https://orcid.org/0000-0002-1155-5816
Abstract
Hypoglycemia is a major barrier to optimal glycemic control in insulin-treated diabetes. Recent guidelines from the American Diabetes Association have subcategorized “non-severe” hypoglycemia into level 1 (<3.9 mmol/L) and 2 (<3 mmol/L) hypoglycemia. Gastric emptying of carbohydrate is a major determinant of postprandial glycemia but its role in hypoglycemia counter-regulation remains underappreciated. “Marked” hypoglycemia (~2.6 mmol/L) accelerates gastric emptying and increases carbohydrate absorption in health and type 1 diabetes, but the impact of “mild” hypoglycemia (3.0-3.9 mmol/L) is unknown.
To determine the effects of 2 levels of hypoglycemia, 2.6 mmol/L (“marked”) and 3.6 mmol/L (“mild”), on gastric emptying in health.
Fourteen healthy male participants (mean age: 32.9 ± 8.3 years; body mass index: 24.5 ± 3.4 kg/m2) from the general community underwent measurement of gastric emptying of a radiolabeled solid meal (100 g beef) by scintigraphy over 120 minutes on 3 separate occasions, while blood glucose was maintained at either ~2.6 mmol/L, ~3.6 mmol/L, or ~6 mmol/L in random order from 15 minutes before until 60 minutes after meal ingestion using glucose-insulin clamp. Blood glucose was then maintained at 6 mmol/L from 60 to 120 minutes on all days.
Gastric emptying was accelerated during both mild (P = 0.011) and marked (P = 0.001) hypoglycemia when compared to euglycemia, and was more rapid during marked compared with mild hypoglycemia (P = 0.008). Hypoglycemia-induced gastric emptying acceleration during mild (r = 0.57, P = 0.030) and marked (r = 0.76, P = 0.0014) hypoglycemia was related to gastric emptying during euglycemia.
In health, acceleration of gastric emptying by insulin-induced hypoglycemia is dependent on the degree of hypoglycemia and baseline rate of emptying.
The rate of gastric emptying is recognized to be both a determinant of, as well as determined by, the blood glucose concentration. In relation to the latter, acute changes in blood glucose have a major effect on gastric emptying in both health and diabetes (1, 2). A number of studies have evaluated the effects of acute hyperglycemia and demonstrated a “dose-dependent” effect to slow gastric emptying (2). Acute hyperglycemia may also impact oral drug absorption, presumably by its effect on gastric emptying; for example, the rate of absorption of the sulfonylurea, glipizide, is inversely related to the blood glucose concentration (3, 4). Moreover, acute hyperglycemia may attenuate the effect of drugs which accelerate gastric emptying, such as erythromycin (5), and potentiate the effect of drugs which slow emptying, such as intravenous glucagon-like peptide-1 (GLP-1) (6). In contrast to the relatively well-characterized effects of acute hyperglycemia, there is little information about the effects of insulin-induced hypoglycemia on gastric emptying in humans, although this is likely to be of major clinical relevance (7). Recovery from hypoglycemia is critical for survival, and a hierarchy of defense mechanisms is elicited in response to low blood glucose levels (8). In health, these include suppression of endogenous insulin secretion and the release of immediate-acting (glucagon and epinephrine) and delayed-acting (growth hormone and cortisol) counter-regulatory hormones (9), accompanied by subjective awareness of early hypoglycemia and a drive to eat (9). It is not widely appreciated that hypoglycemia accelerates gastric emptying. An early study in the 1920s in dogs reported increased gastric contractility after insulin administration (10). Studies in rats in the 1980s and 1990s demonstrated an acceleration in gastric emptying during insulin-induced hypoglycemia (11, 12). In humans, we (1) and others (13, 14) have shown that gastric emptying is accelerated markedly in response to acute hypoglycemia in both health and type 1 diabetes (T1D), and that gastric emptying of an oral glucose load is inversely related to the plasma glucose concentration (15). We have also recently reported that this gastrokinetic response to hypoglycemia is not attenuated by an antecedent episode of hypoglycemia (16) but is diminished in health by intravenous infusion of GLP-1 (17). These studies have all examined the effect of relatively low plasma glucose concentrations (~2.2-2.8 mmol/L) on gastric emptying. Recently, the International Hypoglycemia Study group recommended subclassification of non-severe hypoglycemia into level 1 (<3.9 mmol/L) and level 2 (<3.0 mmol/L), which is reflected in the American Diabetes Association’s annual Standards of Care publication (18). Mild hypoglycemia (ie, blood glucose between 3.1 and 3.9 mmol/L) is very common in clinical practice—on average, individuals with T1D have 1 to 2 episodes of mild/non-severe hypoglycemia each week (19, 20), which affects daily activities such as driving, employment, exercise, and travel (21, 22). However, the gastric emptying response during mild hypoglycemia has not been assessed in either health or diabetes. Accordingly, we have evaluated the effects of “mild” (3.6 mmol/L) when compared with “marked” hypoglycemia (2.6 mmol/L) and “euglycemia” (6 mmol/L) on gastric emptying in healthy humans, to establish proof-of-principle.
