GRAPHICAL ABSTRACT

Highlights

• miR-374 suppressed PGC-1α, upregulated in β-cells and hepatocytes in diabetes.

• Adenoviral miR-374 improves β-cell function and suppresses gluconeogenesis in db/db mice.

• Ex-4-CCL enhanced miR-374 delivery efficiency to pancreatic β-cells in vivo.

• Ex-4-CCL improved function in human iPSC-derived insulin-producing cells.

INTRODUCTION

Type 2 diabetes mellitus (T2DM) is multi-organ disorder characterized by hyperglycemia. During the development of T2DM, increased adiposity elevates serum free fatty acids levels which triggers insulin resistance in liver and muscle. When insulin resistance persists, pancreatic β-cells fails to compensate the increased insulin demand and hyperglycemia develops [1]. In turn, hyperglycemia aggravates β-cell decompensation and forms a vicious cycle to accelerate β-cell failure [2,3]. When β-cell failure progress, patients with T2DM are bound to relative insulin deficiency which aggravates hepatic insulin resistance [4]. Therefore, salvaging β-cells from glucotoxicity and suppressing hepatic glucose overproduction is important to prevent the progression of T2DM.

Peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1α) is a transcriptional coactivator that plays a key role in regulating energy metabolism, exercise and mitochondrial biogenesis in multiple tissues [5-8]. Cumulative evidence suggests the role of PGC-1α in T2DM by regulating the function of pancreatic islets and liver. Human genetic variants of PGC-1α correlate with the onset of T2DM and insulin resistance, suggesting that PGC-1α is involved in the development of diabetes mellitus [9]. Moreover, PGC-1α was reported to be upregulated in liver and pancreas of T2DM mouse model [10]. PGC-1α has been shown to stimulate hepatic gluconeogenesis and suppress glucose stimulated insulin secretion (GSIS) in pancreatic islets [11-13]. Therefore, PGC-1α is an attractive target for the treatment of T2DM by regulating metabolism of liver and pancreatic islets. However, attempts for PGC-1α targeted therapy has been limited mostly due to lack of optimal candidates and delivery methods.

Coated cationic lipoparticles (CCLs) are lipid bilayer structure composed of phospholipid hydrophilic head in the outer layer and cationic head in the inner layer to form aqueous core. Because nucleic acids are negatively charged and hydrophilic, CCLs are attractive source of vehicle to deliver nucleic acids. Recently, lipid nanoparticles were widely applied to humans as a vehicle to coronavirus disease 2019 (COVID-19) vaccinations and proved their safety and efficacy. MicroRNA (miRNAs) are small noncoding approximately 19 to 24 nucleotide RNA molecules that negatively regulates gene expression and are powerful cellular regulators and attractive therapeutic targets [14]. Therefore, miRNA and nanoparticle can be an safe and efficient method to alleviate glucotoxicity and rescue diabetic condition in pancreatic β-cells and hepatocytes by targeting PGC-1α .

In this study, we demonstrate miR-374 mediated PGC-1α regulation can ameliorate hyperglycemia in diabetic animal mouse model by restoring β-cell dysfunction in mouse and human β-like cells differentiated from human induced pluripotent stem cells (iPSCs) and suppressing hepatic gluconeogenesis. We further demonstrate that peripheral vein injection of exendin-4 (Ex-4) tagged CCLs encapsulation can enhance the miRNA delivery to the target tissues with preserved therapeutic effects.

METHODS

Synthesis and characterization of Ex-4-CCL-miRNA

CCLs encapsulating 1 nmol of miRNA were prepared by dissolving 1 nmol of miRNA in 500 μL of distilled deionized water supplemented with 8.6 μg of DOTAP (Avanti Polar Lipids, Alabaster, AL, USA) in 500 μL of chloroform and 1,040 μL of methanol to obtain a single phase. After 30 minutes of incubation at room temperature, 500 μL of distilled water and 500 μL of chloroform were added to form two separate phases. The solution was vortexed and centrifuged at 1,000 ×g for 10 minutes at 4°C. The organic phase containing DOTAP-miRNA complex was extracted and added to the dried lipid film composed of hydrogenated soy phosphatidylcholine (HSPC):1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol₂₀₀₀ (PEG2k):Chol:DSPE-PEG2k-maleimide (5.8:0.04:3.8:0.497 μmol, Avanti Polar Lipids). Next, 500 μL of double distilled water was added to the mixture and vortexed for 20 seconds and emulsified by sonication for 2 minutes. After evaporating organic phase using a desiccator, the solution was extruded with 100 nm polycarbonate membrane (Whatman, Maidstone, UK) at 55°C using an extrusion kit (Avanti Polar Lipids). To conjugate peptides (Peptron, Daejeon, Korea), 6 nmol of peptide was mixed with nanoparticles, followed by removal of unencapsulated peptides via dialysis overnight. The hydrodynamic size and zeta potential were measured with a dynamic light scattering device (Malvern, Malvern, UK).

