Fasting and postprandial concentrations of GLP-1 in intestinal lymph and portal plasma: evidence for selective release of GLP-1 in the lymph system

Glucagon like peptide 1 (GLP-1) is an intestinal hormone that plays an important role in glucose metabolism. GLP-1 is released from mucosal L cells following nutrient ingestion and contributes to the incretin effect, with the enhancement of insulin secretion occurring with enteral compared with intravenous glucose administration. The mechanisms linking nutrient absorption and GLP-1 secretion are unknown, and studies addressing this topic, particularly in small animal models, have been hampered by the relatively low concentrations of GLP-1 in the circulation. We hypothesized that GLP-1 levels would be higher in samples of intestinal lymph compared with plasma and could provide a novel system in which to study meal-induced hormone secretion. We addressed this hypothesis in conscious rats with indwelling catheters in the portal vein and distal intestinal lymph duct. These animals had plasma and lymph sampled before and for 240 min after instillation of a liquid meal in the gastrointestinal tract. Lymph contained detectable concentrations of glucose, insulin, and GLP-1 that were reliably measured using our assays. Before and after the Ensure feeding, plasma insulin levels were approximately two times as high in portal plasma as intestinal lymph. In marked contrast, GLP-1 levels were five to six times higher in lymph relative to portal plasma following nutrient administration. This relative difference in GLP-1 levels was even greater when lymph was compared with peripheral plasma and dramatically exceeded the ratio of lymph to plasma peptide tyrosine-tyrosine concentrations. This is the first observation of a gastrointestinal hormone being disproportionately transported in lymph. The remarkable levels of GLP-1 in intestinal lymph demonstrate the potential for lymphatic sampling as a more sensitive means of studying the secretory physiology of this hormone in vivo. In addition, these data raise the possibility that intestinal lymph may serve as a specialized signaling conduit for regulatory peptides secreted by gastrointestinal endocrine cells.     

GLP-1 reduces intestinal lymph flow, triglyceride absorption, and apolipoprotein production in rats

Glucagon-like peptide 1 (GLP-1) is a gastrointestinal hormone secreted in response to meal ingestion by enteroendocrine L cells located predominantly in the lower small intestine and large intestine. GLP-1 inhibits the secretion and motility of the upper gut and has been suggested to play a role in the "ileal brake." In this study, we investigated the effect of recombinant GLP-1-(7-36) amide (rGLP-1) on lipid absorption in the small intestine in intestinal lymph duct-cannulated rats. In addition, the effects of rGLP-1 on intestinal production of apolipoprotein (apo) B and apo A-IV, two apolipoproteins closely related to lipid absorption, were evaluated. rGLP-1 was infused through the jugular vein, and lipids were infused simultaneously through a duodenal cannula. Our results showed that infusion of rGLP-1 at 20 pmol.kg(-1).min(-1) caused a dramatic and prompt decrease in lymph flow from 2.22 +/- 0.15 (SE) ml/h at baseline (n = 6) to 1.24 +/- 0.06 ml/h at 2 h (P < 0.001). In contrast, a significant increase in lymph flow was observed in the saline (control) group: 2.19 +/- 0.20 and 3.48 +/- 0.09 ml/h at baseline and at 6 h of lipid infusion, respectively (P < 0.001). rGLP-1 also inhibited intestinal triolein absorption (P < 0.05) and lymphatic apo B and apo A-IV output (P < 0.05) but did not affect cholesterol absorption. In conclusion, rGLP-1 dramatically decreases intestinal lymph flow and reduces triglyceride absorption and apo B and apo A-IV production. These findings suggest a novel role for GLP-1 in lipid absorption.     

Priming effect of glucagon-like peptide-1 (7-36) amide, glucose-dependent insulinotropic polypeptide and cholecystokinin-8 at the isolated perfused rat pancreas.

