Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity

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Abstract

Non-alcoholic fatty liver disease (NAFLD) represents a key feature of obesity-related type 2 diabetes with increasing prevalence worldwide. To our knowledge, no treatment options are available to date, paving the way for more severe liver damage, including cirrhosis and hepatocellular carcinoma. Here, we show an unexpected function for an intracellular trafficking regulator, the small Rab GTPase Rab24, in mitochondrial fission and activation, which has an immediate impact on hepatic and systemic energy homeostasis. RAB24 is highly upregulated in the livers of obese patients with NAFLD and positively correlates with increased body fat in humans. Liver-selective inhibition of Rab24 increases autophagic flux and mitochondrial connectivity, leading to a strong improvement in hepatic steatosis and a reduction in serum glucose and cholesterol levels in obese mice. Our study highlights a potential therapeutic application of trafficking regulators, such as RAB24, for NAFLD and establishes a conceptual functional connection between intracellular transport and systemic metabolic dysfunction.

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Fig. 1: RAB24 upregulation in patients with NAFLD and regulated glucose tolerance and serum lipid parameters in mice.
Fig. 2: Liver-specific knockdown of Rab24 induces upregulation of mitochondrial proteins.
Fig. 3: Rab24 depletion leads to an increase in mitochondrial mass and function.
Fig. 4: Rab24 knockdown increases mitochondrial area and connectivity.
Fig. 5: Rab24 knockdown increases mitochondrial connectivity by interfering with the fission machinery.
Fig. 6: Rab24 knockdown causes a reduction in mitophagy and an increase in autophagic flux.
Fig. 7: Depletion of Rab24 in mice fed an HFD improves serum lipid and glucose parameters.
Fig. 8: Knockdown of Rab24 in a NASH mouse model ameliorates liver steatosis and inflammation.

Data availability

The proteomics data generated or analysed during this study are included in this published article (and its Supplementary Information files). Additional data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data for Figs. 18, Extended Data Figs. 8 and 9, and Supplementary Figs. 1 and 3 are available online.

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Acknowledgements

We acknowledge T. Schwarz-Romond, A. M. Cuervo, H. M. McBride, L. Pellegrini, M. Jastroch and L. Scorrano for helpful discussions. We thank A. Takacs, J. Biebl, S. Krämer, Q. Reinold and S. Ambos for mouse care and injections. We thank A. Georgiadi and C. Glantschnig for scientific help. The authors thank N. Itho (Kyoto University Graduate School of Pharmaceutical Sciences) for providing the FGF21 knockout mice. This work was financially supported by DFG grant nos. ZE1037/1-1 and ZE1037/3-1, DZD grant no. 920.561-82DZD0032G, EFSD grant no. 01KU1501C and BMBF grant no. 031L0114A to A.Z., AMPro funding (ZT-0026) from the Helmholtz Association to S.H., and the DFG within the framework of the Munich Cluster for Systems Neurology (EXC2145 SyNergy), the Collaborative Research Center (CRC1177) and DFG Schwerpunktprogramm 1629 ThyroidTransAct (TS 226/3-1) and DFG (SFB TRR 152/2, P23) awarded to T.D.M., and DFG (SFB 1321/1, P08) awarded to K.S., as well as by the Boehringer Ingelheim Foundation to C.B. J.G. was supported by the Institut National de la Santé et de la Recherche Médicale, Université Côte d’Azur and ANR Young Investigator Program (no. ANR18-CE14-0035-01-GILLERON). N.K. is funded by Emmy-Noether DFG (KR5166/1-1).

Author information

A.Z. designed and directed the project. S.S., G.H. and M.B.D. designed and performed the animal and in vitro experiments. Y.K. developed the skeletonization and image analysis of the mitochondrial network. J.J. and C.B. completed the GST Rab24 pulldown experiments. R.S. performed the triglyceride assays. N.K. and M.M. conducted the proteomics analysis. B.N. performed part of the immunoblot analysis. A.L. and S.H. analysed and blotted the proteomics data and provided information on the MCD-HFD studies. S.G. and M.R. provided samples of patients with fatty liver disease and NASH and performed the correlation analysis. A.F. helped with sectioning, staining and quantification of liver tissue. K.S. and T.D.M. provided the FGF21 knockout mice. M.H.D.A. supervised the Rab24 knockout mouse experiments. M.B. provided the expression data and correlation analysis of patients who were obese and patients with type 2 diabetes. J.G. performed the electron microscopy experiments and analysis. A.Z. and S.S. wrote the manuscript. J.G., A.L., C.B., G.H. and S.H. edited the manuscript.

