Gut hormone multi-agonists for the treatment of type 2 diabetes and obesity: advances and challenges.

The rapidly rising incidence of obesity, coupled with type 2 diabetes mellitus (T2DM), is a growing concern. Glucagon-like peptide-1 (GLP-1), an endogenous peptide secreted by enteroendocrine L-cells, demonstrates exceptional pharmacological potential for the treatment of T2DM and obesity. Considering its proven efficacy in glucoregulation and weight loss, GLP-1 receptor agonists (GLP-1RAs) have emerged as a revolutionary breakthrough in the arena of diabetes management and weight control. Additional gastrointestinal hormones, such as glucose-dependent insulinotropic peptide (GIP) and glucagon, exhibit structural similarities to GLP-1 and work synergistically to lower blood glucose levels or aid in weight loss. Today, various classes of gut hormone receptor multiple agonists are steadily progressing through development and clinical trials, including dual GLP-1/glucagon receptor agonists, dual GLP-1/GIP receptor agonists, and triple GLP-1/GIP/glucagon receptor agonists. The GLP-1/GIP receptor co-agonist, tirzepatide, was approved by the US Food and Drug Administration (FDA) for the treatment of T2DM, outperforming basal insulin or selective GLP-1RAs by providing superior HbA1c reductions. Remarkably, tirzepatide also facilitated unprecedented weight loss of up to 22.5% in non-diabetic individuals living with obesity. This result is comparable to those achieved with certain types of bariatric surgery. Therefore, the advent of gut hormone multi-agonists signifies the dawn of an exciting new era in peptide-based therapy for obesity and T2DM. This review offers a comprehensive summary of the various types of gut hormone multiple agonists, including their discovery, development, action of mechanisms, and clinical effectiveness. We further delve into potential hurdles, limitations, and prospective advancements in the field.

Hsa_circ_0007590/PTBP1 complex reprograms glucose metabolism by reducing the stability of m6A-modified PTEN mRNA in pancreatic ductal adenocarcinoma.

The role of circular RNAs (circRNAs) in glucose metabolism in pancreatic duct adenocarcinoma (PDAC) remains elusive. Through RNA sequencing of cells cultured under conditions of glucose deprivation, we identified hsa_circ_0007590. Sanger sequencing and RNase R and Act D treatments were performed to confirm the circular RNA features of hsa_circ_0007590. RNA in situ hybridization (RNA-ISH) and quantitative reverse transcription PCR (qRT-PCR) were used to estimate hsa_circ_0007590 expression in PDAC clinical specimens and cell lines. hsa_circ_0007590 expression was higher in PDAC patients and closely related to the clinicopathological characteristics of the disease. Cytoplasm‒nuclear fractionation and FISH assays demonstrated that hsa_circ_0007590 was located in the nucleus. Gain-of-function and loss-of-function assays were performed to assess the biological behaviors of PDAC cells. Seahorse XF assays were performed to validate the Warburg effect. hsa_circ_0007590 facilitated the proliferation, migration, and invasion of PDAC cells and promoted the Warburg effect. Mass spectrometry, RNA pulldown, RNA immunoprecipitation (RIP), RNA m6A quantification, m6A dot blot, MeRIP, and Western blotting were conducted to investigate the detailed mechanism through which hsa_circ_0007590 produces these effects. Mechanistically, hsa_circ_0007590 targeted PTBP1 and increased the expression of the m6A reader protein YTHDF2, leading to PTEN mRNA degradation and PI3K/AKT/mTOR pathway activation. Overall, hsa_circ_0007590, which targets PTBP1, reprograms glucose metabolism by attenuating the stability of m6A-modified PTEN mRNA and holds potential promise as a therapeutic target for PDAC.

CircMYOF triggers progression and facilitates glycolysis via the VEGFA/PI3K/AKT axis by absorbing miR-4739 in pancreatic ductal adenocarcinoma.

Emerging evidence has demonstrated that circular RNAs (circRNAs) take part in the initiation and development of pancreatic ductal adenocarcinoma (PDA), a deadly neoplasm with an extremely low 5-year survival rate. Reprogrammed glucose metabolism is a key feature of tumour development, including PDA. In this research, we evaluated the role of circRNAs in reprogrammed glucose metabolism in PDA. RNA sequencing under various glucose incubation circumstances was performed. A new circMYOF was identified. Sanger sequencing and RNase R treatment confirmed its circular RNA characteristics. Real-time PCR indicated that it was highly expressed in PDA clinical specimens and cell lines. Gain-of- and loss-of-function assays showed that circMYOF induced progression in PDA. Mechanistically, RNA pull-down and luciferase reporter experiments elucidated that circMYOF, as a competing endogenous RNA for miR-4739, facilitated glycolysis via the VEGFA/PI3K/AKT pathway. Taken together, our findings indicate that circMYOF may work as a desirable biomarker and therapeutic target for PDA patients.

SLC45A4 promotes glycolysis and prevents AMPK/ULK1-induced autophagy in TP53 mutant pancreatic ductal adenocarcinoma.

