Ⅰ. INTRODUCTION
Animal models have long been used to elucidate the pathogenic mechanisms of diabetes mellitus and to identify molecular regulators involved in disease progression1. Among these, the streptozotocin (STZ)-induced diabetic mouse model has been widely utilized because STZ selectively damages pancreatic β-cells through its cytotoxic action, leading to insulin deficiency and persistent hyperglycemia2. Although this model primarily resembles type 1 diabetes mellitus, it has also been extensively applied to study common downstream events associated with diabetes, including metabolic dysregulation, oxidative stress, and inflammatory activation3,4. Importantly, STZ-induced diabetes is not merely a consequence of direct β-cell destruction but is also accompanied by robust inflammatory responses within the pancreas and peripheral metabolic tissues5,6. Necrotic cell death induced by STZ releases intracellular components that amplify local inflammation, promote immune cell recruitment, and enhance cytokine production, thereby accelerating the progression of diabetic pathology.
Inflammation is increasingly recognized as a central mechanism in the development and progression of diabetes mellitus. Pro-inflammatory mediators produced by innate immune cells and damaged parenchymal cells can exacerbate β-cell dysfunction, intensify tissue injury, and promote systemic metabolic imbalance7. Periodontitis, a chronic infection-driven inflammatory disease of the supporting tissues of the teeth, represents a major source of persistent low-grade systemic inflammation8. It has been associated with elevated circulating inflammatory mediators and impaired glycemic control, suggesting a potential link between chronic inflammatory burden and metabolic regulation9,10. Therefore, genes that regulate inflammatory signaling are of particular interest, as they may provide a mechanistic connection between local inflammatory conditions such as periodontitis and systemic metabolic dysfunction.
Cysteine-rich intestinal protein 1 (CRIP1) was originally described as a zinc-binding protein, but its biological function has not yet been fully defined11. Recent evidence suggests that it may play a more active role in immune regulation and inflammatory responses than previously appreciated12. In our previous study, we found that CRIP1 expression was elevated in immune cell populations from patients with periodontitis, suggesting a potential association with immune activation and inflammatory tissue responses13,14. Furthermore, CRIP1-deficient experimental models have demonstrated altered cytokine production and dysregulated inflammatory responses, particularly in hepatic tissues, supporting a role for CRIP1 in the modulation of inflammatory signaling pathways14. Collectively, these findings raise the possibility that CRIP1 functions as a regulator of inflammatory homeostasis rather than merely serving structural or transport-related roles.
Importantly, although CRIP1 has been implicated in several malignancies, including leukemia, hepatocellular carcinoma, and gastric cancer, its role in metabolic disease and inflammation-associated disorders remains poorly defined15,16,17,18. Given that diabetes mellitus is strongly influenced by inflammatory signaling19,20,21, it is plausible that CRIP1 contributes to disease progression by modulating immune cell activity, cytokine production, and tissue-specific inflammatory responses. We therefore hypothesized that CRIP1 influences the development and progression of diabetes mellitus through regulation of inflammation. More specifically, we propose that CRIP1 participates in the inflammatory microenvironment associated with STZ-induced tissue injury, thereby affecting β-cell damage, immune cell infiltration, and the severity of hyperglycemia. In this context, inflammation serves as a key mechanistic link between metabolic disease and chronic inflammatory conditions such as periodontitis, providing a rationale for linking CRIP1 to both metabolic and chronic inflammatory conditions such as periodontitis. In this study, we aimed to investigate the role of CRIP1 in the pathogenesis of diabetes mellitus using an STZ-induced mouse model. We sought to determine whether CRIP1 modulates inflammatory responses during diabetic tissue injury and whether its dysregulation influences the onset and progression of hyperglycemia. By elucidating the functional relationship between CRIP1 and inflammation in diabetes, this study may provide novel insight into the molecular mechanisms underlying periodontitis-associated metabolic disease and identify CRIP1 as a potential mediator linking oral inflammation to systemic metabolic dysregulation.
