaffect the expression of WdrlS, PrkabZ, Akt2 and Dnajc27 but inhibited the expression of Zdhhc7. However, upon transfection of p-cells with miR-642a-3p, a statistically significant inhibition in the expression of WdrlS, Zdhhc7, Dusp4 and Dnajc27 was observed and a trend towards decreased expression of Cmtm6 and Akt2 (Fig. 3 J). To further validate that the inhibition of these genes could phenocopy the protective effects observed with miR-642a-3p, WdrlS, Zdhhc7, Dusp4, Dnajc27 and Cmtm6 were individually knockdown using short-interference RNAs (siRNAs) under conditions similar to those reported for the primary screening and subsequent validation assays. These results showed that only the inhibition of Cmtm6 was able to recapitulate the protective effect observed with miR-642a-3p (Fig. 3K). In silica prediction pairing between the hsa-miR-642a-3p and the 3'- UTR region of rat Cmtm6 mRNA is shown in Figure 4. Overall, these findings suggest that Cmtm6 could be one of the targets of miR-642a-3p.miR-642a-3p restores the expression of (3-cell identity markers after exposure to GLT and activates prosurvival signalling pathways

[0110] In an embodiment, the impact of GLT and miR-642a-3p in the expression of genes related to p-cell identity (Foxa2, Nkx6.1, Mafa and Pdxl) was analysed (Fig.5 A-D). The RNA-Seq results indicated that exposure of p-cells to GLT downregulated the expression of Foxa2 and that miR-642a-3p restored its expression. However, in an independent experiment and using RT-PCR analysis, no differences in the expression of Foxa2 were observed (Fig. 5 A). Compared to the control, exposure of p-cells to GLT significantly downregulated the expression of Nkx6.1 and Mafa whereas transfection with miR-642a-3p was sufficient to maintain the expression of these genes (Fig. 5 B-C). Moreover, compared to the BSA control and the GLT condition, miR-642a-3p significantly upregulated the expression of Pdxl (Fig. 5 D).Interestingly, inhibition of Cmtm6 followed by exposure to GLT also resulted in the upregulation of the p-cell identity gene Pdxl, but not Nkx6.1, Foxa2 and Mafa (Fig.6). This evaluation was extended to genes involved in lipid metabolism (fatty acid synthase (Fasn)), oxidative stress (catalase (Cat)) and thioredoxinbinding protein (Txnip)) and p-cell function (Paired Box 6 (Pax6)) (Fig. 7). RNA-Seq analysis revealed that exposure of p-cells to GLT resulted in the downregulation of Fasn, Txnip and Pax6 genes. Remarkably, transfection with miR-642a-3p increased the expression of Fasn, Cat and Pax6 compared to the GLT condition. By RT-PCR analysis, and compared to the control, a statistically significant downregulation of Fasn following exposure to GLT was demonstrated and transfection with miR-642a-3p was unable to rescue Fasn expression. In the case of Cat, compared to the control, there was a significant upregulation upon exposure to GLT which was further enhanced upon transfection with miR-642a-3p. In the case of Txnip, results showed no significant changes in expression upon exposure to GLT whereas transfection with miR-642a-3p led to a significant increase in Txnip expression. Compared to the control, a downregulation of Pax6 following exposure to GLT was observed, and, upon transfection with miR-642a-3p, Pax6 expression was rescued. Given the crucial role of glucagon-like peptide-1 receptor (Glplr) in p-cell survival and proliferation27it was showed that transfection of p-cells with miR-642a-3p, in the presence of GLT, led to a significant upregulation of Glplr (Fig.8).

[0111] In an embodiment, considering that miR-642a-3p protects p-cells from GICD, and since the PI3K signalling pathway - a well-established pro-survival pathway - was shown to be modulated in GO enrichment analysis, further analysis was conducted to dissect the role of this pathway. Compared to the control, the transfection of p-cells with miR-642a-3p downregulated PTEN expression and increased the phosphorylation of the downstream targets AKT and p44 / 42 MAPK, both previously downregulated by GLT exposure (Fig. 5 E-F). These results showed that exposure of p-cells to GLT did not change the cytoplasmic:nuclear ratio of FOXO1 (a well-established AKT target) but, upon transfection of p-cells with miR-642a-3p, a 1.7-fold increase in cytoplasmic:nuclear ratio of FOXO1 (Fig. 5 G-H) was observed. Collectively, these results demonstrate the capacity of miR642a-3p to modulate classical pro-survival pathways ultimately protecting p-cells from GICD.miR-642a-3p increases lipid accumulation in p-cells