The priming effect of glucagon-like peptide-1 (7-36) amide (GLP-1 (7-36) amide), glucose-dependent insulin-releasing polypeptide (GIP) and cholecystokinin-8 (CCK-8) on glucose-induced insulin secretion from rat pancreas was investigated. The isolated pancreas was perfused in vitro with Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose. After 10 min this medium was supplemented with GLP-1 (7-36) amide, GIP or CCK-8 (10, 100, 1000 pmol/l) for 10 min. After an additional 10 min period with 2.8 mmol/l glucose alone, insulin secretion was stimulated with buffer containing 10 mmol/l glucose for 44 min. In control experiments the typical biphasic insulin response to 10 mmol/l glucose occurred. Pretreatment of the pancreas with GIP augmented insulin secretion: 10 pmol/l GIP enhanced only the first phase of the secretory response to 10 mmol/l glucose; 100 and 1000 pmol/l GIP stimulated both phases of hormone secretion. After exposure to CCK-8, enhanced insulin release during the first (at 10 and 1000 pmol/l CCK-8) and the second phase (at 1000 pmol/l) was observed. Priming with 100 pmol/l GLP-1 (7-36) amide significantly amplified the first and 1000 pmol/l GLP-1 (7-36) amide both secretion periods, 10 pmol/l GLP-1 (7-36) amide had no significant effect. All three peptide hormones influenced the first, quickly arising secretory response more than the second phase. Priming with forskolin (30 mM) enhanced the secretory response to 10 mM glucose plus 0.5 nM GLP-1 (7-36) amide 4-fold. With a glucose-responsive B-cell line (HIT cells), we investigated the hypothesis that the priming effect of GLP-1 (7-36) amide is mediated by the adenylate cyclase system. Priming with either IBMX (0.1 mM) or forskolin (2.5 microM) enhanced the insulin release after a consecutive glucose stimulation (5 mM). This effect was pronounced when GLP-1 (7-36) amide (100 pM) was added during glucose stimulation. Priming capacities of intestinal peptide hormones may be involved in the regulation of postprandial insulin release. The incretin action of these hormones can probably, at least in part, be explained by these effects. The priming effect of GLP-1 (7-36) amide is most likely mediated by the adenylate cyclase system.     

The regulation of the lymphatic secretion of glucagon-like peptide-1 (GLP-1) by intestinal absorption of fat and carbohydrate

Glucagon-like peptide-1 (GLP-1) is an important incretin produced in the L cells of the intestine. It is essential in the regulation of insulin secretion and glucose homeostasis. Systemic GLP-1 concentrations are typically low in rodents, so it can be difficult to assay physiological levels or detect changes in response to nutrients. We have established a method of assaying GLP-1 in response to nutrients using the intestinal lymph fistula model. Intraduodenal infusion of Intralipid (4.43 kcal/3 ml) induced a significant increase of lymphatic GLP-1 concentration compared with saline control at the peak of 30 min. (P < 0.001). Isocaloric and isovolumetric treatment with dextrin, a glucose polymer, also caused a significant fourfold increase in peak concentration at 60 min (P = 0.001). These findings indicate that intestinal lymph contains high concentrations of postprandial GLP-1. Second, they reveal that GLP-1 secretion into lymph occurs in response to both enteral carbohydrate and fat, but the response to dextrin occurs later than to Intralipid with peak times at 60 and 30 min, respectively. Third, the combination of Intralipid plus dextrin demonstrated an additive effect in the stimulation of GLP-1 with peak at 30 min. These results indicate that assessment of levels in lymph is a novel and powerful means of studying the secretion of GLP-1 and potentially other gastrointestinal hormones in vivo. Furthermore, the lymph fistula rat model provides insight into the gut hormone concentrations to which the neurons and cells in the lamina propria of the gut are likely exposed.     

Important species differences regarding lymph contribution to gut hormone responses

IntroductionGLP-1 is secreted from the gut upon nutrient intake and stimulates insulin secretion. The lymph draining the intestine may transport high levels of GLP-1 to the systemic circulation before it is metabolized by DPP-4. The aims of this study were to investigate to what extent the lymphatic system might contribute to the final level(s) of systemic circulating intact GLP-1 and, in addition, whether secretory profiles in intestinal lymph might reflect lamina propria levels of GLP-1 i.e. before capillary uptake and degradation by endothelial dipeptidyl peptidase-4 (DPP-4).Method7 pigs of the YDL-strain were catheterized in the portal vein, carotid artery and cisterna chyli (lymph). Neuromedin C (NC) was infused through an ear vein catheter, before and after injection of a selective DPP-4 inhibitor (vildagliptin). Total and intact GLP-1 levels were measured throughout the 150 min experiments using specific sandwich ELISAs. DPP-4 activity was measured spectrophotometrically.ResultsConcentrations of both total and intact GLP-1 were markedly lower in lymph compared to plasma samples, and did not increase significantly in response to stimulation with NC in the absence/presence of vildagliptin. In contrast, total and intact GLP-1 levels increased significantly in the portal vein and carotid artery. DPP-4 activity was lower in lymph than plasma, and was reduced further by vildagliptin.ConclusionOur observations indicate that the lymphatic system does not transport high levels of intact GLP-1 to the systemic circulation, and that GLP-1 levels in cisternal lymph do not reflect the hormone levels in the intestinal lamina propria.     

