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. 2023 May 26;8(1):190.
doi: 10.1038/s41392-023-01415-6.

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγ Ser273 phosphorylation in type 2 diabetic mice

Yudian Zhang #  1 , Zihua Li #  2 , Xinyi Liu #  1 , Xinyu Chen  1 , Shujie Zhang  1 , Yuemeng Chen  1 , Jiangnan Chen  1 , Jin Chen  1 , Fuqing Wu  1 , Guo-Qiang Chen  3   4   5
Affiliations

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγ Ser273 phosphorylation in type 2 diabetic mice

Yudian Zhang et al. Signal Transduct Target Ther. .

Abstract

3-Hydroxybutyrate (3HB) is a small ketone body molecule produced endogenously by the body in the liver. Previous studies have shown that 3HB can reduce blood glucose level in type 2 diabetic (T2D) patients. However, there is no systematic study and clear mechanism to evaluate and explain the hypoglycemic effect of 3HB. Here we demonstrate that 3HB reduces fasting blood glucose level, improves glucose tolerance, and ameliorates insulin resistance in type 2 diabetic mice through hydroxycarboxylic acid receptor 2 (HCAR2). Mechanistically, 3HB increases intracellular calcium ion (Ca2+) levels by activating HCAR2, thereby stimulating adenylate cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) concentration, and then activating protein kinase A (PKA). Activated PKA inhibits Raf1 proto-oncogene serine/threonine-protein kinase (Raf1) activity, resulting in a decrease in extracellular signal-regulated kinases 1/2 (ERK1/2) activity and ultimately inhibiting peroxisome proliferator-activated receptor γ (PPARγ) Ser273 phosphorylation in adipocytes. Inhibition of PPARγ Ser273 phosphorylation by 3HB altered the expression of PPARγ regulated genes and reduced insulin resistance. Collectively, 3HB ameliorates insulin resistance in type 2 diabetic mice through a pathway of HCAR2/Ca2+/cAMP/PKA/Raf1/ERK1/2/PPARγ.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
3HB Treatment Reduces Insulin Resistance in T2D Mice. a Body weights of db/db mice (n = 10). b Overnight fasted blood glucose level of db/db mice (n = 10). c The glucose tolerance test of db/db mice. Glucose (2 g/kg body weight) was intraperitoneally (i.p.) administered in overnight-fasted db/db mice. (n = 5) d The area under the curve (AUC) in c. e Fasting serum insulin levels of db/db mice after 8 weeks of indicated treatment. (n = 6) f A homeostasis model for assessment of the insulin resistance index (HOMA-IR) of the mice in e. g Body weights of STZ induced T2D mice. (n = 10) h Overnight fasted blood glucose level of STZ mice. (n = 10) i The glucose tolerance test of STZ mice. Glucose (2 g/kg body weight) was intraperitoneally (i.p.) administered in overnight-fasted STZ mice. (n = 5–9) j The area under the curve (AUC) in i. k Fasting serum insulin levels of STZ mice after 8 weeks of indicated treatment. (n = 5–9). l A homeostasis model for assessment of the insulin resistance index (HOMA-IR) of STZ mice. (n = 5–9) Data reported as mean ± SD, *p < 0.05, **p < 0.001, and ***p < 0.0001
Fig. 2
Fig. 2
Transcriptomics Analysis for Adipose Tissue of T2D Mice. a After 8 weeks of indicated treatment, the adipose tissue of db/db mice was collected and studied the differential gene expression analysis (p < 0.05) by RNA sequencing. b Downstream genes of PPARγ in differentially expressed genes of db/db mice. c Gene ontology (GO) enrichment analysis of db/db mice. The related biological processes (BP), cellular component (CC) and molecular function (MF) were analyzed. d Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of db/db mice. e After 8 weeks of indicated treatment, the adipose tissue of STZ induced T2D mice was collected and conducted the differential gene expression analysis (p < 0.05) by RNA sequencing. f Downstream genes of PPARγ in differentially expressed genes of STZ mice. g GO enrichment analysis of STZ mice. The related biological processes (BP), cellular component (CC) and molecular function (MF) were analyzed. h KEGG enrichment analysis of STZ mice