Ⅱ. Materials and Methods
Experimental animals and housing conditions
Eight-week-old male wild-type (WT) and knockout (KO) mice on a C57BL/6 background were used in this study. The mice were obtained from Macrogen Inc. (Seoul, Korea). All animals were maintained under controlled environmental conditions at 22 ± 1°C with a 12-h light/dark cycle and were provided sterile food and water ad libitum.
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Pusan National University and were performed in accordance with the relevant institutional guidelines. The approval number was LT2024-07-A1C0(0), approved in December 2024.
Induction of type 1 diabetes
Type 1 diabetes was induced by intraperitoneal administration of streptozotocin (STZ; Sigma-Aldrich, USA). STZ was freshly dissolved in 0.1 M citrate buffer (pH 4.5) immediately before injection. Mice received divided doses of STZ at 50 mg/kg on day 1 and 100 mg/kg on day 2. Because STZ is unstable in aqueous solution, the STZ solution was prepared immediately before use and administered promptly. All procedures before and after STZ preparation were performed rapidly under light-protected conditions.
Measurement of body weight and fasting blood glucose
Body weight and fasting blood glucose levels were measured once during the acclimatization period and approximately twice per week during the experimental period at a consistent time of day. Blood glucose levels were measured from tail vein blood using a glucometer.
Sample collection and tissue processing
At the end of the experimental period, mice were euthanized under inhalation anesthesia. Blood was collected from the aorta and kept on ice for plasma separation. Plasma was obtained by centrifugation at 3,000 rpm for 10 min at 4°C and stored until further analysis.
A portion of each tissue sample was fixed in 4% paraformaldehyde for histological analysis. The remaining tissues and plasma samples were rapidly frozen in liquid nitrogen and stored at −80°C until use.
The spleen was rapidly excised, washed with phosphate-buffered saline (PBS) to remove residual blood, and gently dried with filter paper. Spleen weight was measured using an electronic balance. To evaluate splenic enlargement or atrophy, the spleen index was calculated as the ratio of spleen weight to body weight immediately before euthanasia, using the following formula:
Analysis of blood biochemical parameters
The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from fasting insulin and fasting glucose levels according to the method described by Matthews et al22.
When fasting glucose is expressed in mmol/L and insulin in μU/mL, HOMA-IR was calculated using the following formula:
Plasma concentrations of leptin, insulin, adiponectin, IL-6, TNF-α, and resistin were measured using a mouse multiplex assay kit based on Luminex technology (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions. Signal detection was performed using the Luminex 200 platform coupled with Bio-Plex software (Bio-Rad, USA).
Histopathological analysis
Spleen and liver tissues fixed in 4% paraformaldehyde were embedded in paraffin and processed into tissue blocks. Tissue sections were cut at a thickness of 3 μm and stained with hematoxylin and eosin (H&E). Morphological changes in the spleen and liver were observed under a light microscope.
Statistical analysis
All statistical analyses were performed using GraphPad Prism software version 5.0. Data are presented as the mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). After assessment of data distribution, non-parametric comparisons among multiple groups were performed using the Kruskal–Wallis test, followed by Dunn’s multiple comparison test for post hoc analysis. For comparisons between two groups, Mann–Whitney U test was used when appropriate. Changes in body weight over time were analyzed by comparing body weight at each time point with the baseline value within each genotype using the Wilcoxon signed-rank test. Baseline was defined as the body weight measured on the day of STZ injection. Statistical significance was defined as .
Ⅲ. Results
STZ injection successfully induced pancreatic dysfunction, as confirmed by markedly slowed body weight gain and even transient body weight loss observed from day 3 post-injection in both WT and KO mice at 8 weeks of age. This phenotype contrasts with the expected continuous weight gain typically observed in young mice approaching growth completion and aligns with extensive prior literature documenting STZ-induced body weight loss, which stems from its well-established mechanism of selectively destroying pancreatic β-cells through DNA alkylation and oxidative stress induction. Notably, while WT mice successfully recovered their body weight to pre-injection baseline levels by day 10 post-injection, reflecting effective metabolic adaptation, CRIP1 KO mice exhibited substantially delayed recovery, requiring up to 17 days to reach baseline. Furthermore, by day 25 post-injection, WT mice demonstrated approximately 10% body weight gain above their pre-injection baseline, whereas KO mice not only failed to recover but showed a persistent slight decrease relative to baseline (Fig. 1A & B). These divergent temporal trajectories between genotypes provide initial evidence that CRIP1 plays a critical role in facilitating metabolic resilience and rebalancing required for re-establishing systemic homeostasis following STZ-induced cytotoxic stress to the pancreas.