High-fat diet effects on gut motility,hormone, and appetite responses to duodenal lipid in healthy men

There is evidence that gastrointestinal function adapts in response to a high-fat (HF) diet. This study investigated the hypothesis that an HF diet modifies the acute effects of duodenal lipid on appetite, antropyloroduodenal pressures, plasma CCK and plasma glucagon-like peptide-1 (GLP-1) levels in humans. Twelve healthy men were studied twice in randomized, crossover fashion. The effects of a 90-min duodenal lipid infusion (6.3 kJ/min) on the above parameters were assessed immediately following 14-day periods on either an HF or a low-fat (LF) diet. After the HF diet, pyloric tonic and phasic pressures were attenuated, and the number of antropyloroduodenal pressure-wave sequences was increased when compared with the LF diet. Plasma CCK and GLP-1 levels did not differ between the two diets. Hunger was greater during the lipid infusion following the HF diet, but there was no difference in food intake. Therefore, exposure to an HF diet for 14 days attenuates the effects of duodenal lipid on antropyloroduodenal pressures and hunger without affecting food intake or plasma hormone levels.     

Peripheral administration of GLP-2 to humans has no effect on gastric emptying or satiety

Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are secreted in parallel to the circulation after a meal. Intravenous (IV) GLP-1 has an inhibitory effect on gastric emptying, hunger and food intake in man. In rodents, central administration of GLP-2 increases satiety similar to GLP-1. The aim of the present study was to assess the effect of IV administered GLP-2 on gastric emptying and feelings of hunger in human volunteers. In eight (five men) healthy subjects (age 31.1+/-2.9 years and BMI 24.1+/-1.0 kg m(-2)), scintigraphic solid gastric emptying, hunger ratings (VAS) and plasma concentrations of GLP-2 were studied during infusion of saline or GLP-2 (0.75 and 2.25 pmol kg(-1) min(-1)) for a total of 180 min. Concentrations of GLP-2 were elevated to a maximum of 50 and 110 pmol l(-1) for 0.75 and 2.25 pmol kg(-1) min(-1) infusion of GLP-2, respectively. There was no effect of GLP-2 on either the lag phase (29.5+/-4.4, 26.0+/-5.2 and 21.2+/-3.6 min for saline, GLP-2 0.75 or 2.25 pmol kg(-1) min(-1), respectively) or the half emptying time (84.5+/-6.1, 89.5+/-17.8 and 85.0+/-7.0 min for saline, GLP-2 0.75 or 2.25 pmol kg(-1) min(-1), respectively). The change in hunger rating after the meal to 180 min was also unaffected by infusion of GLP-2. GLP-2 does not seem to mediate the ileal brake mechanism.     

Impact of gender on the cardiac autonomic response to angiotensin II in healthy humans

Premenopausal women have a lower risk of cardiovascular disease (CVD) compared with men of a similar age. Furthermore, the regulation of factors that influence CVD appears to differ between the sexes, including control of the autonomic nervous system (ANS) and the renin-angiotensin system. We examined the cardiac ANS response to angiotensin II (Ang II) challenge in healthy subjects to determine whether differences in women and men exist. Thirty-six healthy subjects (21 women, 15 men, age 38 ± 2 years) were studied in a high-salt balance. Heart-rate variability (HRV) was calculated by spectral power analysis [low-frequency (LF) sympathetic modulation, high-frequency (HF) parasympathetic/vagal modulation, and LF:HF as a measure of overall ANS balance]. HRV was assessed at baseline and in response to graded Ang II infusions (3 ng·kg(-1)·min(-1) × 30 min; 6 ng·kg(-1)·min(-1) × 30 min). Cardiac ANS tone did not change significantly in women after each Ang II dose [3 ng·kg(-1)·min(-1) mean change (Δ)LF:HF (mean ± SE) 0.5 ± 0.3, P = 0.8, vs. baseline; 6 ng·kg(-1)·min(-1) ΔLF:HF (mean ± SE) 0.5 ± 0.4, P = 0.4, vs. baseline], whereas men exhibited an unfavorable shift in overall cardiac ANS activity in response to Ang II (ΔLF:HF 2.6 ± 0.2, P = 0.01, vs. baseline; P = 0.02 vs. female response). This imbalance in sympathovagal tone appeared to be largely driven by a withdrawal in cardioprotective vagal activity in response to Ang II challenge [ΔHF normalized units (nu), -5.8 ± 2.9, P = 0.01, vs. baseline; P = 0.006 vs. women] rather than an increase in sympathetic activity (ΔLF nu, -4.5 ± 5.7, P = 0.3, vs. baseline; P = 0.5 vs. women). Premenopausal women maintain cardiac ANS tone in response to Ang II challenge, whereas similarly aged men exhibit an unfavorable shift in cardiovagal activity. Understanding the role of gender in ANS modulation may help guide risk-reduction strategies in high-risk CVD populations.     