Cell culture and glucotoxicity

Insulin 1 (INS-1) cells were maintained in RPMI1640 medium supplemented with 11.1 mmol/L glucose, 10% fetal bovine serum (FBS), β-mercaptoethanol and antibiotics. We cultured INS-1 cells and islets in 5.5 mmol/L glucose for 3 days as the normal condition. Exposure to glucotoxic conditions was instigated by treatment for 3 days with 33.3 mmol/L glucose in a culture medium containing 10% FBS.

miRNA and gene array hybridization and data analysis

We performed global miRNA gene expression analyses using an Agilent Rat miRNA Microarray (8X15K, Agilent design IDs019159, Agilent Technologies, Amstelveen, The Netherlands). Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer. RNA quality was assessed with an Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies), and quantity was determined with an ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Each RNA sample (100 ng) was processed using the protocol recommended by the manufacturer (Agilent) (miRNA Microarray System with miRNA Complete Labeling and Hyb Kit protocol 2.1). As input, 100 ng of total RNA was used for the labeling reaction, and labeled miRNA was hybridized to the array for 20 hours at 55°C and 20 rpm as described in the protocol. After hybridization, the chips were washed and scanned with an Agilent DNA Microarray Scanner. Biotin-labeled cDNA was synthesized using a GeneChip Whole Transcript Sense Target Labeling Assay (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Following fragmentation, cDNAs were hybridized for 17 hours at 45°C on Affymetrix rat Gene 1.0 ST Arrays. Arrays were washed and stained in the GeneChip Fluidics Station 450 (Affymetrix) and scanned on a GeneChip Scanner 3000 7G (Affymetrix). Data intensities were log-transformed and normalized with a quantile normalization method using Robust Multiarray Average (RMA).

Inhibition and overexpression of miRNAs

The 2’-O-methyl oligonucleotides were synthesized by ST Pharm (Seoul, Korea). The 2’-O-methyl antisense oligonucleotides were transfected into INS-1 cells using Lipofectamine plus (Invitrogen). The shRNA structure of miR-374 was inserted into the EcoRI/XhoI sites of the pAdTrack-CMV shuttle vector. The recombinants were amplified in HEK-293 cells and isolated and purified via CsCl (Sigma, St. Louis, MO, USA) gradient centrifugation. The titers were determined using Adeno-X Rapid Titer (BD Bioscience, San Jose, CA, USA) according to the manufacturer’s protocol. INS-1 cells were infected with adenovirus-mediated miR-374 (Ad-miR-374) at a multiplicity of infection of 100, which was sufficient to infect 80% of the cells as determined by fluorescence. The miR-374 RNA precursor (pre-miR-374) was purchased from Applied Biosystems (Carlsbad, CA, USA).

Western blotting analysis

Western blot was performed as previously described [15]. In brief, total protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with the following primary antibodies and horseradish peroxidase-conjugated secondary antibodies: anti-PGC-1α (1:500, ab54481, Abcam, Cambridge, MA, USA), anti-phosphorylated adenosine monophosphate-activated kinase (AMPK, 1:500, #2531), anti-phosphorylated insulin receptor substrate-1 (IRS-1, 1:1,000, #2385), anti-phosphorylated phosphoinositide 3-kinase (PI3 Kinase, 1:500, #4228), anti-phosphorylated AKT (1:500, #9271, Cell Signaling Technology, Danvers, MA, USA) or anti-β-actin (1:5,000, A5441, Sigma-Aldrich). Proteins were visualized using enhanced chemiluminescence in accordance with the manufacturer’s recommendations.

In vivo study

The db/db mice, which are obese and diabetic due to a leptin receptor mutation on the C57BLKS/J genetic background, were 49 days old and weighed approximately 37 g at the beginning of the experiments; groups were normalized for age and gender. Age-matched heterozygotes (db/dm), a nonpenetrant genotype, were used as the control animals. The mice were housed in pathogen free facility where 12-hour light/dark cycle, temperature and humidity controlled. All procedures were preapproved by the Institutional Animal Care and Use Committee at the Catholic University of Korea. The db/db mice were randomly allocated to control (n=6), Ad-green fluorescent protein (GFP) (n=4) and Ad-miR-374 (n=8) treatment groups by intraperitoneal glucose tolerance test (IP-GTT) and were then anesthetized with ketamine and rompun (5:1), followed by treatment with 1×10⁹ plaque-forming units of Ad-miR-374 or Ad-GFP adenovirus and 500 μL of 90% NaCl injected systemically into the celiac artery with a 26-G needle. The non-fasting glucose concentrations and body weights were measured every other day, and animals were sacrificed after 20 days.

Study approval

Every experiments followed the relevant guidelines and regulations. The Institutional Review Board of Catholic University of Korea (CUMS-2019-0025-04) approved this study.

Statistical analysis

The results are expressed as the mean±standard error of at least three independent experiments. Analysis of variance was used to compare different groups. Statistical significance was determined using Student’s t-test and P<0.05 was considered significant.

RESULTS

Identification of miR-374 as a potential therapeutic target

To establish an ex vivo model that physiologically recapitulates glucotoxic conditions, we first exposed primary rat islets to euglycemic (5.5 mmol/L glucose) and hyperglycemic (33.3 mmol/L glucose) environments. Under hyperglycemic conditions, we observed impaired GSIS, accompanied by decreased mRNA expression of insulin, pancreatic and duodenal homeobox 1 (Pdx-1), and neuronal differentiation (NeuroD), while islet viability was maximally preserved after 3 days of culture in 33.3 mmol/L glucose (Supplementary Fig. 1). To identify miRNAs that can restore pancreatic β-cell function under glucotoxic condition, we performed miRNA chip analysis in primary rat islets cultured under euglycemic and hyperglycemic conditions for 3 days. Among the changes, we candidated 15 miRNAs (eight upregulated and seven downregulated, fold change ≥1.5, P<0.05) that were differentially expressed in euglycemic and glucotoxic conditions (Fig. 1A). Among these changes, miR-374 was down regulated in islets exposed under hyperglycemic condition which was known to regulate PGC-1α in pancreatic β-cells and hepatocytes (Supplementary Methods).

