High-Intensity Exercise as a Dishabituating Stimulus Restores Counterregulatory Responses in Recurrently Hypoglycemic Rodents
Abstract
Hypoglycemia is a major adverse effect of insulin therapy for people with type 1 diabetes (T1D). Profound defects in the normal counterregulatory response to hypoglycemia explain the frequency of hypoglycemia occurrence in T1D. Defective counterregulation results to a large extent from prior exposure to hypoglycemia per se, leading to a condition called impaired awareness of hypoglycemia (IAH), the cause of which is unknown. In the current study, we investigate the hypothesis that IAH develops through a special type of adaptive memory referred to as habituation. To test this hypothesis, we used a novel intense stimulus (high-intensity exercise) to demonstrate two classic features of a habituated response, namely dishabituation and response recovery. We demonstrate that after recurrent hypoglycemia the introduction of a novel dishabituating stimulus (a single burst of high-intensity exercise) in male Sprague-Dawley rats restores the defective hypoglycemia counterregulatory response. In addition, the rats showed an enhanced response to the novel stimulus (response recovery). We make the further observation using proteomic analysis of hypothalamic extracts that high-intensity exercise in recurrently hypoglycemic rats increases levels of a number of proteins linked with brain-derived neurotrophic factor signaling. These findings may lead to novel therapeutic approaches for individuals with T1D and IAH.
Introduction
Impaired awareness of hypoglycemia (IAH), defined as “a diminished ability to perceive the onset of acute hypoglycemia,” affects 20–25% of all people with type 1 diabetes (T1D) and increases the risk of severe hypoglycemia sixfold (1). IAH develops primarily in response to repeated episodes of hypoglycemia (2). Individuals with or without T1D, who experience one or more episodes of hypoglycemia have impaired hormonal and symptomatic counterregulatory responses (CRRs) during a further episode of hypoglycemia, with the extent of suppression dependent on the depth, duration, and frequency of antecedent hypoglycemia (2). Conversely, the CRR can be restored if hypoglycemia is avoided (3). The mechanisms underpinning the development of IAH remain unknown but are likely a result of changes in specialized glucose-sensing regions of the body such as those found in distinct brain regions like the ventromedial hypothalamus (VMH) (4).
Habituation is a type of adaptive memory that occurs in many organisms in response to a repeated, often stressful stimulus (5). Habituation is defined as a “reduction of the psychological, behavioral or physiological response to a stimulus as a result of repeated or prolonged exposure” (5,6). Aspects of the physiological responses to repeated hypoglycemia, such as the progressive diminishment of CRR to hypoglycemia after repeated exposure, are consistent with the principal features of a habituated response (5–7), suggesting that this may provide an explanation for IAH. In this study, we directly address this hypothesis in a rodent model of recurrent hypoglycemia (RH) by demonstrating one of the defining features of a habituated process, namely that it is possible to rapidly restore the habituated response by dishabituation, the introduction of a novel strong stimulus (5,6).
Research Design and Methods
Animals
Male Sprague-Dawley rats (250–300 g; Harlan UK) maintained on a 12-h day/night cycle and provided with food and drinking water ad libitum. Experimental procedures were approved by the University of Dundee Ethical Review Process and performed in accordance with U.K. Home Office regulations.
Experiment 1: Dishabituation With High-Intensity Exercise After RH
To test our hypothesis in rodents, high-intensity (HI) exercise was used as a dishabituating stimulus with RH as the habituated response. After 2 weeks of daily handling, rats underwent insulin-induced (0.75–1 units/kg i.p., Novorapid; NovoNordisk Ltd) RH (N = 24) or intraperitoneal volume-matched saline injections (Control; N = 16) three times weekly for 4 weeks (Fig. 1A and B). Animals were familiarized (5 m/min for 15 min daily) with the treadmill during the last 2 weeks, with exercise training in the morning and intraperitoneal injection in the late afternoon. Subsequently, vascular catheters were inserted under general anesthesia as previously described (8), and animals recovered over 5 days. On day 5 postsurgery, a further insulin or control injection was administered, and then on day 6, after feeding freely overnight, the animals were allocated to one of the following groups: 1) no exercise, 2) low-intensity (LI) exercise (15-min “walking” pace of 5 m/min with a 10% incline), or 3) HI exercise (5 min at 5 m/min followed by an incremental increase in speed at 2 m/min intervals to 5 min of “running” at 15 m/min). Where required, exercise was encouraged using a bottlebrush to the tail. Animals were then returned to their home cages and fed ad libitum. The following day, all animals underwent a hyperinsulinemic-hypoglycemic (2.8 mmol/L) clamp (8), with sampling for CRR hormones under euglycemic and hypoglycemic conditions (Fig. 1C). Animals were euthanized on completion of the study, and brains were removed for later analysis.
