Hyperglycemia alters the gene and protein expression of CDC42 in small and large intestine of Sprague-Dawley rats
Marija Stojanovic, Yssel Mendoza-Mari, Vikrant Rai, Devendra K. Agrawal
TL;DR
This study shows that high blood sugar in diabetic rats changes CDC42 gene and protein levels in the intestines, with differences between small and large intestines and between genders.
Contribution
The study reveals gender-specific and tissue-specific changes in CDC42 expression in diabetic rats, linking hyperglycemia to intestinal alterations.
Findings
CDC42 gene expression increased in the small and large intestine of diabetic rats, especially in females.
Protein levels of CDC42 decreased in the colon of diabetic rats.
Inflammatory cell infiltration was observed in both ileum and colon of diabetic rats.
Abstract
Diabetes mellitus (DM) is associated with gastrointestinal complications, including structural and functional changes in both small and large intestine. CDC42, a Rho GTPase, plays a critical role in maintaining epithelial integrity through regulation of tight junctions and cytoskeletal organization. Moreover, CDC42 expression has been reported in inflammatory bowel disease (IBD). However, its expression patterns and regulatory mechanisms in the diabetic gut remain poorly defined, particularly in the context of DM - IBD comorbidity. Our study aimed to evaluate histological changes and CDC42 gene and protein expression in the small intestine (ileum) and large intestine (colon) of streptozotocin-induced female and male Sprague-Dawley rats. Rats were divided in control (n = 10) and diabetic (n = 12) group. Histological analysis was based on hematoxylin-eosin staining sections. CDC42 gene…
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TopicsBarrier Structure and Function Studies · Pancreatic function and diabetes · Metabolism, Diabetes, and Cancer
Introduction
Diabetes mellitus (DM), characterized by persistent hyperglycemia, refers to a metabolic disorder affecting different organ systems. Epidemiological data indicate an increasing number of newly diagnosed cases of DM, especially due to lifestyle habits such as lack of physical activity and poor quality diet leading to obesity [1]. It is expected that by 2030, 439 million adults will be diagnosed with DM [2]. DM is typically classified as Type 1 (insulin-dependent) and Type 2 (insulin-independent), presented with various symptoms depending on the type and the stage of the disease. Untreated long-lasting hyperglycemia can lead to numerous complications such as diabetic retinopathy, nephropathy, or neuropathy [3].
It has been reported that DM is one of the most frequent comorbidities presented in patients with ulcerative colitis (UC). UC, along with Crohn’s disease (CD), represents a clinical entity known as inflammatory bowel disease (IBD), characterized by intestinal wall inflammation mediated by innate and adaptive immune system disturbances [4]. It has been suggested that both environmental factors (smoking, dietary habits, the use of nonsteroidal anti-inflammatory drugs (NSAIDs), and genetic predisposition represent risk factors for IBD development, leading to chronic inflammation (Fig. 1) [5]. A chronic inflammatory response is also observed in diabetes mellitus, primarily driven by cytokines released from excess adipose tissue associated with obesity. These findings indicate that inflammation associated with obesity might play a role in the development of IBD, while the elevated levels of pro-inflammatory cytokines seen in IBD could, in turn, promote systemic inflammation and potentially contribute to insulin resistance and DM [6].
Fig. 1. Illustration of the interplay between principal factors contributing to the pathophysiology of IBD and DM, including obesity, smoking, diet, drugs, genetics, sedentary lifestyle, and systemic inflammation
From a clinical perspective, corticosteroid therapy is widely used for the acute treatment of IBD; however, corticosteroid-induced diabetes and hyperglycemia are among the frequent complications in IBD patients receiving this therapy. This poses significant challenges in treating DM patients with concomitant IBD during exacerbations. A recent study by Bezzio et al. reported a common genetic background between DM type 1 and IBD, identifying susceptibility genes such as ORMDL3, HERC2, TNFAIP3, IL-10, IL-26, and IL-27 [4]. Our previous study, based on network analysis and a literature review, identified ORMDL3 as a gene interacting with various factors contributing to inflammation, impaired autophagy, and mitochondrial dysfunction related to IBD [7]. From the aspect of inflammation, the NLRP3 inflammasome is a critical mediator connecting diabetes mellitus and inflammatory bowel disease [6].
Whether and to what extent the cell division cycle 42 (CDC42) molecule contributes to IBD and hyperglycemia is our current area of interest. CDC42 is a member of the RHO family of small GTPases and regulates epithelial polarity, as well as fundamental cellular biological functions such as growth, migration, and cellular trafficking [8]. In addition, CDC42 is important for maintaining an intact intestine epithelial barrier, by regulating tight junctions between intestinal epithelial cells [9].