Growth hormone-mediated breakdown of body fat: insulin and leptin responses to GH are modulated by diet composition and caloric intake in old rats.

This work was performed to elucidate whether growth hormone (GH)-mediated loss of adipose tissue and responses in plasma insulin and leptin are modulated by diet composition. 12-month-old rats were first fed a high-fat (HF) diet or a low-fat (LF) diet for 14 weeks. After that, GH or saline was administered to rat groups that were maintained on either HF or LF diets or that were switched from the HF to the LF diet. All 6 groups had free access to food. One additional saline group was pair-fed with the GH group that was switched from the HF to the LF diet. The caloric consumption of this latter group was also translated to yet another GH group receiving restricted amounts of the HF diet. GH was given in a total dose of 4 mg/kg/d for three weeks. After sacrifice, blood was collected and tissues were excised. In groups injected with saline, the weight of excised adipose tissue was 60 +/- 4.7, 41 +/- 3.8 and 50 +/- 4.5 g in animals that continued with the HF diet, LF diet, or that were switched from HF to LF, respectively. Corresponding figures after GH treatment were significantly (p < 0.05) decreased to 38 +/- 2.7, 30 +/- 2.3, and 31 +/- 2.7 g, respectively. Pair-feeding had no effect, whereas only 26 +/- 3.0 g of adipose tissue was retrieved in rats fed restricted amounts of HF diet while receiving GH. In this group, plasma insulin and leptin were also significantly (p < 0.05) depressed compared with other GH groups, especially to the group fed the unrestricted HF diet (203 +/- 35 vs. 1345 +/- 160 pmol/l and 9.3 +/- 1.2 vs. 31 +/- 4.4 micro g/l). In conclusion, this study shows that GH mediates breakdown of adipose tissue under a variety of dietary conditions, and that induction of hyperinsulinemia can be prevented if GH treatment is combined with restricted feeding of a diet which is relatively low in carbohydrates and rich in fat. This will also promote a fall of plasma leptin.     

Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are both incretin hormones regulating postprandial insulin secretion. Their relative importance in this respect under normal physiological conditions is unclear, however, and the aim of the present investigation was to evaluate this. Eight healthy male volunteers (mean age: 23 (range 20-25) years; mean body mass index: 22.2 (range 19.3-25.4) kg/m2) participated in studies involving stepwise glucose clamping at fasting plasma glucose levels and at 6 and 7 mmol/l. Physiological amounts of either GIP (1.5 pmol/kg/min), GLP-1(7-36)amide (0.33 pmol/kg/min) or saline were infused for three periods of 30 min at each glucose level, with 1 h "washout" between the infusions. On a separate day, a standard meal test (566 kcal) was performed. During the meal test, peak insulin concentrations were observed after 30 min and amounted to 223+/-27 pmol/l. Glucose+saline infusions induced only minor increases in insulin concentrations. GLP-1 and GIP infusions induced significant and similar increases at fasting glucose levels and at 6 mmol/l. At 7 mmol/l, further increases were seen, with GLP-1 effects exceeding those of GIP. Insulin concentrations at the end of the three infusion periods (60, 150 and 240 min) during the GIP clamp amounted to 53+/-5, 79+/-8 and 113+/-15 pmol/l, respectively. Corresponding results were 47+/-7, 95+/-10 and 171+/-21 pmol/l, respectively, during the GLP-1 clamp. C-peptide responses were similar. Total and intact incretin hormone concentrations during the clamp studies were higher compared to the meal test, but within physiological limits. Glucose infusion alone significantly inhibited glucagon secretion, which was further inhibited by GLP-1 but not by GIP infusion. We conclude that during normal physiological plasma glucose levels, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide contribute nearly equally to the incretin effect in humans, because their differences in concentration and potency outweigh each other.     

