Discussion
This untargeted metabolic profiling of individuals with T1D under a hyperinsulinemic-normoglycemic and hypoglycemic clamp condition showed dynamic changes in several metabolites that increased following antecedent hypoglycemia. On day 1, we found that an event of hypoglycemia decreased the BCAAs: isoleucine and leucine. A subsequent hypoglycemic event 24 hours later resulted in a decrease in additional three amino acids (phenylalanine, valine, and methionine) and an increase in two fatty acids (myristic and oleic acids). Despite this suggestion of an adaptation toward a more dynamic metabolic response to hypoglycemia on day 2, there were no statistically significant differences in the responses of the single metabolites between the 2 days. Interestingly, in this study, alanine, an important energy fuel for muscle and other tissues,23 is unaltered during hypoglycemia on both days. However, our study cannot exclude other possibilities; one suggestion could be that BCAAs are converted into alanine, maintaining plasma alanine concentration while also being a factor in the decrement of the plasma BCAA levels.24
The changes in amino acids correspond to the findings in healthy human studies, which have shown a reduction in BCAA concentration during acute hyperinsulinemic hypoglycemia.5 25 Thus, the decrease in plasma amino acids could reflect increased utilization of amino acids as substrates for ketone bodies and gluconeogenesis.26 Battezzati et al have shown that the decrease in plasma concentrations of leucine and phenylalanine was significantly smaller when comparing individuals with T1D and healthy controls,8 suggesting an impairment of this counter-regulatory response in T1D. Our study is not suited to support their findings; however, exploring this would be very interesting. Furthermore, the metabolomics platform employed in our study could only detect 12 out of the 20 standard proteinogenic amino acids, as shown in tables 2 and 3. Therefore, future studies using metabolomics should use platforms capable of identifying all amino acids. Doing so would provide a more comprehensive understanding of the alterations in amino acid levels associated with hypoglycemia.
On day 2, alongside the downregulation of five amino acids, we also observed increased levels of two fatty acids, tetradecanoic acid and oleic acid, which could suggest an increase in lipolysis. However, in our study, glycerol, another product of lipolysis, did not change in plasma levels during hypoglycemia.27 Therefore, our study cannot determine whether the elevation in Free Fatty Acids (FFAs) results from increased lipolysis or suppression of the utilization of FFAs, which in both cases would lead to an increase in plasma FFA. However, one study examining lipolysis and hypoglycemia in T1D found an elevation in lipolysis and an increase in FFAs,9 indicating that our findings could also result from increased lipolysis. Additionally, our data explored the fingerprint of the changes in FFA during hypoglycemia and showed a faster response in specific FFAs, oleic acid and tetradecanoic acid.
FFAs are important substrates during fasted conditions. FFAs are substrates for hepatic β oxidation providing energy in the form of ATP and forming ketone bodies which can be transported to other extrahepatic tissues via the circulation.28 Although β oxidation does not produce any substrates for gluconeogenesis, the ATP formed by β oxidation is used in facilitating gluconeogenesis.28 Our study found that 3-hydroxybutyric acid (3-OHB), a ketone body, was unaltered during hypoglycemia on both days (tables 2 and 3), indicating that ketogenesis does not seem to increase during hypoglycemia in our study. Another study examining healthy participants during hypoglycemia used a bolus insulin injection to induce either hypoglycemia or normoglycemia randomly and found that while FFA increased immediately after inducing hypoglycemia, 3-OHB levels from the hypoglycemia-induced participants did not increase until plasma insulin levels were down to baseline.29 In contrast, we used a continuous insulin infusion which probably severely suppressed ketogenesis.
The metabolic responses to hypoglycemia are primarily stimulated by the counter-regulatory hormonal response.30 As mentioned, the hormonal responses in this study have previously been presented by Sejling et al.14 Briefly, all counter-regulatory hormones (glucagon, epinephrine, cortisol and growth hormone) increased on both days. There were no differences between the 2 days for the hormonal responses except for growth hormone, which was lower during the second day. In T1D, the glucagon response to glucagon is severely diminished,31 while growth hormone and cortisol do not seem to have an effect during the first hour of hypoglycemia.32 33 These factors make epinephrine the pivotal counter-regulatory hormonal response during the acute phase. Previous studies investigating epinephrine infusion in healthy subjects and patients with T1D have shown a similar fall in amino acid plasma levels.34 35 Although we did not find a statistically significant difference in amino acids between the 2 study days, we did observe more amino acids downregulated on day 2 than on day 1. This alteration in metabolic response could be a result of a change in the cellular response to epinephrine since the counter-regulatory response from epinephrine did not differ between the first and subsequent episodes of hypoglycemia.14
Previous studies have shown diverging results regarding the physiological responses to epinephrine. Guy et al found that the physiological response to epinephrine is altered in T1D, finding lower glucose levels and cardiovascular responses. However, they did see a greater increase in lipolytic response compared with healthy subjects.36 Hypoglycemia also seems to impact the physiological response of epinephrine, reducing the cardiovascular, hepatic, and adipose tissue responses to epinephrine.37 Another study, however, did not see the same changes.38 All mentioned studies did not investigate the changes in different metabolites as we did; however, due to the limitation of our study, which did not specifically focus on metabolic responses to epinephrine, further studies looking at the amino acid and lipid fluxes are needed to elaborate our findings.
Although the glucagon response to hypoglycemia is deficient in T1D,31 we did observe an increase in our study.14 Previous studies investigating the effects of glucagon on protein metabolism in healthy subjects have shown that glucagon may lower leucine concentration but not plasma concentration of phenylalanine.39 Another study, however, did not find a significant alteration in the plasma concentration of BCAAs in response to glucagon.40 Thus, it is still debatable whether the changes we see in the plasma concentrations of the BCAAs are influenced by glucagon. In our study, this is further supported by the minimal glucagon responses.14
Another important hormone is insulin, which alters protein metabolism, lowers the plasma concentration of amino acids41 42 and stimulates lipogenesis, thus decreasing the plasma concentration of fatty acids.43 In our study, however, baseline normoglycemic plasma samples and plasma taken in hypoglycemia were the same during a constant hyperinsulinemic conditions, only altering the glucose infusion. Thus, the observed metabolic changes should not be a result of insulin. However, as discussed, the continuous insulin infusion in our study seems to suppress some metabolic pathways and to fully understand the metabolic consequences of the alterations, future studies aimed at investigating how these changes could facilitate energy metabolism should include experiments using bolus insulin to induce hypoglycemia, thus limiting the influence of the continuous hyperinsulinemic condition found in a clamp study.
There were no differences in the metabolic reactions of people with normal hypoglycemia awareness and unawareness on either of the days. This could be explained by the indifferent hormonal response between the two groups, reported previously by Sejling et al.14 Similar findings were also observed in a previous study investigating metabolic responses to hypoglycemia in T1D.9
In conclusion, this study shows a significant metabolic response to hypoglycemia in people with long-standing T1D and indicates that recurrent hypoglycemia alters the metabolic response, particularly within protein and lipid metabolism. Our results could indicate that while the counter-regulatory hormonal response attenuates to recurrent hypoglycemia, the metabolism may try to adapt to these conditions. However, future studies are needed to confirm and elaborate our findings and explore the consequences. The strengths of this study include a considerable sample size and a rigorous clamp protocol. However, to further understand metabolic responses to hypoglycemia in T1D, patients with newly onset diabetes duration and healthy controls could have been included.