BACKGROUND: Somatic mutations of the TP53 gene occur frequently in pancreatic ductal adenocarcinoma (PDA). Solute carrier family 45 member A4 (SLC45A4) is a H+ -dependent sugar cotransporter. The role of SLC45A4 in PDA, especially in TP53 mutant PDA, remains poorly understood. METHODS: We explored the TCGA datasets to identify oncogenes in TP53 mutant PDA. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], colony formation and 5-ethynyl-2'-deoxyuridine (Edu) assays were performed to investigate the function of SLC45A4 in vitro. Glucose consumption, lactate production and ATP production were detected to evaluate glucose utilization. Extracellular acidification rate and oxygen consumption rate assays were used to evaluate glycolysis and oxidative phosphorylation. The subcutaneous xenotransplantation models were conducted to explore the function of SLC45A4 in vivo. RNA-sequencing and gene set enrichment analysis were employed to explore the biological alteration caused by SLC45A4 knockdown. Western blotting was performed to evaluate the activation of glycolysis, as well as the AMPK pathway and autophagy. RESULTS: SLC45A4 was overexpressed in PDA for which the expression was significantly higher in TP53 mutant PDA than that in wild-type PDA tissues. Moreover, high level of SLC45A4 expression was tightly associated with poor clinical outcomes in PDA patients. Silencing SLC45A4 inhibited proliferation in TP53 mutant PDA cells. Knockdown of SLC45A4 reduced glucose uptake and ATP production, which led to activation of autophagy via AMPK/ULK1 pathway. Deleting SLC45A4 in TP53 mutant HPAF-II cells inhibited the growth of xenografts in nude mice. CONCLUSIONS: The present study found that SLC45A4 prevents autophagy via AMPK/ULK1 axis in TP53 mutant PDA, which may be a promising biomarker and therapeutic target in TP53 mutant PDA.

Antimicrobial characterization of site-directed mutagenesis of porcine beta defensin 2.

Porcine ß defensin 2 (pBD2) is a small, cationic and amphiphilic antimicrobial peptide. It has broad antimicrobial activities against bacteria and plays an important role in host defense. In order to enhance its antimicrobial activity and better understand the effect of positively charged residues on its activity, we substituted eight amino acid residues with arginine or lysine respectively. All mutants were cloned and expressed in BL21 (DE3) plysS and the mutant proteins were then purified. These mutant versions had higher positive charges but similar structural configurations compared to the wild-type pBD2. Moreover, these mutant proteins showed different antimicrobial activities against E. coli and S. aureus. The mutant I4R of pBD2 had the highest antimicrobial activity. In addition, all the mutants showed low hemolytic activities. Our results indicated that the positively charged residues were not the only factor that influenced antimicrobial activity, but other factors such as distribution of these residues on the surface of defensins might also contribute to their antimicrobial potency.

Effect of p38 mitogen-activate protein kinase on MUC5AC protein expression of bile duct epithelial cells in hepatolithiasis patients.

Primary hepatolithiasis is a common bile duct disease with benign nature but complicated mechanisms. Current studies have revealed its correlation with cytokine release by chronic inflammation, which also increased mucin (MUC) synthesis. This study investigated the role of p38 mitogen-activated protein kinase (MAPK) in regulating cytokine release and mucin synthesis, in an attempt to elucidate the role of p38 signaling molecule in the pathogenesis of hepatolithiasis. In human intrahepatic bile duct endothelial cells (HIBECs), lipoprotein (LPS) was used to induce the high expression of MUC. Small interference RNA (siRNA) was then used to silencing p38 gene expression. Cytokines including interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were measured, along with MUC5AC protein and mRNA expression assay. The interference of p38 gene expression inhibited the release of IL-1ß and TNF-α in cultured cells. It also depressed both mRNA and protein levels of MUC5A. P38 MAPK signal pathway may be involved in the formation and progression of hepatolithiasis. This study provides potential new strategy for treating hepatolithiasis using p38 MAPK signal pathway as the drug target.

Molecular cloning, expression and characterization of the porcine ß defensin 2 in E. coli.

Porcine ß defensin 2(pBD2)is a cationic 37-amino acid antimicrobial peptide with disulfide bonds. Synthetic pBD2 had broad antimicrobial activity against pathogenic bacteria, and thus pBD2 could be a good candidate as a bactericidal agent for pigs. This study reported the successful recombinant expression of pBD2 in Escherichia coli and analysis of its antimicrobial activity, its hemolytic activity, salt-tolerance and thermal stability as well. The pBD2 gene, obtained by RT-PCR using the tongue total RNA as a template and cloned into pET30a expression vector, was transformed into E. coli BL21 (DE3) plysS. The recombinant pBD2 was expressed after induction by IPTG and purified by His tag affinity column with 95% purity. The recombinant pBD2 exhibited antimicrobial activity against both Gram-positive S. aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentration (MIC) of recombinant pBD2 against tested bacteria was 10 µg/mL, and the recombinant pBD2 could kill 50% E. coli at 14.39 µg/mL and S. aureus at 21.1 µg/mL. In addition, pBD2 showed low hemolytic activity, salt-tolerance and thermal stability, the properties would be important for its application in practice.

Cloning, expression and characterization of antimicrobial porcine ß defensin 1 in Escherichia coli.

Porcine ß defensin 1 (pBD1) is a cationic antimicrobial peptide with three pairs of disulfide bonds. When expressed in insect cells, two polypeptides of different length (pBD1(38) and pBD1(42)) accumulated, which differed by N-terminal truncation. However, only pBD1(42) was found in pigs. pBD1(42) had stronger antimicrobial activity than pBD1(38), and thus could be a good candidate as a bactericidal agent for pigs. In this study, pBD1(42) gene, obtained by RT-PCR using the tongue total RNA as a template, was cloned into pET30a expression vector and transformed into Escherichia coli BL21 (DE3) plysS. The recombinant pBD1(42) was expressed after induction by IPTG and purified by His tag affinity column with 90% purity. The recombinant pBD1(42) exhibited antimicrobial activity against both Gram-positive Staphylococcus aureus and Gram-negative E. coli including the multi-resistant E. coli. The minimum inhibitory concentrations (MICs) of recombinant pBD1(42) against tested bacteria were 100 µg/mL for E. coli and 80 µg/mL for S. aureus. In addition, pBD1(42) showed low hemolytic activity and high thermal stability. These properties are relevant for the biotechnological applications of the peptide.