Study design. A: Study design for experiments 1 and 2. Experiment 2 followed exactly the same protocol as experiment 1, with the exception that animals underwent blood sampling on day 6 after HI or LI exercise and did not proceed to the hyperinsulinemic hypoglycemic (Hypo) clamp study on day 7 postsurgery. B: Mean (SEM) weekly glucose level at baseline and after insulin injection during the RH protocol. After 3 weeks, the dose of insulin was reduced from 1 to 0.75 units/kg to prevent the development of severe hypoglycemia where animals might require rescue. At the end of each hypoglycemia period, the rats were given free access to food. The glucose levels of sham-injected control rats did not differ significantly from basal levels in the RH group (data not shown). C: Hyperinsulinemic clamp protocol. Fasted rats were stabilized for 90 min after catheter opening before insulin (20 mU ⋅ kg−1 ⋅ min−1) was infused. Once animals reached their target glucose level, a variable 10% dextrose infusion was started and adjusted based on bench-side glucose readings every 5–10 min. Glucagon (G), epinephrine (E), and corticosterone (C) were sampled at baseline and during the hypoglycemia clamp.
Experiment 2: Response Recovery to HI Exercise After RH
To examine for response recovery, an enhanced response to the novel stimulus and to gain insight into the potential mechanism, a further two groups of male Sprague-Dawley rats (n = 12–18 per group) were studied, and the impact of each exercise modality on counterregulatory hormones, cytokines, brain-derived neurotrophic factor (BDNF), and lactate was measured. The induction of hypoglycemia, familiarization with the treadmill, surgical procedures, and exercise were identical to the protocol described for experiment 1. However, immediately after each of the three exercise protocols blood samples were obtained from the carotid artery (N = 6–8 per group). The animals were then euthanized.
Hormone, Cytokine, and Metabolite Analysis
Hormones, cytokines, and metabolites were assessed as follows: glucose (Analox GM9 glucose analyzer; Analox Instruments Ltd.); insulin and glucagon (multiplex ELISA, Bio-Plex; Bio-Rad); epinephrine and corticosterone (ELISA; ALPCO and Demeditic Diagnostics, respectively); BDNF (DuoSet ELISA; R&D Systems); lactate levels (Sigma-Aldrich, Irvine, U.K.); and cytokines [V-PLEX Proinflammatory Panel 2 (rat) ELISA; Meso Scale Diagnostics].
Proteomic Analysis
Proteins extracted from each VMH sample were reduced, alkylated, and subjected to trypsin digestion. The tryptic peptides from each animal were labeled with iTRAQ (AB Sciex) labeling according to the manual of the manufacturer. All labeled samples were then pooled together. One-half of the pooled sample was subjected to high-pH off-line C18-based fractionation (eight fractions), and the other half was subjected to strong cation exchanger fractionation (five fractions). Liquid chromatography tandem mass spectrometry was carried out as previously described (9). iTRAQ quantification was carried out using Peaks software (Bioinformatics Solutions Inc.) with the International Protein Index rat database (2012–09–27). All samples were randomized, and the analyst was blinded to sample grouping during the processing.
Statistical Analysis
All results are expressed as the mean ± SEM. Statistical analyses were performed using SPSS (version 21; SPSS). Data were analyzed by one-way ANOVA or repeated-measures ANOVA followed by post hoc testing (Bonferroni test) to localize significant effects. Statistical significance was set at P < 0.05.
Results
Dishabituation With HI Exercise After RH
CRRs to hypoglycemia after LI exercise or no exercise in both control and RH animals did not differ significantly (Supplementary Data) so were combined into the LI exercise grouping. Plasma glucose profiles were matched during hypoglycemia in all groups (Fig. 2A) (F = 0.4, df [2,39], P = NS). In contrast, mean glucose infusion rate (GIR) (F = 9.2, df [2,39], P < 0.05), epinephrine level (F = 34.5, df [2,39], P < 0.05), and glucagon level (F = 10.4, df [2,39], P < 0.05) differed significantly between groups. Post hoc analysis revealed that CRRs were significantly suppressed in RH and LI exercise rats compared with Control and LI exercise rats during hypoglycemia (Fig. 2B–E). In contrast, the mean (SEM) values for GIR (2.9 [0.6] vs. 4.4 [1.0] mg ⋅ kg−1 ⋅ min−1, Control and LI vs. RH and HI exercise rats; P = NS), epinephrine (7.9 [0.4] vs. 6.8 [0.6] ng ⋅ mL−1, respectively; P = NS), and glucagon (245 [18] vs. 219 [31] ng ⋅ L−1, respectively; P = NS) CRRs were not different between Control and LI exercise and RH and HI exercise groups (Fig. 2B–E). Corticosterone responses to the hypoglycemic challenge were not affected by any intervention. These findings indicate that a single episode of HI exercise can restore the CRR in rats exposed to RH over 4 weeks.
