Divisions of 1Endocrinology, Metabolism, and Internal Medicine and Nutrition and 2Gastroenterology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Submitted 6 February 2003 ; accepted in final form 19 March 2003
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ABSTRACT |
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portal signal; splanchnic glucose extraction; glucose uptake; endogenous glucose production
The mechanism by which the portal vein senses glucose is an area of active investigation. Burcelin et al. (57), in an elegant series of experiments, have strongly implicated GLUT2 transporters in this process. They have shown that infusion of glucose in the portal vein of mice at a rate that approximated basal endogenous glucose production (EGP) markedly increased peripheral glucose clearance (7). This effect did not occur in mice in whom the GLUT2 transporter was knocked out and could be abolished by concurrent infusion of either somatostatin or the glucagon-like peptide (GLP)-1 antagonist exendin (57). In addition to providing interesting mechanistic insights, these studies were both surprising and provocative since intraportal glucose infusion caused a paradoxical fall in the plasma glucose concentration to hypoglycemic levels (57).
We have demonstrated previously that peripheral and splanchnic glucose
uptake were the same in nondiabetic humans when glucose was infused
intraduodenally or intravenously at a rate of 4 mg ·
kg-1 · min-1
(24). However, in those
experiments, glucose was clamped at 150 mg/dl and insulin at
400
pmol/l to stimulate hepatic and peripheral glucose uptake. Somatostatin had to
be given to inhibit endogenous insulin secretion so as to ensure comparable
portal insulin concentrations on both occasions. Somatostatin also has been
given for the same reason in the animal experiments that have shown that
intraportal glucose infusion decreases rather than increases extrahepatic
glucose uptake (2,
8,
10,
18). Therefore, it is possible
that use of somatostatin in ours and other investigators' experiments either
dampened or ablated the response of the putative portal glucose sensor.
Alternatively, the response to intraportal glucose delivery in dogs and humans
may differ from that in mice.
The present experiments sought to address this question by testing the hypothesis that intraduodenal infusion of glucose (and its resultant selective delivery in the portal vein) at rates approximating EGP results in hypoglycemia in humans. We further hypothesized that hypoglycemia (if it occurred) would be the result of a combination of inappropriately elevated rates of glucose uptake and inappropriately suppressed rates of EGP. To examine this question, we infused glucose at rates of 1.55 and 3.1 mg · kg-1 · min-1 id for 7 h and compared the results with those observed when either saline or trace (0.1 mg · kg-1 · min-1) amounts of glucose were infused intraduodenally for the same length of time. We report that intraduodenal glucose infusion at rates approximating EGP does not cause hypoglycemia in humans.
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RESEARCH DESIGN AND METHODS |
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Experimental design. Each subject was studied on two occasions
separated by at least 1 wk. On the afternoon before each study, subjects were
taken to the fluoroscopy unit on the Mayo General Clinical Research Center
where an 8-Fr Flexi-flo enteral feeding tube (Ross Laboratories, Columbus, OH)
was passed under fluoroscopic guidance via the nasopharynx in the fourth part
of the duodenum. On average, 15 min were required for tube placement and <2
min of fluoroscopy for verification of the tube's location. The subjects were
then admitted to the Mayo General Clinical Research Center where they ate a
standard 10 kcal/kg meal (55% carbohydrate, 30% fat, and 15% protein) between
1700 and 1730. The subjects took nothing by mouth after supper with the
exception of small amounts of water until the end of study. At 0430 on the
following morning, a forearm vein was cannulated with an 18-gauge needle to
allow infusion of [6,6-2H2]glucose. In addition, an
18-gauge cannula was inserted retrogradely in a vein of the contralateral
hand. The hand was then placed in a Plexiglas box maintained at 55°C to
allow sampling of arterialized venous blood. At 0500, a primed continuous