Research Design and Methods
Subjects
Participants were recruited from existing databases and via advertisements placed at the Royal Adelaide Hospital and city university campuses. Fifteen healthy males (mean age 32.9 ± 8.3 years, mean body mass index of 24.5 ± 3.4 kg/m2 and mean glycated hemoglobin A1c [HbA1c] of 5.26% ± 0.01%) who met inclusion criteria following screening were included. Females were excluded to avoid the potential confounding effect of the menstrual cycle on gastric emptying. Participants with a history of diabetes (HbA1c ≥6.5 %), gastrointestinal disease (including gastroparesis, peptic ulcer disease, significant upper or lower gastrointestinal symptoms, or previous gastrointestinal surgery other than uncomplicated appendicectomy or cholecystectomy), a history of migraine or panic attacks, other significant illness (including epilepsy, cardiovascular, or respiratory disease; impaired renal or hepatic function), evidence of iron deficiency, and those with requirement for medication known to influence gastrointestinal function, were excluded. Written, informed consent was obtained from each participant before enrollment. The research protocol was approved by the Human Research Ethics Committee (HREC) of the Central Adelaide Local Health Network (CALHN).
Protocol
Each participant was studied on 3 occasions, during euglycemia, and mild and marked hypoglycemia, in single-blinded randomized order, with individual studies separated by at least 3 days. On each study day, the participant attended the Royal Adelaide Hospital at 8:00 a.m. after an overnight fast (12 hours for solids and liquids). Intravenous cannulae were inserted into the antecubital/cephalic vein of each arm, one for infusion of insulin and 25% dextrose and the other for blood sampling. A heat pad was applied to the blood-sampling arm to achieve arterialization of venous blood samples. The insulin infusion was prepared by diluting 0.5 mL insulin (Actrapid, Novo Nordisk Pharmaceuticals, Auckland, New Zealand) (100 units/mL) with 49.5 mL 0.9% saline to achieve a concentration of 1 unit/mL. After baseline blood samples were obtained at t = −30 minutes, 25% glucose and insulin infusions were commenced. Insulin was infused intravenously with the initial rate of 125 mU/m2/min and then titrated to a maintenance rate of 40 mU/m2/min for 10 minutes (16, 17) with a concurrent continuous intravenous infusion of 25% glucose between 10 and 500 mL/h (16, 23) in order to maintain blood glucose concentrations at either 6 mmol/L [108 mg/dL] (euglycemia), 3.6 mmol/L [64.8 mg/dL] (mild hypoglycemia), or 2.6 mmol/L [46.8 mg/dL] (marked hypoglycemia) from t = −15 minutes to t = 60 minutes, and then at 6 mmol/L from t = 60 to 240 minutes on all 3 days. The test meal was ingested from t = −5 to 0 minutes, and its emptying monitored from t = 0 to 120 minutes. Symptoms related to hunger (measured on a linear scale from 1 “no hunger at all” to 7 “very severe hunger”) and hypoglycemia were recorded using validated questionnaires (visual analog scale) every 15 minutes during the period from t = −30 minutes to t = 120 minutes (24). Venous blood was sampled at 5-minute intervals until t = 60 minutes and then every 15 minutes until t = 240 minutes and analyzed immediately for blood glucose concentrations (YSI Life Sciences, Yellow Springs, Ohio, USA). Samples taken every 15 minutes were initially stored on ice and plasma stored at −80 °C for subsequent measurement of glucagon concentrations by radioimmunoassay (GL-32K, Millipore, Billerica, MA, USA (25). At t = 240 minutes, each participant was provided with lunch prior to leaving the laboratory (Fig. 1).
