Restoring glucose balance: Conditional HMGB1 knockdown mitigates hyperglycemia in a Streptozotocin induced mouse model

Abstract

Diabetes mellitus (DM) poses a significant global health burden, with hyperglycemia being a primary contributor to complications and high morbidity associated with this disorder. Existing glucose management strategies have shown suboptimal effectiveness, necessitating alternative approaches. In this study, we explored the role of high mobility group box 1 (HMGB1) in hyperglycemia, a protein implicated in initiating inflammation and strongly correlated with DM onset and progression. We hypothesized that HMGB1 knockdown will mitigate hyperglycemia severity and enhance glucose tolerance. To test this hypothesis, we utilized a novel inducible HMGB1 knockout (iHMGB1 KO) mouse model exhibiting systemic HMGB1 knockdown. Hyperglycemic phenotype was induced using low dose streptozotocin (STZ) injections, followed by longitudinal glucose measurements and oral glucose tolerance tests to evaluate the effect of HMGB1 knockdown on glucose metabolism. Our findings showed a substantial reduction in glucose levels and enhanced glucose tolerance in HMGB1 knockdown mice. Additionally, we performed RNA sequencing analyses, which identified potential alternations in genes and molecular pathways within the liver and skeletal muscle tissue that may account for the in vivo phenotypic changes observed in hyperglycemic mice following HMGB1 knockdown. In conclusion, our present study delivers the first direct evidence of a causal relationship between systemic HMGB1 knockdown and hyperglycemia in vivo, an association that had remained unexamined prior to this research. This discovery positions HMGB1 knockdown as a potentially efficacious therapeutic target for addressing hyperglycemia and, by extension, the DM epidemic. Furthermore, we have revealed potential underlying mechanisms, establishing the essential groundwork for subsequent in-depth mechanistic investigations focused on further elucidating and harnessing the promising therapeutic potential of HMGB1 in DM management.

Keywords: Diabetes mellitus; HMGB1; Hyperglycemia; Inflammation; RNA sequencing.

Fig. 1

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Fig. 2

Development and efficacy of TMX-induced conditional knockdown of HMGB1 (A) Representative gel images of PCR products obtained from the tails of iHMGB1 KO TMX mice, HMGB1 Flox TMX mice, control Flox mice, and C57/BL6 mice. Our results showed that the HMGB1 gene was absent only in iHMGB1 KO mice that received TMX injection. (B) Plasma samples obtained from iHMGB1 KO TMX STZ and HMGB1 Flox TMX STZ mice were analyzed for HMGB1 concentration via ELISA. We observed a significant decrease in circulating HMGB1 in iHMGB1 KO TMX STZ mice. (C) Representative Western blot images and quantification of HMGB1 levels in different tissues (liver, kidney, heart, and lung) harvested from iHMGB1 KO TMX STZ and HMGB1 Flox TMX STZ mice showing a significant decrease in HMGB1 expression in iHMGB1 KO TMX STZ mice. (D) Immunoblot summary data of quantification of HMGB1 knockdown indicates at least 70 % of HMGB1 knockdown across all tissues following TMX injection (Full uncropped gels for protein (Fig. 2A–B) and PCR (Fig. 2C) are included as Supplemental Fig. 6 Blot Images for Fig. 2A,C). N of 4 per group were used for t-test analysis. Significance levels: *:<0.05, **: 0.001–0.01, ***: 0.0001–0.001, ****: <0.0001.

Fig. 3

Impact of HMGB1 Knockdown on Hyperglycemia Development and Glucose Tolerance (A) Glucose levels in iHMGB1 KO TMX STZ and HMGB1 Flox TMX STZ mice show no significant difference pre- and post-TMX injection. (B) Body weight measurements of mice 10 weeks after induction of the hyperglycemia phenotype (N:7–8). (C) Longitudinal blood glucose level analysis in HMGB1 Flox TMX STZ and iHMGB1 KO TMX STZ mice following STZ injection (iHMGB1 KO TMX STZ N = 7, HMGB1 Flox TMX STZ N = 8). (D) Percent decrease in blood glucose levels compared between HMGB1 KO TMX STZ and HMGB1 Flox TMX STZ mice at various time points during hyperglycemia phenotype development. (E) Glucose tolerance test outcomes for HMGB1 KO TMX STZ and HMGB1 Flox TMX STZ mice, with 0 min denoting baseline condition and time of oral glucose administration (N = 4 per group). (F) Area under the curve (AUC) for the glucose tolerance test, normalized to baseline difference. Significance levels: *:<0.05, **: 0.001–0.01, ***: 0.0001–0.001, ****: <0.0001.

Fig. 4

RNA sequencing analysis identifies key genes and pathways influenced by HMGB1 knockdown in the liver of hyperglycemic mice. A) Volcano plot and (C) heat map demonstrate the up- and down-regulated genes of interest upon HMGB1 knockdown, with red indicating up-regulation and blue indicating down-regulation. (B) Ingenuity Pathway Analysis (IPA) was used to identify highly enriched pathways as a result of HMGB1 knockdown using DEGs (Differentially Expressed Genes). (D) IPA analysis also revealed predictive changes in diseases/phenotypes associated with the identified DEGs, with the corresponding genes responsible shown on the left and the predicted direction of the disease/phenotype identified indicated by positive or negative Z scores shown on the right. (E) Gene ontology (GO) analysis was performed, and genes and pathways of interest are linked in a chord graph to direct further gene specific pathway studies.

Fig. 5

RNA sequencing analysis identifies the genes and pathways associated with DM and hyperglycemia that are impacted by HMGB1 knockdown in skeletal muscle A) A volcano plot and (C) a heat map displays the up- and down-regulated DEGs (Differentially Expressed Genes) of particular interest upon HMGB1 knockdown in hyperglycemic mice, with red indicating up-regulation and blue indicating down-regulation. (B) Ingenuity Pathway Analysis (IPA) was utilized to identify highly enriched pathways that are altered by HMGB1 knockdown in skeletal muscle. (D) Further pathway analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database was performed to identify additional pathways and genes of interest that are directly influenced by HMGB1 knockdown. A chord graph is used to link genes and pathways of interest, directing further studies.

figs1

Conditional knockout of HMGB1 in diabetic mice protects from liver damage. iHMGB1-KO TMX STZ mice have decreased liver damaged evidenced by decreased AST and AST/ALT ratio (A-C), decrease inflammatory infiltrates (black arrowheads) and lipid droplets (orange arrowheads) (D), decrease glycogen storage (PAS staining) (E) and ORO staining (F) compared to HMGB1 flox TMX STZ mice.