[0112] In an embodiment, it was investigated whether palmitate (PA) influences cellular lipid dynamics and if the protective effects observed upon transfection of p-cells with miR-642a-3p could involve changes in lipid metabolism. Compared to the control, the results demonstrated a statistically significant increase (3.8-fold) in lipid droplets (LD) accumulation upon exposure of miR-642a-3p transfected p-cells to GLT (Fig.9 A-B). It was also shown that exposure of p-cells to GLT in the presence of oleate recapitulated the increase in LD accumulation observed upon transfection with miR-642a-3p. Of note, LD accumulation did not increase in p-cells exposed to 0.5 mM PA and 25 mM glucose. Given the involvement of Plin2 and Plin5 in lipid metabolism28, their expression in the RNA-Seq dataset previously presented. These results showed that, compared to the control, miR-642a-3p increased the expression of Plin2 and Plin5 (Fig.9 C).Taken together, these results suggest that both oleate and miR-642a-3p exert a protective effect on p-cells exposed to GLT, likely by improving their capacity to accumulate lipids intracellularly. Similarly to the results obtained for miR-642a-3p, the increase in lipid accumulation by oleate was accompanied by an increase in cell survival (Fig.9 D).miR-642a-3p protects cells against GLT-induced apoptotic cell death

[0113] miRNAs can act as crucial post-transcriptional regulators of mitochondrial function, influencing metabolism, dynamics, apoptosis, and redox signaling (PMID: 39627218). Vital epifluorescence imagingof cells loaded with the AQjmt-sensitive fluorescent dye TMRM was carried out to evaluate the effects of miR-642a-3p in mitochondrial network morphology. I an embodiment, both GLT and miR-642a-3p+GLT induced mitochondrial hyperpolarization (Fig. 10A and Fig. 10A i). Morphometric analysis revealed that the GLT condition decreased mitochondrial circularity (Fig. 10A iii) while increasing area (Fig. 10A iv) and Feret's diameter (Fig. 10A vi). In contrast, miR-642a-3p overexpression increased the number of mitochondrial particles (Fig. 10A ii), area (Fig. 10A iv), aspect ratio (Fig. 10A v), and Feret's diameter (Fig.10A vi), while decreasing circularity (Fig. 10A iii), indicating changes in mitochondrial morphology and network organization. To investigate the effects of miR-642a-3p on mitochondrial biogenesis, we measured mitochondrial DNA copy number. As shown in Fig. 10B, mitochondrial DNA content was increased in both GLT and GLT+miR-642a-3p conditions. Mitochondrial bioenergetic function was further evaluated using Seahorse XFe96 Extracellular Flux Analyzer. GLT treatment significantly reduced oxygen consumption rate (OCR) (Fig. IOC), including basal respiration (Fig. IOC i), ATP-linked OCR (Fig. IOC ii), and maximal respiration (Fig. IOC iii). In contrast, GLT+miR-642a-3p significantly increased these parameters compared with the GLT group, indicating improved mitochondrial biogenesis and respiratory capacity (Fig. IOC). Mitochondrial oxidative stress and quality control pathways, including apoptosis and lysosomal activity, were assessed. GLT treatment increased mitoSOX fluorescence intensity (Fig. 10D) and caspase-3 / 7 activity (Fig. 10F), while no significant changes were observed in Lysotracker Red fluorescence (Fig.10E). In the GLT+miR-642a-3p group, caspase-3 / 7 activity was significantly reduced (Fig. 10F), accompanied by increased Lysotracker Red fluorescence (Fig. 10E). Collectively, these results reveal that miR-642a-3p protects cells against GICD by promoting mitochondrial biogenesis and mitophagy, thereby facilitating the removal of damaged mitochondria and the restoration of functional mitochondrial networks.Cmtm6 knockdown protects against GICD in a mitochondrial-independent manner