Literature findings showed the downregulation of CDC42 in patients with active forms of IBD [10]. In mouse model, CDC42 deficiency has been associated with enhanced susceptibility to chemically induced colitis and increased risk of dysplastic changes due to chronic inflammation and impaired mucosal repair [11]. In addition, another IBD mouse model showed significantly elevated levels of inflammatory cytokine in the colon tissue and serum of IBD mice; however, treatment with CDC42 was able to reduce these elevated cytokine levels [12]. Under in vitro hyperglycemic conditions, CDC42 overexpression has been shown to promote F-actin polymerization, alter cytoskeletal organization, and consequently reduce the migration of human umbilical vein endothelial cells (HUVECs) [13]. In addition, modulation of F-actin cytoskeletal organization by CDC42 mitigates intestinal injury, as reported in acute pancreatitis [14]. CDC42 was also identified as one of the P21-activated kinase (PAK1) upstream regulators. According to the literature reports, TNF-α is an important mediator of PAK1 activation in IBD [15]. Considering the latter, it can be stated that CDC42 might participate in intestinal inflammation driven by TNF-α activation.
There is scarcity of literature on the association of CDC42 pathways in relation to IBD and DM. Our recent in silico analysis predicted CDC42 interactions related to IBD and hyperglycemia emphasizing the following genes: LGR4, CCND1, RSPO3, and DOCK8, which are regulated by transcriptional factors such as TFAP2A, NFIC, E2F1, and PAX2 [16].
Gaining a deeper understanding of how CDC42 contributes to the pathogenesis of inflammatory bowel disease (IBD) and diabetes mellitus (DM) may reveal novel therapeutic targets for the treatment of these conditions. Based on the considerations outlined above, this study sought to investigate the expression of the CDC42 gene in small and large intestine tissues of diabetic rats. In this context, we hypothesized that chronic hyperglycemia dysregulates CDC42 expression and contributes to intestinal epithelial dysfunction.
Materials and methods
Animals and study design
Male and female, 8–10 weeks old, Sprague Dawley rats (Charles River Laboratories, USA) were used in this study, according to guidelines of the National Institutes of Health and USDA for the care and use of experimental animals. The experimental research protocol (R24IACUC013) was approved by the Institutional Animal Care and Use Committee (IACUC) of Western University of Health Sciences, Pomona, CA, USA for an ongoing study. The animals were housed at constant room temperature (22 °C) with a 12 h light/dark cycle. They were divided into two groups: the control group (n = 12) and the diabetic group (n = 12). During the course of the protocol, two animals from the control group died. Consequently, data collection and analysis were conducted on the remaining ten control animals (n = 10). Animals in the control group were fed a normal diet (20% protein, 70% carbohydrate, 10% fat; #D12450B; Research Diet Inc) and water ad libitum. Animals in the diabetic group received a high-fat diet (HFD; 45% fructose (carbohydrate), 5% protein, 45% fat; #D12451; Research Diet Inc) and water ad libitum. To induce diabetes mellitus type II, after 6 weeks of HFD, rats in the diabetic group were injected i.p. with a single dose of streptozotocin (25 mg/kg) dissolved in 0.1 M sodium citrate buffer at pH 4.4. At the same time, animals in the control group received i.p. injection of vehicle citrate buffer (0.25 ml/kg). The random blood sugar was measured by Alphatrak glucometer using tail vein blood. Blood glucose levels > 300 mg/dL for a minimum of two weeks were considered as confirmation of the diabetic model. Four weeks after the streptozotocin injection, animals were sacrificed. Small (ileum) and large (colon) intestine tissue samples were collected in 10% formalin (6764254, ThermoFisher Scientific, Waltham, Massachusetts, USA) for histological analysis, in RNA later (AM7021, ThermoFisher Scientific, Waltham, Massachusetts, USA) for RNA extraction, and tissues were also stored at −80 °C for protein isolation.
Histology processing and staining
Small intestine and large intestine tissue were prepared using a Tissue-Tek VII system (Sakura Finetek, Torrance, CA, USA) tissue processor. The samples underwent sequential immersion in ethanol, xylene, and paraffin wax for processing. After embedding in paraffin, tissue blocks were sectioned into 7 μm slices using a Leica RM2265 rotary microtome (Leica™, Wetzlar, Germany). These slices were mounted onto glass slides and incubated at 60 °C for one hour. Standard deparaffinization and rehydration protocols were followed prior to staining and immunofluorescence analysis. For hematoxylin and eosin (H&E) staining, slides were treated with hematoxylin for one minute and eosin for two minutes. After staining, sections were coverslipped using Cytoseal 60, a xylene-based mounting medium (23–244257, Thermo Fisher Scientific, Waltham, MA, USA). Images were acquired using a Leica DM6 light microscope (Leica™, Wetzlar, Germany) at a magnification scale of 100 μm. Morphometric evaluation of ileal sections included measurements of villus height and mucosal thickness, whereas crypt depth and mucosal thickness were assessed in colonic sections, with all parameters expressed in micrometers (µm). A minimum of three consecutive sections per sample were analyzed, with 3–5 imaging fields evaluated for each section.