Correspondence to Anja Zeigerer.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 RAB24 expression is positively correlated with fat accumulations in humans.

(a, b) Spearman’s correlation analysis of RAB24 in the liver of a cohort of obese patients versus healthy controls for BMI and Clamp glucose infusion rate (GIR). (N=33 patients) (c, d) Spearman’s correlation analysis of RAB24 in liver of patients with NALFD w/ and w/o steatosis and NASH patients versus healthy controls for M-value and liver fat (HCL) levels (N=44 patients). P-value: two-tailed paired Student’s t-test.

Extended Data Fig. 2 Rab24 depletion caused an increase in glycolysis.

(a) Seahorse measurements (N=10 wells per condition) of the extracellular acidification rate (ECAR) and their corresponding metabolic rates in (b). N=30 wells (glycolysis) and N=29 wells (glycolytic capacity) per condition. (c) Abundance of glucokinase (Gck), phosphoglucomutase (Pgm), pyruvate kinase (Pklr) and pyruvate dehydrogenase complex (Pdha1, Pdhb) by proteomics after in vivo Rab24KD. N=6 animals per condition. (d) Glucose uptake assay under basal, oligomycin (2 μM), antimycin A (AA 1 μM) + rotenone (Rot 1 μM) and Glucose (25 mM) conditions. N=5 independent wells per condition. Relative expression of GLUT1 and GLUT2 in vitro (N=4 wells per condition) (e) and in livers of control and Rab24 KD mice (N=6 animals per condition) (f). (g) Lactate secretion in primary hepatocytes analyzed from N=8 (CTR) and N=9 (KD) independent wells. All in vitro experiments measured after 3 days after RNAi (40 nM) in primary hepatocytes. (h) ipPTT (2 g/kg) of control (N=5) and Rab24 KD (N=6) mice after 16 h of fasting. All animals treated with control (CTR) and Rab24 (KD) LNPs (0.5 mg/kg) (mean +/- SEM). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by two-tailed unpaired Student’s t-test. Only data that reached statistical significance are indicated.

Extended Data Fig. 3 Rab24 reduction led to an increase in mitochondrial area.

Electron micrographs of mitochondria in primary hepatocytes of control (a, c) and Rab24KD (b, d) cells; higher magnifications shown in c and d. The images are representative of 12 independent wells of a 24-well plate. Scale bar 5 µm. Zoom scale bar 500 nm. Quantification of mitochondria morphology by morpho-EM for mitochondria density (e), surface area (f), perimeter or contour (g), form factor or complexity (h) and circularity (i) of control and Rab24KD cells. N=7 (CTR) and N=12 (KD) cells quantified in (e). N=329 (CTR) and N=453 (KD) in (f), N=108 (CTR) and N=(209) in (g, h, i) mitochondria analyzed. All measured after 3 days of RNAi (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, ***P<0.001, by two-tailed unpaired Student’s t-test.

Extended Data Fig. 4 Rab24KD enhanced mitochondrial connectivity.

(a) Representative confocal images (single confocal section) of cultured primary hepatocytes after 3 days of RNAi (40 nM) stained with dapi (blue), phalloidin (green) and Tom20 (grey) as single section, deconvolved, zoomed and skeletonized with Fiji. The images are representative of three independent wells of a 24-well plate. (b-d) Quantification of number of branches, number of junctions per area and mean length of the branches of (a) with Fiji from N=10 (CTR) and N=9 (KD) cellular regions. The experiment was done twice with similar results. (e) Representative confocal images (single confocal section) of HFD liver sections stained with dapi (blue), phalloidin (green) and Tom20 (grey) as single section, deconvolved, zoomed and skeletonized with Fiji. The images are representative of four independent biological samples, which give rise to the quantifications in f-h. (f-h) Quantification of number of branches, number of junctions per area and mean length of the branches of (e) using Fiji from N=4 animals per condition. Measured after 14 days of KD with LNPs (weekly injection; 0.5 mg/kg) and 6 h starvation in 15-week HFD mice. Scale bar 20 µm (mean +/- SEM). **P<0.01, ***P<0.001, ****P<0.0001 by two-tailed unpaired Student’s t-test.

