Evaluation of the Effect of Enteral Lipid Sensing on Endogenous Glucose Production in Humans

Endogenous glucose production (EGP), mainly by the liver, plays an important role in regulating glucose homeostasis. In patients with type 2 diabetes, impaired insulin action in insulin-sensitive tissues such as the liver, skeletal muscle, and adipose tissue contributes to hyperglycemia. Hepatic glucose production is inappropriately elevated and is a major determinant of fasting hyperglycemia in this condition (1).

Studies in rodents suggest that EGP is subject to neuronal regulation involving nutrient sensing in the hypothalamus (2,3) and in the small intestine (4). Intraduodenal administration of lipids, particularly long-chain fatty acids (LCFAs), has been shown to reduce food intake in both rodents and humans (5,6) and to suppress EGP profoundly and rapidly in rats under experimental conditions. EGP was suppressed by >50% during a 50-min intraduodenal infusion of Intralipid under conditions of a pancreatic insulin clamp, and plasma glucose concentration was lowered by ∼20% 15 min after intraduodenal infusion of Intralipid in nonclamped conditions (4). This effect occurred prior to significant absorption of the lipids, as evidenced by the absence of elevations in plasma free fatty acid (FFA) or triglyceride (TG) levels (4). Based on studies in rats, upper intestinal lipid sensing lowers EGP via a gut-brain-liver axis (4,7,8). Since this occurred at a time when insulin levels at the liver were lower than would be present during a meal, the physiological role of this pathway in regulating glucose homeostasis during food consumption remains unclear.

The role of upper intestinal lipid sensing and the potential existence of a gut-brain-liver axis in the regulation of glucose homeostasis have not previously been investigated in humans, hence the aim of the current study. We assessed EGP during infusion of either normal saline or Intralipid into the duodenum in healthy men. Intralipid is an emulsion consisting of predominantly long-chain polyunsaturated fatty acids that has been used to demonstrate a gut-brain-liver axis in rats (4). The choice of infusion rate could not easily be extrapolated from previous rat studies. We chose to infuse Intralipid at a rate that has been demonstrated in humans to inhibit food intake and induce cholecystokinin (CCK) release (9), which is believed to mediate the gut-brain signaling effect in the regulation of satiety in humans (10,11). Higher doses of Intralipid could cause nausea and raise plasma FFA concentrations. As in previously published rat studies, in order to control for potential fluctuations of hormones in response to enteral nutrient infusion, we performed our studies under conditions of a pancreatic clamp with the infusion of somatostatin and insulin. Since somatostatin also inhibits the secretion of glucagon and growth hormone, in our study these hormones were also replaced.

Nine healthy men participated in the study. Their demographic and biochemical parameters are shown in Table 1. Participants had no existing medical illnesses, were not taking any medications, and had normal glucose tolerance. Each participant was studied on two occasions (receiving intraduodenal infusion of Intralipid or normal saline), in random order, 4–6 weeks apart, in a single-blind crossover design.

The study outline is depicted in Fig. 1. Participants were admitted to the Metabolic Test Centre of the Toronto General Hospital after an overnight fast. A radio-opaque polyvinyl feeding tube was inserted into the first part of the duodenum under fluoroscopic guidance. Two intravenous catheters were inserted into superficial forearm veins in opposite arms: one for infusion of hormones and isotopically enriched glucose and the other for blood sampling. Blood sampling from a forearm vein may systematically underestimate arterial plasma glucose. Since the same protocol was used for both study arms, this is not expected to affect the conclusion of the study. At 1300 h (referred to as t = −120 min), a pancreatic clamp, with infusion of somatostatin (30 μg/h), insulin (0.05 mU/kg/min), glucagon (0.65 ng/kg/min), and growth hormone (3.0 ng/kg/min), was started and continued for the remainder of the study as previously described (12). Concurrently, a primed, constant infusion (22.5 µmol/kg bolus followed by 0.25 μmol/kg/min) of d-[6,6-²H₂]glucose (D2-glucose; Cambridge Isotope Laboratories) was started and continued until the end of the study. Two hours later, i.e., at 1500 h (referred to as t = 0 min), infusion into the duodenum was started for delivery of either Intralipid (20%, duodenal fat infusion study [DF]) or normal saline (NS) at the rate of 30 mL/h and continued for 90 min. Blood samples were drawn at baseline (t = −120 min), every 30 min before intraduodenal infusion (t = −90, −60, and −30 min), and every 10 min thereafter until the end of the study (t = 90 min). A dextrose (20%) solution was infused to maintain euglycemia if blood glucose declined to <4.0 mmol/L. Plasma was immediately separated from blood samples into tubes containing aprotinin, sodium azide, and tetrahydrolipstatin and stored for future analysis.

