MiRNA and associated inflammatory changes from baseline to hypoglycemia in type 2 diabetes

Abstract

ObjectiveHypoglycemia in type 2 diabetes (T2D) increases morbidity and mortality but the underlying physiological response is still not fully understood, though physiological changes are still apparent 24 hours after the event. Small noncoding microRNA (miRNA) have multiple downstream biological effects that may respond rapidly to stress. We hypothesized that hypoglycemia would induce rapid miRNA changes; therefore, this pilot exploratory study was undertaken.MethodsA pilot prospective, parallel study in T2D (n=23) and controls (n=23). Insulin-induced hypoglycemia (2mmol/l: 36mg/dl) was induced and blood sampling performed at baseline and hypoglycemia. Initial profiling of miRNA was undertaken on pooled samples identified 96 miRNA that were differentially regulated, followed by validation on a custom designed 112 miRNA panel.ResultsNine miRNAs differed from baseline to hypoglycemia in control subjects; eight were upregulated: miR-1303, miR-let-7e-5p, miR-1267, miR-30a-5p, miR-571, miR-661, miR-770-5p, miR-892b and one was downregulated: miR-652-3p. None of the miRNAs differed from baseline in T2D subjects.ConclusionA rapid miRNA response reflecting protective pathways was seen in control subjects that appeared to be lost in T2D, suggesting that mitigating responses to hypoglycemia with blunting of the counter-regulatory response in T2D occurs even in patients with short duration of disease.Clinical trial registrationhttps://clinicaltrials.gov/ct2/show/NCT03102801?term=NCT03102801&draw=2&rank=1, identifier NCT03102801.

MicroRNA Changes Up to 24 h following Induced Hypoglycemia in Type 2 Diabetes

“…This study extends the work already published from the same clinical study and performed in the same patient population that reported changes in miRNAs from baseline to the point of hypoglycemia [ 23 ] and, further, extends the work of others [ 24 ]. Our hypothesis that hypoglycemia would induce miRNA was demonstrated with the primary outcome.…”

“…A previous small hypoglycemia study restricted to 14 patients with type 2 diabetes alone using selective miRNA analysis based on platelet expression revealed that an upregulation of the miRNA may be seen at 1 day and 7 days, though an earlier time course miRNA analysis within the first 24 h period was not undertaken [ 24 ]; unfortunately, it is not possible to directly compare our study with this study as the degree and duration of hypoglycemia differed between studies. Hypoglycemia in normal control subjects is an unphysiological event but, as was previously reported, the miRNA changes were pronounced at the point of hypoglycemia [ 23 ], with more miRNA altered in the controls (nine miRNAs) than in type 2 diabetes (no miRNA changes). This suggests that the physiological response in the T2D subjects in this early phase had been blunted and delayed despite the relatively short duration of disease, which is a feature reported to correspond to a long duration of disease for other physiological parameters [ 25 ].…”

“…As described previously [ 23 ], “Total RNA was extracted using the MagMAX™ mir Vana™ RNA Isolation Kit (Thermo Fischer Scientific, Waltham, MA, USA) on an automated KingFisher instrument (Thermo Fischer Scientific, Waltham, MA, USA). The MagMAX™ mir Vana™ RNA isolation kit is a magnetic bead-based kit that uses MagMAX magnetic-bead technology for efficient isolation of total RNA from plasma samples from 100 µL of human plasma”.…”

“…As detailed previously [ 23 ], “There was no information regarding changes in miRNA on which to base a sample size calculation. For such pilot studies, Birkett and Day [ 49 ] suggest a minimum of 20 degrees of freedom to estimate variance from which a larger trial could be powered, hence 23 subjects in each group were recruited.…”

“…For example, within diabetes, miR-126 participates in vascular repair [ 18 ], miR-146 is involved with oxidative stress and proinflammatory factors [ 19 ], the knockdown of miR-185 may provoke oxidative stress [ 20 , 21 ], and miR-503 inhibition restores endothelial dysfunction [ 22 ]. In a recent publication from our group relating to the same clinical study performed on the same patients but reporting only changes in miRNA from baseline to hypoglycemia, no changes in miRNA that passed the false detection rate were seen at the point of hypoglycemia, though the top altered miRNAs were associated with neurological disease and organismal abnormalities [ 23 ]. In a small hypoglycemia study restricted to 14 patients with type 2 diabetes, a selective miRNA analysis (chosen based upon platelet expression, namely, hsa-miR-129-2-3p, hsa-miR-223-5p, hsa-miR-223-3p, hsa-miR-16-5p, hsa.miR-15b-5p, hsa-miR-15b-3p, hsa-miR-15a-5p, hsa-miR-126-5p, hsa-miR-126-3p, and hsa-miR-106a.5p) was performed; the results at 1- and 7-days following the hypoglycemic event revealed increased levels of miR-126, miR-223, miR-16-5p, miR-15a, miR-15b, and miR-106a-5p [ 24 ].…”