Active ghrelin levels across time and associations with leptin and anthropometrics in healthy ache Amerindian women of Paraguay

Active (acylated) ghrelin is a peptide hormone secreted primarily by the stomach, positively associated with fasting, orexigenic, and promotes growth hormone secretion. It is therefore important to energy intake management. The objective of this pilot research was to (1) compare active ghrelin with previous measurements of leptin and anthropometrics; (2) assess the consistency of active ghrelin across time in this population; (3) extend our understanding of potential population variation in active ghrelin. Two serum samples separated by 10 days at the same time between meals were collected from healthy Ache women (n = 12, mean age 32.2 ± 14.0 SD) to determine consistency over time, associations with leptin, and anthropmetric values. Mean active ghrelin was 72.9 ± 23.0 pg/ml, highly correlated (r² = 0.95, P < 0.0001) between collections, and showed no paired mean differences (P < 0.18). There was no significant correlation with leptin, age, or anthropometric measures. Active ghrelin appears to be consistent over time in this population, perhaps reflecting regimented meal schedules and less interpopulation variation compared to leptin. Am. J. Hum. Biol., 2008. © 2007 Wiley‐Liss, Inc.     

Decreased food intake following overfeeding involves leptin-dependent and leptin-independent mechanisms

After a period of forced overfeeding, many individuals actively compensate for this weight gain by reducing food intake and maintaining this state of hypophagia well into the post-overfeeding period. Our central goal is to define the mechanism underlying this adaptive reduction in food intake. When male Long Evans rats were implanted with indwelling gastric cannula and overfed a liquid low-fat (10% fat) diet for 17 days, overfed rats exhibited increased weight gain (P < 0.01) but decreased food intake, and this hypophagia persisted for 4-6 days post-overfeeding (P < 0.05). Leptin levels were increased 8-fold by overfeeding (P < 0.01), yet returned to baseline within 2 days post-overfeeding, despite the persistent hypophagia. Energy expenditure and oxygen consumption (VO2) were increased on the first day post-overfeeding (P < 0.05), but subsequently normalized prior to the normalization of food intake. Lastly, in leptin receptor deficient Obese Zucker (fa/fa) rats, overfeeding produced a significant decrease in food intake during active overfeeding. However, food intake returned to near baseline levels within one day post-overfeeding. Contrastingly, food intake remained suppressed in lean controls for 6 days post-overfeeding. Thus intact leptin signaling is not required for the decrease in food intake that occurs during overfeeding, but the ability to maintain this hypophagia is substantially impaired in the absence of leptin signaling. In addition, this post-overfeeding leptin effect appears to occur despite the fact that leptin levels normalize relatively rapidly post-overfeeding.     

Ghrelin and PYY levels in adolescents with severe obesity: effects of weight loss induced by long-term exercise training and modified food habits

This study investigated (a) changes in ghrelin and peptide YY (PYY) concentrations during a weight reduction programme and (b) baseline ghrelin and PYY levels as predictors of weight loss in 32 severely obese adolescents (BMI z score = 4.1). Subjects spent an academic year in an institution for childhood obesity. Fasting ghrelin and PYY, leptin, insulin levels and insulin resistance were measured at baseline (month 0) and during the programme (months 3, 6, 9). In addition, 15 normal-weight teenagers served as reference for the baseline assessments. At baseline, obese teenagers had lower ghrelin and PYY concentrations than normal-weight adolescents (P < 0.05). Moreover, they showed significantly higher leptin, insulin levels and homeostasis model assessment (HOMA) (P < 0.0001). During the lifestyle modification, there was a significant decrease in body weight among obese teenagers, associated with an increase in ghrelin (apparent from month 6; P < 0.05), a decrease in leptin (from month 3; P < 0.05) and a decrease in insulin and HOMA (from month 3; P < 0.0001), without any significant change in PYY. Anthropometrical changes were correlated neither with baseline ghrelin levels nor with changes in ghrelin and PYY after the lifestyle modification. However, higher baseline PYY tended to correlate with greater anthropometrical changes (P < 0.1). In adolescents with severe obesity, a long-term combination of supervised aerobic exercises and a balanced diet led to weight reduction and increased ghrelin concentrations, without any change in PYY concentrations. Moreover, baseline PYY concentrations might be considered as predictors of weight loss.     