Schematic representation of the study protocol. Blood glucose concentrations were stabilized using a glucose/insulin clamp at either 6.0 mmol/L (euglycemia), 3.6 mmol/L (mild hypoglycemia), or 2.6 mmol/L (marked hypoglycemia) from t = −15 to t = 60 minutes, and then at 6.0 mmol/L from t = 60 to t = 240 minutes. Subjects ingested a radiolabeled meal from t = −5 to t = 0 minutes, and gastric emptying was measured scintigraphically from t = 0 to t = 120 minutes.
Measurement of gastric emptying
The test meal consisted of a 100 g cooked lean minced beef patty, radiolabeled with 20 MBq technetium Tc 99m (99mTc)-sulfur colloid (Pharmalucence Inc., Bedford, MA, USA) and was consumed with 150 mL water (26). Subjects lay semi-recumbent (upper body at an angle of 45°), with the gamma camera (Digirad, Poway, CA, USA) placed over their abdomen to obtain a left anterior oblique image to allow correction for gamma-ray attenuation (1). Scintigraphic data were acquired in 1-minute frames for the first hour, followed by 3-minute frames for the subsequent hour, and were corrected for radionuclide decay and participant movement (26). Data were analyzed by 2 experienced investigators who were blinded to the study conditions. Regions-of-interest were drawn around the total stomach and gastric emptying curves, expressed as intragastric retention over time, were derived. The intragastric retention at 100 minutes (T100) and area under the curve (AUC0-120min) were calculated, and the lag phase (time until activity was seen first seen in the proximal small intestine) was derived. Delayed gastric emptying was defined as a T100 > 66% based on an established normal range (26).
Statistical analysis
We calculated that 15 participants completing all 3 study days would provide 80% power at P < 0.05 to detect a 10% difference in gastric retention at 100 minutes (T100) between glycemic conditions, using our previous data relating to the effects of insulin-induced hypoglycemia on gastric emptying in health (1, 16). Coefficients of variation (CV) for blood glucose at steady state (t = −15 to +60 minutes) were determined for euglycemia and hypoglycemia clamps and expressed as a percentage. Data are presented as mean values ± standard error of the mean (SEM) unless otherwise stated and evaluated using 2-way repeated-measures analysis of variance (ANOVA), with Bonferroni-Holm–adjusted post hoc tests for multiple comparisons. The relationship between the change in gastric retention during hypoglycemia and gastric retention during euglycemia was evaluated using linear regression analysis. Statistical analyses were performed using SPSS 26.0 software (SPSS, Chicago, IL, USA).
Results
All participants tolerated the studies well and there were no serious adverse events. One participant was determined during analysis not to have fasted before all study days and accordingly, was excluded, leaving 14 participants in the final analysis. Mean fasting blood glucose concentrations were comparable on all study days (4.79 ± 0.29 mmol/L for euglycemia, 4.64 ± 0.17 mmol/L for mild and 4.59 ± 0.17 mmol/L for marked hypoglycemia; P = NS) and blood glucose concentrations closely approximated the desired levels during clamps, that is, between t = −15 and t = 60 minutes on each study day (marked hypoglycemia: 2.8 ± 0.1 mmol/L; mild hypoglycemia: 3.6 ± 0.0 mmol/L; euglycemia: 5.8 ± 0.1 mmol/L) (Fig. 2A). The CV at steady state during the marked hypoglycemia, mild hypoglycemia, and euglycemia clamps were 10%, 7%, and 7%, respectively. Participants reported mild characteristic symptoms during hypoglycemia, including hunger, palpitations, and sweating. Baseline hunger scores were similar on the 3 study days (marked hypoglycemia: 3 ± 0.4; mild hypoglycemia: 4 ± 0.4; euglycemia: 3 ± 0.4, P = NS). Between t = 0 and t = 60 minutes, the cumulative hunger score was greater during marked, compared with mild, hypoglycemia (marked hypoglycemia: 18 ± 2; mild hypoglycemia: 15 ± 2, P < 0.0001) and euglycemia (marked hypoglycemia: 18 ± 2; euglycemia: 12 ± 1, P < 0.0001).