miR-374 regulates PGC-1α in glucotoxicity-induced β-cells and hepatocytes

To investigate the physiologic relevance of miR-374 in diabetic condition we first attempted to understand how miRNA expression changes in islets exposed under hyperglycemic milleu. We examined the expression of miR-374 from high-fat diet (HFD) fed mice and db/db mice. miR-374 expression was significantly downregulated in the pancreas and liver of db/db mice when compared to control mice (approximately 0.5- and 0.8-fold, respectively) (Supplementary Fig. 2A). Similarly, miR-374 was downregulated in the pancreas and liver of mice exposed to HFD when compared to normal chow fed control mice (approximately 0.6- and 0.7-fold, respectively) (Supplementary Fig. 2A). Conversely, the expression of Pgc-1α was increased in the pancreas and liver of HFD and db/db mice compared with the control mice (Supplementary Fig. 2B). As such, miR-374 was down regulated while Pgc-1α was upregulated in pancreas and liver of diabetic condition.

To validate whether miR-374 can regulate the expression of PGC-1α in glucotoxic condition, we performed gain (pre-miR-374) and loss (anti-miR-374) of function experiments in primary islets and hepatocytes under euglycemic and glucotoxic conditions. Under euglycemic conditions, anti-miR-374 treatment dramatically increased the basal expression levels of Pgc-1α in islets (Fig. 1B) and hepatocytes (Fig. 1C). In contrast, miR-374 overexpression suppressed the glucotoxicity-induced Pgc-1α expression (Fig. 1B and C). We also confirmed similar expression pattern in protein levels by western blot analyses (Fig. 1D and E). Likewise, the number of expressing PGC-1 α expressing cells increased after exposure to glucotoxicity in islets and hepatocytes which was reduced by miR-374 treatment (Fig. 1F and G). Taken together, miR-374 regulates PGC-1α in β-cells and hepatocytes under glucotoxic condition.

miR-374 interacts directly with the 3’-untranslated region of PGC-1α

Next we wanted to understand how miR-374 regulate PGC-1α in β-cells and hepatocytes. We identified seven nucleotides length sequence in the miR-374 which complementarily binds to the 3’ region of Pgc-1α mRNA (Supplementary Fig. 3). We transfected a GFP-PGC-1α construct and an antisense oligonucleotide against miR-374 in islets and hepatocytes. Overexpression of miR-374 reduced the luciferase activity when the reporter construct contained the PGC-1α 3’-untranslated region (3’-UTR). In contrast, when antisense sequence (3’-UAUUAUAA-5’) in PGC-1α 3’-UTR region was mutated (3’-UAUUAUAA-5’), luciferase activity was not affected both in islets (Fig. 2A) and hepatocytes (Fig. 2B). These findings suggest that miR-374 regulates the expression of Pgc-1α by directly interacting to their 3’UTR.

miR-374 improves pancreatic β-cell function and suppresses gluconeogenic genes in hepatocytes under glucotoxic condition

To investigate the functional role of miR-374, we examined the miR-374 treated β-cells and hepatocytes under glucotoxic condition in vitro. In islets, glucotoxicity induced repression of mature β-cell genes (Pdx-1, β-cell E-box transcription factor [BETA2]/NeuroD, and Insulin) was mimicked by anti-miR-374 treatment and was restored by miR-374 precursor (pre-miR-374) treatment (Fig. 2C). Functionally, anti-miR-374 treatment dramatically decreased GSIS even in euglycemic state (Fig. 2E and FF). Importantly, pre-miR-374 partially restored the glucotoxicity induced GSIS suppression and insulin contents in islets (Fig. 2E-G). In hepatocytes, anti-miR-374 dramatically increased the gluconeogenesis gene (glucose-6-phosphatase [G6Pase], phosphoenolpyruvate carboxykinase [PEPCK], pyruvate carboxylase [PC]) expression while pre-miR-374 treatment and repressed the glucotoxicity induced gluconeogenesis genes overexpression (Fig. 2D). Functionally, anti-miR-374 treatment increased the glucose production under euglycemic condition, while pre-miR-374 treatment significantly decreased the glucotoxicity induced glucose overproduction in hepatocytes (Fig. 2H). These findings suggest the functional role of miR-374 in β-cells and hepatocytes under glucotoxic condition in vitro.

Ad-miR-374 treatment suppresses PGC-1α expression in islets and hepatocytes and alleviates hyperglycemia in db/db mice

