Yeungnam Univ J Med.  2019 Jan;36(1):26-35. 10.12701/yujm.2019.00059.

Telmisartan increases hepatic glucose production via protein kinase C ζ-dependent insulin receptor substrate-1 phosphorylation in HepG2 cells and mouse liver

• Cho KW
• Cho DH

• KMID: 2434096
• DOI: http://doi.org/10.12701/yujm.2019.00059

Abstract

BACKGROUND
Dysregulation of hepatic glucose production (HGP) contributes to the development of type 2 diabetes mellitus. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), has various ancillary effects in addition to common blood pressure-lowering effects. The effects and mechanism of telmisartan on HGP have not been fully elucidated and, therefore, we investigated these phenomena in hyperglycemic HepG2 cells and high-fat diet (HFD)-fed mice.
METHODS
Glucose production and glucose uptake were measured in HepG2 cells. Expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase α (G6Pase-α), and phosphorylation levels of insulin receptor substrate-1 (IRS-1) and protein kinase C ζ (PKCζ) were assessed by western blot analysis. Animal studies were performed using HFD-fed mice.
RESULTS
Telmisartan dose-dependently increased HGP, and PEPCK expression was minimally increased at a 40 μM concentration without a change in G6Pase-α expression. In contrast, telmisartan increased phosphorylation of IRS-1 at Ser302 (p-IRS-1-Ser302) and decreased p-IRS-1-Tyr632 dose-dependently. Telmisartan dose-dependently increased p-PKCζ-Thr410 which is known to reduce insulin action by inducing IRS-1 serine phosphorylation. Ectopic expression of dominant-negative PKCζ significantly attenuated telmisartan-induced HGP and p-IRS-1-Ser302 and -inhibited p-IRS-1-Tyr632. Among ARBs, including losartan and fimasartan, only telmisartan changed IRS-1 phosphorylation and pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not alter this effect. Finally, in the livers from HFD-fed mice, telmisartan increased p-IRS-1-Ser302 and decreased p-IRS-1-Tyr632, which was accompanied by an increase in p-PKCζ-Thr410.
CONCLUSION
These results suggest that telmisartan increases HGP by inducing p-PKCζ-Thr410 that increases p-IRS-1-Ser302 and decreases p-IRS-1-Tyr632 in a PPARγ-independent manner.

Keyword

Hepatic glucose production; IRS-1; Phosphorylation; PKCζ; Telmisartan

MeSH Terms

Animals
Blotting, Western
Diabetes Mellitus, Type 2
Diet, High-Fat
Ectopic Gene Expression
Glucose*
Glucose-6-Phosphatase
Hep G2 Cells*
Insulin Receptor Substrate Proteins*
Insulin*
Liver*
Losartan
Mice*
Peroxisomes
Phosphoenolpyruvate
Phosphorylation
Protein Kinase C*
Protein Kinases*
Receptor, Angiotensin, Type 1
Receptor, Insulin*
Serine
Glucose
Glucose-6-Phosphatase
Insulin
Insulin Receptor Substrate Proteins
Losartan
Phosphoenolpyruvate
Protein Kinase C
Protein Kinases
Receptor, Angiotensin, Type 1
Receptor, Insulin
Serine