The inhibitory mechanism of GLP-1, but not glucagon, on fasted gut motility is dependent on the L-arginine/nitric oxide pathway

Effects of glucagon-like peptide-1 (GLP-1) and glucagon on fasted gut motility in conscious rats were investigated as regards dependence on nitric oxide (NO). Small bowel motility was studied by electromyography and a jugular vein catheter was implanted for administration of drugs. GLP-1 (5-40 pmol x kg(-1) x min(-1)) prolonged the cycle length and abolished phase III of the migrating myoelectric complex (MMC) (P<0.01). Low doses of GLP-1 did not affect duration, propagation velocity or calculated length of phase III. At 1 mg x kg(-1) N(omega)-nitro-L-arginine (L-NNA) blocked the GLP-1 response up to a dose of 10 pmol x kg(-1) x min(-1) (P<0.05), while higher doses were able to overcome L-NNA-induced disinhibition of the GLP-1 response (P<0.05). Similarly, L-arginine at 300 mg x kg(-1) prevented L-NNA-induced disinhibition of the GLP-1 response (P<0.05). Glucagon (200-1000 pmol x kg(-1) x min(-1)) prolonged the cycle length and abolished phase III of MMC (P<0.01) independent of NO. Again, low doses of glucagon did not affect duration, propagation velocity or calculated length of phase III. In conclusion, inhibition of fasted motility by GLP-1 at low doses is dependent on NO, while high doses of GLP-1 and glucagon exert effects on motility independently from NO.     

A novel monoclonal antibody specific for lymphatic endothelium

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G₁ immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gall-bladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.     

Exogenously imposed postprandial-like rises in systemic glucose and GLP-1 do not produce an incretin effect, suggesting an indirect mechanism of GLP-1 action

The insulinotropic intestinal hormone GLP-1 is thought to exert one of its effects by direct action on the pancreatic beta-cell receptors. GLP-1 is rapidly degraded in plasma, such that only a small amount of the active form reaches the pancreas, making it questionable whether this amount is sufficient to produce a direct incretin effect. The aim of our study was to assess, in a dog model, the putative incretin action of GLP-1 acting directly on the beta-cell in the context of postprandial rises in GLP-1 and glucose. Conscious dogs were fed a high-fat, high-carbohydrate meal, and insulin response was measured. We also infused systemic glucose plus GLP-1, or glucose alone, to simulate the meal test values of these variables and measured insulin response. The results were as follows: during the meal, we measured a robust insulin response (52 +/- 9 to 136 +/- 14 pmol/l, P < 0.05 vs. basal) with increases in portal glucose and GLP-1 but only limited increases in systemic glucose (5.3 +/- 0.1 to 5.7 +/- 0.1 mmol/l, P = 0.1 vs. basal) and GLP-1 (6 +/- 0 to 9 +/- 1 pmol/l, P = 0.5 vs. basal). Exogenous infusion of systemic glucose and GLP-1 produced a moderate increase in insulin (43 +/- 5 to 84 +/- 15 pmol/l, 43% of the meal insulin). However, infusion of glucose alone, without GLP-1, produced a similar insulin response (37 +/- 6 to 82 +/- 14 pmol, 53% of the meal insulin, P = 0.7 vs. glucose and GLP-1 infusion). In conclusion, in dogs with postprandial rises in systemic glucose and GLP-1, the hormone might not have a direct insulinotropic effect and could regulate glycemia via indirect, portohepatic-initiated neural mechanisms.     