Quantitative real-time polymerase chain reaction (RT-qPCR)
Approximately 50 mg of both small intestine and large intestine tissue per sample were utilized for total RNA isolation using TRIZOL reagent (T9424, Millipore Sigma, Burlington, MA, USA), following the manufacturer’s guidelines. The isolated RNA was resuspended in 30 µL of nuclease-free water (BP561-1, ThermoFisher Scientific, Waltham, MA, USA), and its concentration was determined using a Nanodrop 2000 Spectrophotometer (ThermoFisher, Waltham, MA, USA). RNA were then reverse transcribed into 2µg cDNA using the AzuraQuant™ cDNA Synthesis Kit (AZ-1996, Azura Genomics Inc., Raynham, MA, USA) following the supplier’s instructions, with reactions conducted on a T100™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The resulting cDNA was diluted at a 1:20 ratio with nuclease-free water. Quantitative real-time PCR (RT-qPCR) was performed in triplicate using AzuraView™ GreenFast qPCR Blue Mix LR (AZ-2350, Azura Genomics Inc., Raynham, MA, USA) in a 10 µL final reaction volume. Amplification was conducted on a C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) under the following cycling conditions: initial denaturation at 95 °C for 3 minutes, followed by 40 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds. Post-amplification, a melting curve analysis was performed. The primers specific to the target genes and the housekeeping gene were obtained from Integrated DNA Technologies (Coralville, IA, USA). Gene expression was normalized to 18S rRNA, and relative expression levels were determined using the 2^−ΔΔCT^ method. The nucleotide sequence for CDC242 was forward: 5’-GATTACGACCGCTGAGTTATCC-3’ and reverse: 5’-GTTATCTCAGGCACCCACTTT-3’ and for 18S was forward: 5’-CCCACGGAATCGAGAAAGAG-3’ and reverse: 5’-TTGACGGAAGGGCACCA-3’.
Western blot analysis
For Western blot analysis, 100 mg of small intestine and large intestine tissue was utilized for total protein extraction. The tissues were homogenized in 1 mL of Radioimmunoprecipitation Assay (RIPA) Lysis and Extraction Buffer (ThermoFisher Scientific, PI89901) containing a protease inhibitor cocktail (Pierce Protease Inhibitor Mini Tablets, A32953, ThermoFisher Scientific) using a PowerGen 125 tissue homogenizer (ThermoFisher Scientific). Once homogenization was complete, samples were centrifuged at 4 °C for 10 min to eliminate insoluble debris. The resulting supernatant was collected, and protein concentration was assessed using the Bradford assay with the Bio-Rad Protein Assay Kit II (5000002, Bio-Rad Laboratories). An amount of 30 µg of total protein was separated on Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) gels (4–15% Mini-PROTEAN Tris-Glycine eXtended (TGX) Precast Gels, 4561084, Bio-Rad) and transferred onto polyvinylidene difluoride (PVDF) membranes (1620177, Bio-Rad). Transfer efficiency was verified with Ponceau Red staining (P7170, Millipore Sigma). Membranes were then blocked for one hour in 1x Tris Buffered Saline (TBS; ThermoFisher, 50–489-119) containing 0.1% Tween 20 (P1379, Millipore Sigma) and 5% skim milk (1706404, Bio-Rad). Primary antibodies (listed in Table 1) were incubated overnight at 4 °C in a blocking buffer. After washing three times with TBS containing 0.1% Tween 20, membranes were incubated with appropriate secondary antibodies (Table 1) for one hour at room temperature. Following additional washes, protein bands were visualized using Pierce Enhanced Chemiluminescence (ECL) substrate (32106, ThermoFisher) and imaged using the ChemiDoc XRS + system (Bio-Rad). After visualization and subsequent washing, stripping was performed under standard conditions (1 h at 37 °C), followed by blocking and overnight incubation with another antibody. Image analysis was performed using ImageJ software (NIH). The housekeeping protein β-actin to mouse was used for small intestine samples while β-actin to rabbit was used for large intestine samples for confirmation of consistent protein loading.
Table 1. Primary and secondary antibodies used for western blot and immunofluorescenceAntibodySupplierCatalog numberDilution in Western blotDilution in immunofluorescencePrimary antibodies CDC42Biorbytorb58311:10001:250 ACTBAbcamab82261:1000- ACTBMybiosource.comMBS8217861:1000-Secondary antibodies Anti-mouseInvitrogenA160111:3000- Anti-rabbitInvitrogenA160231:2000- Anti-rabbitAbcamab1500651:500ACTB antibody for β-actin
Immunofluorescence staining protocol
Tissue sections underwent deparaffinization, rehydration, and antigen retrieval before proceeding with immunofluorescent staining. Following these preparatory steps, the slides were rinsed with phosphate-buffered saline (PBS). Antigen retrieval was performed using a heat-induced epitope retrieval (HIER) buffer (ab208572) followed by cooling to room temperature. The tissues were circled with Pap Pen and incubated for one hour in a blocking/permeabilization buffer. This buffer consisted of 5% (v/v) appropriate normal serum, 0.25% Triton X-100, and 0.1% bovine serum albumin (BSA). After blocking, the sections were incubated with the primary antibody overnight at 4 °C at a titrated dilution: rabbit anti-CDC42 (1:250; Biorbyt, orb5831). After overnight incubation, the sections were washed with PBS 3 times, each wash lasting for five minutes, and incubated with donkey polyclonal antibody to rabbit Alexa Flour 488 (green) - conjugated secondary antibody (abcam; ab150065) for 30 min (1:500 dilution) at room temperature. Slides were washed in 1xPBS 3 times for 5 min each, and incubated in Sudan Black B for 5 min. After washing the slides with tap water to remove Sudan Black, the slides were dried at room temperature before mounting. Nuclei were counterstained using DAPI (4′,6-diamidino-2-phenylindole). Fluorescence imaging was conducted using a Leica DM6 microscope at a scale of 100 μm. To ensure specificity, isotype control antibodies were included as negative controls. For quantitative analysis, three representative images were captured per section. These were used to assess fluorescence intensity for CDC42 using ImageJ software (version 1.54 J, NIH, USA). The mean fluorescence intensity was calculated as the average of the three measurements for each group.