Fig. 3
Fig. 3
3HB Inhibits Phosphorylation of PPARγ Ser273 in vitro and in vivo. a TR-FRET PPARγ ligand displacement assay of 3HB and positive control rosiglitazone. b Influence on PPARγ transcriptional activity of 3HB or 0.5 μM rosiglitazone was measured using a 293 T cells based on the luciferase reporter assay. c Oil red-O staining of mature 3T3-L1 adipocytes after 8 days of 3HB treatment. d Quantification of the staining results in c was presented relative to the control. e 3T3-L1 adipocytes with or without siRNA knockdown of HCAR2 were pretreated with 3HB or 0.5 μM rosiglitazone for 24 h. After 10 min stimulation of insulin, the glucose uptake of cells was measured by a bioluminescent assay. f 3T3-L1 adipocytes with or without siRNA knockdown of HCAR2 were treated with TNFα, followed by treatment with 3HB or rosiglitazone (Rosi) for 1 h. Phosphorylated PPARγ at Ser273 and total PPARγ were detected using anti-pPPARγ or anti-PPARγ antibodies, respectively. h–i pPPARγ (Ser273) and total PPARγ of adipose tissue in 8-week treated db/db mice, STZ induced T2D mice and STZ induced HCAR2-/- T2D mice, respectively. j–m The amounts of pPPARγ (Ser273) and total PPARγ in f–i were quantified using ImageLab, and pPPARγ/ PPARγ ratio was calculated. The results (expressed as the mean ± SD, n ≥ 3) were presented relative to the control (3HB 0 mM, db/db-Ctrl, STZ-Ctrl or STZ-HCAR2-/--Ctrl), *p < 0.05, **p < 0.001, and ***p < 0.0001, **** p < 0.00001
Fig. 4
Fig. 4
3HB Inhibits Phosphorylation of PPARγ Ser273 Through HCAR2/Ca2+/cAMP/PKA/Raf1/ERK1/2 Pathway. a 3T3-L1 adipocytes with or without siRNA knockdown of HCAR2 were treated with 3HB or 100 μM forskolin for 1 h. Intracellular cAMP concentration was detected using an ELISA kit. b Intracellular Ca2+ levels of 3T3-L1 adipocytes treated with 3HB or 1 μM ATP for 1 h were detected with Fura-2/AM probe kit. The results were presented relative to 3HB 0 mM. c The kinase activity of ERK1/2 of 3T3-L1 adipocytes treated with 3HB or 10 μM SCH779284 for 1 h was measured using an ERK1/2 kinase Activity Quantitative detection Kit. d 3T3-L1 adipocytes with or without siRNA knock down of HCAR2 were treated with 3HB or 0.5 μM rosiglitazone (Rosi) for 1 h. Phosphorylated PKA at Thr197, Raf1 at Ser259, ERK1/2 at Thr202/Tyr204, PPARγ at Ser273, and total PKA, Raf1, ERK1/2, PPARγ, HCAR2, GAPDH were studied using corresponding antibodies. e–h The amounts of phosphorylated and total proteins in d were quantified using ImageLab, and pPKA/PKA, pRaf1/Raf1. pERK1/2/ERK1/2 and pPPARγ/ PPARγ ratio was calculated. The results (expressed as the mean ± SD, n = 3) were presented relative to 3HB 0 mM, *p < 0.05, **p < 0.001, and ***p < 0.0001
Fig. 5
Fig. 5
3HB Affects the Expression of Genes Regulated by PPARγ Ser273 Phosphorylation in 3T3-L1 Adipocytes. a, b Gene expression of adipocytes with or without siRNA knockdown of HCAR2 after 6 h 5 mM 3HB or 0.5 μM rosiglitazone (Rosi) treatment. c, d Gene expression of adipocytes treated with 5 mM 3HB or 0.5 μM for 6 h in the presence of 10 μM SCH779284 or 20 μM GW9662. e Relative HCAR2 expression of adipocytes with or without siRNA knockdown of HCAR2. f 3T3-L1 adipocytes with or without siRNA knockdown of HCAR2 were treated with 3HB or 0.5 μM rosiglitazone (Rosi) for 48 h in the absence or presence of 10 μM SCH779284 or 20 μM GW9662. Adiponectin, GLUT4, HCAR2 and GAPDH were detected by corresponding antibodies. g Adiponectin and GLUT4 protein expression of db/db mice adipose tissue. h Schematic diagram of the mechanism by which 3HB ameliorates insulin resistance in type 2 diabetic mice. The results (expressed as the mean ± SD, n = 3) were presented relative to Ctrl groups for each gene, *p < 0.05, and ***p < 0.0001
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