infusion of [6,6-2H2]glucose was started and continued
until the end of the study. At 0630, the position of the nasoduodenal
tube was confirmed by a portable abdominal radiography. At 0800 (0 min), a
primed (12 µCi) continuous infusion (0.12 µCi/min) of
[3-3H]glucose was infused in the duodenum via the nasoduodenal tube
for the next 7 h. The [3-3H]glucose infusion also contained
unlabeled glucose. In five subjects, glucose was infused in the duodenum at a
rate of 3.1 mg · kg-1 ·
min-1 on one occasion and 0.1 mg ·
kg-1 · min-1 on the other
occasion. In nine subjects, glucose was infused in the duodenum at a rate of
1.55 mg · kg-1 ·
min-1 on one occasion and either saline (n = 4)
or glucose (n = 5) at a rate of 1.22 mg ·
kg-1 · min-1 (in error
rather than saline) on the other occasion. The results of 1.22 mg ·
kg-1 · min-1 infusion did
not differ substantially from the 1.55 mg · kg-1
· min-1 infusion and therefore are not presented
as part of the current study but are available upon request. In addition,
because results did not differ during the 0.1 mg ·
kg-1 · min-1 id or saline
alone infusions, these results were combined for purposes of analysis. Blood
samples were collected at -180, -30, 0, 60, 120, 180, 210, 220, 230, 240, 270,
300, 330, 360, 390, and 420 min for analysis of tracer, hormone, and substrate
concentrations.
Analytical determinations. Arterialized venous plasma samples were placed on ice, centrifuged at 4°C, and separated and stored at -20°C until assay. C-peptide and glucagon concentrations were measured using reagents purchased from Linco Research (St. Louis, MO). Insulin and growth hormone were measured using a chemiluminescence assay with reagents obtained from Beckman (Access Assay; Beckman, Chaska, MN). Plasma glucose concentration was measured using a Yellow Springs glucose analyzer. Plasma [3-3H]glucose specific activity and [6,6-2H2]glucose enrichment were measured by liquid scintillation counting and gas chromatography-mass spectrometry (16, 19). Percent body fat and fat-free mass were measured using a dual-energy X-ray absorptiometry scan QDR4500 with fan scan technology.
Calculations. Rates of glucose appearance and disappearance were calculated using Steele's non-steady-state equations (22). A pool volume of 200 ml/kg and a pool fraction of 0.65 were assumed. The systemic rate of appearance of the intraduodenally infused [3-3H]glucose was also calculated using Steele's steady-state equations in which the plasma [3-3H]glucose concentration was substituted for the unlabeled glucose concentration (4). The systemic rate of appearance of [3-3H]glucose (in dpm · kg-1 · min-1) was divided by the specific activity of the intraduodenally infused glucose (in dpm/mg) to convert the rates to milligrams per kilogram per minute. EGP was calculated by subtracting the systemic rate of appearance of the intraduodenally infused glucose from the total rate of appearance of glucose. Initial splanchnic glucose extraction was calculated as 1 minus the systemic rate of appearance of [3-3H]glucose divided by the intraduodenal infusion rate of [3-3H]glucose.
Statistical analysis. Data in the text and Figs. 1, 2, 3 are expressed as means ± SE. All rates are expressed as milligrams per kilogram total body weight per minute. ANOVA followed by a signed-rank test was used to compare the results of the different studies. A P value <0.05 was considered statistically significant.
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RESULTS |
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Fasting plasma insulin concentrations (Fig. 1B) did not differ on the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id study days (5 ± 1 vs. 5 ± 1 vs. 4 ± 1 µU/ml). Insulin concentrations fell during the 7 h of the 0/0.1 mg · kg-1 · min-1 id study (to 3 ± 1 µU/ml). In contrast, insulin increased (P < 0.05) during the 1.55 and 3.1 mg · kg-1 · min-1 infusions, reaching a peak of 10 ± 2 and 18 ± 5 µU/ml, respectively, by 2 h (P = 0.07; 1.55 vs. 3.1 mg · kg-1 · min-1 infusions). Insulin concentrations then fell back to concentrations that no longer differed by study end (7 ± 1 vs. 8 ± 1 µU/ml). Of note, the plasma insulin concentrations of one individual were three SDs greater than those of the other subjects in that group both before and during the 1.55 mg · kg-1 · min-1 infusion and therefore were excluded from analysis.
