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[Preprint]. 2024 Aug 3:2024.07.31.605332.
doi: 10.1101/2024.07.31.605332.

Secreted chemokines reveal diverse inflammatory and degenerative processes in the intervertebral disc of the STZ-HFD mouse model of Type 2 diabetes

Christian E Gonzalez  1 , Rachana S Vaidya  2 , Sade W Clayton  2 , Simon Y Tang  1   3   4   2
Affiliations

Secreted chemokines reveal diverse inflammatory and degenerative processes in the intervertebral disc of the STZ-HFD mouse model of Type 2 diabetes

Christian E Gonzalez et al. bioRxiv. .

Abstract

The chronic inflammation present in type 2 diabetes causes many chronic inflammatory comorbidities, including cardiovascular, renal, and neuropathic complications. Type 2 diabetes is also associated with a number of spinal pathologies, including intervertebral disc (IVD) degeneration and chronic neck and back pain. Although confounding factors such as obesity are thought to increase the loads to the musculoskeletal system and subsequent degeneration, studies have shown that even after adjusting age, body mass index, and genetics (e.g. twins), patients with diabetes suffer from disproportionately more IVD degeneration and back pain. Yet the tissue-specific responses of the IVD during diabetes remains relatively unknown. We hypothesize that chronic diabetes fosters a proinflammatory microenvironment within the IVD that accelerates degeneration and increases susceptibility to painful disorders. To test this hypothesis, we evaluated two commonly used mouse models of diabetes - the leptin-receptor deficient mouse (db/db) and the chronic high-fat diet in mice with impaired beta-cell function (STZ-HFD). The db/db is a genetic model that spontaneous develop diabetes through hyperphagia, while the STZ-HFD mouse first exhibits rapid obesity development under HFD and pronounced insulin resistance following streptozotocin administration. Both animal models were allowed to develop sustained diabetes for at least twelve weeks, as defined by elevated hemoglobin A1C, hyperglycemia, and glucose intolerance. Following the twelve-week period, the IVDs were extracted in quantified in several measures including tissue-specific secreted cytokines, viscoelastic mechanical behavior, structural composition, and histopathologic degeneration. Although there were no differences in mechanical function or the overall structure of the IVD, the STZ-HFD IVDs were more degenerated. More notably, the STZ-HFD model shows a significantly higher fold increase for eight cytokines: CXCL2, CCL2, CCL3, CCL4, CCL12 (monocyte/macrophage associated), IL-2, CXCL9 (T-cell associated), and CCL5 (pleiotropic). Correlative network analyses revealed that the expression of cytokines differentially regulated between the db/db and the STZ-HFD models. Moreover, the STZ-HFD contained a fragmented and modular cytokine network, indicating greater complexities in the regulatory network. Taken together, the STZ-HFD model of type 2 diabetes may better recapitulate the complexities of the chronic inflammatory processes in the IVD during diabetes.

Keywords: chronic inflammatory cytokines; intervertebral disc degeneration; leptin receptor deficiency; streptozotocin-high-fat-diet; type 2 diabetes.

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Conflict of interest statement

Conflicts of Interest Authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Experimental design, animal model, and workflow of the current study.
(A) The db/db model arises due to a point mutation in the leptin receptor gene, while the STZ-HFD model develops diabetes through a pro-glycemic diet and beta cell impairment. Both models present symptoms of obesity, chronic hyperglycemia, and insulin resistance, though the magnitude can vary both between and within each model. (B) The experimental timeline outlines the progression of the study for both models. The db/db mice are acquired at skeletal maturity (12 weeks old) and sacrificed after metabolic measurements are collected. For the STZHFD model, mice undergo a lead-in phase of 4-6 weeks on a HFD followed by a single low dose of STZ. Subsequently, the experimental phase for the STZ-HFD mice continues with HFD for 12 weeks, with periodic assessments of fasting blood glucose, A1c levels, and glucose tolerance. (C) After sacrifice, FSUs, consisting of the intervertebral disc and the adjacent vertebral bodies, are extracted from mice. FSUs are then utilized for terminal measures pictured above.
Figure 2.
Figure 2.. Both db/db and STZ-HFD mice represent a characteristically T2D phenotype.
(A) AUC GTT shows elevated glucose intolerance in db/db and STZ-HFD mice, with a highly significant difference between Con+Veh and STZ-HFD (p < 0.0001). (B) Terminal body weights indicate db/db mice are significantly heavier than db/+ and STZ-HFD mice. (C) Terminal fasting blood glucose levels are significantly higher in STZ-HFD mice compared to Con+Veh. (D) HbA1c levels indicate chronic hyperglycemia in both db/db and STZ-HFD mice, with no difference between them, but significant differences from their respective controls.
Figure 3.
Figure 3.. STZ-HFD mice exhibit more severe histopathological IVD degeneration compared to db/db mice.
(A) Comparison of histological phenotypes in db/+, db/db, Con+Veh, and STZ-HFD groups, showing best, median, and worst samples. (B) Histopathological scoring shows significantly greater IVD degeneration in STZ-HFD mice versus controls. (C) Con+Veh samples have healthy lamellae (▾➨); STZ-HFD samples show degenerate, disorganized lamellae (➪▽).
Figure 4.
Figure 4.. Comparative analysis of IVD structure, mechanics, and composition are similar between db/db and STZ-HFD mice.
(A)-(C) NPVF, NIDI, and DHI indicate few significant differences in structural integrity between the models. (D)-(F) Load slope, energy dissipated, and phase shift demonstrating no significant variations in viscoelastic mechanical behavior. (G)-(I) Biochemical content measurements, including collagen, s-GAG, and AGEs, show no significant differences.
Figure 5.
Figure 5.. STZ-HFD IVD Produces a More Pro-Inflammatory Microenvironment than the db/db IVD in Comparative Analysis of Cytokine Expression.
(A) The STZ-HFD model shows a significantly higher fold increase for eight cytokines: CXCL2, CCL2, CCL3, CCL4, CCL12 (monocyte/macrophage associated), IL-2, CXCL9 (T-cell associated), and CCL5 (pleiotropic). (B) The STZ-HFD model encompasses a broader and more pronounced cytokine response compared to the db/db model, highlighting the extensive upregulation of inflammatory cytokines in the STZ-HFD model.
Figure 6.
Figure 6.. STZ-HFD IVD Invokes Unique Inflammatory Signaling Pathways in Networks of Cytokine Expression.
(A) The STZ-HFD model shows a distinct network structure, demonstrating the unique upregulation of various inflammatory pathways (B) The STZ-HFD and db/db networks each rely on a number of unique (red/blue) and shared (purple) cytokines, indicating both leptin-dependent and leptin-independent inflammatory signaling cascades (C) The STZ-HFD mouse model displays a fragmented and modular cytokine network, indicating the parallel signaling of multiple signaling pathways.
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