To further investigate the anti-diabetic effect of miR-374 in a physiological context, we examined the metabolic profile of miR-374 treated db/db mice. Seven-week-old db/db mice were randomly allocated to control (Ad-GFP-injected group) and Ad-miR-374-injected group based on non-fasting glucose levels. To maximize the delivery of vectors to the pancreas and liver, adenovirus was delivered to the mice via celiac artery. Five days after adenovirus was injected, the morning non-fasting glucose level was significantly lower in the Ad-miR-374-injected mice compared with the Ad-GFP-injected mice (Fig. 3A). There was minimal body weight changes between the Ad-miR-374-injected and control db/db mice (Fig. 3B). Moreover, the mean area under the glucose curve during an IP-GTT was lower in the Ad-miR-374-injected group than in the control group (Fig. 3C and D). The glucose lowering effect of Ad-miR-374 lasted even 20 days after the single injection (Fig. 3A). At 20 days after injection, the plasma insulin level of the Ad-miR-374-injected group was significantly lower than that of the Ad-GFP-injected group (Fig. 3E) but the insulinogenic index, which represents the β-cell function, was higher in the Ad-miR-374-injected group compared with the Ad-GFP-infected group (Fig. 3F). These data imply that miR-374 ameliorates glucose intolerance and restores insulin secretory capacity in diabetic mice model. We also checked the miR-374 expression levels in the liver and pancreas 20 days after the adenoviral injection. In accordance to the long lasting metabolic effect of adenoviral injection, miR-374 expression was persistently upregulated in Ad-miR-374 treated db/db mice at 20 days after the injection (Fig. 3G). Moreover, upregulation of Pgc-1α mRNA expression in db/db mice was partially suppressed in the pancreas and liver of mice injected with Ad-miR-374 (Fig. 3H). We also performed immunohistochemical staining in islets and liver to determine the protein expression level of PGC-1α (islets, liver), Insulin (islets), and PEPCK (liver) in each group of mice. In islets, increased immunoreactivity of PGC-1α was decreased while decreased Insulin immunoreactivity was increased in db/db mice injected with Ad-miR-374 (Fig. 3I). In hepatocytes, increased immunoreactivity of PGC-1α and PEPCK was decreased in in Ad-miR-374 injected db/db mice (Fig. 3J). In alignment to the ex vivo studies (Fig. 2C and D), the suppression of mature β-cell genes (Pdx-1, BETA2/NeuroD, and Insulin) was restored in the pancreas of Ad-miR-374-injected db/db mice (Supplementary Fig. 4A). Ad-miR-374-injection also suppressed the upregulation of gluconeogenesis genes (G6Pase, PEPCK and PC) upregulation in hepatocytes (Supplementary Fig. 4B). Taken together, adenoviral delivery of miR-374 through celiac artery injection ameliorated diabetic conditions in db/db mice by restoring β-cell function and suppressing gluconeogenesis in the liver which seems to be mediated by the PGC-1α regulation.

Synthesis and characterization of Ex-4-CCL-miR-374

The delivery route (celiac artery injection) and vehicle (adenoviral) adopted in the previous experiment can be critical technical barriers for the clinical application of miRNA treatment. Especially, safe and sufficient delivery to pancreatic β-cells is challenging when particles are systematically delivered.

To deliver miRNAs safely and effectively, we developed a novel strategy to deliver miRNAs using CCL conjugated with β-cell specific surface marker [16,17]. We conjugated target peptide, polymer and lipid (lipo-polyethylene glycol [PEG]-peptide) within the outer leaflet. With this strategy, the toxicity of the particle was minimized, the stability of the miRNAs was preserved and loading efficiency and efficacy was maximized [18,19]. In search of optimal β-cell specific surface markers, we candidated ATP binding cassette subfamily C member 8 (ABCC8; SUR1) and Ex-4 (9-30, 9-39). Liposomes conjugated with PEG-candidate peptides were tagged with red fluorescent protein (RFP) and were delivered to immortalized pancreatic β-cell lines (INS-1). Ex-4 (9-30) conjugated liposomes exhibited highest uptake to INS-1 cell lines compared to Ex-4 (9-39) or ABCC8 (Supplementary Fig. 5A). We did not observe any liposome accumulation in controls (Supplementary Fig. 5A). From these results, we confirmed that Ex-4 (9-30) tagging can efficiently deliver lipo-PEG to pancreatic β-cell surface. miR-374 was encapsulated into a formulation of DOTAP:DSPEPEG2k: HSPC: chol (9.3:3.1:52.6:35, molar ratio) in a multistep process (Fig. 4A). Ex-4-CCLs encapsulating miR-374 (Ex-4-CCL-miR-374) had an average diameter of 294.8 nm and zeta potential of -12.3 mV, confirming the successful coating of the cationic lipid (Fig. 4B). To further validate the selectivity of Ex-4-CCL-miR-374 to pancreatic β-cells, we examined the probe uptake in various cells such as 3T3L1, fibroblasts as well as INS-1 cells. Among the cell lines, we observed accumulation of the Ex-4-CCL-miR-374 probe only in INS-1 cells (Supplementary Fig. 5B). We further explored whether delivered Ex-4-CCL-miR-374 can be internalized into INS-1 cells and regulate their gene expressions. miR-374 labeled with Alexa555 was encapsulated into CCL-miR-374 as a non-targeting control. Compared with CCL-miR-374, Ex-4-CCL-miR-374 was abundantly observed in the cytosol of INS-1 cells (Supplementary Fig. 6A). We next performed GSIS test and real-time polymerase chain reaction (RT-PCR) in Ex-4-CCL-miR-374 or CCL-miR-374 treated primary rat islets cultured under euglycemic or hyperglycemic conditions. The GSIS was suppressed under glucotoxic condition which was restored by Ex-4-CCL-miR-374 but not in negative controls (NCs) (Supplementary Fig. 6B). The expression level of insulin gene, which had been suppressed by glucotoxicity and Ex-4-CCL-miRNA NC, was normalized in Ex-4-CCL-miR-374 treated group (Supplementary Fig. 6C). On the other hand, glucotoxicity-induced Pgc-1α gene expression was suppressed by Ex-4-CCL-miR-374 (Supplementary Fig. 6D). In summary, we have developed cationic lipid nanoparticle tagged with Ex-4 (9-39) on the surface which are able to deliver and internalize miR-374 to β-cells to exert molecular effect in vitro.