Acute exposure to HI exercise restores the defective CRR to RH. A: Plasma glucose level during hyperinsulinemic hypoglycemic clamp study. GIRs (B) and mean GIR (C) during stable hypoglycemia (90–150 min). Plasma epinephrine (D), glucagon (E), and corticosterone (F) responses during hypoglycemia. Control and LI exercise animals, white circles/bars; RH and LI exercise animals, black squares/bars; RH and HI exercise animals, gray diamonds/bars. Values are the mean ± SEM. **P < 0.05.
Response Recovery to HI Exercise After RH
HI exercise resulted in significantly higher levels of epinephrine, glucagon, and lactate in both the Control and HI exercise and RH and HI exercise groups compared with their respective LI exercise studies (Figs. 3A–C). Moreover, consistent with response recovery after habituation, the mean (SEM) epinephrine (2.2 [0.3] vs. 1.4 [0.1] ng ⋅ mL−1; RH and HI exercise vs. Control and HI exercise, P < 0.05), glucagon (46 [2] vs. 38 [2] pg ⋅ mL−1; P < 0.05), and lactate (88 [3] vs. 77 [2] ng ⋅ μL−1, P < 0.05] responses to HI exercise were all significantly greater after RH (Fig. 3A–C). Interestingly, the profile of cytokine release differed with the expected increase in cytokine release with HI exercise in Control groups (Fig. 3E–I) but had a more varied response after RH when the release of broadly proinflammatory cytokines (interleukin [IL]-6, IL-1β, and tumor necrosis factor-α) after HI exercise was significantly suppressed compared with Control and HI exercise, whereas the release of anti-inflammatory cytokines (IL-4 and IL-13) was generally increased; the latter increase also was evident after low-stress LI exercise (Figs. 3H and I).
Hormone, metabolic, and cytokine responses evoked by LI or HI exercise in RH or control rats. Mean ± SEM epinephrine (A), glucagon (B), lactate (C), BDNF (D), IL-6 (E), IL-1β (F), tumor necrosis factor-α (G), IL-4 (H), and IL-13 (I). Control and LI exercise studies, white bars; control and HI exercise studies, light gray bars; RH and LI exercise studies, black bars; RH and HI exercise studies, dark gray bars. δ = P < 0.05, Li vs. HI exercise; ε = P < 0.05, control and HI exercise vs. RH and HI exercise; β = P < 0.05, control and LI exercise vs. RH and LI exercise; ND, not detectable. Values are the mean ± SEM.
Hypothalamic Mechanisms of Dishabituation
Proteomic analysis of VMH samples from RH and LI exercise versus RH and HI exercise rats (Fig. 4A and B) revealed that DNAJB1 (or Hsp40) and Bassoon (Bsn) were most significantly affected by HI exercise (Fig. 3B), which was confirmed by Western blot analysis (Fig. 4C). Bsn is involved in glutamatergic signaling, as is BDNF (10), and BDNF levels in humans are significantly elevated in response to exercise (11). To determine whether BDNF may be involved in dishabituation, plasma BDNF level was measured and found to be augmented by HI exercise in RH animals (Fig. 3D). In addition, VMH expression of BDNF, TrkB, and pCREB were all significantly increased after RH and HI exercise (Supplementary Data).
Potential mechanisms underlying dishabituation of VMH glucose sensing. A: Investigation of the effects of HI exercise to VMH proteome using an iTRAQ-based proteomic workflow. Proteins extracted from each VMH sample were reduced, alkylated, and subjected to trypsin digestion. The tryptic peptides from each animal were labeled with iTRAQ labeling reagents, where report ions 113, 115, 117, and 119 were used to label peptide sample from the RH and LI exercise groups, and 114, 116, 118, and 121 were used from the RH and HI exercise group. The samples were combined and analyzed using nano–liquid chromatography (LC) high-resolution tandem mass spectrometry (HR-MS/MS). B: Volcano plot showing the differences of the VMH proteome between the LI and HI exercise groups. Quantification data were available on 1,866 proteins where unique peptides/quantifiable reporter ions are detected. The top differentially expressed proteins with a fold change >30% and a P value <0.05 (Bonferroni-Hochberg method) are labeled in the chart. C: Western blotting of Bsn and Dnajb1 in VMH lysates from RH animals after LI or HI exercise. D: A potential model explaining the dishabituation effect. Acute hypoglycemia results in increased glutamatergic synaptic activity, which is suppressed after RH. The dishabituating stimulus then acts to restore glutamatergic synaptic activity, perhaps via BDNF/Bsn, leading to restoration of the CRR. SAX, strong anion exchange.
