Novel GLP-1R/GIPR co-agonist "twincretin" is neuroprotective in cell and rodent models of mild traumatic brain injury

Abstract

Several single incretin receptor agonists that are approved for the treatment of type 2 diabetes mellitus (T2DM) have been shown to be neuroprotective in cell and animal models of neurodegeneration. Recently, a synthetic dual incretin receptor agonist, nicknamed "twincretin," was shown to improve upon the metabolic benefits of single receptor agonists in mouse and monkey models of T2DM. In the current study, the neuroprotective effects of twincretin are probed in cell and mouse models of mild traumatic brain injury (mTBI), a prevalent cause of neurodegeneration in toddlers, teenagers and the elderly. Twincretin is herein shown to have activity at two different receptors, dose-dependently increase levels of intermediates in the neurotrophic CREB pathway and enhance viability of human neuroblastoma cells exposed to toxic concentrations of glutamate and hydrogen peroxide, insults mimicking the inflammatory conditions in the brain post-mTBI. Additionally, twincretin is shown to improve upon the neurotrophic effects of single incretin receptor agonists in these same cells. Finally, a clinically translatable dose of twincretin, when administered post-mTBI, is shown to fully restore the visual and spatial memory deficits induced by mTBI, as evaluated in a mouse model of weight drop close head injury. These results establish twincretin as a novel neuroprotective agent and suggest that it may improve upon the effects of the single incretin receptor agonists via dual agonism.

Keywords: Glucagon-like peptide-1; Glucose-dependent insulinotropic peptide; Incretin; Incretin mimetic; Neurodegeneration; Traumatic brain injury.

Fig. 1

Twincretin has activity at both the GLP-1R and GIPR in human neuroblastoma cells. (a) The levels of cAMP in SH-SY5Y cells treated with 10 µM Ex 9–39 and in cells treated with 10 µM Pro3GIP are statistically equivalent to the levels of cAMP in control cells (n=6). (b) The cAMP levels in SH-SY5Y cells coincubated with 1 nM twincretin and 100 nM Ex 9–39 are significantly lower than the levels of cAMP in cells incubated with 1 nM twincretin alone. Additionally, 1 nM twincretin alone caused significant increases in cAMP levels as compared to sham control (n=4). (c) The cAMP levels in SH-SY5Y cells coincubated with 10 nM twincretin and 1 µM Pro3GIP are significantly lower than the levels of cAMP in cells incubated with 10 nM twincretin alone. Also, 10 nM twincretin alone significantly increased cAMP levels as compared to sham control (n= 3; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2

Twincretin is neurotrophic in human neuroblastoma cells. (a) The levels of cAMP signicantly and dose-dependently increased in SH-SY5Y cells exposed to 1–1000 nM twincretin over the course of one hour (n=5). (b) The fraction of pCREB/CREB significantly increases in SH-SY5Y cells exposed to 100 nM TCT for 1 h as compared to control SH-SY5Y cells that were not exposed to twincretin (n= 4). (c) 1–1000 nM twincretin increased cell viability by approximately 5% as measured via MTS assay, and decreased LDH release into the extracellular space by approximately 20% as compared to controls, although neither trend was significant (n = 10). (d) In SH-hGLP-1R#9 cells, which are SH-SY5Y cells that overexpress the human GLP-1R, 1–1000 nM twincretin treatment increased cell viability by approximately 20% and reduced LDH release by approximately 20% as compared to controls (n = 10; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3

Twincretin is neuroprotective in human neuroblastoma cells and rat primary midbrain neurons. (a) 1–1000 nM twincretin pretreatment dose-dependently increased the viability of SH-SY5Y cells exposed to 100 mM glutamate as compared to cells that only received the toxic insult (n=10). (b) The cell viabilities of SH-SY5Y cells pretreated with 1–1000 nM twincretin before exposure to 200 µM H₂O₂ were statistically equivalent to control cell viability, while the cell viability of cells that received the toxic insult alone were significantly reduced (n=10). (c) 1–1000 nM twincretin pretreatment of SH-hGLP-1R#9 cells prior to 100 mM glutamate exposure fully prevented the reduction in cell viability caused by glutamate insult alone. The viabilities of cells pretreated with twincretin were statistically equivalent to control cell viability (n=10). (d) 1–100 nM twincretin pretreatment significantly increased the viability of SH-hGLP-1R#9 cells exposed to 600 µM H₂O₂ as compared to cells exposed to insult alone. The viability of cells pretreated with 10 nM twincretin were statistically equivalent to control viability (n = 10). (e) TH immunoreactivity significantly increased in rat primary midbrain neurons treated with 10–1000 nM twincretin after 100 µM 6OHDA insult as compared to cells that only received the insult (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4

Twincretin provides maximal cAMP production and neurotrophic effects in human neuroblastoma cells as compared to equimolar Ex-4 and GIP. (a) Treatment with 1 nM twincretin and 1 nM Ex-4 significantly raised the levels of cAMP in SH-SY5Y cells as compared to sham control cells over the course of one hour. 1 nM GIP treatment did not significantly alter the levels of cAMP as compared to control (n=5). (b) Only SH-SY5Y cells treated with 1 nM twincretin had significantly increased cell viability as compared to controls. The viability of cells treated with 1 nM Ex-4 and 1 nM GIP were statistically equivalent to controls (n = 15; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 5

Twincretin administration (50 µg/kg once daily × 7 days, s.c.) prevents deficits in visual and spatial memory in mice at 7 and 30 days post-mTBI. (a) The preference index of mTBI mice in the novel object recognition test was significantly reduced at both and 7 and 30 days post insult, as compared to sham control. Mice that received twincretin treatment after mTBI demonstrated equivalent preference indices to sham controls (n=9–12). (b) The preference index of mTBI mice in the Y-maze paradigm was significantly reduced as compared to sham control at 7 and 30 days post-mTBI. mTBI + twincretin mice did not demonstrate these reductions in preference indices at either 7 or 30 days post-mTBI (n=12–16). (c) All conditions of mice spent equivalent times in the open arm of the elevated plus maze at both 7 and 30 days post-mTBI (n= 9–14; *p < 0.05, **p < 0.01, ***p < 0.001).