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. 2022 Dec;13(6):2697-2711.
doi: 10.1002/jcsm.13061. Epub 2022 Aug 12.

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity

Lai Wang  1 , Zuguo Zheng  1 , Lijun Zhu  1 , Lingchang Meng  1 , Hanling Liu  1 , Keke Wang  1 , Jun Chen  1 , Ping Li  1 , Hua Yang  1
Affiliations

Geranylgeranyl pyrophosphate depletion by statins compromises skeletal muscle insulin sensitivity

Lai Wang et al. J Cachexia Sarcopenia Muscle. 2022 Dec.

Abstract

Background: Statins are widely prescribed cholesterol-lowering drugs but have been shown to increase the risk of type 2 diabetes mellitus. However, the molecular mechanisms underlying the diabetogenic effect of statins are still not fully understood.

Methods: The effects of geranylgeranyl transferase I and II (GGTase I and II) inhibition on insulin-stimulated glucose uptake and GLUT4 translocation, and the dependence of these effects on insulin signalling were investigated in skeletal muscle cells. The protective effects of geranylgeranyl pyrophosphate (GGPP) and its precursor geranylgeraniol (GGOH) on simvastatin-induced insulin resistance were evaluated in vitro and in vivo. The effect of GGTase II inhibition in skeletal muscle on insulin sensitivity in vivo was confirmed by adeno-associated virus serotype 9 (AAV9)-mediated knockdown of the specific subunit of GGTase II, RABGGTA. The regulatory mechanisms of GGTase I on insulin signalling and GGTase II on insulin-stimulated GLUT4 translocation were investigated by knockdown of RhoA, TAZ, IRS1, geranylgeranylation site mutation of RhoA, RAB8A, and RAB13.

Results: Both inhibition of GGTase I and II mimicked simvastatin-induced insulin resistance in skeletal muscle cells. GGPP and GGOH were able to prevent simvastatin-induced skeletal muscle insulin resistance in vitro and in vivo. GGTase I inhibition suppressed the phosphorylation of AKT (Ser473) (-51.3%, P < 0.01), while GGTase II inhibition had no effect on it. AAV9-mediated knockdown of RABGGTA in skeletal muscle impaired glucose disposal without disrupting insulin signalling in vivo (-46.2% for gastrocnemius glucose uptake, P < 0.001; -52.5% for tibialis anterior glucose uptake, P < 0.001; -17.8% for soleus glucose uptake, P < 0.05; -31.4% for extensor digitorum longus glucose uptake, P < 0.01). Inhibition of RhoA, TAZ, IRS1, or geranylgeranylation deficiency of RhoA attenuated the beneficial effect of GGPP on insulin signalling in skeletal muscle cells. Geranylgeranylation deficiency of RAB8A inhibited insulin-stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells (-42.8% for GLUT4 translocation, P < 0.01; -50.6% for glucose uptake, P < 0.001).

Conclusions: Geranylgeranyl pyrophosphate regulates glucose uptake via GGTase I-mediated insulin signalling-dependent way and GGTase II-mediated insulin signalling-independent way in skeletal muscle. Supplementation of GGPP/GGOH could be a potential therapeutic strategy for statin-induced insulin resistance.

Keywords: GGPP; Insulin resistance; RAB8A; RhoA; Skeletal muscle; Statin.

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Conflict of interest statement