Extended Data Fig. 5

Rab24KD enhanced mitochondrial connectivity without affecting the fusion machinery. (a) Abundance of fusion and fission regulators by proteomics. N=6 animals per condition. (b) Representative confocal images (single middle confocal sections) of primary polarized hepatocytes in collagen sandwich stained for Tom20 (green), KDEL (red), actin (grey) and dapi. Higher zoom shown in the middle insert to better observe co-localization. The images are representative of three independent wells of a 24-well plate. Person’s and Spearman’s correlation analyzed with Fiji representing overlap between the green and red channel on the right and quantification of PSC in (c) analyzed from N=3 wells per condition. The experiment was done twice with similar results. (d, e) Representative confocal images (single confocal sections) of primary hepatocytes stained for Mfn1 (d) and Mfn2 (e). The images are representative of three independent wells of a 24-well plate. (f) Quantification of the mean fluorescent intensity per cell from (d, e) using Fiji from N=22 (CTR) and N=27 (KD) (Mfn1) and N=24 (CTR) and N=29 (KD) (Mfn2) cells per condition. Scale bar = 20 μm. All measured after 3 days of RNAi (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, *****P<0.00001 by two-tailed unpaired Student’s t-test. Only data that reached statistical significance are indicated.

Extended Data Fig. 6 Mild KD of Fis1 mimics Rab24’s effect on mitochondrial connectivity and respiration.

(a) Relative expression of Fis1 in cultured primary hepatocytes after RNAi treatment. 9 wells pooled into N=3 replicates per condition. (b) Seahorse measurements of the oxygen consumption rate (OCR) in primary hepatocytes (N=10 wells per time point) and their corresponding metabolic rates in (c-e) after 3 days of KD. N=3 time points with 10 wells/time point (c, d). N=8 (CTR) and N=9 (KD) wells per condition from the first time point after FCCP treatment, representing maximal respiration (e). (f) Representative confocal images (single confocal section) of cultured primary hepatocytes stained with dapi (blue) and Tom20 (grey) as single section, deconvolved, zoomed and skeletonized with Fiji. The images are representative of three independent wells of a 24-well plate. (g-j) Quantification of mean Tom20 intensity per cell, number of branches, number of junctions per area and mean length of the branches of (f) with Fiji analyzed from N=14 (CTR) and N=17 (KD) for (g), N=10 (CTR) and N=11 (KD) for (h, i), N=9 (CTR) and N=10 (KD) for (j) cellular regions (multiple cells per region). Scale bar 20 µm. All measured after 3 days of RNAi (0.1 nM). (mean +/- SEM). *P<0.05, **P<0.01, ****P<0.0001, *****P<0.00001 by two-tailed unpaired Student’s t-test.

Extended Data Fig. 7 Rab24 depletion reduced fluorescence overlap between mitochondria and lysosomes.

(a) Mitophagy flux assay in primary hepatocytes incubated for 2 h with 20 µM FCCP w/ or w/o 20 µM chloroquine. N=6 wells per condition. (b) Representative confocal images (single middle confocal sections) of primary polarized hepatocytes in collagen sandwich stained for Tom20 (green), Lamp1 (red) and dapi. Higher zoom shown as insert. Scale bar 20 µm. The images are representative of three independent wells of a 24-well plates. Person’s and Spearman’s correlation analyzed with Fiji representing overlap between the green and red channel in (c) analyzed from N=19 (CTR) and N=16 (KD) cellular regions (multiple cells per region). The experiment was done twice with similar results. All measured after 3 days of RNAi (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, **P<0.01 by two-tailed unpaired Student’s t-test.

Extended Data Fig. 8 Rab24 KD caused an increase in p62 independent autophagic flux during fasting.