Statistical analysis
Data were analyzed using GraphPad Prism 10 for Windows (version 10.1.1) and presented as mean ± standard deviation for normally distributed variables, or median with interquartile range (10–90 percentile) for data not following a normal distribution. The normality of data was verified by Shapiro Wilk’s test. For data showing normal distribution statistical comparisons between the groups were performed using Student’s t-test, while Mann Whitney-U test was used to compare the data with non-normal distribution. Linear regression analysis was performed to assess the relationship between CDC42 relative gene expression (ΔΔCt) and both, weight gain and glucose levels in female and male rats. Differences were considered significant at p < 0.05.
Results
Body weight and glucose levels measurements
Body weight measurements at two time points, along with percentage (%) of weight gain and fasting blood glucose levels increase in diabetic group in comparison to the control values, are summarized in Table 2. Male diabetic rats exhibited a statistically significant increase in baseline weight (196.0 g; 186.4–198.0 percentile; p < 0.01), final weight at sacrifice (473.8 g; 454.8–516.0 percentile; p < 0.01), and overall percentage (%) of weight gain (49.89 ± 22.77; p < 0.05) compared to female diabetic rats. In the female group, baseline weight was 145.5 g (141.6–156.3 percentile), sacrifice weight was 320.0 g (301.8–326.0 percentile), and weight gain was 25.03 ± 1.80%. Although diabetic female rats had higher percentage of glucose levels increase, compared to the control, (261.5 ± 76.76%) than males (230.9 ± 18.00%), this difference was not statistically significant.
Table 2. Body weight measurements and % increase compared to the control in fasting blood glucose levels in female and male diabetic ratsParametersFemale ratsMale ratsWeight at baseline (g)145.5 (141.6–156.3.6.3)196.0 (186.4–198.0) **Weight at sacrifice (g)320.0 (301.8–326.0)473.8 (454.8–516.0) **Percent weight gain25.03 ± 1.8049.89 ± 22.77 *Percent increase in blood glucose levels261.5 ± 76.76230.9 ± 18.00Data are presented as mean ± SD for normally distributed variables, or as median (10th–90th percentile) for non-normally distributed variables. Student’s t-test (normal distribution), or Mann Whitney-U test (non-normal distribution); *p < 0.05; **p < 0.01 vs. Female group; female (n = 6); male (n = 6)
Small intestine
Histological analysis
Hematoxylin and eosin (H&E) staining of ileal sections from diabetic rats revealed notable pathological changes, particularly an increase in cellular infiltration within the lamina propria of the mucosal layer, compared to non-diabetic controls. These alterations observed in diabetic animals suggest the presence of an inflammatory response, accompanied by Goblet cell hypoplasia. In addition, intestinal villi of diabetic animals exhibited marked architectural alterations, including villus shortening, blunting, and disruption. In contrast, the control group exhibited a well-preserved intestinal architecture, with all characteristic histological layers clearly distinguishable. These included regularly shaped villi extending into the lumen, a continuous epithelial lining within the tunica mucosa, a defined submucosa, and properly organized circular and longitudinal layers of smooth muscle (Fig. 2A and B). Figure 2C and D represent quantification data fot the ileum tissue samples in the control and diabetic group. Statistically significant decrease in villus height was shown in the diabetic group in comparison to the control (p < 0.0001) (Fig. 2C). No significant changes were found in the mucosal thickness of the ileum tissue samples between the groups (Fig. 2D).
Fig. 2. Hematoxylin and eosin staining in the small intestine (ileum) tissues of control (A) and diabetic rats (B). An asterisk indicates inflammatory infiltration in the lamina propria tunicae mucosae. 1- tunica mucosa (ileal villi); 2- tunica submucosa; 3- tunica muscularis. Images are representative of all H&E analyses for n = 10 (control) and n = 12 (diabetic). Images were taken at 20x magnification and scale bar 100 μm. Quantification of villus height (C) and mucosal thickness (D) in the ileum of control and diabetic rats. Data are presented as mean ± SD. Student’s t-test; ****p < 0.0001
Gene expression analysis by RT-PCR in the small intestine
As shown in Fig. 3A, CDC42 expression was significantly increased in the small intestine of diabetic rats compared to control (p < 0.01). When analyzed by gender, female rats in the diabetic group demonstrated statistically highly significant (p < 0.001) increase in CDC42 expression relative to the control (Fig. 3B). In male rats, a similar trend of increased CDC42 expression was observed in the diabetic group; however, this difference did not reach statistical significance (p > 0.05; Fig. 3C).