A, Blood glucose concentrations on the 3 study days in 14 healthy male subjects (mean ± SEM). These were stabilized using a glucose/insulin clamp to a target of either 6.0 mmol/L (euglycemia, filled triangles), 3.6 mmol/L (mild hypoglycemia, filled squares), or 2.6 mmol/L (marked hypoglycemia, filled circles) from t = −15 to t = 60 minutes, and then a target of 6.0 mmol/L from t = 60 to t = 240 minutes. Subjects ingested a radiolabeled meal between t = −5 to t = 0 minutes. B, Gastric retention (AUC0-120min) of 100 g minced beef labeled with 20 MBq technetium Tc 99m (99mTc)-sulfur colloid during euglycemia (filled triangles), mild hypoglycemia (filled squares), and marked hypoglycemia (filled circles) in 14 healthy male subjects. Data are mean ± SEM. Abbreviations: Eu, euglycemia; Mild, mild hypoglycemia; Marked, marked hypoglycemia.
Gastric emptying
No participant had abnormally delayed or rapid gastric emptying during euglycemia and in all cases gastric emptying approximated an overall linear pattern after an initial lag phase (Fig. 2B). Both the lag phase (28 ± 7 minutes during euglycemia vs 22 ± 5 minutes during mild vs 14 ± 3 minutes during marked hypoglycemia; P = 0.003) and T100 (48% ± 8% for euglycemia, 32% ± 6% for mild, and 16% ± 5% for marked hypoglycemia; P < 0.001) were reduced during hypoglycemia. Pairwise comparisons demonstrated a shorter lag phase during marked hypoglycemia vs euglycemia (P = 0.006) and mild vs marked hypoglycemia (P = 0.031), with a nonsignificant difference between mild hypoglycemia vs euglycemia (P = 0.062). The total AUC0-120 for gastric emptying was less during both mild (P = 0.011) and marked (P = 0.001) hypoglycemia when compared with euglycemia, and it was lower during marked when compared with mild hypoglycemia (P = 0.008) (Figs. 2B and 3).
Scintigraphic images demonstrating gastric emptying of the radiolabeled meal (left to right) from t = 0 to t = 120 minutes during euglycemia (6.0 mmol/L), mild hypoglycemia (3.6 mmol/L) and marked hypoglycemia (2.6 mmol/L) in a sample participant. At t = 0 minutes, the radiolabeled food is entirely contained within the stomach (outlined by a tracing) on all clamp days. At t = 120 minutes, the maximum gastric retention was observed during euglycemia and minimal during marked hypoglycemia.
Relationship between gastric emptying at euglycemia and the magnitude of acceleration during hypoglycemia
The changes in the intragastric retention at T100 during both mild (r = 0.57, P = 0.030; Fig. 4A) and marked (r = 0.76, P = 0.0014; Fig. 4B) hypoglycemia were related directly to gastric retention at 100 minutes during euglycemia (Fig. 4).
Relationship between the change in gastric retention (%) during A, mild (3.6mmol/L); and B, marked (2.6mmol/L) hypoglycemia, and gastric retention at 100min during euglycemia (6.0mmol/L) in 14 healthy male participants.
Plasma glucagon
Baseline glucagon concentrations were comparable on the 3 days (euglycemia: 37.49 ± 3.54; mild hypoglycemia: 40.74 ± 3.29; and marked hypoglycemia: 40.52 ± 3.0; P = NS). Plasma glucagon (AUC−30-120min) decreased during euglycemia and increased in response to both mild (P = 0.004) and marked (P < 0.001) hypoglycemia (Fig. 5), with a greater increase during the latter (P < 0.001).
Plasma glucagon concentrations during euglycemia (filled triangles), mild hypoglycemia (filled squares) and marked hypoglycemia (filled circles) in 14 healthy male participants. Data are mean ± SEM. Abbreviations: Eu, euglycemia; Mild, mild hypoglycemia; Marked, marked hypoglycemia.
