Hybrid protein-peptide system for the selective pH-dependent binding and photodynamic ablation of cancer cells.

Creating new tools for the early diagnosis and treatment of cancer is one of the most important and intensively developing areas of modern medicine. Currently, photodynamic cancer therapy (PDT) is attracting increasing attention as a unique modality of minimally invasive treatment and due to the absence of acquired resistance. However, PDT is associated with undesirable activities, such as non-specific photodynamic effects of sunlight on healthy tissues. Therefore, an important fundamental task is the development of improved PDT agents that selectively act on the affected areas. Here, we report the development of a hybrid protein-peptide system for the selective pH-dependent binding and subsequent photodynamic cancer cells ablation. It is known that a distinctive feature of cancer cells is a decreased pH level in the extracellular space. In this study we exploited a peptide fragment (pHLIP) as a targeting module, which spontaneously binds and embeds into the cell membrane when pH decreases below neutral. A mutant of miniSOG protein fused to pHLIP was used as a photosensitizing constituent. We demonstrate that this protein-peptide photosensitizing system selectively binds to HeLa cells at pH below 6.8 and kills them when exposed to light. These findings demonstrate the feasibility of using genetically encoded MiniSOG fusions with pHLIP for the targeted delivery of PSs to cancer cells and subsequent highly precise photodynamic therapy.

Regulation of Glucose Transport in Adipocytes by Interleukin-4.

Metabolic abnormalities such as obesity, insulin resistance, and type 2 diabetes mellitus are known to be associated with adipose tissue inflammation and impaired secretion of cytokines. Anti-inflammatory cytokine interleukin-4 (IL-4) was found to promote insulin sensitivity, glucose tolerance, and reduce lipid accumulation in vivo through multiple mechanisms, including direct regulation of lipolysis in adipocytes. However, little is known about its role in adipocyte glucose metabolism. This study reveals that IL-4 upregulates glucose uptake in adipocytes without additional activation of the insulin-dependent IRS1 (insulin receptor substrate 1)-Akt (protein kinase B) pathway. Moreover, the main transcription factor STAT6 (signal transducer and activator of transcription 6), regulated by IL-4, was not involved in adipocyte glucose uptake. The proteomic results showed that IL-4 upregulates expression of proteins involved in mitochondrial biogenesis, renewal, and glucose oxidation. Our study provides a new hypothesis, explaining protective effects of IL-4 against metabolic abnormalities through activation of adipocytes glucose utilization and maintenance of mitochondrial function under metabolic overload conditions.

NDRG1 Activity in Fat Depots Is Associated With Type 2 Diabetes and Impaired Incretin Profile in Patients With Morbid Obesity.

Objective: We aimed to investigate insulin-, mTOR- and SGK1-dependent signaling basal states in morbidly obese patients' fat. We analyzed the correlation between the signaling activity, carbohydrate metabolism, and incretin profiles of patients. Methods: The omental and subcutaneous fat was obtained in patients with obesity. The omental study included 16 patients with normal glucose tolerance (NGT) and 17 patients with type 2 diabetes mellitus (T2DM); the subcutaneous study included 9 NGT patients and 12 T2DM patients. Insulin resistance was evaluated using the hyperinsulinemic euglycemic clamp test and HOMA-IR index. The oral glucose tolerance test (OGTT) for NGT patients and mixed meal tolerance test (MMTT) for T2DM patients were performed. The levels of incretins (GLP-1, GIP, oxyntomodulin) and glucagon were measured during the tests. Signaling was analyzed by Western blotting in adipose tissue biopsies. Results: We have shown equal levels of basal phosphorylation of insulin- and mTOR-dependent signaling in omental fat depot in NGT and T2DM obese patients. Nevertheless, pNDRG1-T346 was decreased in omental fat of T2DM patients. Correlation analysis has shown an inverse correlation of pNDRG1-T346 in omental fat and diabetic phenotype (HbA1c, impaired incretin profile (AUC GLP-1, glucagon)). Moreover, pNDRG1-T346 in subcutaneous fat correlated with impaired incretin levels among obese patients (inverse correlation with AUC glucagon and AUC GIP). Conclusions: According to results of the present study, we hypothesize that phosphorylation of pNDRG1-T346 can be related to impairment in incretin hormone processing. pNDRG1-T346 in adipose tissue may serve as a marker of diabetes-associated impairments of the systemic incretin profile and insulin sensitivity.

Direct Effect of the Synthetic Analogue of Glucagon-Like Peptide Type 1, Liraglutide, on Mature Adipocytes Is Realized through Adenylate-Cyclase-Dependent Enhancing of Insulin Sensitivity.

Incretin hormones analogues, including glucagon-like peptide type 1 (GLP-1), exhibit complex glucose-lowering, anorexigenic, and cardioprotective properties. Mechanisms of action of GLP-1 and its analogues are well known for pancreatic ß-cells, hepatocytes, and other tissues. Nevertheless, local effects of GLP-1 and its analogues in adipose tissue remain unclear. In the present work effects of the GLP-1 synthetic analogue, liraglutide, on adipogenesis and insulin sensitivity of the 3T3-L1 adipocytes were examined. Enhancement of insulin sensitivity of mature adipocytes by the GLP-1 synthetic analogue liraglutide mediated by adenylate cyclase was demonstrated. The obtained results imply existence of the positive direct insulin-sensitizing effect of liraglutide on mature adipocytes.

Grain-Based Dietary Background Impairs Restoration of Blood Flow and Skeletal Muscle During Hindlimb Ischemia in Comparison With Low-Fat and High-Fat Diets.

Background: Among vascular pathologies associated with obesity, peripheral artery disease (PAD) occupies the important position. In clinical practice, nutritional interventions are recommended for patients with PAD. In this work, we investigated how the different dietary backgrounds affect the regeneration rate of ischemic hindlimb in mice. Methods: Male C57BL/6J mice were housed on three types of diet: low-fat (LFD), high-fat (HFD), and grain-based diet (GBD) for 13 weeks. Metabolic parameters including FBG level, ITT, and GTT were evaluated. The blood flow was assessed by laser Doppler scanning on 7, 14, and 21 days after hindlimb ischemia. Necrotic area of m.tibialis, macrophage infiltration, and angiogenesis/arteriogenesis were evaluated by histology. Glucose uptake in recovered skeletal muscle was analyzed using [3H]-2-deoxyglucose, and GLUT1 and GLUT4 expression were assessed by Western blotting. Results: In our work, we developed three experimental groups with different metabolic parameters: LFD with normal glucose metabolism, GBD with mild hyperglycemia, and HFD with impaired glucose tolerance. GBD-fed mice had a tendency to increase necrosis of m. tibialis and significantly higher macrophage infiltration than LFD and HFD groups. Moreover, GBD-fed mice had a trend to decreased blood flow recovery and significantly impaired arteriogenesis. Recovered skeletal muscle of GBD-fed mice had lower glucose uptake and decreased level of GLUT4 expression. Conclusion: Thus, we conclude that dietary background and metabolic status determine the rate of post-ischemic regeneration including angiogenesis, skeletal muscle recovery and metabolic activity. The most effective regeneration is supported by LFD, while the lowest rate of regeneration occurs on GBD.