Fasting plasma C-peptide concentrations (Fig. 1C) also did not differ on the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 study days (0.4 ± 0.0 vs. 0.4 ± 0.1 vs. 0.3 ± 0.0 nmol/l). Plasma C-peptide concentrations fell (P < 0.001) to 0.3 ± 0 nmol/l during the 7 h of the 0/0.1 mg · kg-1 · min-1 id study. In contrast, C-peptide increased (P < 0.01) during 3.1 mg · kg-1 · min-1 id infusion to 0.8 ± 0.1 nmol/l. C-peptide tended to increase (to 0.8 ± 0.2 nmol/l) during the 1.55 mg · kg-1 · min-1 id infusion. Plasma C-peptide concentrations did not differ (P = 0.5) during the final hour of the 1.55 and 3.1 mg · kg-1 · min-1 id infusions (0.8 ± 0.2 and 0.8 ± 0.1 nmol/l).
Fasting plasma glucagon concentrations (Fig. 1D) did not differ on the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id study days (134 ± 13 vs. 146 ± 8 vs. 115 ± 6 pg/ml). Glucagon concentrations fell (P < 0.01) during the 3.1 mg · kg-1 · min-1 id infusion to 100 ± 6 ng/l and during the 1.55 mg · kg-1 · min-1 infusion to 128 ± 6 ng/l and remained unchanged during the 0/0.1 mg · kg-1 · min-1 id infusion (125 ± 8). Growth hormone concentrations also did not differ on the three study days either before or during the intraduodenal infusions (data not shown).
Plasma [6,6-2H2]glucose enrichment and [3-3H]glucose specific activity. Plasma [6,6-2H2]glucose enrichment (Fig. 2A) remained essentially unchanged during the 7 h of the 0/0.1 mg · kg-1 · min-1 id infusion. Plasma [6,6-2H2]glucose enrichment fell during the first 2 h of the 1.55 and 3.1 mg · kg-1 · min-1 id glucose infusions and changed minimally thereafter. The fall in plasma [6,6-2H2]glucose enrichment was greater during the 3.1 than during the 1.55 mg · kg-1 · min-1 id infusions, indicating a higher systemic rate of appearance of unlabeled glucose in the former.
The ratio of [6,6-2H2]glucose to [3-3H]glucose fell during the first 3 h of the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id infusions, indicating progressive absorption of the intraduodenally infused tracer (Fig. 2B). The ratio of [6,6-2H2]glucose to [3-3H]glucose changed minimally thereafter.
Systemic rate of appearance of intraduodenally infused glucose, EGP, and glucose disappearance. The systemic rate of appearance of intraduodenally infused glucose increased during the first 3 h of the 1.55 and 3.1 mg · kg-1 · min-1 id infusions, reaching a plateau thereafter (Fig. 3A). The appearance of intraduodenal glucose was greater (P < 0.001) during the final hour of the 3.1 than during the 1.55 mg · kg-1 · min-1 id infusion (3.66 ± 0.2 vs. 2.65 ± 0.03 mg · kg-1 · min-1). No intraduodenal glucose appeared during 0/0.1 mg · kg-1 · min-1 id infusions. Systemic appearance of intraduodenally infused tracer averaged 105 ± 14, 108 ± 3, and 113 ± l8% during the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id infusions, respectively, indicating negligible splanchnic tracer extraction.
Fasting EGP (Fig. 3B) did not differ on the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id study days (2.34 ± 0.1 vs. 2.30 ± 0.1 vs. 2.35 ± 0.1 mg · kg-1 · min-1). EGP suppressed to 1.98 ± 0.2 mg · kg-1 · min-1 during the final hour of the 0/0.1 mg · kg-1 · min-1 id infusion (P < 0.05); however, EGP suppressed to a greater degree during the final hour of the 1.55 and 3.1 mg · kg-1 · min-1 id infusions to 0.84 ± 0.1 and 0.63 ± 0.1 mg · kg-1 · min-1 (P < 0.001). However, EGP did not differ during the final hour of the 1.55 and 3.1 mg · kg-1 · min-1 id infusions.