Intravenous delivery of Ex-4-CCL-miR-374 ameliorates hyperglycemia in diabetic mice model

In vitro results encouraged us to examine the therapeutic potential of Ex-4-CCL-miR-374 in vivo. First, we examined the distribution of nanoparticles throughout the organs 48 hours after the tail vein injection in C57BL/6J mice. Although immunofluorescence was most highly enriched in liver and spleen, the pancreas uptake was about 2-fold increased in Ex-4-CCL-miRNA treated mice compared with the CCL-miRNA controls (Fig. 4C, Supplementary Fig. 6A-C). From these results, we tail vein injected Ex-4-CCL-miR-374 or Ex-4-CCL-miR control to the diabetic mice model (db/db) four times periodically and followed up for 19 weeks and examined whether Ex-4-CCL-miR-374 injection can change their metabolic profiles. Non-fasting glucose concentration of the Ex-4-CCL-miR-374-injected mice were significantly lower than the db/db control and Ex-4-CCL-miRNA-injected mice (Fig. 4D) while body weight was comparable among the groups (Fig. 4E). At 19 weeks after the injection, IP-GTT was markedly improved in Ex-4-CCL-miR-374-injected group compared with the db/dbs or vehicle controls (Fig. 4F and G). Intraperitoneal insulin tolerance test (IP-ITT) was significantly improved in Ex-4-CCL-miR-374-injected group compared to the controls (Fig. 4H and I). Fasting plasma insulin level was decreased (Fig. 4J) while insulinogenic index was increased in Ex-4-CCL-miR-374-injected group compared with the controls (Fig. 4K). These data imply that Ex-4-CCL-miR-374 improves glucose intolerance and insulin sensitivity and restores insulin secretory function in vivo. To provide mechanical insight of metabolic impact of Ex-4-CCL-miR-374, we tested whether Ex-4-CCL carrying miR-374 was delivered to β-cells 19 weeks after the injection. In immunofluorescence staining, RFP+/INS+ co-positive cells were commonly observed in Ex-4-CCL-miR-374 treated islets suggesting that Ex-4-CCL-miR-374 was incorporated to β-cells (Fig. 5A). Grossly, nanoparticles were highly uptaken not only in pancreas but also in liver (Supplementary Fig. 7). Indeed, immunofluorescence staining revealed that Ex-4-CCL-miR-374 were incorporated to hepatocytes (Fig. 5B). Likewise, miR-374 expression was increased in the pancreas and liver of the Ex-4-CCL-miR-374-injected db/db mice compared with the Ex-4-CCL-miRNA NC-injected group (Fig. 5C). We also confirmed that the Pgc-1α mRNA expression in db/db mice was down regulated in the pancreas and liver of Ex-4-CCL-miR-374 injected mice (Fig. 5D).

We also performed immunohistochemistry staining to determine the expression pattern of PGC-1α, INSULIN, and PEPCK in each group at protein level. Similarly observed in previous adenoviral injection data (Fig. 3I and J), the number of PGC-1α expressing cells increased in pancreatic islets and liver of db/db mice compared with control db/dm mice. Ex-4-CCL-miR-374 treatment decreased the number of PGC-1α expressing cells in both islets and liver while control Ex-4-CCL-miR could not change the number of PGC-1α expressing cells (Fig. 5E and F). In liver, Ex-4-CCL-miR-374 treatment decreased the number of PEPCK expressing cells compared with the control Ex-4-CCL-miR treated db/db mice (Fig. 5F). In alignment to the adenoviral and in vitro studies, Ex-4-CCL-miR-374 restored the suppression of mature β-cell genes (Pdx-1, BETA2/NeuroD, Insulin) in pancreas and gluconeogenesis genes (G6Pase, PEPCK, PC) in liver (Supplementary Fig. 8). As such, intravenous delivery of Ex-4-CCL-miR-374 ameliorated hyperglycemia in diabetic mouse model. Ex-4 tagging facilitated the delivery of CCL-miR-374 to pancreatic β-cells and restored insulin secretory function while additional delivery to liver suppressed gluconeogenesis to exert glucose lowering effect.

Ex-4-CCL-miR-374 restores the functional impairment of human β-like-cells under glucotoxic condition

To further pursue therapeutic potential of Ex-4-CCL-miR-374 in human, we treated Ex-4-CCL-miR-374 in glucotoxicity exposed human insulin producing cells. Umbilical cord blood mononuclear cell derived human iPSCs were differentiated to insulin producing cells (β-like-cells) in a multistep process as described previously (Fig. 6A) [20-22]. After the differentiation step, β-like-cells were treated with Ex-4-CCL-miR-374 or Ex-4-CCL-miR control and exposed under euglycemic or glucotoxic conditions for 3 days (Fig. 6B). In accordance to rodent data, Ex-4-CCL-miR-374 treatment restored glucotoxicity induced downregulation of Insulin gene expression and suppressed glucotoxicity induced upregulation of Pgc-1α (Fig. 6C and D). Glucotoxicity induced impairment of GSIS in β-like-cells were restored in Ex-4-CCL-miR-374 treatment group (Fig. 6E and F).

DISCUSSION

In this study, we thoroughly demonstrate that miR-374 can simultaneously ameliorate β-cell and hepatocyte dysfunction observed in T2DM by targeting PGC-1α from in vitro to diabetic mice model. We further increase the applicability of miRNA therapy by developing novel miRNA vehicle (Ex4-CCL) and validating its’ efficacy in human β-like-cells. To our best knowledge, PGC1-α targeted miRNA therapy to restore diabetic condition by simultaneously targeting pancreatic β-cells and liver has not been reported.