Discussion
Our study evaluated the effects of marked (2.6 mmol/L) and mild (3.6 mmol/L) hypoglycemia on gastric emptying in healthy males. The key observations are that: (i) both mild and marked hypoglycemia accelerate gastric emptying substantially; and (ii) the magnitude of this acceleration is greater during marked hypoglycemia and dependent on the rate of emptying during euglycemia.
Hypoglycemia is the most feared complication of insulin and sulfonylurea therapy and is widely regarded as a major barrier to achieving optimal glycemic control. While mild hypoglycemia is common, it can be difficult to quantify accurately and may often be underreported due to poor recall (27). Nevertheless, recognition of mild hypoglycemia is important, as repeated episodes predispose to impaired awareness and severe hypoglycemia in T1D (28, 29). While a sequential counter-regulatory response to hypoglycemia, characterized by suppression of endogenous insulin release, stimulation of glucagon and catecholamines, and a subjective desire to eat, is well established, the “gastric” response, ie, acceleration in gastric emptying, has been less well appreciated. This is surprising given that this phenomenon was first documented as early as 1924 (10), although in the last 2 decades it has been demonstrated more precisely with the use of scintigraphy to quantify gastric emptying and glucose-insulin clamps, by our group and others, in both health and T1D (1, 13, 14). These studies, however, have all evaluated the effects of marked hypoglycemia, in the range 2.2 to 2.8 mmol/L. We have now demonstrated that a clinically meaningful (~20%) acceleration in gastric emptying of solids occurs during mild hypoglycemia (3.6 mmol/L) in health, albeit less than the acceleration observed during marked hypoglycemia (2.6 mmol/L). The magnitude of the latter was consistent with the reports of Russo et al and Schvarcz et al (1, 13, 14). While the mechanisms underlying the acceleration of gastric emptying during an acute hypoglycemic event are uncertain, the interplay between gastric inhibitory and excitatory vagal circuits is likely to be important (30).
There is a wide inter-individual variation in rate of gastric emptying in health, ie, between 1 and 3 kcal/min (31); this is even wider in diabetes as a substantial proportion have abnormally delayed (32), and a minority, accelerated (33), gastric emptying. We demonstrated that the magnitude of acceleration by both “mild” and “marked” hypoglycemia was inversely related to the rate of emptying during euglycemia, consistent with our previous study in T1D (1), so that when gastric emptying was relatively slower, albeit within the normal range, the acceleration was greater. This suggests that the “gastric” counter-regulatory response may be intricately linked to the overall counter-regulation during hypoglycemia. It would, therefore, be of interest to evaluate the gastric emptying response in patients with diabetes, particularly those with gastroparesis, although we found that T1D patients with delayed emptying (ie, gastroparesis) still exhibited acceleration during marked hypoglycemia (1).
We do not know whether the “gastric” counter-regulatory response occurs concomitantly with the better-known hormonal counter-regulation. The glycemic thresholds for hormonal counter-regulatory responses to hypoglycemia in health (eg, plasma glucagon, catecholamines, growth hormone, and cortisol) have been well-characterized (9). As anticipated, plasma glucagon was stimulated by marked and, to a lesser degree, mild hypoglycemia.
The impact of multiple hypoglycemic episodes on the gastrokinetic response cannot, of course, be ascertained from this study. Our previous study, however, indicates that antecedent hypoglycemia (three 45-minute periods of a blood glucose of 2.8 mmol/L in a 12-hour period), which affects hormonal counter-regulatory responses, does not attenuate the acceleration of gastric emptying by hypoglycemia in health (16).
Strengths of our study are the use of the “gold-standard” technique for measurement of gastric emptying (scintigraphy), and that blood glucose concentrations were maintained in the desired range using a glucose/insulin clamp. However, in interpreting our observations, the following limitations should also be considered: (i) The study employed healthy participants to establish “proof-of-principle”; hence, a similar study in people with T1D or T2D is indicated; (ii) we did not explore the mechanism(s) by which hypoglycemia accelerates gastric emptying; (iii) hypoglycemia was maintained for only 60 minutes postprandially and the effects of more sustained hypoglycemia on gastric emptying are not known; (iv) we studied only healthy men to avoid the potential confounding impact of phases of menstrual cycle on gastric emptying in women; however, there is no reason to believe that there would be a gender disparity.