Fasting glucose disappearance (Fig. 3C) did not differ on the 0/0.1, 1.55, and 3.1 mg · kg-1 · min-1 id study days (2.32 ± 0.3 vs. 2.1 ± 0.2 vs. 2.3 ± 0.2 mg · kg-1 · min-1). Glucose disappearance fell slightly, but not significantly, to 2.3 ± 0.1 during the 0/0.1 mg · kg-1 · min-1 id infusions. Glucose disappearance during the final hour of the 1.55 and 3.1 mg · kg-1 · min-1 id infusions (4.47 ± 0.2 vs. 2.6 ± 0.1 vs. 2.3 ± 0.1 mg · kg-1 · min-1) was greater (P < 0.001) than that observed over the same interval during the 0/0.1 mg · kg-1 · min-1 id infusions. Glucose disappearance was higher (P < 0.001) during the final hour of the 3.1 mg · kg-1 · min-1 id infusion than during the final hour of the 1.55 mg · kg-1 · min-1 infusion.
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DISCUSSION |
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The present experiments were undertaken to determine whether low rates of
delivery of glucose in the portal vein also resulted in hypoglycemia. To do
so, we infused glucose in the duodenum at rates of 1.55 and 3.1 mg ·
kg-1 · min-1. We chose
these rates since we wanted to bracket the normal postabsorptive rate of EGP
(i.e., 2 mg · kg-1 ·
min-1). Both infusions stimulated insulin secretion;
however, in contrast to what was observed in mice
(57),
glucose concentrations rose rather than fell. The increase in glucose was
evident whether considered as the change from the basal value or compared with
the glucose concentrations observed over the same interval during the 0/0.1 mg
· kg-1 · min-1 id
glucose infusions. The rise in glucose concentration continued until glucose
disappearance increased sufficiently to equal the sum of the systemic rate of
appearance of the intraduodenally infused glucose and EGP. The increase in
glucose and insulin concentrations during the 1.55 and 3.1 mg ·
kg-1 · min-1 infusions
resulted in equivalent suppression of EGP. However, despite comparable glucose
and insulin concentrations, glucose disappearance increased more during the
3.1 than during the 1.55 mg · kg-1 ·
min-1 id infusions, thereby compensating for the higher
systemic rate of appearance of the intraduodenal glucose. Thus selective
intraportal infusion of glucose in humans did not cause hypoglycemia but may
have enhanced glucose disposal.
It is interesting to speculate why glucose disappearance was higher during
the 3.1 than during the 1.55 mg · kg-1 ·
min-1 id infusion. Burcelin et al.
(6,
7) observed greater rates of
glucose clearance during intraportal than during intravenous glucose infusion
in mice. This effect did not occur in the GLUT2 knockout mice, suggesting a
role for a portal glucose sensor
(6). Activation of a portal
glucose sensor that in turn enhanced extrahepatic glucose disposal also may
have occurred in the present experiments. The higher rates of glucose
disappearance during the 3.1 than during the 1.5 mg ·
kg-1 · min-1 id infusion
despite comparable glucose, insulin, and C-peptide concentrations is
consistent with this possibility. On the other hand, we have previously shown
that glucose disappearance was the same when glucose was infused
intraduodenally or intravenously in nondiabetic volunteers at a rate of 4.0 mg
· kg-1 · min-1
(24). If the higher rates of
glucose disappearance were mediated by a glucose sensor, this would imply a
narrow dose-response curve, since this effect was absent during the current
1.55 and the past 4.0 mg · kg-1 ·
min-1 glucose id infusions but present during the
current 3.1 mg · kg-1 ·
min-1 glucose infusion. On the other hand, glucose was
clamped at 150 mg/dl in our previous experiments, and somatostatin was
infused to inhibit endogenous insulin secretion
(24). Because somatostatin,
perhaps by inhibiting GLP-1 secretion
(12), blunts the "portal
signal" in mice (5), our
previous experiments could have missed a stimulatory effect of intraportal
glucose delivery on glucose disappearance
(24).
