HMGB1 Regulates Adipocyte Lipolysis via Caveolin-1 Signaling: Implications for Metabolic and Cardiovascular Diseases

Figures

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  Figure 1. HMGB1 expression increased in parallel with the expression of adipogenic markers during 3T3-L1 adipogenic differentiation. During adipogenic differentiation, 3T3-L1 preadipocytes were harvested for mRNA, protein, and culture medium collection at the indicated time points. (A) HMGB1 mRNA levels were assessed using RT-PCR. (B) Intracellular HMGB1 and α-tubulin protein levels were analyzed using western blotting. (C) Secreted HMGB1 (sHMGB1) protein levels in the culture medium were measured using western blot analysis. (D) Western blot analysis of pre-SREBP-1, m-SREBP-1, PPARγ, aP-2, and α-tubulin protein levels. (E) Quantitative results of panel D. All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the 0 min time point.

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  Figure 2. Recombinant HMGB1 treatment does not affect preadipocyte proliferation or adipogenic differentiation. (A) 3T3-L1 preadipocytes were treated with various concentrations of recombinant HMGB1 (rHMGB1; 0, 125, 250, and 500 nM) for 24 h, and cell proliferation was assessed using the MTT assay. (B) Western blot analysis of m-SREBP-1, PPARγ, aP-2, and α-tubulin during adipogenic differentiation. (C) Quantitative results of panel B. All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the vehicle group.

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  Figure 3. CL-316,243 induces lipolysis in differentiated 3T3-L1 adipocytes via β3-adrenoreceptor activation. (A) Western blot analysis of HSL and α-tubulin during 3T3-L1 adipogenic differentiation. (B,C) Differentiated 3T3-L1 adipocytes were treated with CL-316,243 (1 µM), a β3-adrenoreceptor agonist, for the indicated time points (0, 5, 10, 15, 30, and 60 min). (B) Glycerol release into the culture medium was quantified using a colorimetric assay. (C) Western blot analysis of HSL phosphorylation at Ser563 and Ser660, total HSL, phosphorylated AMPK, total AMPK, phosphorylated ERK1/2, total ERK1/2, and α-tubulin. (D) Quantitative results of panel C. All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the 0 min time point.

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  Figure 4. HMGB1 inhibits CL-316,243-induced lipolysis by reducing HSL and ERK phosphorylation. (A,B) After 7-day of differentiation, 3T3-L1 adipocytes were treated with (A) various concentrations of recombinant HMGB1 (rHMGB1; 0, 125, 250, 500, and 1000 nM) or (B) 500 nM HMGB1 for the indicated times (0, 3, 6, 9, 12, and 24 h). Glycerol release into the culture medium was measured. (C,D) 3T3-L1 adipocytes were pretreated with 500 nM HMGB1 for 30 min, followed by stimulation with CL-316,243 for 1 h (C) or 5 min (D). (C) Glycerol release into the culture medium was assessed. (D) Intracellular cAMP levels were measured using ELISA. (E) 3T3-L1 adipocytes were pretreated with 500 nM HMGB1 for 30 min, followed by CL-316,243 treatment for 5 or 30 min. Western blot analysis was performed to assess HSL phosphorylation at Ser563 and Ser660, total HSL, phosphorylated AMPK, total AMPK, phosphorylated ERK1/2, total ERK1/2, and α-tubulin. (F) Quantitative results of panel E. All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the 0 min time point or vehicle group. # p < 0.05 versus the CL-316,243-treated group.

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  Figure 5. Role of c-Src/CAV1 signaling in HMGB1 secretion from 3T3-L1 adipocytes. (A) Western blot analysis of CAV1, phosphorylated CAV1 (pCAV1), and α-tubulin during 3T3-L1 preadipocyte differentiation. (B) Quantitative results of panel (A). (C) Immunoprecipitation of cell lysates from undifferentiated and 5-day differentiated 3T3-L1 adipocytes with an anti-HMGB1 antibody, followed by immunoblotting with anti-pCAV1, CAV1, and HMGB1 antibodies. (D) Quantitative results of panel (C). (E) Undifferentiated and 5-day differentiated 3T3-L1 adipocytes were treated with increasing concentrations of PP1 (0, 0.1, 1, and 10 µM), a Src kinase inhibitor, for 24 h. Western blot analysis was performed to assess pCAV1 and α-tubulin, as well as secreted HMGB1 (sHMGB1) in the culture medium. (F) Quantitative results of panel (E). All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the 0 min time point or vehicle-treated group. # p < 0.05 versus the untreated group (without PP1).

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  Figure 6. CAV1 deletion increases HMGB1 secretion and reduces lipolysis. Mouse embryonic fibroblasts (MEFs) from wild-type (WT) and CAV1-deficient (CAV1−/−) mice were differentiated into adipocytes for 9 days. (A) Western blot analysis of secreted HMGB1 (sHMGB1). (B) Glycerol levels were measured as an indicator of lipolysis. (C) WT and CAV1−/− mice were fasted for 18 h, and serum samples were collected to assess sHMGB1 by western blot analysis. (D) WT mice received intraperitoneal injections of either HMGB1 antibody (10 µg) or immunoglobulin G (IgG, 10 µg). After 18-hour fasting, blood samples were collected, and serum glycerol levels were measured. All data are presented as mean ± SEM from four independent experiments. * p < 0.05 versus the WT 0-day or IgG group. # p < 0.05 versus the WT 9-day group.

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  Figure 7. Schematic illustration of the proposed mechanisms by which HMGB1 regulates lipolysis in adipocytes. HMGB1 is a key lipid metabolism regulator through the c-Src/CAV1 signaling pathway. HMGB1 does not affect adipogenesis, including preadipocyte proliferation or differentiation, but significantly inhibits lipolysis in mature adipocytes. This inhibition occurs through the suppression of HSL phosphorylation and ERK activation, without altering upstream cAMP signaling, and acts downstream of β3-adrenoreceptor activation. In mature adipocytes, HMGB1 physically interacts with CAV1, whose phosphorylation by Src kinase negatively regulates HMGB1 secretion. As a result, HMGB1 serves as a negative regulator of lipolysis through CAV1-dependent mechanisms, highlighting a potential therapeutic target for obesity-related cardiovascular diseases.