Plasma was deproteinized, delipidated, dried, and derivatized with acetic anhydride. Derivatized samples were analyzed with gas chromatography–mass spectrometry (Agilent 5975/6890N) with electron impact ionization using helium as the carrier gas. Selective ion monitoring at m/z = 242 and 244 was performed. Atom percent excess (APE) fraction was calculated for each sample as APE = tracer/(tracer + tracee). Commercial kits were used to measure TG (Wako), FFA (Wako), insulin (Millipore), and glucagon (Millipore).

EGP was estimated as previously described (13). Briefly, during steady state, glucose R_a = glucose R_d, where R_a = tracer infusion rate/APE fraction, and EGP = R_a – glucose infusion rate.

Results are presented as mean ± SEM. Paired t test was used to compare mean plasma concentrations of glucose, TG, FFA, insulin, and glucagon and EGP. A P value <0.05 was considered significant.

Baseline (before clamp, t = −120 min) levels of plasma TG were lower in DF compared with NS (P = 0.02), which may have contributed to a trend toward lower mean TG in DF versus NS during the 30 min prior to intraduodenal infusion (P = 0.078) and lower mean TG in DF versus NS 30 min after intraduodenal infusion (P = 0.047) (Fig. 2A). Intraduodenal infusion of Intralipid did not elevate plasma TG. Plasma FFA levels in DF and NS were similar at baseline, declined similarly in both arms prior to intraduodenal infusion possibly owing to mild arterial hyperinsulinemia, and were not significantly different between DF and NS after intraduodenal infusion (Fig. 2B). The absence of TG and FFA elevation indicates no significant absorption of luminal lipids in DF. Plasma concentrations of insulin and glucagon were similar between DF and NS both before and after intraduodenal infusion (Fig. 2C and D).

Plasma glucose concentrations were not significantly different between treatments at baseline (Fig. 3A). There was a small but significant increase in both groups (P < 0.05) after the commencement of the pancreatic clamp, but plasma glucose levels at the start of intraduodenal infusion were not significantly different between treatments (NS 7.00 ± 0.49 vs. DF 6.60 ± 0.53 mmol/L, P = 0.30). During the first 30 min of intraduodenal infusion, mean plasma glucose concentrations were not significantly different between treatments (NS 6.82 ± 0.52 vs. DF 6.37 ± 0.53 mmol/L, P = 0.20). Only one subject required dextrose infusion to maintain euglycemia with identical requirements during both the NS and DF arms of the study.

Calculated APE (Fig. 3B), a measure of glucose isotopic enrichment, and EGP (Fig. 3C) were not significantly different between treatments during the period before intraduodenal infusion. During the intraduodenal infusion period, APE was also superimposable between NS and DF (Fig. 3B). No significant differences between NS and DF were seen in EGP (NS 13.47 ± 1.36 vs. DF 12.84 ± 1.72 μmol/kg/min, P = 0.37) in the first 30 min after the start of the intraduodenal infusion (Fig. 3C and D) (P = 0.34). EGP declined to a similar extent in both NS and DF during the study, possibly due to lower FFA or hepatic hypoglucagonemia during the clamp, since the latter restores systemic but not hepatic glucagon levels or both. Since only one subject required dextrose infusion in both the NS and DF visits, R_d was not significantly different between treatments either during the basal period or during the intraduodenal infusion period (not shown).

Rodent studies suggest that administration of nutrients, such as lipids, in the proximal intestine evokes a gut-brain-liver circuitry to lower EGP in the preabsorptive state (4,14). Intraduodenal infusion of lipids also has been shown to suppress food intake in both rodents and humans (5,6). In light of these findings, it is important to examine whether a similar gut-brain-liver axis for regulation of EGP is operative in humans. In the current study in humans, we infused Intralipid into the duodenum and measured EGP using stable isotope enrichment techniques under pancreatic clamp conditions. Under these experimental conditions, no detectable effect of this dose and rate of intraduodenal Intralipid infusion on glucose concentrations or EGP was observed in healthy men.