Previously, our group demonstrated that β-cell dysfunction induced by glucolipotoxic stress can be reversed by siPGC-1α–mediated suppression of overexpressed PGC-1α. In this study, we suppressed upregulated PGC-1α in diabetic milieu by identifying a novel miRNA. miRNAs play a key role in metabolic homeostasis by regulating the cellular response to changes in nutrient levels or fluctuations in various metabolic substrates. Several studies have reported miRNAs that can regulate PGC-1α gene in various tissues [23-26]. By analyzing miRNA expression in hyperglycemic environment, we suggest that miR-374 was potentially related to PGC-1α in pancreatic islets and hepatocytes. Previously, miR-374a/b was reported to regulate CCAAT/enhancer-binding protein-β (C/EBP-β) expression in porcine adipocytes, Brn3b expression in retinal ganglion cell development and vascular endothelial growth factor receptor 1 (VEGFR1)-mediated regression pathway in cardiac hypertrophy [27-29]. However, the physiological relevance of miR-374 in diabetes—particularly its relationship with PGC-1α—has been poorly studied. In this study, we focused on miR-374 as it may influence PGC-1α expression during glucotoxicity in pancreatic β-cells and hepatocytes. We first confirmed the expression of miR-374 and PGC-1α in various tissues of diabetic animal models. miR-374 was specifically downregulated in pancreas and liver of db/db and HFD mice. However, the expression of PGC-1α was inversely correlated with miR-374 expression in pancreas and liver of diabetic mice. Therefore, we assumed that overexpression of miR-374 regulated the PGC-1α expression under diabetic conditions.

Systemic insulin secretion and glucose homeostasis are regulated by both β-cell mass and function [30]. To assess whether miR-374–mediated effects were associated with changes in β-cell mass, we evaluated β-cell proliferation (Ki-67) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) in Ad-miR-374 and Ad-GFP treated db/db mice, and observed no significant differences between the groups (Supplementary Fig. 9). These findings suggest that the improved insulin secretion observed with miR-374 treatment is likely due to enhanced β-cell function, rather than an increase in their mass—possibly mediated through the BETA2/NeuroD pathway. mRNA-sequencing in miR-374 treated islets of db/db mice would have provided in depth molecular insights how miR-374 improved β-cell function. However, we could not do such experiment since db/db mice islets are destructive and technically challenging to isolate their islets.

In this study, we aimed to target PGC-1α in the pancreatic β-cells and hepatocytes under hyperglycemic condition. This has not only been due to the importance of islets and liver in glucose homeostasis but also because PGC-1α exerts complicating effects among tissues. PGC-1α whole body knockout mice exhibited enhanced insulin sensitivity under metabolic challenge [31]. In liver, PGC-1α has been shown to stimulate hepatic gluconeogenesis [12]. In β-cells, PGC-1α suppressed β-cell energy metabolism and insulin secretion in mice [13]. Paradoxically, transgenic expression of PGC-1α in fat and skeletal muscle leads to robust mitochondrial biogenesis but also causes insulin resistance, likely the result of imbalance of lipid uptake and oxidation [13]. Therefore, tissue specific PGC-1α targeted therapy is important to maximize the metabolic efficacy of the treatment. For this reason, we first delivered ad-miR-374 via celiac artery to db/db mice to maximize their delivery to pancreas and liver. We further demonstrate that tissue selective PGC-1α targeted therapy can be achieved with Ex-4 tagged CCL vehicle even delivered intravenously.

Although adenoviral vector miR-374 delivery successfully exerted metabolic effect in diabetic mice model, virus associated side effects and delivery route (celiac artery) remained as a critical issue for clinical application. Therefore, we selected lipid nanoparticles as a safer and more effective way to transport miR-374. Nanoparticles has recently widely adopted as a vehicle for COVID-19 vaccination and proved their safety in human. We further increased the transport efficiency of nanoparticles to pancreatic β-cells by conjugating Ex-4 to CCL. Ex-4, a 39-amino acid (AA) peptide, is a long-acting agonist of the glucagon-like peptide-1 (GLP-1) receptor and is resistant to dipeptidyl peptidase-IV. GLP-1 receptor is highly expressed on the surface of pancreatic β-cells, and GLP-1 receptor-targeting nanoparticle has previously shown to successfully image pancreatic islets [32]. The C-terminal region of Ex-4 showed high affinity binding with the N-terminal domain of the GLP-1 receptor, whereas Ex-4 (9-30) containing only the core alpha-helix region was higher than GLP-1 [33]. Therefore, Ex-4 (9-30) was used for nanoparticles in miRNA transport to β-cells. Ex-4 increases insulin secretion under glucotoxic conditions; however, Ex-4 (9-30) was not involved in insulin secretion under glucotoxic conditions (Supplementary Fig. 10). Therefore, we speculate that the metabolic effect of Ex-4-CCL-miR-374 was mediate by miR-374 rather than by Ex-4 (9-30).

Previous literatures suggest complex role of PGC-1α in β-cells and T2DM. PGC-1α Gly482Ser variant has been shown to be associated with increased risk of T2DM [34]. β-cell specific PGC-1α/PGC-1β knockout mice exhibit diabetic phenotype with impaired insulin secretory function [35]. Conversely, Pgc-1α overexpression impaired the insulin secretory function of rat islets [13,15]. Therefore, we speculate optimal level of PGC-1α is necessary to maintain the function of pancreatic β-cells. Although the precise mechanism is not fully understood, this ambivalent nature of PGC-1α might have been due to PGC-1α’s role as a mitochondrial regulator [35]. In this study, miR-374 delivery partially suppressed the level of PGC-1α expression which was increased by hyperglycemic condition. Importantly, PGC-1α levels of Ex-4-CCL-miR-374 treated groups were comparable or slightly higher than those of the basal euglycemic state. We speculate this optimal restoration of PGC-1α expression level would have contributed anti-diabetic effect of miR-374.

