Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 15:15:96.
doi: 10.1186/s12933-016-0410-9.

Effect of the long-acting insulin analogues glargine and degludec on cardiomyocyte cell signalling and function

Thorsten Hartmann  1 , Sabrina Overhagen  2 , D Margriet Ouwens  2   3   4 , Silja Raschke  1 , Paulus Wohlfart  5 , Norbert Tennagels  5 , Nina Wronkowitz  1 , Jürgen Eckel  6   7
Affiliations

Effect of the long-acting insulin analogues glargine and degludec on cardiomyocyte cell signalling and function

Thorsten Hartmann et al. Cardiovasc Diabetol. .

Abstract

Background: The effects of insulin on cardiomyocytes, such as positive inotropic action and glucose uptake are well described. However, in vitro studies comparing long-acting insulin analogues with regard to cardiomyocyte signalling and function have not been systematically conducted.

Methods: Insulin receptor (IR) binding was assessed using membrane embedded and solubilised IR preparations. Insulin signalling was analysed in adult rat ventricular myocytes (ARVM) and HL-1 cardiac cells. Inotropic effects were examined in ARVM and the contribution of Akt to this effect was assessed by specific inhibition with triciribine. Furthermore, beating-rate in Cor.4U(®) human cardiomyocytes, glucose uptake in HL-1 cells, and prevention from H2O2 induced caspase 3/7 activation in cardiac cells overexpressing the human insulin receptor (H9c2-E2) were analysed. One-way ANOVA was performed to determine significance between conditions.

Results: Insulin degludec showed significant lower IR affinity in membrane embedded IR preparations. In HL-1 cardiomyocytes, stimulation with insulin degludec resulted in a lower Akt(Ser(473)) and Akt(Thr(308)) phosphorylation compared to insulin, insulin glargine and its active metabolite M1 after 5- and 10-min incubation. After 60-min treatment, phosphorylation of Akt was comparable for all insulin analogues. Stimulation of glucose uptake in HL-1 cells was increased by 40-60 %, with a similar result for all analogues. Incubation of electrically paced ARVM resulted for all insulins in a significantly increased sarcomere shortening, contractility- and relaxation-velocity. This positive inotropic effect of all insulins was Akt dependent. Additionally, in Cor.4U(®) cardiomyocytes a 10-20 % increased beating-rate was detected for all insulins, with slower onset of action in cells treated with insulin degludec. H9c2-E2 cells challenged with H2O2 showed a fivefold increase in caspase 3/7 activation, which could be abrogated by all insulins used.

Conclusions: In conclusion, we compared for the first time the signalling and functional impact of the long-acting insulin analogues insulin glargine and insulin degludec in cardiomyocyte cell models. We demonstrated similar efficacy under steady-state conditions relative to regular insulin in functional endpoint experiments. However, it remains to be shown how these results translate to the in vivo situation.

