Introduction
Until a few decades ago, diabetes was considered a disease essentially driven by pancreatic beta cell failure. While this concept holds true for type 1 diabetes, in type 2 diabetes insulin resistance develops long before beta cell failure and overt hyperglycaemia [
1‐
3] and, thus, it is now recognised as the primary defect leading to type 2 diabetes.
Bariatric surgery has demonstrated the central role played by the small intestine in insulin resistance. It is of note that type 2 diabetes remission and insulin resistance reversal [
4‐
10] following proximal gut bypass, such as in bilio-pancreatic diversion (BPD) and Roux-en-Y gastric bypass (RYGB), occur within a few days after the operation, when body weight is not yet significantly reduced [
11,
12]. Despite intensive scientific research on the relationship between gut function and glucose homeostasis [
13‐
15], this topic is still a matter of debate.
In the 1960s, it was shown that oral administration of 20 g of glucose in normal adults resulted in a much larger rise of plasma insulin compared with the administration of the same amount of glucose intravenously [
16]. This ‘incretin effect’ was thereafter shown to depend on the secretion of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), which stimulate insulin secretion [
17]. Already in the first publication [
16], it was observed that, notwithstanding higher plasma insulin concentrations, glucose clearance rates were similar after oral and intravenous glucose administration. It therefore seems likely that some form of suppression of insulin activity is at play after oral glucose dosing.
We hypothesised that a high flow of carbohydrates through the duodenum and upper jejunum, eliciting a high insulin secretory response, may also induce insulin resistance as a protective mechanism against hypoglycaemia. This mechanism is consistent with the ‘foregut hypothesis’, which holds that surgical exclusion of the proximal gut reduces intestinal factor/s that impair the action of insulin [
18,
19].
The present study aims at making a significant step forward following on from our previous investigation [
20]. Using a mathematical model, the previous study examined the impact of oral, as opposed to intravenous, glucose administration while holding insulin sensitivity constant. In this case, the subject would undergo hypoglycaemia [
20]. That study [
20] estimated the glucose absorption rate (rate of appearance;
Ra) of a single 75 g dose of glucose, while the present study measures it directly using stable isotopes and with increasing doses of glucose. To evaluate whether different degrees of insulin sensitivity coupled with a different insulin secretory efficiency could elicit distinct responses to the oral vs the intravenous route of glucose administration, we extended our previous investigation to different glycaemic states. Therefore, we enrolled individuals with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), which is considered to be a transition state between NGT and diabetes, and with type 2 diabetes. In addition, metabolomics was performed in order to investigate the effects of insulin in suppressing proteolysis, ketogenesis, lipolysis and glucagon levels.
Methods
Participants
The primary endpoint of this proof-of-concept study was the difference in insulin-mediated glucose metabolic clearance rate (MCR/I) between the oral and intravenous method of glucose administration. Secondary endpoints were differences in the insulin effect on proteolysis, ketogenesis, lipolysis and glucagon levels.
The sample size was calculated according to the MCR/I data reported in Gastaldelli et al. [
21]. Assuming a 30% higher glucose MCR/I during isoglycaemic intravenous glucose infusion (IGIVI) (4.6 for oral vs 5.98 ml min
−1 kg
−1 per nmol/l for IGIVI, each SD = 0.9), α = 0.05 and power = 0.90, 24 participants (eight with NGT, eight with IGT and eight with type 2 diabetes) were required, using a more conservative
t test for independent samples and considering a 25% attrition rate.
Inclusion criteria were: normal glucose tolerance or impaired glucose tolerance or type 2 diabetes; BMI> 30 kg/m2; age between 18 and 65 years; both sexes; and capacity to give informed consent. Exclusion criteria were: liver, kidney, cardiac or respiratory failure; major endocrine diseases requiring treatment; active cancer (surgical or medical treatment in the 5 years preceding the enrolment); HbA1c ≥ 10% (85.5 mmol/mol) for participants with type 2 diabetes. Participants, caregivers, people doing measurements or examinations, and people assessing the outcomes were unblinded to group assignment.
The study was conducted at the University Hospital Policlinico Gemelli at Rome, Italy between July 2017 and July 2019. One participant initially allocated to the IGT group had NGT after re-examining the OGTT results and one participant with type 2 diabetes refused to undergo the intravenous study, and thus was excluded from the study. Therefore, nine participants with NGT, seven with IGT and seven with type 2 diabetes underwent oral and, after 7–10 days, intravenous glucose tests following a 12 h overnight fast on each occasion. Diabetes duration was 2–4 years and all patients were receiving oral hypoglycaemic agents (metformin alone or plus sodium–glucose cotransporter 2 inhibitors), which were discontinued 24 h before the studies.
The protocol was approved by the ethics committee of Catholic University of Rome, Italy. All participants provided written informed consent. Details on inclusion and exclusion criteria are reported in the ESM Methods.
Biochemical measurements
To collect arterialised venous blood, a retrograde catheter was inserted in a dorsal hand vein, with the hand kept in a warming blanket. A forearm vein of the contralateral arm was catheterised for the infusions.