[0114] Afterwards, the effects of Cmtm6 knockdown on mitochondrial function were assessed, as Cmtm6 is a predicted target of miR-642a-3p. Vital epifluorescence imaging of cells loaded with the AQjmt-sensitive fluorescent dye TMRM was carried out to evaluate the impact of Cmtm6 knockdown on mitochondrial network morphology. In an embodiment, while GLT induced mitochondrial hyperpolarization, Cmtm6 knockdown restored mitochondrial membrane potential to control levels (Fig. HAi).Morphometric analysis revealed that GLT treatment decreased mitochondrial circularity (Fig. 11A iii) while increasing area (Fig. 11A iv) and Feret's diameter (Fig. 11A vi). In contrast, Cmtm6 knockdown reduced mitochondrial area (Fig. 11A iv), and circularity (Fig. 11A iii), indicating significant alterations in mitochondrial morphology and network organization. To assess the effects of Cmtm6 knockdown on mitochondrial biogenesis, mitochondrial DNA copy number was measured. As shown in Fig. 11B,mitochondrial DNA content was increased under GLT conditions but not in the Cmtm67“+GLT group, revealing that Cmtm6 may be required for GLT-induced mitochondrial biogenesis. Mitochondrial bioenergetic function was further evaluated using Seahorse XFe96 Extracellular Flux Analyzer. Both GLT treatment and Cmtm6 knockdown significantly reduced oxygen consumption rate (OCR) (Fig. 11C), including basal respiration (Fig. 11C i), ATP-linked respiration (Fig. 11C ii), and maximal respiration (Fig.11C iii), indicating impaired mitochondrial respiratory capacity in both conditions. Mitochondrial oxidative stress and quality control pathways were examined, including apoptosis and lysosomal activity. GLT treatment increased mitoSOX fluorescence intensity (Fig. 11D) and caspase-3 / 7 activity (Fig. 11F), while no significant changes were observed in Lysotracker Red fluorescence (Fig. HE). In the GLT+Cmtm67“ group, mitoSOX fluorescence was further increased compared with both control and GLT conditions, whereas caspase-3 / 7 activity was significantly reduced relative to GLT alone (Fig. 11F). Collectively, these results indicate that Cmtm6 knockdown confers protection against GLT-induced apoptotic cell death despite exacerbating mitochondrial oxidative stress and respiratory dysfunction. This reveals that the anti-apoptotic effect of Cmtm6 depletion is likely mediated through mitochondria-independent survival pathways or alternative stress-response mechanisms rather than through direct improvement of mitochondrial function.miRNA-mediated protection in pancreas and liver of hypercaloric-fed mice

[0115] In an embodiment, the selection of GLT-protective miRNAs identified in the screening for further experiments was guided by comprehensive literature mining of the transcriptome and proteome datasets from palmitate-treated INS1E cells and / or human islet preparations. The expression of, miR-17-5p, miR-20a-5p, miR-20b-5p and miR-432-5p in the pancreas and liver of mice fed with a hypercaloric diet was investigated (Fig. 12 A). Overall, the results showed that compared to control mice, the expression of the selected miRNAs was downregulated, without reaching statistical significance, in mice fed with a hypercaloric diet, both in the liver and pancreas (Fig. 12 B-C). These results suggest that mice fed with a hypercaloric diet showed a reduction in the levels of GLT-protective miRNAs. Since miR-642-3p is not conserved in mice, analysed publicly available datasets were analysed to assess its expression in human islets from control and T2DM donors. Analysis of two independent datasets revealed a downregulation of miR-642-3p in T2DM-derived islets, supporting the findings that it may play a protective role in this context (Fig. 12 D).miRNA-EV signature in plasma of patients with obesity undergoing BS

[0116] In an embodiment, given that GLT leads to p-cell dysfunction and death, phenomena frequently associated with obese and / or diabetic patients, the expression of the same five GLT-protective miRNAs mentioned above (miR-642a-3p, miR-17-5p, miR-20a-5p, miR-20b-5p and miR-432-5p) were analysed in EVs isolated from the plasma of patients undergoing BS (Fig. 13 A). These results showed that, before surgery, the diabetic group had 7.25% HbAlc whereas the non-diabetic group had 5.6%. As expected, BSresulted in a reduction in body mass index (BMI), decreasing from 44.62 before surgery to 35.48 Kg / m2one-month post-surgery (Fig. 13 B). Importantly, these results showed that prior to surgery, obese nondiabetic patients exhibited higher levels of GLT-protective miRNAs in plasma-derived EVs, in contrast to their obese diabetic counterparts. Moreover, the number of copies of miR-642a-3p per EV were statistically significant higher in non-diabetic patients (1.26xl0-5± 2.05xl0-5) compared to diabetic patients (1.65xl0-6± 2.81xl0-6) before surgery. Interestingly, for both groups of patients, there was an increase in the expression of GLT-protective miRNAs between pre-surgery and one month after surgery (Fig. 13 C). Overall, these results may suggest that elevated levels of GLT-protective miRNAs play a protective role in obese patients against diabetes.miR-372 protects cells against GICD