In interpreting the results of this study, it should be recognized that only healthy participants were recruited. The next step would be to evaluate the response in people with T1D and insulin-treated type 2 diabetes (T2D). Several important questions remain unanswered: (i) is a similar “dose-dependent” response evident in diabetes? (ii) what is the impact of factors including the duration of diabetes, glycemic control, presence of autonomic dysfunction or gastroparesis on the response? (iii) is the acceleration of gastric emptying by hypoglycemia influenced by the rate of decline in glucose?
It would also be of interest to know whether the gastric response is intact or modified in individuals in whom the awareness of hypoglycemia is impaired. While it has been clearly established that autonomic counter-regulation is abnormal in people with T1D and impaired awareness of hypoglycemia, there is no information about gastric emptying in this group, including the response to hypoglycemia. It might be expected that the delayed and diminished neuroendocrine response to hypoglycemia characteristic of impaired awareness of hypoglycemia will extend to an impaired gastric emptying response. If so, this would be intuitively likely to delay recovery driven by ingested glucose, thereby prolonging exposure to hypoglycemia and increasing risks of both a severe episode and of impaired responses to a subsequent event. Similarly, the gastric emptying response during acute hypoglycemia in T2D has neither been quantified nor compared to T1D. If differences in the glycemic threshold for eliciting gastric responses between T1D and T2D exist, they might be of relevance to the observed lower prevalence of hypoglycemia, including severe hypoglycemia, in the latter (34). Finally, commonly prescribed glucose-lowering medications which slow gastric emptying substantially, such as “short-acting” GLP-1 agonists and pramlintide, are available for treatment in combination with basal insulin. The potential impact of the combination of agents with opposing effects on gastric emptying should clearly be evaluated to identify those patients in whom such therapy might be best avoided (for example, those with gastroparesis).
In conclusion, both “mild” and “marked” hypoglycemia accelerate gastric emptying substantially in health, the response is dependent on the baseline rate of emptying, and the effect of “marked” hypoglycemia is greater.
Abbreviations
- AUC
area under the curve
- CV
coefficient of variation
- GLP-1
glucagon-like peptide-1
- HbA1c
glycated hemoglobin A1c
- T1D
type 1 diabetes
- T2D
type 2 diabetes
Acknowledgments
We thank the staff of the Intensive Care Unit Research Group (L.Weinel, A. Emmerich, R. Louis), Centre for Research Excellence in Translating Nutritional Science to Good Health, Faculty of Health and Medical Sciences, University of Adelaide and the Department of Nuclear Medicine, PET and Bone Densitometry, of the Royal Adelaide Hospital for facilitating the studies.
Financial Support: This study was supported by a Royal Adelaide Hospital (Clinical Project Grant Ref: 8556). T.A.M. is supported by a RTP University of Adelaide Postgraduate Scholarship, S.H. by The Hospital Research Foundation, L.S.C. and C.S.M. by an NHMRC Early Career Fellowship and K.L.J. by the University of Adelaide William T Southcott Research Fellowship. The gamma camera was purchased by funding support from The Hospital Research Foundation, the University of Adelaide, and The Ian Potter Foundation.
Author Contributions: T.A.M. performed the experiments, analyzed data, and wrote the manuscript. J.G. performed the experiments and collected gastric emptying data. S.H. analyzed gastric emptying data. M.C., L.C., and C.K.R. reviewed the manuscript and provided intellectual inputs. J.S. contributed to study design. C.H.M. designed the gastric emptying program and reviewed the manuscript. M.H. contributed to study design, data analysis, and wrote the manuscript. K.L.J. contributed to study design, gastric emptying analysis, and reviewed the manuscript. C.S.M. contributed to study design, performed the experiments, analyzed data, and wrote the manuscript. C.S.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.
Additional Information
Disclosure Summary: The authors have no relevant conflicts of interest to disclose.
Data Availability
Some or all data generated or analyzed during this study are included in this published article or in the data repositories listed in References.
References