Quantification of LC3-II/VCP levels in primary hepatocytes (a) and liver tissue (b) in fed, starved or chloroquine treated conditions (mean +/- SD). Cells were kept in full medium (fed) or serum starved for 12 h (fasted), followed by 20 µM chloroquine treatment for 3 h (fasted & CQ). Mice were either fed ad libitum, fasted for 12 h, or fasted for 12 hours followed by 100 mg/kg chloroquine treatment for 3 h (fasted & CQ). (c) Steady state levels (fed condition) of LC3-II staining in primary hepatocytes (mean +/- SEM, N=14 for CTR and N=12 for KD cellular regions (multiple cells per region) analyzed). (d) Representative Western blots and quantification thereof for p62 in primary hepatocytes (e) and liver tissue (f) in fed, starved or chloroquine treated conditions (mean +/- SD). The quantifications in (a) and (e) are from six independent wells of a 24-well plate pooled into N=2 replicates. The quantifications in (b) and (f) and the Western blot in (d) are from N=2 animals per condition. Both experiments were done twice with similar results. (g) Representative confocal images (three merged confocal section) of primary hepatocytes stained with dapi and p62 (grey) and quantification thereof with Fiji in (h) after treatment with full medium (fed) or serum starved for 12 h (fasted), followed by 20 µM chloroquine treatment for 3 h (fasted & CQ). The images are representative of N=12 (fed), N=20 (starved), N=12 (starved & CQ) for CTR and N=8 (fed), N=20 (starved), N=12 (starved & CQ) cellular regions (multiple cells per region) analyzed. (i) Representative confocal images (single middle confocal sections) of primary polarized hepatocytes in collagen sandwich stained for LAMP1 (grey), phalloidin (green), and dapi (blue) and quantification of the mean fluorescent intensity of LAMP1 per cell using Fiji in j. Scale bar = 20 μm. The images are representative of three independent wells of a 24-well plate. N=40 (CTR) and N=29 (KD) cells per condition. The in vitro experiment was repeated twice with similar results. All measured after 3 days of RNAi against Rab24 (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, ****P<0.0001 by two-tailed unpaired Student’s t-test. Source data

Extended Data Fig. 9 Rab24 reduction in HFD mice improved liver steatosis and mitochondrial respiration.

(a) Body weight over 13 weeks of HFD treatment. (b) Fed blood levels and relative expression of Rab24 (c) in the liver at 13 weeks of HFD. (d) Western blot analysis and quantification thereof from livers lysates of mice after control and Rab24KD. Body weight (e) and Triglycerides levels (f) after Rab24KD. N=6 animals per condition (a-e). N=4 for LFD CTR, N=5 for HFD CTR and N=6 for HFD KD (f) animals per condition. (g) Representative confocal images (single confocal sections) of control or Rab24 KD liver slices labeled with (3.8 μM) Bodipy and quantification thereof for number of lipid droplets (h), area of lipid droplets (i) and diameter of lipid droplets (j) using Fiji. Scale bar = 60 μm. The images are representative of four independent biological samples, which give rise to the quantifications in h-j. All animals received 15 weeks of low fat (LFD) or high fat diet (HFD) and were treated with control and Rab24 LNPs for the last 2 weeks with a weekly injection of LNPs (0.5mg/kg) (mean+/-SEM). (k) Representative confocal images (single confocal sections) of primary hepatocytes treated with BSA or BSA complexed with oleate and palmitate for 3 days and stained for Plin2. Scale bar = 20 μm. The images are representative of three independent wells of a 24-well plates. The in vitro experiment (k) was repeated twice with similar results. (l) Seahorse measurements of the oxygen consumption rate (OCR) after KD upon oleate and palmitate treatment (N=5 wells per condition). All measured after 3 days after RNAi (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, ***P<0.001, ****P<0.0001 by two-tailed unpaired Student’s t-test. Only data that reached statistical significance are indicated. Source data

Extended Data Fig. 10 Rab24 KD in LFD mice improve liver and serum lipid content.

Serum parameters for total Cholesterol (a), LDL (b), ApoB (c) and ALT (d) in LFD mice. (e) H&E staining of control and Rab24 KD liver sections of LFD mice and quantification thereof for hepatic lipid accumulations (% steatosis) (f). Scale bar 200 µm. The images are representative of five independent biological samples. (g) Liver/body weight ratio of LFD control and Rab24 KD mice. All measured after 14 days of KD with LNPs (weekly injection; 0.5 mg/kg) and 6 h starvation in 15-week LFD mice. Fasted blood glucose levels (h) after 4 weeks of KD with LNPs (weekly injection; 0.5 mg/kg) in 17-week LFD mice. (mean +/- SEM; N=4 (a-d), N=5 (f, g) and N=6 (h) animals per condition for LFD CTR; N=6 (a-c, f-h) and N=5 (d) animals per condition for LFD KD). *P<0.05, **P<0.01 by two-tailed unpaired Student’s t-test. Only data that reached statistical significance are indicated.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Table 3

Reporting Summary

Supplementary Table 1

Proteomics datasets

Supplementary Table 2

Enrichment analysis for mitochondrial proteins and proteins of carbon metabolism

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Seitz, S., Kwon, Y., Hartleben, G. et al. Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity. Nat Metab 1, 1009–1026 (2019) doi:10.1038/s42255-019-0124-x

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