Lai Wang, Zuguo Zheng, Lijun Zhu, Lingchang Meng, Hanling Liu, Keke Wang, Jun Chen, Ping Li, and Hua Yang declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Geranylgeranyl pyrophosphate (GGPP) depletion by statins inhibits glucose uptake in skeletal muscle cells in vitro and in vivo. (A) Mevalonate pathway. (B) C2C12 myotubes were pretreated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for 24 h, then cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (C) C2C12 myotubes were treated with 10 μM GGPP, 10 μM simvastatin, and 10 μM GGPP combined with 10 μM simvastatin for 24 h, then cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (D–F) Male C57BL/6J mice (20 ± 2 g) were randomly grouped (n = 6). After administration of geranylgeraniol (GGOH) (25 mg/kg/day), simvastatin (40 mg/kg/day), and GGOH combined with simvastatin for 3 weeks, mice were subjected with experiments below. (D) Glucose tolerance test (GTT) and GTT AUC. (E) Insulin tolerance test (ITT) and ITT AUC. (F) After the measurement of GTT and ITT, mice were fasted for 16 h, and then mice were administrated with 2‐DG (2 g/kg body weight) via intraperitoneal injection. Mice were sacrificed 30 min later after the injection. The uptake of 2‐DG in gastrocnemius, tibialis anterior, soleus, and extensor digitorum longus was measured using Glucose Uptake Colorimetric Assay Kit. Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 2
Figure 2
Impaired GLUT4 translocation to plasma membrane caused by simvastatin is restored by geranylgeranyl pyrophosphate (GGPP). (A) C2C12 myotubes were pretreated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for 24 h. Then cells were incubated with or without 100 nM insulin for 30 min, total protein and plasma membrane protein were harvested, and GLUT4 expression was analysed by western blot, with GAPDH as the loading control (n = 3). (B) C2C12 myotubes were previously transfected with siRNAs targeting GGPS1, PGGT1B, and RABGGTA, respectively, for 48 h. Then cells were incubated with or without 100 nM insulin for 30 min, then total protein and plasma membrane protein were harvested, and GLUT4 expression was analysed by western blot, with GAPDH as the loading control (n = 3). (C) C2C12 myotubes were pretreated with 10 μM GGPP, 10 μM simvastatin, and 10 μM GGPP combined with 10 μM simvastatin for 24 h, and then cells were treated with 100 nM insulin for 30 min. Total protein samples and plasma membrane protein samples were harvested, and GLUT4 expression was analysed by western blot, with GAPDH as the loading control (n = 3). (D, E) Male C57BL/6J mice (20 ± 2 g) were randomly grouped (n = 6). After administration of geranylgeraniol (GGOH) (25 mg/kg/day), simvastatin (40 mg/kg/day), and GGOH combined with simvastatin for 3 weeks, mice were sacrificed and membrane GLUT4 expression in gastrocnemius (D) and tibialis anterior (E) was analysed by western blot, with GAPDH as the loading control (n = 3). Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns denotes no significance.
Figure 3
Figure 3
Insulin signalling is not necessary for simvastatin‐caused inhibition of insulin‐stimulated glucose uptake in skeletal muscle cells. (A) C2C12 myotubes were pretreated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for 24 h. Then cells were incubated with or without 100 nM insulin for 30 min, total protein was harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). Long exposure of p‐AKT (Ser473) for 30 s and short exposure of p‐AKT (Ser473) for 5 s were shown. (B) C2C12 myotubes were previously transfected with siRNAs targeting GGPS1, PGGT1B, and RABGGTA, respectively, for 48 h. Then cells were incubated with or without 100 nM insulin for 30 min, total protein was harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). Long exposure of p‐AKT (Ser473) for 30 s and short exposure of p‐AKT (Ser473) for 5 s were shown. (C) C2C12 myotubes were previously transfected with or without 1 μg Myr‐AKT plasmid using Lipofectamine 3000 for 48 h. Then the cells were treated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for another 24 h. Cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (D) C2C12 myotubes were previously transfected with 1 μg Myr‐AKT plasmid using Lipofectamine 3000 for 48 h. Then the cells were treated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for another 24 h. Cells were incubated with 100 nM insulin for 30 min, then protein samples were harvested, and the expression of indicated proteins was checked by western blot, with GAPDH as the loading control (n = 3). (E) C2C12 myotubes were previously transfected with or without 1 μg Myr‐AKT plasmid using Lipofectamine 3000 for 48 h. Then cells were transfected with siRNAs targeting GGPS1, PGGT1B, and RABGGTA, respectively, for another 48 h. Cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (F) C2C12 myotubes were previously transfected with or without 1 μg Myr‐AKT plasmid using Lipofectamine 3000 for 48 h. Then cells were transfected with siRNAs targeting GGPS1, PGGT1B, and RABGGTA, respectively, for another 48 h. Cells were incubated with 100 nM insulin for 30 min, protein samples were harvested, and the expression of indicated proteins was checked by western blot, with GAPDH as the loading control (n = 3). Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns denotes no significance.