Fig. 3RT-PCR data for mRNA transcripts of CDC42 in the small intestine-ileum (SII) of control and diabetic rats relative to the housekeeping gene 18s. Data are presented as mean ± SD for normally distributed variables, or as median (10th–90th percentile) for non-normally distributed variables. A- animals of both sexes included; B- only female rats; C- only male rats. Student’s t-test (normal distribution), or Mann Whitney-U test (non-normal distribution). **p < 0.01; ***p < 0.001 diabetic vs. control; n = 10 (control); n = 12 (diabetic)
Correlation between CDC42 relative gene expression and both blood glucose levels and weight gain in the small intestine
Correlation analysis between CDC42 relative expression (ΔΔCt) and weight gain revealed a moderate positive correlation in female rats (R² = 0.48), suggesting that increased weight gain may be associated with increased ΔΔCt and, consequently, decreased CDC42 expression. In contrast, no meaningful correlation was observed in male rats (R² = 0.18), indicating no clear association between the two parameters in this group (Fig. 4A and B). Furthermore, no significant correlation was found between glucose levels and CDC42 in either female (R² = 0.03) or male rats (R² = 0.18) (Fig. 4C and D).
Fig. 4. Correlation between both weight gain (g) and blood glucose levels (mg/dL) and CDC42 relative expression (ΔΔCt) in the small intestine -ileum (SII) of female (n = 6) (A, C) and male (n = 6) (B, D) diabetic rats
Western blot analysis of CDC42 expression in the small intestine
According to the bands obtained from western blot imaging from ileum tissue samples CDC42 was detected in both control and diabetic group at 25 kDa, as expected according to its predicted molecular weight of 25 kDa (Fig. 5A). Densitometric analysis, with mean intensity normalized to β-actin, showed a statistically significant (p < 0.05) increase in CDC42 expression in diabetic group, compared to the control (Fig. 5B). Unlike expected at 42 kDa, housekeeping protein used in this experiment (β-actin) was observed at 25 kDa, presented with consistent bands in both control and diabetic group, as shown in Fig. 5A.
Fig. 5. Western blot analysis of CDC42 in the ileum tissue samples of control and diabetic rats. A- Representative β-actin and CDC42 western blot bands in control and diabetic group B- Immunoblots quantification of CDC42. All bands were quantified and normalized by β-actin. Data are presented as mean ± SD; Student’s t-test; *p < 0.05 diabetic vs. control; n = 7 (control); n = 8 (diabetic)
Immunofluorescence analysis of CDC42 expression in the small intestine
Immunofluorescence analysis revealed a statistically highly significant increase (p < 0.001) in CDC42 expression in the small intestine of diabetic rats compared to control (Fig. 6A). In female rats, CDC42 mean fluorescence intensity (MFI) was significantly higher in the diabetic group compared to controls (p < 0.05; Fig. 6B), indicating increased protein expression. In male rats, although a similar trend of increased CDC42 MFI was observed in the diabetic group, the difference was not statistically significant (p > 0.05; Fig. 6C).
Fig. 6. Immunofluorescence (IF) staining of small intestine from control and diabetic rats. Images are representative of all IF analyses for n = 10 (control) and n = 12 (diabetic). All images were taken at 20x magnification and scale bar 100 μm (A). Comparisons between control and diabetic groups (B) were performed using Student’s t-test (normal distribution), or Mann Whitney-U test (non-normal distribution), showing mean fluorescence intensity (MFI). Data are presented as mean ± SD for normally distributed variables, or as median (10th–90th percentile) for non-normally distributed variables. *p < 0.05; ***p < 0.001 diabetic vs. control; n = 10 (control); n = 12 (diabetic)
Large intestine
Histological analysis
Histological analysis of large intestine tissue (colon) stained with hematoxylin and eosin revealed distinct morphological alterations in diabetic group compared to the control. Prominent inflammatory infiltration was presented in the submucosal layer of diabetic rats. As shown in Fig. 7B. disorganized connective tissue and signs of inflammation are present. The overlying mucosal architecture, including the crypts, was altered, displaying an irregular epithelial lining. In addition, goblet cell depletion was evident in certain regions, suggesting compromised mucosal barrier integrity. As shown in Fig. 7B, increased thickness of mucosal layer is presented. Quantification data presented in Fig. 7C and D showed a significant increase in crypt depth (p < 0.01) and mucosal thickness (p < 0.05) in colon tissue samples from diabetic group in comparison to the control.
Fig. 7. Hematoxylin and eosin staining in the large intestine (colon) tissues of control (A) and diabetic rats (B). Circle indicates inflammatory infiltration. 1- tunica mucosa 2-tunica submucosa. Images are representative of all H&E analyses for n = 10 (control) and n = 12 (diabetic). Images were taken at 20x magnification and scale bar 100 μm. Quantification of crypt depth (C) and mucosal thickness (D) in the colon of control and diabetic rats. Data are presented as mean ± SD. Student’s t-test; *p < 0.05; **p < 0.01
Gene expression analysis by RT-PCR in the large intestine
Based on the RT-PCR analysis of large intestine samples, CDC42 relative expression was significantly higher (p < 0.05) in diabetic group in comparison to the control, as presented in Fig. 8A. Gender-specific analysis revealed significantly increased (p < 0.01) CDC42 expression in female diabetic rats, in comparison to the control ones (Fig. 8B). CDC42 expression was higher in male diabetic rats versus control, however these changes didn’t reach statistical significance (p > 0.05; Fig. 8C).