To elucidate the impact of CRIP1 genotype on pancreatic tissue damage, a hallmark of STZ toxicity mediated through selective DNA alkylation and oxidative stress in pancreatic β-cells, histopathological examination of pancreatic sections was performed. Unexpectedly, KO mice displayed a significantly greater number of intact islets of Langerhans than WT mice following STZ exposure (Fig. 2A). In addition, the remaining islets in WT mice were smaller and showed greater atrophy than those in KO mice (Fig. 2B). This paradoxical preservation of islet architecture in CRIP1-deficient mice indicates that the absence of CRIP1 confers partial resistance to STZ-mediated β cell destruction, potentially through mechanisms involving attenuated inflammatory amplification rather than direct enhancement of cellular repair pathways.
To investigate the immunological involvement of CRIP1, spleen weight was measured as a proxy for systemic immune activation, and the spleen index (spleen weight normalized to body weight) was calculated. Both parameters trended lower in KO mice compared to WT mice, although these differences did not achieve statistical significance, likely due to inherent biological variability (Fig. 3).
Complementary analysis of circulating inflammatory mediators revealed that tumor necrosis factor-α (TNF-α) levels were significantly reduced in STZ-treated KO mice relative to their WT counterparts, while interleukin-6 (IL-6) and resistin concentrations remained comparable between genotypes (Fig. 4). Collectively, these observations position CRIP1 as a positive regulator of STZ-induced inflammatory responses, particularly those driven by TNF-α signaling, whose absence mitigates pancreatic inflammation and thereby contributes to the observed β cell preservation.
Despite this relative sparing of pancreatic islets, KO mice did not exhibit the anticipated improvements in glycemic control and fasting blood glucose concentrations were higher in KO mice (Fig. 5A & B). Rather, serum insulin levels were paradoxically lower in KO mice compared to WT mice, and the homeostatic model assessment of insulin resistance (HOMA-IR) was elevated in KO mice, although neither the differences reached statistical significance (Fig. 5C & D). To further characterize systemic metabolic changes, leptin, adiponectin, and the leptin-to-adiponectin ratio were measured. Adiponectin and leptin levels both tended to be lower in KO mice compared with WT mice, although differences were not prominent. Consistently, the leptin-to-adiponectin ratio showed no marked genotype-dependent difference (Fig. 6). These results suggest that CRIP1 deficiency has only a limited effect on circulating adipokine profiles at this stage of STZ-induced injury.
In summary, these analyses suggest that CRIP1 plays a multifaceted and context-dependent role in orchestrating the pancreatic and systemic responses to cytotoxic injury. While CRIP1 deficiency dampens TNF-α-mediated inflammation to preserve islet architecture, it simultaneously impairs the coordinated metabolic adaptations necessary for weight recovery and glycemic homeostasis, culminating in prolonged cachexia-like phenotypes and insulin resistance. These findings highlight CRIP1 as a novel molecular nexus linking inflammation, β cell survival, and metabolic resilience, meriting further mechanistic dissection in future studies.