Glucagon-like peptide 1 (GLP-1) suppresses ghrelin levels in humans via increased insulin secretion

INTRODUCTION: Ghrelin is an orexigenic peptide predominantly secreted by the stomach. Ghrelin plasma levels rise before meal ingestion and sharply decline afterwards, but the mechanisms controlling ghrelin secretion are largely unknown. Since meal ingestion also elicits the secretion of the incretin hormone glucagon-like peptide 1 (GLP-1), we examined whether exogenous GLP-1 administration reduces ghrelin secretion in humans. PATIENTS AND METHODS: 14 healthy male volunteers were given intravenous infusions of GLP-1(1.2 pmol x kg(-1) min(-1)) or placebo over 390 min. After 30 min, a solid test meal was served. Venous blood was drawn frequently for the determination of glucose, insulin, C-peptide, GLP-1 and ghrelin. RESULTS: During the infusion of exogenous GLP-1 and placebo, GLP-1 plasma concentrations reached steady-state levels of 139+/-15 pmol/l and 12+/-2 pmol/l, respectively (p<0.0001). During placebo infusion, ghrelin levels were significantly reduced in the immediate postprandial period (p<0.001), and rose again afterwards. GLP-1 administration prevented the initial postprandial decline in ghrelin levels, possibly as a result of delayed gastric emptying, and significantly reduced ghrelin levels 150 and 360 min after meal ingestion (p<0.05). The patterns of ghrelin concentrations in the experiments with GLP-1 and placebo administration were inversely related to the respective plasma levels of insulin and C-peptide. CONCLUSIONS: GLP-1 reduces the rise in ghrelin levels in the late postprandial period at supraphysiological plasma levels. Most likely, these effects are indirectly mediated through its insulinotropic action. The GLP-1-induced suppression of ghrelin secretion might be involved in its anorexic effects.     

Isolation and purification of afferent lymph dendritic cells that drain the skin of cattle

Dendritic cells (DCs) are central to the induction of immune responses and are a pivotal control point that determines the outcome of infectious challenge. Cannulation of afferent lymphatic vessels allows the isolation of large numbers of lymph DCs. First, lymph nodes that are draining the skin are surgically removed (takes approximately 1 h). Over a period of 6-8 weeks, afferent lymphatic vessels re-anastomose with the efferent duct, forming larger 'pseudoafferent' lymphatic vessels that can be surgically cannulated. Surgical cannulation takes 2 h to perform; daily maintenance of the catheter requires 30 min. Isolation of lymph cells requires 1 h and an additional 60-180 min to enrich or purify the DCs. The lymph can be harvested for up to 1 month, with relatively constant cell numbers and subset distribution throughout this period. This technique, although technically demanding, facilitates studies of DCs and other cells that traffic in the lymph in both the steady state and following antigenic exposure.     

Insulin transport by thoracic duct of male rabbits with CCI4-induced liver cirrhosis.

Insulin concentration in the peripheral blood and the thoracic duct lymph from male rabbits with CCl4-induced liver cirrhosis was measured using a radioimmunoassay technique. Although insulin concentration in the lymph was lower in the cirrhotic animals than in the control ones, the hourly transport of the hormone by the lymphatic vessel of the cirrhotic animals markedly increased in company with the higher flow rate of the lymph.     

Absence of a memory effect for the insulinotropic action of glucagon-like peptide 1 (GLP-1) in healthy volunteers.

BACKGROUND/AIMS: The term memory effect refers to the phenomenon that B cell stimuli retain some of their insulinotropic effects after they have been removed. Memory effects exist for glucose and sulfonylureas. It is not known whether there is a B-cell memory for incretin hormones such as GLP-1. SUBJECTS/METHODS: Eight healthy young volunteers were studied on four occasions in the fasting state. In one experiment, placebo was administered (a). in three more experiments (random order), synthetic GLP-1 (7 - 36 amide) at 1.2 pmol/kg/min was administered over a period of three hours. At 0 min, a bolus of glucose was injected intravenously (0.33 g/kg body weight). GLP-1 was infused from (b). - 60 to 120 min, (c). - 210 to - 30 min, or (d). - 300 to - 120 min. Glucose (glucose oxidase), insulin, C-peptide, GLP-1, and glucagon (immunoassays) were determined. Statistical analysis was carried out by ANOVA and appropriate post hoc tests. RESULTS: GLP-1 plasma levels during the infusion periods were elevated to 89 +/- 9, 85 +/- 13, and 89 +/- 6 pmol/l (p < 0.0001 vs. placebo, 10 +/- 1 pmol/l). Glucose was eliminated faster (p < 0.0001), with an enhanced negative rebound (p = 0.014), and insulin and C-peptide increments were greater after intravenous glucose administration (p < 0.0001) if GLP-1 was administered during the injection of the glucose bolus, but not if GLP-1 had been administered until 120 or 30 min before the glucose load. There was a trend towards higher insulin concentrations (p = 0.056) five minutes after glucose with GLP-1 administered until - 30 min before the glucose load. Glucagon was suppressed by exogenous glucose, but increased significantly (p = 0.013) during the induction of reactive hypoglycemia after glucose injection during GLP-1 administration. CONCLUSION: 1). No memory effect appears to exist for insulinotropic actions of GLP-1, in line with clinical data. 2). Reactive hypoglycemia causes a prompt rise in glucagon despite pharmacological circulating concentrations of GLP-1. 3). Similar studies should be performed in Type 2-diabetic patients, because exposure to GLP-1 might recruit dormant pancreatic B cells to become glucose-competent, and this might contribute to the overall antidiabetogenic effect of GLP-1 in such patients.     