Fig. 8RT-PCR data for mRNA transcripts of CDC42 in the large intestine (colon) of control and diabetic rats relative to the housekeeping gene 18s. A- animals of both sexes included; B- only female rats; C- only male rats. Data are presented as mean ± SD. Student’s t-test; *p < 0.05; **p < 0.01 diabetic vs. control; n = 10 (control); n = 12 (diabetic)
Correlation between CDC42 relative gene expression and both blood glucose levels and weight gain in the large intestine
Correlation analysis between CDC42 relative expression (^ΔΔ^Ct) and weight gain in the large intestine showed a moderate positive correlation in female rats (R² = 0.46), suggesting that increased weight gain may be associated with increased ^ΔΔ^Ct and, consequently, decreased CDC42 expression (Fig. 9A). Similarly, in male rats, a positive correlation was observed between weight gain and CDC42 ^ΔΔ^Ct (R² = 0.32) (Fig. 9B). In contrast, negative correlation was found between glucose levels and CDC42 ^ΔΔ^Ct in both female (R² = 0.59) and male rats (R² = 0.09) (Fig. 9C and D).
Fig. 9. Correlation between both weight gain (g) and blood glucose levels (mg/dL) and CDC42 relative expression (ΔΔCt) in the large intestine (colon) of female (n = 6) (A, C) and male (n = 6) (B, D) diabetic rats
Western blot analysis of CDC42 expression in the large intestine
CDC42 was detected at a molecular weight of approximately 63 kDa in the large intestine of both control and diabetic rats (Fig. 10A). Quantitative analysis, normalized to β-actin, demonstrated a significantly decreased expression of CDC42 in the diabetic group compared to the control (p < 0.001) (Fig. 10B).
Fig. 10. Western blot analysis of CDC42 in the colon tissue samples of control and diabetic rats. A- Representative β-actin and CDC42 western blot bands in control and diabetic group B- Immunoblots quantification of CDC42. All bands were quantified and normalized by β-actin. Data are presented as mean ± SD; Student’s t-test; *p < 0.001 diabetic vs. control; n = 7 (control); n = 7 (diabetic)
Immunofluorescence analysis of CDC42 expression in the large intestine
Immunofluorescence analysis revealed a significant decrease (p < 0.01) in CDC42 expression in the large intestine of diabetic rats compared to control (Fig. 11C). In male rats, CDC42 mean fluorescence intensity (MFI) was significantly lower in the diabetic group compared to controls (p < 0.0001; Fig. 11E). In female rats, although a similar trend of decreased CDC42 MFI was observed in the diabetic group, the difference was not statistically significant (p > 0.05; Fig. 11D).
Fig. 11. Immunofluorescence (IF) staining of large intestine (colon) from control and diabetic rats. Images are representative of all IF analyses for n = 10 (control) and n = 12 (diabetic). All images were taken at 20x magnification and scale bar 100 μm (A). Comparisons between control and diabetic groups (B) were performed using Student’s t-test (normal distribution), or Mann Whitney-U test (non-normal distribution), showing mean fluorescence intensity (MFI). Data are presented as mean ± SD for normally distributed variables, or as median (10th–90th percentile) for non-normally distributed variables. **p < 0.01; ****p < 0.0001 diabetic vs. control; n = 10 (control); n = 12 (diabetic)
Discussion
Accumulating evidence from experimental models indicates that the CDC42 signaling pathway contributes to the pathogenesis of IBD, primarily through its role in epithelial barrier integrity, immune modulation, and intestinal stem cell regulation (Fig. 12) [12, 17]. On the other hand, CDC42 has been shown to influence pancreatic β-cells’ function and insulin secretion [18]. Additionally, emerging evidence highlights CDC42 as a key regulatory molecule implicated in the development and progression of diabetes [19]. However, the literature has yet to fully elucidate the expression pattern of CDC42 in the context of comorbidity between IBD and DM. Therefore, the primary goal of our study was to investigate CDC42 gene and protein expression levels in the small and large intestine of the diabetic rats.
Fig. 12. Schematic representation of CDC42 function in the intestinal epithelium under normal and pathological conditions. Under physiological conditions, CDC42 supports actin cytoskeleton integrity and tight junction formation, maintaining intestinal barrier function. In diabetes and IBD, CDC42 expression or localization is disrupted, leading to cytoskeletal disorganization, impaired junctional integrity, and increased immune cell infiltration, contributing to chronic inflammation
Our study included both female and male rats. As previously described, including both sexes in this type of research study may reveal gonadal hormone influence on gene expression [20]. According to our results, male diabetic rats exhibited a greater percentage increase in body weight but a smaller percentage increase in blood glucose levels compared to female diabetic rats. These differences may be partly explained by hormonal variations between genders. In male rats, the greater percentage of weight gain is expected, at least in part, due to their higher initial body weight compared to females, which may reflect the anabolic effects of testosterone on muscle mass. Even though glucose levels were higher in female rats compared to the male’s, these changes didn’t reach statistical sinificance. Estrogen in female rats could contribute to altered glucose metabolic pathway and therefore higher increase in glucose levels. It has been reported that estrogen plays a critical role in regulating glucose homeostasis through both central and peripheral mechanisms. Disruption of estrogen signaling, whether due to deficiency or functional impairment, has been linked to insulin resistance and metabolic imbalance, thereby promoting the onset of type 2 diabetes (T2D) and obesity [21–24]. In line with our findings, study by Cortright et al., reported no significant difference in blood glucose levels between female and male diabetic rats. The latter study demonstrated a greater reduction in body weight in male diabetic rats compared to the control than females, suggesting that male rats may be more susceptible to diabetes-induced body mass loss [25].