In this study, we extended the translational relevance of our findings by demonstrating the effects of miR-374 in human insulin-producing cells. Notably, the complementary target sequence of miR-374 for PGC-1α is conserved between rat mice and humans, supporting the cross-species applicability of our results. Therefore, we propose that our findings provide strong evidence for the therapeutic potential of miR-374 as a novel treatment strategy for diabetes. Although we observed changes in gluconeogenic gene expression in hepatocytes, this study lacks in vivo functional analyses of miR-374’s effects on liver function. Exploring the role and mechanisms of miR-374 in hepatic metabolism would be an important and interesting subject to explore in the future.

In conclusion, we demonstrate that miR-374 mediated PGC1-α down regulation can enhance β-cell function and suppress hepatic gluconeogenesis in diabetic animal mouse model. We further increase the therapeutic applicability of miR-374 by developing a safe and efficient vehicle, Ex-4 tagged CCL, and validate its’ efficacy in human β-like-cells. Future clinical studies to further validate the safety and efficacy of Ex-4-CCL-miR374 should be followed.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: J.W.K., Y.H.Y., H.S.P., K.H.Y.

Acquisition, analysis, or interpretation of data: J.W.K., Y.H.Y., C.H.O., H.S.P., J.H.P., K.H.Y.

Drafting the work or revising: J.W.K., J.L., E.Y.L., S.H.L., S.H.K., K.H.Y.

Final approval of the manuscript: all authors.

FUNDING

This study was supported by the National Research Foundation of Korea funded by the Korean government (to Ji-Won Kim, No. 2016R1D1A1A09918219, RS-2022-NR067389 to Joonyub Lee No. 2022R1I1A1A01068401, RS-2025-00555223), Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (to Joonyub Lee No. RS-2022-KH127044, RS-2024-00408915, and RS-2024-00404132), and from the Korean Diabetes Association (Ji-Won Kim, 2019F-3).

ACKNOWLEDGMENTS

The authors are grateful to Dr. V. Narry Kim (Seoul National University) for helpful discussion regarding this study. We appreciate the expert technical assistance provided by Heon-Seok Park and Dong-Sik Ham.

Fig. 1.

miR-374 regulates proliferator-activated receptor γ coactivator-1 (PGC-1α) in islets and liver under diabetic conditions. (A) Heatmap of differentially expressed microRNAs microarray in primary rat islets exposed to euglycemic and glucotoxic conditions. Low: euglycemic (glucose 5.5 mmol/L), glucotoxic (GTx) (glucose 33.3 mmol/L), fold change ≥1.5, P<0.05. The mRNA levels of Pgc-1α in (B) islets and (C) hepatocytes treated with scrambled miRNA, pre-mir-374, or anti-miR-374 (n=4 per group). The mRNA levels of genes were normalized by the mRNA level of β-actin. Western blot analyses were performed to determine the PGC-1α protein level in (D) insulin 1 (INS-1) cells and (E) hepatocytes (n=3 per group). Immunofluorescence staining of PGC-1α in pancreatic (F) islets and (G) hepatocytes in response to miR-374 regulation using antisense or overexpression of miR374. Islets and hepatocytes were transfected with scrambled miRNA, anti-miR-374, or pre-miR-374 under glucotoxic conditions (mean±standard error). NC, negative control; DAPI, 4ʹ,6-diamidino-2-phenylindole. ^aP<0.05, ^bP<0.01.

Fig. 2.

miR-374 directly interacts with the 3’-untranslated region (3’ UTR) of proliferator-activated receptor γ coactivator-1 (PGC-1α) and regulates metabolic function of islets and liver under glucotoxic condition. Incorporation of the PGC-1α 3’-UTR into a luciferase reporter construct validated luciferase activity compared with a reporter lacking the PGC-1α 3’-UTR upon transfection of (A) islets and (B) hepatocytes (n=5, mean±standard error [SE]). The levels of mRNA gene expression in primary (C) rat islets and (D) hepatocytes treated with pre-miR-374 or anti-miR-374. The mRNA levels of genes were normalized to β-actin mRNA (n=4, mean±SE). (E, F) Glucose-stimulated insulin secretion; (G) insulin contents in primary islets; and (H) glucose production in hepatocytes after miRNA administration. Functional analysis was performed on 10 islets or 1.5×10⁶ hepatocytes in each group from 10 independent experiments (n=10, mean±SE). Pdx-1, pancreatic and duodenal homeobox 1; BETA2, beta cell E-box transcription factor; NeuroD, neuronal differentiation; G6Pase, glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; PC, pyruvate carboxylase. ^aP<0.05, ^bP<0.01.

Fig. 3.