Figure

• 

  Fig. 1. Telmisartan increases glucose production by increasing p-IRS-1-Ser302 and decreasing p-IRS-1-Tyr632 in hyperglycemic HepG2 cells. (A) HepG2 cells were treated with various doses of telmisartan (0, 10, 20, or 40 μM) for 24 h in the presence of 5.5 mM or 25 mM D-glucose and then glucose production assay was performed as described in the Methods. (B−D) HepG2 cells were treated with various doses of telmisartan (0, 10, 20, or 40 μM) for 24 h in the presence of 25 mM D-glucose and then with or without 100 nM insulin for an additional 10 min. Total proteins were obtained, and levels of PEPCK, G6Pase-α, p-IRS-1-Ser302, p-IRS-1-Ser318, p-IRS1-Ser1101, p-IRS-Tyr632, and p-IRS-Tyr896 were detected using western blot analysis. Nitrocellulose membranes were reprobed using antibody to detect actin or total IRS-1 to monitor equal sample loading. Densitometry was used to quantify PEPCK, G6Pase-α, p-IRS-1-Ser302, and p-IRS-Tyr632 relative to the actin or the IRS-1 bands, respectively. Blots represent at least four experiments. Bar graphs show mean fold alterations above/below control (±SD). Differences were statistically significant at a)p<0.05, b)p<0.01. IRS-1, insulin receptor substrate-1; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase-α, glucose-6-phosphatase α.

• 

  Fig. 2. Telmisartan-induced p-PKCζ-Thr410 mediates IRS-1 phosphorylation changes that increase glucose production. (A) HepG2 cells were treated with various concentrations of telmisartan (0, 10, 20, or 40 μM) for 24 h in the presence of 25 mM D-glucose and then 100 nM insulin for an additional 10 min. Western blot analyses were performed as described in Fig. 1. (B) After HA-tagged dn-PKCζ (K281R) constructs were transfected into HepG2 cells as described in the Methods, cells were treated with 40 μM telmisartan or vehicle for 24 h in the presence of 25 mM D-glucose and then 100 nM for an additional 10 min. Western blot analyses were performed as described in Fig. 1. (C) HepG2 cells were transfected with dn-PKCζ constructs, treated with 40 μM telmisartan for 24 h in the presence of 25 mM D-glucose, and glucose production assay was performed as described in the Methods. (D) After transfection as described above, HepG2 cells were treated with 40 μM telmisartan for 24 h in the presence of 25 mM D-glucose and then 100 nM insulin for an additional 10 min. Western blot analyses were performed as described in Fig. 1. Blots represent at least four experiments. Bar graphs show mean fold alterations above/below control (±SD). Differences are statistically significant at a)p<0.05, b)p<0.01. n.s., not significant. PKCζ, protein kinase C ζ; IRS-1, insulin receptor substrate-1; HA, hemagglutinin.

• 

  Fig. 3. Among tested ARBs, only telmisartan induces p-IRS-1-Ser302 and represses p-IRS-1-Tyr632 via PPARγ-independent pathway. (A) HepG2 cells were treated with various ARBs (telmisartan, losartan, or fimasartan at 40 μM) for 24 h in the presence of 25 mM D-glucose and then 100 nM insulin for further 10 min. Western blot analyses were performed as described in Fig. 1. (B) After pretreatment with 5 μM GW9662 for 1 h, HepG2 cells were treated with 40 μM telmisartan for 24 h in the presence of 25 mM D-glucose and then 100 nM insulin for further 10 min. Western blot analyses were performed as described in Fig. 1. Blots represent at least four experiments. Bar graphs show mean fold alterations above/below control (±SD). Differences were statistically significant at b)p<0.01. n.s., not significant. ARB, angiotensin II type 1 receptor blocker; IRS-1, insulin receptor substrate-1; PPARγ, peroxisome proliferator-activated receptor γ.

• 

  Fig. 4. Telmisartan induces p-IRS-1-Ser302, represses p-IRS-1-Tyr632, and increases p-PKCζ-Thr410 in livers of HFD-fed mice. (A) Schematic diagram of animal experiments. Hyperglycemic mouse models were established as described in the Methods. (B‒D) Mice were euthanized, their livers were dissected, total liver proteins were extracted, and then subjected to western blot analysis as described in Fig. 1. Blots represent at least five livers from each mouse group. Bar graphs show mean fold alterations above/below control (±SD). Differences are statistically significant at a)p<0.05, b)p<0.01. IRS-1, insulin receptor substrate-1; PKCζ, protein kinase C ζ; HFD, high-fat diet.

Reference

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