Discussion
This study demonstrates in an animal model of IAH that diminished CRR can be rapidly restored by a single bout of HI exercise. This finding supports the hypothesis that diminished CRR after RH results from habituation. Habituation is a form of adaptive learning where there is a decrease or cessation of responses to a stimulus after repeated presentations. Habituation is usually considered in the context of innate behaviors and a reduced response to repeated external or internal stimuli (5,6). A classic model of habituation is the gill-withdrawal reflex in Aplysia (12). When the gill-withdrawal reflex is repeatedly evoked by a tactile stimulus to the siphon, the amplitude of the response shows a marked decrement (habituation). The habituated response can then restored by presenting a strong tactile stimulus to another part of the animal (dishabituation) (12). Habituation also occurs after repeated physiological stress such as endotoxin exposure or in models of shock (13). In the context of hypoglycemia, low glucose level can be considered the internal stimulus, which leads to a reflex CRR. RH then leads via habituation to a progressive diminution of this response. This concept of hypoglycemia as an internal sensory cue stimulating a counterregulatory reflex (motor) response is consistent with our current understanding of the neural circuitry of brain glucose sensing (14).
In the current study, HI exercise was chosen as the dishabituating stimulus, but other novel stressors may prove equally effective. For instance, CRRs to hypoglycemia and cold exposure have been shown to influence each other (15). The present findings differ from previous studies where antecedent exercise produced further suppression of CRR to hypoglycemia (e.g., [16]). However, these used continuous aerobic exercise providing a moderate stimulus. Stimulus generalization (or cross-tolerance) between different moderate stressors is a feature of habituation (5,6), distinct from dishabituation, which requires a novel strong stimulus. Interestingly, prior RH did not suppress the CRR to exercise, as reported by others (17), but did lead to a change in the inflammatory response to both exercise modalities. The differing intensity of exercises used in the current study likely explains the contrasting findings. This also suggests that the impact of exercise on the CRR to hypoglycemia may vary depending on the type and duration of exercise performed.
To gain some understanding of the mechanisms by which HI exercise acutely restored the CRR to hypoglycemia, an exploratory proteomic analysis of VMH tissue was performed. A number of candidate proteins emerged such as Glycogenin 1 and adenylate cyclase 10 that play roles in glycogen turnover, which may be relevant to glucose sensing (18), whereas dipeptidyl peptidase 10 affects Kv4 channel gating (19). DNAJB1 encodes for Hsp40, which helps to protect proteins during cellular stress (20). Bsn, which was most affected by HI exercise, is a presynaptic cytomatrix protein that contributes to synaptic plasticity and learning (10). Bsn colocalizes with BDNF in glutamatergic presynapses, and increased BDNF release helps to maintain levels of neural excitation (10). Loss of VMH glutamatergic Steroidogenic Factor 1 (SF1) activity suppresses the CRR to hypoglycemia (21), while specific deletion of BDNF in VMH SF1 neurons also results in a reduced CRR to hypoglycemia (22). VMH SF1 neurons also appear to contribute to the metabolic benefits of endurance exercise (23). Taken together with the findings in the current study, it is possible to speculate that dishabituation may act via BDNF-mediated restoration of glutamatergic activity (Fig. 3D). These findings are, however, observational and need to be directly tested in further studies. In addition, we cannot exclude the possibility that dishabituation occurs in other brain regions not studied.
Overall, the current study supports the hypothesis that suppressed CRR after RH in T1D is a result of habituation. Consistent with this hypothesis, we have demonstrated that the introduction of a dishabituating stimulus, HI exercise, acutely restores the CRR to subsequent hypoglycemia. If confirmed in humans, these findings may lead to an improved understanding of IAH in T1D and the development of novel treatment strategies designed to restore hypoglycemia awareness.
Article Information
Acknowledgments. The authors thank the staff of the Biomarker and Drug Analysis Core Facility, University of Dundee, who analyzed all of the blood and hypothalamic samples, for their support.
Funding. This work was supported by an award to R.J.M. from JDRF (1-2011-574).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. A.D.M., J.R.G., and J.T.-J.H. performed the experiments and wrote the manuscript. M.L.J.A. wrote the manuscript. R.J.M. designed the studies and wrote the manuscript. R.J.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Prior Presentation. Parts of this study were presented in abstract form at the 75th Scientific Sessions of the American Diabetes Association, Boston, MA, 5–9 June 2015.
Footnotes
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1533/-/DC1.
- Received December 12, 2016.
- Accepted February 21, 2017.
- © 2017 by the American Diabetes Association.
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals.org/content/license.
References
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