Adenovirus-mediated miR-374 (Ad-miR-374) treatment effectively suppresses proliferator-activated receptor γ coactivator-1 (PGC-1α) and ameliorates hyperglycemia in db/db mice. Adenovirus-green fluorescent protein (Ad-GFP) or Ad-miR-374 was injected into the celiac artery of db/db mice aged 7 weeks (n=4–6 per group). (A) Morning non-fasting glucose levels and (B) body weights were measured for 19 days (Ad-GFP vs. Ad-miR-374). (C, D) The intraperitoneal glucose tolerance test and area under the glucose curve (AUCg) were measured 20 days after the injection (Ad-GFP vs. Ad-miR-374). (E) Plasma insulin level and (F) insulinogenic index measured in the Ad-miR-374-injected group were compared with the Ad-GFP-infected group. The expression level of (G) miR-374 and (H) Pgc-1α mRNA was measured in the pancreas and liver of the Ad-GFP and Ad-miR-374-injected groups (n=4, mean±standard error). (I) PGC-1α and insulin in pancreatic islets. (J) PGC-1α and phosphoenolpyruvate carboxykinase (PEPCK) in livers were immunohistochemically stained in miR-374-overexpressing mice and wild-type littermate controls (representative figure of n=3, scale bar=100 μm). ^aP<0.05, ^bP<0.01.

Fig. 4.

Exendin-4 (Ex-4) (9-30)-coated cationic lipoparticle (CCL)-miR-374 ameliorates hyperglycemia and protects β-cell dysfunction in db/db mice treated via tail vein injection. (A) A schematic representation of the synthesis of Ex-4-CCL-miR-374. (B) Diameter and zeta potential of nontargeted-CCL-miR-374 and Ex-4-CCL-miR-374. (C) Ex vivo fluorescence images of the pancreas at 48 hours after tail vein injection in C57BL/6J mice. (D) Morning non-fasting glucose values and (E) body weights were measured for 19 weeks. (F) Intraperitoneal glucose tolerance test (IP-GTT) was performed in week 19 after Ex-4 (9-30)-CCL-miR-374 administration. (G) The area under the curve (AUC) of glucose in IP-GTT at week 19 after Ex-4 (9-30)-CCL-miR-374 treatment. (H) Intraperitoneal insulin tolerance test (IP-ITT) was measured at week 19 after Ex-4 (9-30)-CCL-miR-374 treatment. (I) The AUC in IP-ITT at week 19 after Ex-4 (9-30)-CCL-miR-374 exposure. Plasma insulin level (J) and insulinogenic index (K) were measured at 19 weeks after Ex-4 (9-30)-CCL-miR-374 administration. Data represent the mean±standard deviation of all mice in each group (n=6−7). DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; HSPC, hydrogenated soy phosphatidylcholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; PEG₂k, poly(ethylene glycol)2000; DW, distilled water; NC, negative control; AUCg, area under the glucose curve; AUCi, area under the insulin curve. ^aP<0.05, ^bP<0.01.

Fig. 5.

Intravenously delivered exendin-4 (Ex-4) (9-30)-coated cationic lipoparticle (CCL)-miR374 regulates proliferator-activated receptor γ coactivator-1 (PGC-1α) in β-cells and hepatocytes in db/db mice. (A) Anti-insulin immunostaining of the pancreas obtained from db/db mice injected with Ex-4 (9-30)-CCL-miR374 or control Ex-4 (9-30)-CCL-miRNA negative control (NC) by tail veins. Liposome (red), insulin (green), and 4ʹ,6-diamidino-2-phenylindole (DAPI) nuclear stain (blue). (B) Anti-albumin immunostaining of the liver obtained from db/db mice injected with Ex-4 (9-30)-CCL-miR374 or control Ex-4 (9-30)-CCL-miRNA NC. Liposome (red), insulin (green), and DAPI nuclear stain (blue). (C) miR-374 expression and (D) PGC-1α expression levels were confirmed in the pancreas and liver of Ex-4 (9-30)-CCL-miRNA NC and Ex-4 (9-30)-CCL-miR374-treated groups (n=6, mean±standard error). Immunohistochemical staining of (E) PGC-1α and insulin in pancreatic islets and (F) PGC-1α and phosphoenolpyruvate carboxykinase (PEPCK) in livers after nanoparticles were delivered. ^aP<0.01.

Fig. 6.

Effect exendin-4 (Ex-4) (9-30)-coated cationic lipoparticle (CCL)-miR374 in human β-like-cells differentiated from human induced pleuripotent stem cells (hiPSCs). (A) Overview of the differentiation protocol of β-like cells from hiPSCs. (B) Fluorescence microscopy images showing delivery of Ex-4 (9-30)-CCL-miR-374 (red) in differentiated human β-like cells. (C, D) mRNA levels of insulin and proliferator-activated receptor γ coactivator-1 (Pgc-1α) in human β-like cells treated with Ex-4 (9-30)-CCL-miR-374 (n=3 per group). (E, F) In vitro glucose-stimulated insulin secretion (GSIS) of human β-like cells treated with or without Ex-4 (9-30)-CCL-miR-374 (n=3 per each group, mean±standard error). NC, negative control. ^aP<0.05, ^bP<0.01.

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• β‐cell recovery revisited: Is prediabetes remission driven by insulin sensitivity or β‐cell function?
  Joonyub Lee, Kun‐Ho Yoon
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About this article

Kim JW, Lee J, You YH, Oh CH, Park HS, Lee EY, Lee SH, Ko SH, Park JH, Yoon KH. Targeting PGC-1α by miRNA-374 Simultaneously Improve β-Cell Dysfunction and Suppress Hepatic Glucose Overproduction. Diabetes Metab J. 2025 Nov 3. doi: 10.4093/dmj.2025.0287. Epub ahead of print.

Received: Apr 05, 2025; Accepted: Aug 24, 2025

DOI: https://doi.org/10.4093/dmj.2025.0287.

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