Various intestinal lipid infusion protocols have been used to elicit suppression of food intake in humans. Reduced food intake was observed with lipid infusion into the duodenum (6), ileum (11), or jejunum (15). The lipid preparations generally included emulsions of fats containing primarily LCFA, including corn oil (10,11), Intralipid (9), olive oil, or oleate (6). Inhibition of food intake by intestinal lipids in humans is believed to be through the release of CCK (10,11), an effect that requires fatty acids with chain length greater than C10 (6,16). In fact, generation of LCFA from fats in the small intestine is required to suppress food intake in humans (6) and inhibit EGP in rats (4). Intralipid is a soybean-based emulsion that consists of mainly LCFA (C16–C18). Infusion of Intralipid (20%) at the rate of 30 mL/h, which delivers 6 g/h lipids, into the duodenum effectively suppressed food intake and elevated plasma CCK in humans (9), providing evidence for a biological effect of this dose of Intralipid. In the current study, with intraduodenal infusion of Intralipid at this identical rate, no detectable effects on plasma glucose concentration or EGP were observed. No study has demonstrated a plasma glucose–lowering effect of intraduodenal administration of lipids in humans. In a recent study in lean men, a much higher Intralipid infusion rate (equivalent to 90 mL/h for 90 min) caused nausea without affecting plasma glucose concentrations in unclamped conditions (17). Since inhibition of food intake occurred after 60 min of lipid infusion in humans (6,10), whereas reductions in food intake and plasma glucose concentrations were observed within 15 min of infusion in rats (4,5), the lack of effect on glucose concentrations and EGP in the current study is not likely due to insufficient delivery rate or short infusion time. We cannot exclude the possibility, however, that different lipid preparations delivered to other intestinal segments may provide a sufficient signal to activate the neuronal circuitry.

The lack of effect of intraduodenal lipid infusion on EGP in this study could in part be due to differences in liver glycogen content between rodents and humans after an overnight fast. An overnight fast dramatically decreases liver glycogen content in rats, whereas significant liver glycogen content is still present in healthy humans (18,19). Whether intraduodenal infusion of Intralipid inhibits EGP after prolonged fasting, or other conditions causing depletion of liver glycogen, is unknown. Another point to note is that suppression of food intake in humans and glucose lowering in rats with intraduodenal Intralipid infusion were observed in unclamped conditions (4,6). It is also noteworthy that EGP was assessed in rats with a somatostatin clamp where only insulin was replaced (4), whereas our pancreatic clamp also included glucagon and growth hormone replacement. It is not known to what extent this methodological difference may explain the different findings between the previous rodent studies and the current study in humans.

In summary, results of the current study indicate that in healthy men delivery of Intralipid to the upper intestine at a rate that has previously been shown to inhibit food intake does not acutely inhibit EGP in the preabsorptive state in contrast with the effect that has previously been shown in rodents. Whether this represents a true interspecies difference or is the result of specific differences in experimental methods used should be the subject of future investigation.

Acknowledgments. The authors are indebted to Brenda Hughes and Patricia Harley (University Health Network) for nursing assistance and Linda Szeto (University Health Network) for technical assistance.

Funding. G.F.L. holds the Sun Life Financial Chair in Diabetes and the Drucker Family Chair in Diabetes Research and is supported by a Canadian Institutes of Health Research Operating Grant. S.D. and C.M. are recipients of postdoctoral fellowship awards from the Banting & Best Diabetes Centre, University of Toronto. S.D. is the recipient of a Focus on Stroke 12 Fellowship Award from the Heart and Stroke Foundation of Canada. K.K. is funded by a JDRF Canadian Clinical Trial Network postdoctoral fellowship.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. C.X., S.D., and G.F.L. designed the study and interpreted data. C.X., S.D., C.M., and K.K. acquired and analyzed data. C.X. and G.F.L. wrote the manuscript. G.F.L. obtained funding and supervised the study. All authors edited the manuscript. G.F.L. 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.