Keywords: Cardiac action; Insulin analogues; Insulin degludec; Insulin glargine.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Binding affinity and Akt signalling of long-acting insulin analogues. a Membrane embedded insulin receptor preparations were used to analyse binding of Ins, IGlaM1 and IDeg in a competition binding assay, as described in [21]. Percentage of binding is normalised to maximum binding of [125I]-labelled human insulin. Data represent mean values ± SEM, n = 4-5. b–d HL-1 cells were used to assess the onset of insulin action by treatment with 200 nM for 5 (b); 10 (c) or 60 min (d) with insulin or insulin analogues. Phosphorylation of Akt(Ser473) was assessed by Western blot analysis. Data are normalised to tubulin levels. Representative blots are shown. Data represent mean values ± SEM, n = 4–5, *p < 0.05 vs. basal, #p < 0.05 vs. IDeg. Regular insulin Ins, insulin glargine IGla, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
Fig. 2
Fig. 2
Effect of insulin and insulin analogues on Akt signalling and cardiac contraction in ARVM. Adult rat ventricular cardiomyocytes (ARVM) were treated for 10 min with 100 nM insulin or insulin analogues to investigate the insulin signalling pathway in these cells (a). ARVM were isolated by enzymatic digestion, starved overnight and treated with 100 nM insulin or insulin analogues or 10 nM isoproterenol as positive control. ARVM were paced at 1 Hz, 15 V, 0.5 ms with the IonOptix Myopacer Cell Stimulator System to assess the sarcomeric shortening (b), departure velocity (c) and return velocity (d). In each condition at least 10 contractions of 10-14 cardiomyocytes were measured under steady-state conditions. Data represent mean values ± SEM, n = 4, *p < 0.05 vs. basal. Regular insulin Ins, isoproterenol iso, insulin glargine IGla, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
Fig. 3
Fig. 3
Impact of Akt inhibition on insulin-induced cardiac contraction. Adult rat ventricular cardiomyocytes (ARVM) were analysed either without pre-treatment (blank bars) or pre-treated with 10 µM of the specific Akt-inhibitor triciribine (filled bars) for 30 min. Subsequently, ARVM were treated with either 100 nM insulin or insulin analogues for 10 min to investigate the insulin signalling pathway after triciribine treatment (a). Furthermore, ARVM were treated with 100 nM of insulin or insulin analogues or 10 nM isoproterenol and paced at 1 Hz, 15 V, 0.5 ms with the IonOptix Myopacer Cell Stimulator System to assess the sarcomeric shortening (b), departure velocity (c) and return velocity (d). In each condition at least 10 contractions of 10–14 cardiomyocytes were measured. Data represent mean values ± SEM, n = 3–5, *p < 0.05 vs. basal (treated). Regular insulin Ins, isoproterenol iso, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
Fig. 4
Fig. 4
Effect of insulin glargine and insulin degludec on the beating-rate of human iPSC-derived cardiomyocytes. Commercially available human iPSC-derived cardiomyocytes (Cor.4U®) were used to assess the long-term effect of insulins on the beating-rate (a–d). Experiments were performed at day 4 after seeding. Cells were treated with 500 nM of insulin or insulin analogues or 100 nM isoproterenol, respectively. The beating-rate was measured for 6 h. a Basal beating-rate prior to substance application. b Beating-rate of the first 30 min after substance application. c 0.5–6 h time course of beating-rate in Cor.4U® cells after substance application. d Area under the curve for the 6 h beating-rate measurement. Data represent mean values ± SEM, n = 4, *p < 0.05 vs. basal. Regular insulin Ins, isoproterenol iso, insulin glargine IGla, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
Fig. 5
Fig. 5
2-deoxy-d-glucose uptake in HL-1 cells after insulin stimulation with regular insulin and long-acting insulin analogues. HL-1 cells were used to assess the effect of insulin, insulin glargine, the active metabolite M1 and insulin degludec on 2-deoxy-d-glucose uptake. The cells were pre-treated with 200 nM of the indicated insulin for 1 h. Subsequently, the cells were exposed to radioactive labelled mix containing 2–deoxy–d–glucose and 2–deoxy–d–[1–14C]glucose for 10 min at 37 °C. Data represent mean values ± SEM, n = 4, *p < 0.05 vs. basal. Regular insulin Ins, insulin glargine IGla, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
Fig. 6
Fig. 6
Anti-apoptotic potency of long-acting insulin analogues in the presence of H2O2. H9c2 cardiomyocytes overexpressing the human insulin receptor (H9c2-E2 cells) were treated for 2 h with 100 nM of insulin and insulin analogues in the presence or absence of 800 µM H2O2 to evaluate the cardio protective effects of insulin and its analogues. Caspase 3/7 activity was measured using the promega caspase 3/7 Glo Assay. Each condition was performed in quadruplicates. Data represent mean values ± SEM, n = 5–6, *p < 0.05 vs. basal. Regular insulin Ins, insulin glargine IGla, active metabolite of insulin glargine IGlaM1, insulin degludec IDeg
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Muniyappa R, Montagnani M, Koh KK, Quon MJ. Cardiovascular actions of insulin. Endocr Rev. 2007;28(5):463–491. doi: 10.1210/er.2007-0006. - DOI - PubMed
    1. Bertrand L, Horman S, Beauloye C, Vanoverschelde JL. Insulin signalling in the heart. Cardiovasc Res. 2008;79(2):238–248. doi: 10.1093/cvr/cvn093. - DOI - PubMed
    1. Klein LJ, Visser FC. The effect of insulin on the heart: part 1: effects on metabolism and function. Neth Heart J. 2010;18(4):197–201. doi: 10.1007/BF03091761. - DOI - PMC - PubMed
    1. ADA Standards of medical care in diabetes–2008. Diabetes Care. 2008;31(Suppl 1):S12–S54. - PubMed
    1. Rodbard HW, Gough S, Lane W, Korsholm L, Bretler DM, Handelsman Y. Reduced risk of hypoglycemia with insulin degludec versus insulin glargine in patients with type 2 diabetes requiring high doses of Basal insulin: a meta-analysis of 5 randomized begin trials. Endocr Pract. 2014;20(4):285–292. doi: 10.4158/EP13287.OR. - DOI - PubMed

Publication types

MeSH terms