Differential effects of methamphetamine on expression of neuropeptide Y mRNA in hypothalamus and on serum leptin and ghrelin concentrations in ad libitum-fed and schedule-fed rats

Relatively little is known concerning the interaction of psychostimulants with hypothalamic neuropeptide systems or metabolic hormones implicated in regulation of energy balance. The present studies tested whether methamphetamine alters the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP), two important orexigenic neuropeptides, or proopiomelanocortin (POMC), the precursor for the anorexigenic peptide alpha-melanocyte-stimulating hormone, or the secretion of leptin, insulin and ghrelin, concomitant with inhibition of food intake. Female rats were either fed ad libitum (AL) or placed on a scheduled feeding (SF) regimen, with access to food limited to 4 h/day. Administration of (+/-)-methamphetamine (7.5 mg/kg, i.p.) 2 h prior to food presentation significantly inhibited food intake in SF animals, but did not affect intake in AL animals. In a separate study, AL and SF animals were killed just prior to expected food presentation, and expression of NPY, AgRP and POMC mRNAs in hypothalamus was determined using in situ hybridisation; concentrations of leptin, insulin and ghrelin in serum were determined with radioimmunoassays. In saline-treated, SF controls, NPY and AgRP mRNA expression in arcuate nucleus and serum ghrelin were significantly elevated, and serum leptin and insulin were significantly reduced. Methamphetamine reversed the up-regulation of NPY mRNA expression observed in the SF condition, without affecting AgRP mRNA or the serum concentrations of metabolic hormones. However, in AL animals, NPY mRNA expression in arcuate and dorsomedial nuclei was significantly increased by methamphetamine, which also reduced serum leptin and insulin and increased serum ghrelin concentrations. These findings suggest that the inhibition of NPY expression in SF animals may be a mechanism underlying the anorexigenic effect of methamphetamine seen in this condition. The increase in NPY expression produced by methamphetamine in AL animals may be mediated by the ability of this drug to decrease secretion of leptin and insulin and increase secretion of ghrelin.     

Resistin as a putative modulator of insulin action in the daily feeding/fasting rhythm

Resistin and adiponectin are adipokines with postulated opposite functions. Resistin has been related with insulin resistance in obesity, while adiponectin could be associated to higher insulin sensitivity. We have determined whether the production of these two adipokines during the day is related to the feeding rhythm in rats. Resistin mRNA levels in adipose tissue correlated positively with the gastric contents and serum insulin concentration, showing higher levels during the dark phase (period of the highest food intake), especially in the mesenteric depot, while levels decreased during the light phase. The diurnal pattern of resistin expression was not directly reflected in the circulating levels, but it showed a 6-h delay and correlated negatively with the gastric contents and serum insulin. Adiponectin expression followed an opposite pattern, not apparently related to feeding or insulin release, and not translated into changes in circulating levels. Moreover, considering that insulin stimulates resistin expression and that circulating resistin follows a contrary circadian pattern in comparison to insulin, resistin, apart from its role in the increased insulin resistance associated to obesity, could also act as a putative modulator of insulin in the daily feeding/fasting rhythm through a negative feedback regulation of its action.     

Ontogenesis of leptin expression in different adipose tissue depots in the rat

Serum leptin levels and leptin mRNA expression by adipose tissue increase with age and are mainly associated with an increase in adiposity. Regional changes in both leptin production and fat distribution contribute to circulating leptin levels and may play a role in the regulation of body weight. a capacity that changes during development. Here, we have studied leptin mRNA expression in four different white adipose tissue depots (epididymal, retroperitoneal, mesenteric, inguinal; namely, EWAT, RWAT, MWAT, IWAT) and in interscapular brown adipose tissue (IBAT). We have also studied their relationship with lipid content and adiposity changes, together with serum leptin levels in male rats at different ages (18, 55, 93, 159, 212, 294 and 355 days). Serum leptin levels increased during development, reaching stable levels at the age of 7 months, and, as expected, were highly correlated with both the adiposity index (r=0.908, P<0.01) and body weight (r=0.906, P<0.01). Leptin mRNA expression also increased with age, following characteristic ontogenic patterns in every adipose tissue depot. The patterns were similar in EWAT and RWAT: leptin expression increased very rapidly during the first 55 days for EWAT and 3 months for RWAT, with a peak in the latter at 7 months, and high expression levels were retained for the rest of the study period. In IWAT and IBAT, leptin expression increased steadily during the 12-month period studied and was significantly lower than levels in EWAT and RWAT. Leptin expression in MWAT increased progressively with age to reach levels close to those of EWAT and RWAT in 10-month-old animals. The pattern of leptin expression in both EWAT and RWAT paralleled their lipid content, and leptin mRNA expression per unit of tissue lipid content was maintained high and constant from a very young age (about 2 and 3 months, respectively). However, the expression of mRNA for leptin (expressed per unit of tissue lipid concentration) in MWAT, IWAT and IBAT increased steadily during the whole period studied, without attaining the maximal levels observed in EWAT and RWAT. MWAT, IWAT and IBAT maintained their capacity to increase leptin mRNA expression in response to an additional accumulation of lipids. Our data demonstrate that there are regional-specific differences and different rates of increase of leptin gene expression within distinct depots of WAT and BAT. These changes cannot be uniquely explained by changes in adiposity or lipid content, implying that there are regional-specific regulatory mechanisms that may depend on the attenuation with age of the beta-adrenergic inhibitory signalling pathway upon leptin expression or on other factors.     