Alternatively, the higher rate of glucose disappearance during the 3.1 than during the 1.55 mg · kg-1 · min-1 id infusion could have been because of the residual effect of antecedent higher insulin concentration combined with the slightly (but not significantly) higher glucose concentration. As is evident from Fig. 1, both insulin and C-peptide concentrations tended to be higher during the 3.1 than during the 1.55 mg · kg-1 · min-1 id infusions, with the differences being most marked immediately after the start of the intraduodenal infusions. Glucose disappearance more closely reflects interstitial than plasma insulin concentrations, and a change in interstitial insulin concentration lags behind a change in plasma insulin concentration (25). Therefore, the higher plasma insulin concentrations during the first few hours of the 3.1 mg · kg-1 · min-1 id infusion could have resulted in higher rates of glucose disappearance several hours later. The present experiments therefore leave open the possibility that selective intraportal infusion at rates bracketing EGP may enhance extrahepatic glucose uptake in humans.
We infused glucose at rates of either 0 (i.e., saline alone) or 0.1 mg · kg-1 · min-1 as control experiments. We included these control experiments since we wanted to be able to determine whether a fall in glucose concentration (if observed) during the 1.55 and 3.1 mg · kg-1 · min-1 id glucose infusions was greater than that which occurred with fasting alone. We infused [3-3H]glucose and carrier unlabeled glucose at a rate of 0.1 mg · kg-1 · min-1 in five of the subjects and [3-3H]glucose with saline alone in four of the subjects. We included the saline-alone experiments since we were concerned that infusion of even a small amount of glucose in the duodenum might evoke an enteral signal, thereby obscuring differences that might occur with the higher intraduodenal glucose infusion rates. This was not the case since the results were the same during infusion of either the 0 (saline) or 0.1 mg · kg-1 · min-1 id infusions. Bureclin et al. (5) reported that the GLP-1 antagonist exendin-(936) prevented hypoglycemia during intraportal glucose infusion in mice, whereas GLP-1 infusion did not. This suggested that the presence of basal levels of GLP-1 acting alone or in combination with other incretin hormones is required for activation of the portal signal in mice. We presume that incretin levels either remained constant or increased slightly during the intraduodenal glucose infusions. Therefore, the lack of hypoglycemia during the intraduodenal glucose infusions is unlikely to be because of a deficiency of incretin hormones.
The present experiments have several limitations. We assumed that essentially all of the glucose that was infused in the duodenum was absorbed within the small intestine or upper colon. If some of the glucose was absorbed by the distal colon, then the rate of glucose entry in the portal circulation may have been slightly lower than the rate of intraduodenal infusion, since at least a portion of the venous drainage of the distal colon directly enters the systemic circulation. However, we doubt if substantial amounts of glucose were absorbed by the distal colon, since we infused glucose in the duodenum at a rate that was well below the absorptive capacity of the small intestine and since we would anticipate that the majority of glucose reaching the colon would be metabolized by colonic bacteria rather than absorbed (26, 27). Glucose was infused in the duodenum rather than directly in the portal vein since the latter is not feasible in healthy human volunteers. It therefore remains possible that activation of other control mechanisms (e.g., enteric nervous system, enteric hormone secretion) prevented the development of hypoglycemia. Future studies will be required to address this question.
In summary, intraduodenal glucose infusion at rates bracketing normal EGP does not cause hypoglycemia in nondiabetic humans. Rather, glucose and insulin concentrations increase slightly. The higher glucose and insulin concentrations appropriately suppress EGP and stimulate glucose disappearance, thereby minimizing the rise in glucose concentration. On the other hand, the higher rates of glucose disappearance during the 3.1 than during the 1.55 mg · kg-1 · min-1 id infusion in the presence of slightly but not significantly higher glucose and insulin concentrations is consistent with, but does not prove the existence of, a portal signal in humans that enhances extrahepatic glucose disposal. Future studies will be required to specifically address this question in humans.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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