Insulin Therapy is Associated With Increased Myocardial Interstitial Fibrosis and Cardiomyocyte Apoptosis in a Rodent Model of Experimental Diabetes

Figures

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  FIGURE 1. Study Timeline. After being maintained on a regular diet for 2 weeks, 4–6-week-old male Wistar rats were divided into three groups: 2 groups were injected with Streptozotocin (65 mg/kg) to induce diabetes and 1 group received a control solution. Eight weeks after the induction of diabetes (10 weeks of study time), saline-delivering pumps were implanted in the control and one of the diabetic groups (untreated diabetics, UDM), while insulin-delivering pumps were installed in the other diabetic group (insulin-treated diabetics, IDM). Therefore, at the end of the experiment at 14 weeks, three cohorts were inspected: controls (top panel), untreated diabetics (UDM, middle panel), and insulin-treated diabetics (IDM, bottom panel).

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  FIGURE 2. Left Ventricle Myocardial Interstitial Fibrosis and Cardiomyocyte Apoptosis (%). (I) Using Picrosirius red staining of collagen in the extracellular matrix, baseline LV fibrosis among controls (a) was measured (scale bar: 50 µm). UDM and IDM rats experienced a 5-fold (b) and 10-fold (c) increase in LV myocardial interstitial fibrosis, respectively. (II) Quantification of the myocardial interstitial fibrosis. (III) Using TUNEL assay, apoptotic material appeared green (white arrowhead). Compared to controls (a), the rate of apoptosis doubled in the left ventricle of untreated diabetic rats (b) and tripled among IDM rats (c). (IV) Quantification of the cardiomyocyte apoptosis in the LV (%).

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  FIGURE 3. In vitro, Insulin Induces Cardiomyocyte Apoptosis and Myofibroblast α-SMA Expression in a Dose-dependent Manner. (I) Images of human cardiomyocytes (AC16) after 48 h of incubation with tissue culture media alone (a), tissue culture media + insulin 0.1 nmol/L (b), and tissue culture media + insulin 10 nmol/L (c). The number of apoptotic cells (white arrowhead) increases with the increasing concentration of insulin (scale bar: 300 µm). (II) Line chart quantifying the trend of apoptosis in human cardiomyocyte AC16 cultures over 48 h under different insulin concentrations. (III) HCF are shown with the nucleus (blue) and α-SMA (red) at baseline among controls (a). There is no significant difference in α-SMA expression in HFC treated with insulin at 1 nmol/L (b). However, the levels of α-SMA almost doubled with insulin treatment at 10 nmol/L (c, scale bar: 50 µm). (IV) Quantification of the α-SMA expression level using immunofluorescence.

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  FIGURE 4. Insulin Induces Collagen Secretion and α-SMA Expression to Levels Similar to TGFβ1 in HCF. (I) Similar to the findings in rat LV tissue, insulin treatment (10 nmol/L) induced extracellular collagen secretion to a comparable degree with the known fibrosis-inducing agent, TGFβ1 (5 ng/ml). (II) Using total cell immunofluorescence, α-SMA levels were inspected among control HCF simply incubated in culture media for 72 h (a). α-SMA levels were upregulated after HCF incubation with TGFβ1 (5 ng/ml) (b), insulin (10 nmol/L) (c), or insulin (10 nmol/L) + TGFβ1 (5 ng/ml) (d) for 72 h in culture media (Scale bar: 50 µm). (III) Quantification of the of α-SMA expression levels using immunofluorescence in HCF among controls, HCF with TGFβ1, HCF with insulin, and HCF with insulin + TGFβ1.

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  FIGURE 5. SMAD2 Protein Expression Level in HCF Treated with Insulin or TGFβ1. (I) In the presence of growth media, HCF were treated separately with control solution, insulin (10 nmol/L), or TGFβ1 (5 nmol/L) for 72 h. Subsequently, SMAD2 protein expression level was assessed using Western blotting, with GAPDH serving as a control for normalization. These experiments were done in triplicates (Trials 1, 2, and 3). (II) Band intensities were quantified using ImageJ software. Insulin increased SMAD2 protein expression level to more than 3.5 × when compared to controls. TGFβ1, serving as a positive control, also increased SMAD2 expression.