Gastrointestinal (GI) complications, especially those affecting the small intestine and colon, are commonly reported in individuals with DM [26]. Considering this, the present study aimed to evaluate the effects of hyperglycemia on regions of the gastrointestinal tract that are characteristically involved in IBD. The experimental model in our study, combining a single dose of streptozotocin (STZ) and a HFD, was designed to reproduce a key features of the metabolic syndrome. A low single dose of STZ was used to induce pancreatic β-cells dysfunction, while HFD promotes insulin resistance. Together, these interventions result in hyperglycemia, dysregulated insulin signaling and systemic inflammation. This inflammatory environment represents a shared pathophysiological substrate for both DM and IBD. Investigating the interplay between pre-existing inflammation and the gene of interest may provide new insights into the potential link between DM and IBD. To this end, tissue segments from the ileum (small intestine) and the colon (large intestine) were analyzed. Histological analysis of small intestine tissue (Fig. 2) showed signs of pathological changes in diabetic group in comparison to the control, in terms of mucosal inflammatory infiltration, in addition to changes in villi morphology and Goblet cells hypoplasia. Inflammatory infiltration suggests an ongoing inflammatory response typically associated with diabetes-induced immune dysregulation [27]. Additionally, the villi exhibited structural disruption and increased thickness, primarily attributed to inflammatory cell infiltration. Previous studies have reported that histomorphological remodeling in DM involves thickening of the mucosal, submucosal, and muscular layers of the small intestine [28]. This phenomenon is thought to result from hyperphagia and elevated nutrient load within the gastrointestinal lumen, which stimulates the release of glucagon-like peptide 2 (GLP-2), a hormone known to promote intestinal epithelial growth and mucosal expansion [29]. Additionally, administration of the GLP-1 receptor agonist - liraglutide has been shown to reduce colonic inflammation in murine models of colitis [30].
The mucosal epithelial barrier is one of the primary structures impaired in the IBD pathogenesis [31]. One of the affected components includes the tight junctions between intestinal epithelial cells [32]. CDC42 plays a critical role in maintaining tight junction integrity and preserving the functional mucosal barrier [9, 33]. Literature findings highlighted the importance of CDC42 in the development of diabetic nephropathy by modulating podocyte cytoskeleton function [34]. When examined in vitro, in the mouse podocyte cell line (MPC5), CDC42 gene expression was upregulated in a hyperglycemic condition [35]. However, there is a lack of sufficient scientific evidence regarding how hyperglycemia influences CDC42 expression levels in intestinal tissue.
In our study, both gene and protein expression levels of CDC42 were significantly elevated in the small intestine of diabetic rats compared to controls (Figs. 3, 5 and 6). As shown in Fig. 5A, a 42kD to 25kD shift in band size was observed, which could be, at least in part, explained by apoptotic pathways activated in diabetic conditions. Namely, this lower molecular weight than expected likely reflects caspase-mediated cleavage of β-actin in the diabetic conditions. Furthermore, when analyzed by gender, a statistically significant increase in CDC42 expression, at both the transcriptional and protein levels, was observed in female rats. These findings suggest a potential gender-specific role of CDC42 signaling in the small intestine, possibly influenced by female-associated physiological or hormonal factors. The increased expression of CDC42 may represent an adaptive response to inflammation associated with diabetes. Oxidative stress and chronic low-grade inflammation induced by diabetic conditions are known to compromise the integrity of the intestinal epithelial barrier, particularly by disrupting tight junctions - intercellular connections regulated by CDC42 [36]. In response to this epithelial damage, upregulation of CDC42 expression may serve as a compensatory mechanism aimed at preserving epithelial barrier function. The question arises how this CDC42 activation is regulated regarding this matter. Potentially, these could be the transcription factors identified in one of our previous studies, which are associated with the CDC42 signaling pathway in the context of hyperglycemia and IBD, such as: FOXC1, CREB1, FOXA1, TFAP2A, NFIC, E2F1, PAX2 [16].
