Treatment with low glucose (LG) elicits a mitochondrial fission response. A: LG induced mitochondrial fragmentation in endothelial cells and inhibition of dynamin-related protein 1 (Drp1) activity with Mdivi-1 (10 μM) attenuated the response (P = 0.003 overall, *P < 0.02 for LG vs. NG and LG + Mdivi-1, N = 6). Scale bars represent 10 μm. B: similarly, when expression of Drp1 was decreased via siRNA knockdown, mitochondrial fragmentation was decreased in LG-exposed cells (P < 0.001 overall, *P = 0.025 for LG + Scramble vs. LG + siDrp1 and NG, N = 6). Scale bars represent 10 μm. C: Human umbilical vein endothelial cells (HUVECs) exposed to siRNA constructs against Drp1 displayed ~50% decrease in protein expression when analyzed by Western blot (*P < 0.05, N = 6). D: proof of concept in translating cellular knockdown of Drp1 expression to our ex vivo preparation of human tissues. Vessels were exposed to siDrp1 constructs per protocol and then analyzed for Drp1 expression via immunohistochemistry. The endothelium of samples exposed to siDrp1 displayed a decrease in Drp1 expression when compared with scrambled control (arrows). Scale bars represent 10 μm.
LG-induced endothelial dysfunction is attenuated through mitochondrial fission inhibition. A: percent vasodilation in response to acetylcholine (ACh) was measured in vessels incubated in either NG or LG buffer for 2 h either with or without addition of Mdivi-1 (10 μmol/l) for the final 30 min of incubation. Overall, LG exposure displayed significant impairment of endothelial function compared with the other groups (P < 0.001 overall, *P < 0.02 at the indicated doses for LG vs. NG, NG+Mdivi-1, and LG+Mdivi-1, N = 5 subjects). Additionally, l-NAME appears to completely abrogate the ameliorative effect of Mdivi-1 on vasodilation under LG conditions (N = 4 subjects, P = 0.01 vs. all other conditions) B: impact of molecular knockdown of Drp1 expression on endothelial function. LG exposure caused impaired vasodilatory response and was improved under knockdown conditions (P < 0.001 overall, *P ≤ 0.002 at indicated ACh doses for LG+Scrambled siRNA vs. all other exposures, N = 3 subjects). C: inhibition of Drp1 activity improves vasofunction in arterioles isolated from clinically diagnosed T2DM subjects displaying glycosylated hemoglobin (HbA1c) levels ranging from 6.5 to 9.3% (mean and SD; 7.3 ± 1.2) and blood glucose levels between 115 and 162 mg/dl (mean and SD; 135 ± 19) (P < 0.005 overall, *P < 0.05 at indicated dose, N = 5 subjects). D: effect of Mdivi-1 on vasofunction of T2DM arterioles (HbA1c: 7.1 ± 1.3; blood glucose: 134 ± 23) were repeated with exposure to LG (P = 0.032 overall, *P < 0.05 at indicated dose, N = 4 subjects). E: microvessels incubated in either NG (N = 3) or LG (N = 4) buffer were exposed to physiologically relevant concentrations of insulin (I, 0.25 nmol/l), epinephrine (E, 5.5 nmol/l), and glucagon (G, 23 nmol/l) to simulate compensatory signaling of the endocrine system to a LG stress. Hormonal effects were negligible where P = 0.937 and P = 0.745 overall for NG and LG groups, respectively. ACh, acetylcholine; PAP, papaverine.
Increased NO bioavailability in human arterioles with pharmacologic inhibition of fission. Human arterioles were incubated in either NG or LG buffer with or without fission inhibitor, Mdivi-1 (10 µmol/l). NO was visualized with the fluorescent NO sensitive fluorescent probe DAF2-DA. LG treatment reduced NO presence in isolated microvessels, and blockade of the fission process with Mdivi-1 increased NO levels in vessels exposed to LG (P < 0.001 overall, *P < 0.05 for LG vs. NG and LG+Mdivi-1, N = 4). Scale bars represent 50 μm.
Fission inhibition depolarizes mitochondrial membrane potential and reduces mitochondrial superoxide in human arterioles. A: human arterioles were incubated in either NG or LG buffer with or without Mdivi-1 (10 μmol/l) to block the fission response (P = 0.008 overall, N = 4). Treatment with LG alone displayed increased membrane hyperpolarization compared with NG alone (*P < 0.02). When LG-treated vessels were coincubated with Mdivi-1, hyperpolarization was reversed (*P < 0.01 vs. LG alone). B: isolated vessels were exposed to either NG or LG buffer with/or without pretreatment with Mdivi-1 (10 μmol/l). MitoSOX was used to detect for the presence of superoxide; greater fluorescence intensity equals more superoxide present. Vessels treated with LG displayed increased superoxide production (P = 0.003 overall, *P < 0.02 for LG vs. NG, N = 5). Pharmacologic inhibition of mitochondrial fission via administration of Mdivi-1 prevented the increase in superoxide production seen in vessels exposed to LG alone (*P = 0.005 for LG vs. LG+Mdivi-1). Scale bars represent 50 μm.
Mitochondrial membrane depolarization improves bioavailability of NO and reduces mitochondrial superoxide in isolated arterioles. A: vessels were incubated with DAF-2DA with or without exposure to carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (100 nmol/l), a membrane depolarizing agent. LG-treated vessels displayed a significant decrease in NO level compared with other treatment groups (P = 0.003 overall, *P < 0.03 for LG vs. all other exposures, N = 5), while membrane depolarization with CCCP increased NO levels approximating that of the NG+CCCP group. B: superoxide was detected with MitoSOX in the presence of CCCP (100 mol/l). LG-treated vessels displayed a significant increase in superoxide compared with other treatment groups based on increased fluorescence, while membrane depolarization with CCCP decreased MitoSox fluorescence (P < 0.001 overall, *P < 0.01 for LG vs. all other exposures, N = 4). Scale bars represent 50 μm.
Posttranslational regulation of Drp1 via LG exposure. A: phosphorylation of Drp1 at Ser 637 acts as an inhibitor of Drp1 activity. Expression of P-S637-Drp1 was analyzed in HUVECs treated with LG via Western blot. Overall, the data appear to support that LG exposure decreases phosphorylation at Ser 637 and may allow for increased Drp1 activity and subsequent fission. B: conversely, phosphorylation of Drp1 at Ser 616 acts as an activating event and LG exposure appears to increase phosphorylation at this site (*P < 0.01, N = 10).
LG does not alter mitochondrial bioenergetics. A: endothelial cells were exposed to either NG or LG media and the rate of oxygen consumption was analyzed with a Seahorse XF96 Analyzer. B: glycolytic activity was also recorded concurrently during the basal data collection period. No significant differences were observed between the two groups. Data represent results from four independent experiments (P = not significant at all data points).
Impairment of endothelial function through LG exposure. Acute LG conditions appear to increase mitochondrial fragmentation by creating a pro-fission state via activation of Drp1. This in turn causes hyperpolarization of the mitochondrial membrane potential, an increase in superoxide production, and subsequent decrease in NO bioavailability. Taken together, these modifications yield an impairment of human microvessels to vasodilate in an endothelium-dependent manner. Blockade of LG-induced mitochondrial fragmentation via Drp1 attenuates the adverse effects and restores vasodilation, indicating the potential of therapeutic targets to improve endothelial function.