Ⅳ. Discussion
In this study, CRIP1 deficiency appears to preserve pancreatic tissue partly through modulation of inflammatory responses. Although spleen weight and spleen index did not differ significantly between WT and KO mice, circulating TNF-α was significantly reduced in CRIP1 KO mice, whereas IL-6 remained unchanged. The reduced TNF-α levels observed in KO mice likely contribute to limiting secondary inflammatory damage following the initial STZ insult. One plausible explanation for the preserved islet morphology in CRIP1-deficient mice is the attenuation of TNF-α, a key amplifier of STZ-induced β-cell cytotoxicity that promotes local inflammation, oxidative stress, and apoptosis. This selective cytokine response may be biologically relevant, as TNF-α has been directly implicated as a diabetogenic inflammatory mediator in STZ-induced diabetes. In multiple low-dose STZ models, inhibition of TNF-α expression attenuated the development of hyperglycemia, and TNF-α has been implicated in cytokine-mediated β-cell injury23,24. In contrast, IL-6 exerts pleiotropic effects on β-cells and may protect against STZ-induced oxidative stress by promoting antioxidant and autophagic responses25. Thus, the selective reduction in TNF-α may reflect modulation of a TNF-α-associated inflammatory pathway rather than generalized suppression of systemic inflammation. In addition, controlled inflammation is increasingly recognized as a necessary component of tissue remodeling and metabolic adaptation, particularly in the context of acute injury26. In our previous study, we observed that hepatic Crp mRNA, one of the most representative inflammatory markers, was significantly lower in KO mice than in WT mice following oral administration of Porphyromonas gingivalis, a major pathogen associated with periodontitis14. In contrast, under basal conditions, KO mice exhibited higher Crp expression than WT mice. These findings led us to propose that CRIP1 deficiency alters the set point of inflammatory responses, possibly through mechanisms related to immune exhaustion or dysregulated responsiveness. Consistent with this hypothesis, the selective reduction in TNF-α observed in the present study further suggests that CRIP1 deficiency alters the regulation of inflammatory responses to STZ rather than uniformly suppressing inflammation.
However, the present study also reveals a seemingly paradoxical role of CRIP1 in STZ-induced metabolic injury, wherein its deficiency simultaneously preserves pancreatic islet architecture while exacerbating systemic metabolic dysfunction. This dissociation between structural integrity and functional outcome suggests that CRIP1 deficiency is not merely involved in β-cell survival through attenuation of inflammatory responses, but rather plays a critical role in coordinating the systemic adaptive response to pancreatic injury. In this context, CRIP1-dependent inflammatory signaling may be required to appropriately activate compensatory pathways necessary for restoring glucose homeostasis and metabolic balance27. This framework may help explain why preservation of islet structure in KO mice does not translate into improved glycemic control. It is possible that the remaining β-cells in CRIP1-deficient mice are functionally compromised, either due to insufficient activation of stress-response pathways or impaired coupling between insulin secretion and systemic metabolic demand28. Alternatively, CRIP1 may exert critical effects in peripheral metabolic tissues such as the liver, skeletal muscle, or adipose tissue, where it could influence insulin sensitivity, substrate utilization, or overall energy expenditure29. Although WT and CRIP1 KO mice showed different patterns of body weight recovery following STZ administration, the genotype-dependent difference did not reach statistical significance and should therefore be interpreted as a trend. Together with the tendency toward increased HOMA-IR, this observation raises the possibility that CRIP1 deficiency may affect systemic metabolic adaptation following pancreatic injury, although further studies are required to confirm this association.
Another important consideration is that the STZ model reflects an acute cytotoxic insult, in which the balance between inflammation-induced damage and inflammation-driven adaptation is highly dynamic and tightly regulated30. In this context, CRIP1 may function as a rheostat that fine-tunes inflammatory intensity to an optimal range sufficient to initiate repair and metabolic reprogramming, but not so excessive as to cause irreversible tissue damage. Loss of CRIP1 may shift this balance toward insufficient inflammatory signaling, thereby protecting β-cell structure while simultaneously impairing the broader physiological recovery process30. Taken together, our findings suggest that CRIP1 serves as a critical integrator of inflammatory and metabolic signals during the response to pancreatic injury, facilitating the transition from acute damage to systemic recovery. Importantly, these results highlight a key conceptual point that attenuation of specific inflammatory pathways does not necessarily translate into improved metabolic outcomes, particularly in the context of acute injury. Future studies should aim to dissect the tissue-specific roles of CRIP1 and determine whether its primary function is more closely linked to immune cell activation, β-cell functional maturation, or peripheral insulin responsiveness.
