Adiponectin mRNA Conjugated with Lipid Nanoparticles Specifically Targets the Pathogenesis of Type 2 Diabetes

Figures

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  Figure 1.. APN evaluation in vitro, with in vivo measurement of IPGTT, ITT, blood glucose and body weight in vivo. (A) C2C12 myotubes viability was assessed using the CCK8 assay. After transfection with APN-mRNA-LNP, the half maximal inhibitory concentration (IC50) was determined after 24 hours. (B) Relative APN Expression in differentiated Myotubes cell C2C12, (C) Relative APN Expression in Kidney mesangial cell SV40-MES13. APN gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. Both biological and technical triplicates were performed in vitro. Values are mean ± SD. n = 9, student t-test. (D) APN protein expression, three separate blot analyzes of the samples were conducted. (E) Optical densitometry was used to semi-quantify the bands, and ImageJ (1.53t) digital imaging processing software was used for analysis. GAPDH has been used as an endogenous control for protein expression. Protein levels are represented as mean ratio values quantified from protein bands of APN versus GAPDH compared to negative control. (n = 3, Mann-Whitney U tests),…

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  Figure 2.. APN in situ expression. (A) APN gene expression in different tissues. Skeletal muscles; Liver; Kidney; White fats; Brown fats. APN gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. In vivo, a total of five biological samples (n = 5) were collected. Of each sample, three replicates were performed technically., ** P < 0.01 vs. PBS control group and ##P <0.01 vs. Empty-LNP group. (B) Immunohistochemical staining for adiponectin in T2D skeletal muscles, liver, and kidney mice in the studied groups. Negative controls were performed by blocking PBS without APN-Ab. Treated groups were performed by blocking with APN-Ab (200X). Labeling index was used to determine APN-Ab percentile expression (the ratio of positively stained cells/total cells × 100). Blinded staining intensity scores were assigned to each specimen based on arbitrary values of 1, 2, or 3 (reflecting weak, intermediate, and bright staining). A total of five biological samples (n = 5) were collected; three sections were taken from each sample. Scale bar= 50 μm.

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  Figure 3.. Relative gene expression in different tissues. (A) Glut-4 gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. (B) DGKd gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, and Kidney. (C) PKCe gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. Gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. Both biological and technical triplicates were performed in vitro. Values are mean ± SD. n = 9, student t-test. In vivo, a total of five biological samples (n = 5) were collected. Of each sample, three replicates were performed technically. (D) Glut-4, DGKd, and PKCe protein expression, three separate blot analysis of the samples were conducted. (E) Optical densitometry was used to semi-quantify the bands, and ImageJ (1.53t) digital imaging processing software was used for analysis. GAPDH has been used as an endogenous control…

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  Figure 4.. Relative IR expression in different studied tissues. (A) Differentiated Myotubes cell C2C12; (B) Kidney mesangial cell SV40-MES13; (C) Skeletal muscles; (D) Liver; (E) Kidney. IR gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. Both biological and technical triplicates were performed in vitro. Values are mean ± SD. n = 9, student t-test. In vivo, a total of five biological samples (n = 5, student t-test) were collected. Of each sample, three replicates were performed technically. Significances are shown above bars.

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  Figure 5.. Pancreas sections in the studied groups. (A) Pancreas sections focusing on islands of Langerhans diameter (H&E stain, 200X & 400X). (B) Islands of Langerhans average diameter; the diameter average was taken from seven fields on the same slid (n = 7, student t-test), Significances are shown above the bars: **P < 0.01 vs. the PBS and ##P < 0.01 vs. Empty-LNP. Scale bar= 50 μm.

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  Figure 6.. EGFR expression in vitro and in vivo. (A) Relative EGFR Expression in different Differentiated Myotubes cell C2C12; (B) Kidney mesangial cell SV40-MES13; (C) kidney. EGFR gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Fold-change values less than one indicate downregulation, whereas values greater than one indicate upregulation. Both biological and technical triplicates were performed in vitro. Values are mean ± SD. n = 9, student t-test. In vivo, a total of five biological samples (n = 5, student t-test) were collected. Of each sample, three replicates were performed technically. Significances are shown above bars. (D) Overall correlation between APN and EGFR gene expression in kidney. (E) Light microscopic appearance in kidney tissue section of the studied groups of DIO mice, (yellow arrow) indicates Hyperplasia of partial cells in the Bowmans capsule of the glomeruli and (Black arrow) indicates mild diffuse mesangial matrix expansion (PAS stain, 1000X). A total of five biological samples (n = 5, student t-test) were collected; three sections were taken from each sample. Scale bar= 50 μm.

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  Figure 7.. Relative gene expression of the pro-inflammatory cytokines. (A) TNFa gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. (B) IL-6 gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. (C) IL-1b gene in differentiated myotubes cell C2C12, Kidney mesangial cell SV40-MES13, Skeletal muscles, Liver, Kidney, White fats, and Brown fats. Gene expression was calculated based on the Law of Fold-change, which is 2-ΔΔCT. Both biological and technical triplicates were performed in vitro. Values are mean ± SD. n = 9, student t-test. In vivo, a total of five biological samples (n = 5) were collected. Of each sample, three replicates were performed technically. (D) Glut-4 TNFa and IL-6 protein expression, three separate blot analysis of the samples were conducted. (E) Optical densitometry was used to semi-quantify the bands, and ImageJ (1.53t) digital imaging processing software was used for analysis. GAPDH has been used as an endogenous control for protein expression. Protein levels are represented as mean ratio values…

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  Figure 8.. Histopathology results in the studied groups. (A) longitudinal section of the skeletal muscles of the femur, ranks score were used to represent the marked focal degeneration of the skeletal muscle bundles, which signed with different amount of “+” depending on how many other categories lay between these border parameters “weak” (+), “moderate” (++), “strong” (+++) and their variations. (B) liver samples; (C) light microscopic appearance in the kidney tissue section (H&E stain, 200X); black arrows indicate fat degenerations, while yellow arrows indicate hydropic degenerations. (D) Percentile of steatosis and hydropic degeneration in the liver of the studied mouse groups; (E) Percentile of tubular degeneration in the kidney of the studied mouse groups. The percent of fat degeneration in different samples and/or hydropic degeneration was calculated depending on Labeling index (the ratio of positively stained cells/total cells×100). A total of five biological samples (n = 5, student t-test) were collected; three sections were taken from each sample. Scale bar= 50 μm.

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  Figure 9.. Conclusion based on our hypothesis. The alpha and beta cells of the islet of Langerhans work together to control and regulate glucose uptake, gluconeogenesis, and lipolysis in normal conditions. There are several abnormal features associated with type 2 diabetes, including increased adiposity, inflammation, insulin resistance, and decreased adipose tissue function. Thus, adiponectin (APN) production is reduced. As a result of the inhibition of adiponectin receptors (AdipoR1/AdipoR2) and subsequent association with adapter proteins, glucose uptake is inhibited by APPL1-Rab5 or APPL1-AMP-AMPK-mediated translocation of glucose transporter 4 (GLUT4), resulting in an activation of insulin resistance signaling [36]. Insulin resistance is commonly characterized by decreased GLUT4-dependent glucose absorption in skeletal muscles and adipose tissue [38]. A temporal increase in intracellular diacylglycerol (DAG) mass is associated with glucose-induced insulin resistance [39]. Prolonged elevation of intracellular DAG activates protein kinase C (PKC) isoforms, leading to insulin resistance, intracellular lipid accumulation, and impaired signal transmission [40, 41]. Glucose transport is…