Minimally invasive quantification of lymph flow in mice and rats by imaging depot clearance of near-infrared albumin

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.     

Green tea extract markedly lowers the lymphatic absorption and increases the biliary secretion of 14C-benzo[a]pyrene in rats

Previously, we have shown that green tea extract (GTE) lowers the intestinal absorption of lipids and lipophilic compounds in rats. This study was conducted to investigate whether GTE inhibits the intestinal absorption and biliary secretion of benzo[a]pyrene (BaP), an extremely lipophilic potent carcinogen, present in foods as a contaminant. Male rats with lymph or bile duct cannula were infused at 3.0 ml/h for 8 h via a duodenal catheter with lipid emulsion containing (14)C-BaP with or without GTE in PBS buffer. Lymph and bile were collected hourly for 8 h. The (14)C-radioactivities in lymph, bile and intestine were determined and expressed as % dose infused. Results showed that GTE drastically lowered the lymphatic absorption of (14)C-BaP (7.6±3.2% in GTE-infused vs. 14.4±2.7% dose/8 h in control rats), with a significantly higher amount of (14)C-radioactivity present in the small intestinal lumen and cecum in rats infused with GTE. GTE also markedly increased the hourly rate (3.9±0.1% dose/h in GTE-infused vs. 3.0±0.1% dose/h in control rats) and the total biliary secretion of (14)C-BaP (31.5±0.8% dose/8 h in GTE-infused vs. 24.3±0.4% dose/8 h in control rats). The findings provide first direct evidence that GTE has a profound inhibitory effect on the intestinal absorption of BaP and promotes the excretion of absorbed BaP via the biliary route. Further studies are warranted to investigate whether green tea could be recommended as a dietary means of ameliorating the toxicity and carcinogenic effect of BaP.     

Soybean Phosphatidylcholine-Induced Enhancement of Lymphatic Absorption of Triglyceride Depends on Chylomicron Formation in Rats

We investigated whether chylomicron formation is involved in the dietary phosphatidylcholine (PC)-induced increase in triglyceride (TG) absorption using an inhibitor of chylomicron formation, pluronic L-81 (L-81). In rats, cannulas were implanted into the duodenum (exps. 1 and 2) and the mesenteric lymph duct (exp. 1), and an emulsified lipid solution containing the test lipids (soybean oil, SO or soybean oil plus phosphatidylcholine, LE) with or without L-81 was infused through a duodenal cannula at a rate 3 ml/h for 2 h, and followed by infusion of a glucose–NaCl solution for 2 h. Mesenteric lymph was collected for 4 h (exp. 1). In exp. 2, the mucosa and contents of the small intestine were collected at 20, 40, or 90 min after the start of duodenal infusion of the test lipid to evaluate accumulation of lipids incorporated into the mucosa in the rats without a lymph cannula. In exp. 1, lymphatic TG outputs rapidly increased with infusion of both test lipids without L-81, but L-81 abolished these increases. TG accumulated in the small intestinal mucosa with L-81 treatment in a time-dependent manner, but the levels of accumulation were similar between the SO and LE groups (exp. 2). There were no differences in the amounts of lipid remaining in the small intestinal lumen between the L-81-treated SO and LE groups. These results indicate that uptake of lipid into the mucosal cells was not increased by LE. We conclude that the formation of chylomicron is responsible for increases in the promotive effect of a high level of dietary PC on the lymphatic absorption of TG.