[0117] Considering the seven miRNAs identified in the primary screening as capable of increasing both metabolic activity and cell mass, the effects of miR-372 on mitochondrial function were further assessed. Vital epifluorescence imaging of cells loaded with the AQjmt-sensitive fluorescent dye TMRM was carried out to evaluate the impact of miR-372 on mitochondrial network morphology. In an embodiment, GLT induced mitochondrial hyperpolarization, whereas miR-372 restored mitochondrial membrane potential to control levels (Fig. 14A i). Morphometric analysis revealed that the GLT condition decreased circularity (Fig. 1A iii) while increasing area (Fig. 1A iv). In contrast, miR-372 decreased area (Fig. 14A iv). No significant effects were observed on mitochondrial number (Fig.14A ii), aspect ratio (Fig.14A v), or Feret's diameter (Fig. 14A vi). To investigate the effects of miR-372 on mitochondrial biogenesis, mitochondrial DNA copy number was measured. As shown in Fig. 14B, mitochondrial DNA content was increased under GLT conditions but significantly reduced in the GLT+miR-372 group, suggesting that miR-372 interferes with GLT-induced mitochondrial biogenesis. Mitochondrial bioenergetic function was further evaluated using Seahorse XFe96 Extracellular Flux Analyzer. Both GLT treatment and miR-372+GLT significantly reduced oxygen consumption rate (OCR) (Fig. 14C), including basal respiration (Fig. 14C i), ATP-linked respiration (Fig. 14C ii), and maximal respiration (Fig. 14C iii), indicating that miR-372 does not reverse GLT-induced impairment of mitochondrial respiratory capacity. Mitochondrial oxidative stress and quality control pathways were evaluated, including apoptosis and lysosomal activity. GLT treatment increased caspase-3 / 7 activity (Fig. 14F), while no significant changes were observed in mitoSOX (Fig. 14D) or Lysotracker Red fluorescence (Fig. 14E). In the GLT+miR-372 group, mitoSOX fluorescence was significantly increased compared with both control and GLT conditions (Fig. 14D), whereas caspase-3 / 7 activity was markedly reduced relative to GLT alone (Fig. 14F). Moreover, miR-372 decreased Lysotracker Red fluorescence compared with both control and GLT groups (Fig. 14E), indicating reduced lysosomal activity. Collectively, these results indicate that miR-372 protects cells against GICD despite promoting mitochondrial oxidative stress, suppressing mitochondrial biogenesis, and failing to restore respiratoryfunction. This reveals that the anti-apoptotic effects of miR-372 are mediated primarily through mitochondria-independent survival pathways or alternative stress-response mechanisms, rather than through direct preservation of mitochondrial integrity or bioenergetic capacity.miR-17 protects cells against GLT-induced apoptotic cell death

[0118] Next, the effects of miR-17-5p on mitochondrial function were assessed. Vital epifluorescence imaging of cells loaded with the Aipmt-sensitive fluorescent dye TMRM was carried out to evaluate the impact of miR-17-5p on mitochondrial network morphology. In an embodiment, GLT induced mitochondrial hyperpolarization, whereas miR-17-5p restored mitochondrial membrane potential to control levels (Fig. 15A i). Morphometric analysis revealed that the GLT condition decreased circularity (Fig.15A iii) while increasing mitochondrial area (Fig. 15A iv). In contract, miR-17-5p decreased circularity (Fig. 15A iii), and area when compared with GLT condition (Fig. 15A iv). No significant effects were observed on mitochondrial number (Fig. 15A ii), aspect ratio (Fig.15A v), or Feret's diameter (Fig.15A vi).To investigate the effects of miR-17-5p on mitochondrial biogenesis, we measured mitochondrial DNA copy number. As shown in Fig. 15B, mitochondrial DNA content was increased both in GLT and GLT+miR-17-5p conditions, suggesting enhanced mitochondrial biogenesis. Mitochondrial bioenergetic function was further evaluated using Seahorse XFe96 Extracellular Flux Analyzer. GLT treatment significantly reduced oxygen consumption rate (OCR) (Fig.15C), including basal respiration (Fig. 15C i), ATP-linked OCR (Fig.15C ii), and maximal respiration (Fig.15C iii). In contrast, GLT+miR-17-5p significantly increased these parameters compared with the GLT group, indicating improved mitochondrial respiratory capacity and bioenergetic function (Fig. 15C). Mitochondrial oxidative stress and quality control pathways, including apoptosis and lysosomal activity. GLT treatment increased caspase-3 / 7 activity (Fig. 15F), while no significant changes were observed in mitoSOX (Fig. 15D) and Lysotracker Red fluorescence (Fig. 15E). In the GLT+miR-17-5p group, Lysotracker Red fluorescence was significantly decreased (Fig. 15E), whereas caspase-3 / 7 activity was markedly reduced relative to GLT alone (Fig. 15F). Collectively, these results indicate that miR-17-5p protects cells against GLT-induced apoptotic cell death by restoring mitochondrial membrane potential, enhancing mitochondrial bioenergetic capacity, and improving cellular stress resilience.