Figure 4
Figure 4
Adeno‐associated virus serotype 9 (AAV9)‐mediated knockdown of RABGGTA in skeletal muscle causes insulin resistance without disturbing insulin signalling in vivo. Mice were subjected a week of adjustable feeding and then were divided into two groups including shControl group and shRABGGTA group (n = 6). Posterior limbs of mice in shControl group and shRABGGTA group were infected with control AAV9 and shRABGGTA AAV9, respectively, through in situ injection. Four weeks after the infection, mice were subjected with experiments as follows. (A) Glucose tolerance test (GTT) and GTT AUC. (B) Insulin tolerance test (ITT) and ITT AUC. (C) Mice were fasted for 16 h before intraperitoneal injection of 2‐DG (2 g/kg body weight). Thirty minutes after the injection, mice were sacrificed and skeletal muscle including gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus was obtained. 2‐DG concentration in each muscle was measured using Glucose Uptake Colorimetric Assay Kit. (D) The mRNA expression of RABGGTA in skeletal muscle was quantified by RT‐qPCR. (E, F) The expression of indicated proteins in gastrocnemius (E) and tibialis anterior (F) was detected by western blot, with GAPDH as the loading control of total protein and ATP1A1 as the loading control of plasma membrane protein (n = 3). Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns denotes no significance.
Figure 5
Figure 5
Geranylgeranyl pyrophosphate (GGPP) reverses simvastatin‐caused inhibition of insulin signalling via recovering RhoA geranylgeranylation‐mediated TAZ/IRS1 axis. (A) C2C12 myotubes were treated with 10 μM simvastatin, 10 μM FTI‐277, 10 μM GGTI‐298, 1.5 mM 3‐PEHPC, and 1 mM perillyl alcohol for 24 h, protein samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (B) C2C12 myotubes were transfected with siRNAs specifically targeting GGPS1, PGGT1B, and RABGGTA, respectively, for 48 h. Protein samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (C) C2C12 myotubes were treated with 10 μM GGPP, 10 μM simvastatin, or 10 μM GGPP combined with 10 μM simvastatin for 24 h. Then cells were treated or not treated with 100 nM insulin for 30 min. Protein samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (D) C2C12 myotubes were previously transfected with NC or siRNA specifically targeting TAZ and IRS1, respectively, for 48 h. Then cells were treated with 10 μM GGPP, 10 μM simvastatin, or 10 μM GGPP combined with 10 μM simvastatin for another 24 h. Before the end of the experiment, cells were incubated with 100 nM insulin for 30 min. Then protein samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (E) C2C12 myotubes were treated with 10 μM GGPP, 10 μM simvastatin and 30 μM Rhosin as indicated for 24 h. Then cells were incubated with 100 nM insulin for 30 min, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (F) C2C12 myotubes were previously transfected with siRNA targeting RhoA for 24 h, and then cells were transfected with vector, RhoA (WT), or RhoA (C190A) plasmids for 48 h. Before the end of the experiment, cells were incubated with 100 nM insulin for 30 min, then total protein samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 6
Figure 6
Geranylgeranylation of RAB8A is critical for insulin‐stimulated GLUT4 translocation and concomitant glucose uptake in skeletal muscle cells. (A) C2C12 myotubes were transfected with siRNAs specifically targeting RAB8A and RAB13, respectively, for 48 h. Then cells were incubated with 100 nM insulin for 30 min. Total protein samples and plasma membrane fraction samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (B) C2C12 myotubes were transfected with siRNAs specifically targeting RAB8A and RAB13, respectively, for 48 h. Then cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (C) RAB8A‐knockout (RAB8A‐ko) C2C12 myotubes were transfected with vector, RAB8A (WT), and RAB8A (C204A) plasmids for 48 h. Then cells were incubated with 100 nM insulin for 30 min. Total protein samples and plasma membrane fraction samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (D) RAB8A‐ko C2C12 myotubes were transfected with vector, RAB8A (WT), and RAB8A (C204A) plasmids for 48 h. Then cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). (E) RAB13‐ko C2C12 myotubes were transfected with vector, RAB13 (WT), and RAB13 (C199A) plasmids for 48 h. Then cells were incubated with 100 nM insulin for 30 min. Total protein samples and plasma membrane fraction samples were harvested, and the expression of indicated proteins was analysed by western blot, with GAPDH as the loading control (n = 3). (F) RAB13‐ko C2C12 myotubes were transfected with vector, RAB13 (WT), and RAB13 (C199A) plasmids for 48 h. Then cells were exposed to 2‐NBDG containing 100 nM insulin for 30 min, and 2‐NBDG uptake was measured by fluorescence detection (n = 3). Data represented the mean ± SEM. Statistical analysis was performed with one‐way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns denotes no significance.
Figure 7
Figure 7
Schematic diagram of proposed statin‐targeted mevalonate pathway regulating insulin‐stimulated glucose uptake.
See this image and copyright information in PMC

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