During the first session, at 08:00 h, [6,6-2H2]glucose was infused (priming: 22 μmol/kg; infusion rate: 0.22 μmol kg−1 min−1) to determine glucose kinetics. After 2.5 h of isotope infusion (basal period), an OGTT was given and consumed over 5 min. The OGTTs consisted of a 25 g solution followed by 75 g after 2 h and by 100 g after a further 2 h. Each OGTT contained 0.9 g of [U-13C6]glucose tracer. Plasma glucose was measured at baseline and every 10 min thereafter until 360 min.
In a different session, at 08:00 h, the participants were infused with a 20% wt/vol. adjustable glucose infusion in order to match the plasma glucose concentrations obtained during the OGTTs. After baseline blood samples were obtained, [6,6-2H2]glucose (22 μmol/kg prime and 0.22 μmol kg−1 min−1 constant infusion) was infused. At 10:30 h, after the basal period was completed, 20% dextrose enriched to approximately 2.5% with [6,6-2H2]glucose to minimise changes in glucose isotopic enrichment, was infused. Plasma glucose was measured every 10 min until 360 min, in order to change the glucose infusion rate to obtain an isoglycaemic pattern.
Plasma insulin, C-peptide, glucagon and GLP-1, as well as metabolites, were measured during fasting and, thereafter, every 20 min up to 360 min after starting the OGTT or the intravenous isoglycaemic infusion.
We will use the terms Time 1, Time 2 and Time 3 throughout the manuscript to indicate the different sub-experiments with increasing oral glucose loads (25, 75 and 100 g) and intravenous glucose infusion time periods performed to mimic the glycaemic response to the oral glucose challenges.
Assays
Plasma glucose concentrations were determined by a glucose oxidase method using a glucose analyser. Insulin and C-peptide were measured by the Architect 1000 SR (Abbott Diagnostics, Abbott Park, IL, USA). Glucagon and total GLP-1 were measured by ELISA (Mercodia, Uppsala, Sweden).
GC/MS analyses of glucose
Isotopic enrichment of [6,6-
2H
2]-glucose and [U-
13C
6]glucose was measured by electron impact ionisation on a GC/MS 5975 (Agilent Technologies, USA) using a 30 m× 0.25 mm HP-5MS column by monitoring ions at
m/
z 202/200 and 205/200, as previously described [
22]. For all GC/MS analyses, instrument response was calibrated using standards of known enrichment. Glucose enrichment was good; the lowest average (mean) tracer-to-tracee ratio was 1%.
Plasma samples were analysed by two global profiling analytical platforms and a targeted profiling platform. Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) was applied to measure small polar metabolites [
23]; and ultra-HPLC coupled with quadrupole mass spectrometry (UHPLC-QTOFMS) global lipid profiling was used to measure lipids [
24,
25].
Stable isotope calculations
Glucose
Ra and rate of disappearance (
Rd) were calculated from changes in glucose enrichment according to the non-steady-state Steele equation and a time-varying glucose distribution volume to reduce the size of the non-steady-state error [
26]. During the OGTTs, total glucose
Ra was calculated by [6,6-
2H
2]glucose tracer/tracee ratio and the oral
Ra by [U-
13C
6]glucose tracer/tracee [
26]. The endogenous glucose production (EGP) was computed as (total
Ra − oral
Ra).
Discussion
Our study focuses on the differential effect of different routes of glucose administration, oral vs intravenous, on insulin sensitivity. We found that insulin sensitivity was significantly lower when glucose was taken orally rather than given intravenously. This effect was independent of the participant’s glycaemic status, although individuals with type 2 diabetes showed a higher insulin resistance when compared with individuals with IGT or NGT. Multiple and increasing glucose loads enhanced glucose disposal.
Not only was peripheral insulin sensitivity, expressed as glucose MCR/I, blunted during the oral vs the intravenous route of glucose administration, but hepatic insulin clearance was also reduced. This latter is, in fact, a typical feature of hepatic insulin resistance [
33].
Our results confirm the observations of Nauck et al. [
34], who showed a significantly reduced fractional hepatic insulin extraction after oral glucose administration (46.9–54.6%) compared with an IGIVI (63.4–76.5%), suggesting higher hepatic insulin resistance when glucose was given orally. However, Nauck et al. [
34] did not use stable isotopes.
The ‘incretin effect’ is the phenomenon by which the same plasma glucose concentration elicits a much higher insulin secretion during oral rather than intravenous glucose administration [
34]. It is, however, unclear how an individual would not develop hypoglycaemia as a consequence of this higher insulin secretory response. The observation that, after oral glucose administration, the anti-lipolytic and anti-proteolytic action of insulin is blunted in the context of matching plasma glucose levels and similar glucose
Ra, and moreover that glucose MCR/I is reduced, points to the development of insulin resistance when glucose is given orally. In fact, should insulin act similarly after oral glucose loads, we would observe stronger anti-lipolytic and anti-proteolytic effects.