[0119] In an embodiment, the miRNA sequences can be selected from a list consisting in the sequences disclosed on Table 2.Table 2: List of the miRNA sequences.

[0120] In an embodiment, the sequences disclosed herein are selected from Table 3.Table 3. Sequences of the present disclosure.

[0121] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.

[0122] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0123] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0124] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0125] As used herein, the term "about" a number refers to that number plus or minus 10% of that number. The term "about" a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0126] As used herein, the terms "pharmaceutically acceptable" and "cosmetically acceptable" are used interchangeably and refer to those compounds, materials, compositions, and / or dosage forms which are,within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. More specifically, pharmaceutically acceptable refers to a material, compound, or composition which is suitable for use in contact with the skin, scalp, or hair. Pharmaceutically acceptable materials are known to those of ordinary skill in the art.

[0127] As used herein, the terms "treatment" or "treating" are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made. Unless otherwise specified, the term encompasses both human and animal subjects.

[0128] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first numerical value in a series of two or more numerical values, the term "at least," "greater than" or "greater than or equal to" applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0129] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0130] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0131] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinaryskill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0132] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0133] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

[0134] The above-described embodiments are combinable.

[0135] The following claims further set out particular embodiments of the disclosure.References1. Eizirik, D.L., Pasquali, L., and Cnop, M. (2020). Pancreatic p-cells in type 1 and type 2 diabetes mellitus: different pathways to failure. Nat. Rev. Endocrinol. 16, 349-362. 10.1038 / s41574-020-0355-7.2. Lytrivi, M., Castell, A.L., Poitout, V., and Cnop, M. (2020). Recent Insights Into Mechanisms of -Cell Lipo- and Glucolipotoxicity in Type 2 Diabetes. J. Mol. Biol. 432, 1514-1534. 10.1016 / j.jmb.2019.09.016.3. Cnop, M., Abdulkarim, B., Bottu, G., Cunha, D.A., Igoillo-Esteve, M., Masini, M., Turatsinze, J.V., Griebel, T., Villate, O., Santin, I., et al. (2014). RNA sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate. Diabetes 63, 1978-1993. 10.2337 / dbl3-1383.4. Lee, S.H., Cunha, D., Piermarocchi, C., Paternostro, G., Pinkerton, A., Ladriere, L., Marchetti, P., Eizirik, D.L., Cnop, M., and Levine, F. (2017). High-throughput screening and bioinformatic analysis to ascertain compounds that prevent saturated fatty acid-induced 3-cell apoptosis. Biochem. Pharmacol. 138, 140-149. 10.1016 / j.bcp.2017.05.007.5. Sandoval, D.A., and Patti, M.E. (2023). Glucose metabolism after bariatric surgery: implications for T2DM remission and hypoglycaemia. Nat. Rev. Endocrinol. 19, 164-176. 10.1038 / s41574-022-00757-5.

Claims

C L A I M S1. An isolated or artificial ribonucleic acid sequence for use in any condition susceptible of being improved or prevented by modulating GLT, comprisinga nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, or SEQ ID NO: 85 to SEQ ID NO: 109;or an isolated double-stranded ribonucleic acid molecule comprising an antisense strand that is complementary to CMTM6 mRNA wherein the isolated double-stranded ribonucleic acid is capable of knockdown of CMTM6 expression.

2. The isolated or artificial ribonucleic acid sequence for use according to the previous claim, wherein the antisense strand comprises a nucleotide sequence at least 91 %, 92%, 93%, 94% or 95% identical to at least one of the following sequences: SEQ ID NQ:110 or SEQ ID NO:111.

3. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims, wherein the antisense strand comprises a nucleotide sequence at least 96%, 97%, 98%, 99%, or 100% identical to at least one of the following sequences: SEQ ID NQ:110 or SEQ ID NO:111.

4. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims comprising a nucleotide sequence at least 91 %, 92%, 93%, 94% or 95% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111.

5. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims comprising a nucleotide sequence at least 96%, 97%, 98%, 99%, or 100% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111.

6. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims comprising a nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably the nucleotide sequence is 91%, 92%, 93%, or 94% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111.

7. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims comprising a nucleotide sequence at least 95% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46 and / or SEQ ID NO: 85 to SEQ ID NO: 111; preferably the nucleotidesequence is 96%, 97%, 98%, 99%, or 100% identical to at least one of the following sequences: SEQ ID NO: 1 to SEQ ID NO: 46, and / or SEQ ID NO: 85 to SEQ ID NO: 111.

8. The isolated or artificial ribonucleic acid sequence according to any of the previous claims, for use in the treatment or prevention of glucolipotoxicity-associated disorders.

9. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims, comprising a nucleotide sequence at least 90% identical to at least one of the following sequences: SEQ ID NO: 16 to SEQ ID NO: 46 and / or SEQ ID NO: 85 to SEQ ID NQ:109; preferably SEQ ID NO: 16 or SEQ ID NO: 46 and / or SEQ ID NO: 85 to SEQ ID NO: 111.

10. The isolated or artificial ribonucleic acid sequence for use according to any of the previous claims, comprising a nucleotide sequence at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 46, SEQ ID NO: 110, or SEQ ID NO: 111 .

11. A vector or a construct for use in any condition susceptible of being improved or prevented by modulating glucolipotoxicity comprising the isolated or artificial ribonucleic acid sequence as described in any of the previous claims.

12. The vector for use according to the previous claim, wherein the vector is selected from the group consisting of: viral vectors, plasmids, delivery systems, extracellular vesicles, nanoparticles, dendrimers, or virus-like particles.

13. A host cell for use in any condition susceptible of being improved or prevented by modulating glucolipotoxicity comprising the isolated or artificial ribonucleic acid sequence as described in any of the previous claims and / or the vector as described in any of the previous claims.

14. The host cell for use according to the previous claim, wherein the cell is a beta-cell, or endothelial cell.

15. A pharmaceutical composition for use in any condition susceptible of being improved or prevented by modulating glucolipotoxicity composition comprising the isolated or artificial ribonucleic acid sequence as described in any of the previous claims and / or the vector as described in any of the previous claims.

16. The pharmaceutical composition for use composition according to the previous claim further comprising at least a solvent, at least one pharmaceutically acceptable excipient.

17. The pharmaceutical composition for use according to any of the previous claims 15-16, wherein the pharmaceutically acceptable excipient is a buffering agent, stabilizer, cryoprotectant, chelating agent, preservative, viscosity modifier, solubilizer, antioxidant, filler, adjuvant, pH adjuster, among others.

18. A kit comprising isolated or artificial ribonucleic acid sequence for use in any condition susceptible of being improved or prevented by modulating glucolipotoxicity as described in any of previous claims, and / or the vector as described in any of the previous claims, and / or the pharmaceutical composition as described in any of previous claims.

19. A use of the isolated or artificial ribonucleic acid sequence according to any of the previous claims as a biomarker to detect or monitor glucolipotoxicity in a sample.

20. The use according to the previous claim, wherein a measured level of the isolated or artificial ribonucleic acid sequence into a sample is compared to a control reference value, and wherein reduction of said measured level relative to said control reference value is indicative of diagnosis or poor prognosis of diabetes.

21. The use according to any of the previous claims 19-20, wherein the sample is a biological sample selected from the list consisting of: interstitial fluid, blood, plasma, serum, or urine.

22. The use of the isolated or artificial ribonucleic acid sequence according to any of the previous claims for the manufacture of a medicament for the treatment of glucolipotoxicity-associated disorders; preferably for the treatment of diabetes and / or obesity.

23. A method for treating or preventing glucolipotoxicity-associated disorders, preferably diabetes and / or obesity, in a subject, the method comprising administering the isolated or artificial ribonucleic acid sequence according to any of the previous claims to the subject.

24. An in vitro or ex vivo method for diagnosis or prognosis of diabetes, comprising the following steps:providing a biological sample of a patient;measuring the content or amount of the isolated or artificial ribonucleic acid sequence described in any of the previous claims;comparing said content or amount to a control reference value;wherein the reduction of the content or amount of the sequence relative to the control reference value is indicative of diagnosis of diabetes.

25. The method according to the previous claim, wherein the biological sample is selected from the list consisting of: interstitial fluid, blood, plasma, serum or urine.

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