Although the gender-specific regulation of CDC42 expression and activity remains poorly defined, existing evidence indicates that sex hormones may play a significant role in modulating CDC42 function [37]. Significantly higher CDC42 expression in female diabetic rats might be explained by modulation of estrogen effects following activation of estrogen receptors - ER. ERα and ERβ are nuclear receptors presented in the intestinal epithelial cells and mainly exhibit protective effects towards inflammation [38]. Moreover, evidence suggests that CDC42 can negatively regulate the transcriptional activity of ERα, potentially influencing estrogen-mediated signaling pathways [39]. Based on the above findings, it can be hypothesized that the increased expression of CDC42 in the small intestine represents an adaptive response to diabetes-induced inflammation and mucosal injury. Subsequently, the upregulation of CDC42 may interfere with estrogen receptor-mediated protective functions in the gastrointestinal tract, potentially contributing to further mucosal impairment. It is also worth noting that the intestinal microbiome may represent an additional contributing factor in this complex pathophysiological setting, as will be discussed below.
Gender specific data from our study based on simple linear regression, indicate moderate (R² = 0.48) positive correlation between CDC42 ΔΔCt values and weight gain in small intestine of female rats (Fig. 4A). This indicates that increased weight gain is associated with reduced CDC42 gene expression. Even though research on the effects of weight gain (obesity) on CDC42 expression remains limited, one study reported that HFD feeding led to significant actin cytoskeleton remodeling and increased CDC42 activity [40].
Our histological findings in colon tissue samples are consistent with the previously discussed alterations observed in the small intestine, further supporting the presence of diabetes-induced morphological changes in terms of inflammatory infiltration and increased mucosal thickness (Fig. 7B). Similarly, one study reported increased thickness of the colon wall, especially mucosal thickness, in addition to increased stiffness of the colon wall in the diabetic rat model [41]. These changes could be the determinants of colon dysfunction and abdominal pain in diabetic patients.
As observed in our study, transcriptional expression of CDC42 in the large intestine was higher in the diabetic group as compared to the control (Fig. 8A). Additionally, according to gender, a significant increase in CDC42 expression was observed in female diabetic rats (Fig. 8B). Linear regression analysis revealed an association between increased weight gain and reduced CDC42 gene expression (Fig. 9A). These findings are in line with previously discussed data obtained from the small intestinal tissue samples, as well as the underlying scientific interpretation based on adaptive response to diabetic tissue damage and ER signaling.
What can be emphasized regarding colon tissue is that ERβ is the main estrogen receptor isoform found in the colon, contributing to the maintenance of epithelial integrity and offering protection from inflammation associated with chronic colitis [42, 43]. Another study showed that intestinal ERβ acts protective against colitis associated adenomas by TNFα/NFκB signaling pathway [44]. Furthermore, CDC42 has been identified as a regulator of immune response through its control of dendritic cells, where it is upregulated by DOCK8 [45]. Since dendritic cells are known to play a role in the pathogenesis of IBD, it would be relevant to investigate whether the DOCK8-CDC42 interaction contributes to the intestinal inflammatory response in diabetes and whether it may be responsible for the observed CDC42 gene expression patterns in our samples.
As presented in Fig. 9C a moderate negative correlation was shown between CDC42 expression and blood glucose levels in female diabetic rats, indicating that higher expression of CDC42 (reflected by lower ΔΔCt values) is associated with lower blood glucose concentrations. ERK1/2–NeuroD1 signaling pathway has been reported to mediate CDC42 regulation of insulin gene expression and insulin secretion [46].
Contrary to the results obtained from the small intestinal tissue samples where CDC42 protein levels expression was higher in diabetic group, in the large intestine protein expression of CDC42 was significantly lower (Figs. 10 and 11A). This differential CDC42 expression at the transcriptional and protein level of the large intestine, could be in part explained by some of the posttranslational modifications. Reactive oxygen species excessively produced in the diabetic condition might contribute to thiol-oxidation of the protein, leading to its impaired function, meaning lower CDC42 protein expression [47].
Furthermore, it is worth mentioning that CDC42 expression in our study was higher in the diabetic group of small intestine tissue when compared to the large intestine (Figs. 3 and 8). These differences could be explained by several contributing factors which differ between the small and large intestine, such as: tissue environment (pH), functional characteristics, nutrient content or microbiome composition. Literature data have shown that commensal intestinal microbiota play a crucial role in regulating epithelial permeability, especially by supporting the restoration and stability of tight junctions [48]. Considering CDC42’s critical function in tight junction regulation, the specific microbial composition of the small intestine may play a role in driving its increased expression in this region, mainly through some of the metabolites. Nevertheless, it has to be taken into account that gut microbiome is not constant throughout life, and that is influenced by dietary habits, age, exercise frequency or antibiotic treatment [49].
In conclusion, our study revealed the altered CDC42 expression in the small and large intestine in the diabetic rat model. This study provides novel evidence that CDC42 signaling is significantly altered in the intestinal tissue of diabetic rats and is associated with intestinal inflammation and morphological alterations. It has been suggested that the higher gene and protein expression of CDC42 has been linked to increased inflammation in the gut. Translational aspects of our research are subjected to potential modification of CDC42 signaling in order to reduce pathological findings and clinical symptoms in patients co-diagnosed with IBD and DM. It has been already shown that CDC42 inhibition by antidiabetic agents and antioxidants may have beneficial effects promoting weight loss in obese patients [50]. Additional therapeutical strategies might include pharmacological interventions by specific CDC42-inhibitors.