The DI, which reflects the ability to respond to insulin resistance by delivering more insulin into the peripheral circulation through increasing insulin secretion and/or reducing hepatic insulin clearance, was significantly lower during the oral procedure because when ISR increases, glucose MCR/I decreases as an adaptive mechanism. However, the DI increased after repeated oral glucose loads in agreement with the Staub–Traugott effect [
35,
36], showing that repeated administrations of glucose facilitate glucose disposal [
35‐
37].
Circulating GLP-1 levels progressively increased with increasing amounts of glucose ingested; this GLP-1 response was more pronounced in participants with NGT than in those with type 2 diabetes. Sjøberg et al. [
38] demonstrated that at physiological levels GLP-1 does not affect whole-body insulin sensitivity. In fact, the GLP-1 analogue, exenatide, improves both hepatic and adipose insulin resistance but at plasma levels ten times higher than the GLP-1 levels elicited by an OGTT [
22]. This suggests that the effects of the oral glucose challenge on insulin secretion and insulin sensitivity are mediated by two different players, one of which is already known, i.e. GLP-1, and the other not yet identified.
The secretion of glucagon was higher during oral glucose administration in participants with type 2 diabetes, suggesting insufficient glucagon secretion suppression by insulin and GLP-1 in these individuals.
Comparison of metabolites in the heat map showed a clear separation between metabolite profiles in relation to the mode of glucose administration. Metabolomics was performed in a non-targeted mode and, thus, statistical results are to be considered merely exploratory. As reported in ESM Table
2, a linear mixed-effects model showed that hydroxybutyric acid, branched chain amino acids (valine, leucine and isoleucine) and the leucine metabolite ketoleucine, as well as fatty acids (oleic, linoleic, palmitic, stearic and myristic acids) varied the most between the two modalities of glucose administration (interaction coefficient Time 3:IGIVI). The importance rank plot of metabolites (Fig.
4c) shows that most of the above metabolites are the most relevant ones in separating oral vs intravenous modalities of glucose administration, with a prediction accuracy of almost 85%.
Even though the circulating levels of insulin were doubled when glucose was given orally, they failed to suppress lipolysis, proteolysis or glucagon secretion, a crucial hormone for maintaining EGP [
39,
40].
The Adipo-IR index [
31] is considered to reflect adipose tissue resistance to the anti-lipolytic effects of insulin. We found that the Adipo-IR index was higher with oral glucose administration, suggesting impaired suppression of lipolysis in the presence of higher insulin levels when glucose was given orally.
Circulating levels of medium-chain fatty acids, docosahexaenoic and dodecanoic acids, increased more when increasing doses of glucose were given intravenously and may account for the higher insulin sensitivity observed during the intravenous infusion of glucose compared with glucose oral administration. Medium-chain fatty acids, in fact, exert beneficial effects on diabetes, obesity and inflammation [
41], reduce body fat [
42], enhance energy-expenditure [
43] and prevent insulin resistance [
43]. Similar to the effect on lipids, the efficacy of insulin in inhibiting proteolysis was decreased after oral glucose loads.
Taken together, these findings suggest that there is a mechanism, somehow triggered by the presence of glucose in the intestinal lumen, directed to counterbalancing incretin action by limiting the effect of the insulin released.
The oral boluses of glucose given in our study are consistent with the levels of glucose consumed in a typical meal by individuals with obesity who eat sweet bakery items and drink glucose-sweetened beverages. In fact, 25 g of glucose corresponds to one large soft drink. Two cake slices (200 g each) plus a small soft drink (250 ml) provide 75 g of glucose, while one and a half slices of a cake plus two large soft drinks deliver around 100 g of glucose.
Some limitations should be recognised in this study. Although the results are consistent, the sample size was limited and, by design, we did not use the euglycaemic−hyperinsulinaemic clamp, which is the gold standard for insulin sensitivity measurement. We also used multiple oral glucose loads to determine the dose–response in two single studies for participant convenience and in order to limit day-to-day variability. A potential bias derives from the fixed sequence of glucose administration, oral first and then intravenous, which was necessary according to the design of the study and use of chronic medications. Furthermore, our patients were affected by morbid obesity and it is not certain that our results can be extrapolated to individuals with lesser degrees of obesity. Finally, the Adipo-IR index is only a surrogate measure of NEFA turnover. Future research would permit the identification of the gut mediator/s of insulin resistance and possibly provide alternatives to surgery.
In conclusion, our study shows that the degree of insulin sensitivity depends on the route of glucose administration. Oral glucose administration leads to increased insulin secretion and compensatory insulin resistance compared with intravenous glucose administration. The MCR/I is significantly enhanced when glucose is administered intravenously rather than orally. EGP tends to increase (although not significantly, possibly due to high inter-individual variability), while insulin clearance is decreased when glucose is given orally rather than intravenously, in spite of similar rates of glucose appearance, pointing toward hepatic insulin resistance. Increased hepatic insulin resistance results in turn in increased circulating levels of gluconeogenetic metabolites. Our findings suggest that, while glucose-mediated incretin release is followed by an increase in insulin release, the effect of the released insulin is limited through an increase in insulin resistance.
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