First Evidence of a Leptin‐Like Peptide in a Cartilaginous Fish

Leptin is a hormone involved in food intake. Although leptin is evolutionarily conserved, no studies have investigated its presence in cartilaginous fish. Here, we report the presence of leptin‐like immunoreactivity in the gastro‐intestinal tract and liver of the cartilaginous fish Scyliorhinus canicula using western blot and immunohistochemical analyses. A leptin‐like immunoreactive band of 16 kDa was detected in the homogenate of the stomach, whereas no immunoreactivity was observed in the intestine or the liver. Immunohistochemistry of the gastric mucosa revealed leptin‐like staining localized to mucous‐secreting cells and endocrine cells. This is the first report of a leptin‐like peptide in a cartilaginous fish. Anat Rec 293:1692–1697, 2010. © 2010 Wiley‐Liss, Inc.     

Leptin amplifies the feeding inhibition and neural activation arising from a gastric nutrient preload

Leptin affects food intake by reducing meal size, suggesting that it may modulate the efficacy of within-meal satiety signals. To assess whether leptin would amplify the feeding inhibitory actions of a nutrient gastric preload, we compared liquid diet food intake and patterns of c-Fos activation in response to intraventricular leptin (3.5 microg), intragastric Ensure (10 ml over 10 min), or their combination. Leptin alone did not affect Ensure intake but significantly increased the suppression of intake produced by the intragastric preload. Within the nucleus of the solitary tract (NTS), leptin alone did not stimulate c-Fos but significantly elevated the number of c-Fos positive cells in response to intragastric Ensure at medial and rostral levels. Within the paraventricular nucleus (PVN), both leptin and the gastric load stimulated c-Fos expression, but the combination resulted in significantly greater number of c-Fos positive cells. These data demonstrate that leptin modulates the feeding inhibition produced by meal-related signals and suggest that this modulation occurs at the levels of the NTS and PVN.     

Effects of chronic central leptin infusion on proopiomelanocortin and neurotensin gene expression in the rat hypothalamus

Leptin signaling in the hypothalamus is critical for normal food intake and body weight regulation. While hyperleptinemia in obese people suggests a state of leptin resistance, the mechanism is not clearly understood. In a rat model of central leptin infusion in which animals develop resistance to the satiety action of leptin, orexigenic peptide producing neuropeptide Y neurons in the hypothalamus develop leptin resistance. However, it is still unknown if increased hypothalamic leptin tone caused by central leptin infusion results in the development of leptin resistance in anorexigenic peptide producing proopiomelanocortin (POMC) and neurotensin (NT) neurons. To this end, male rats were infused chronically with leptin (160 ng/h) or vehicle into the lateral cerebroventricle for 16 days. On day 4 of leptin infusion when food intake was decreased, POMC and NT mRNA levels, as determined by RNAse protection assay, were significantly increased as compared to control. By contrast, on day 16 of leptin infusion, when food intake was mostly normalized, both POMC and NT mRNA levels remained unchanged compared with control. These findings suggest the development of leptin resistance in the POMC and NT neurons following chronic elevation of hypothalamic leptin tone, which may be involved in the development of resistance to the satiety action of leptin following central infusion of this peptide hormone.     

ESTABLISHMENT OF A HIGHLY SENSITIVE LEPTIN RADIOIMMUNOASSAY AND DETECTION OF INCREASED LEPTIN LEVELS IN HYPERLIPIDEMIA AND PREGNANCY

ABSTRACT

The highly effective antibody has been obtained by immunizing rabbits with recombinant leptin many times. The leptin is iodinated with the chloramine-T method and purified with a Sephadex-G25 chromatography column. The reaction between antigen and antibody is carried out by a one-step balance method and cultured at 4°C for 24 h; the binding and free antigen was then separated by PR reagent. The determining range of this method is about 0.5–24 ng/mL; limited detection level is 0.45 ng/mL, relative standard deviation in a group, and among groups, are less than 5.4% and 8%, respectively. The level of blood leptin in 277 samples of normal persons, in 112 samples of overweight persons (weight/hieght m² ≥ 25) and 224 samples of hyperlipidemic patients have been measured by this method. It is demonstrated that the level of blood leptin in males is much lower than that of the females, and becomes elevated with increased age. Serum leptin level in overweight persons and hyperlipidemic patients is also much higher than that of normal groups (P<0.01). Serum leptin of 21 workers in our lab at 8:00AM and 4:00PM has been tested. It was found that there are no differences between the two time points. The same results are obtained within age groups. Leptin levels of pregnant women's serum is higher than those of the control group (P<0.001). Leptin in newborn's serum is significantly lower than those of mothers (P<0.01). There is no obvious correlation between leptin level of mother and newborns by correlation analysis (r = 0.19, P>0.05). The body weight and body weight index of pregnant women are well correlated with their serum leptin levels (r = 0.33 and 0.35, P<0.05). The body weight and body weight index of newborns are well correlated with their serum leptin levels (r = 0.54 and 0.49, P<0.001). The serum leptin level of pregnant women is not correlated with newborn's body weight (r = 0.10). These results have shown that the proposed method is stable, simple, and specific, being sensitive enough to determine leptin levels in human serum or plasma.     

The influence of increased training volume on cytokines and ghrelin concentration in college level male rowers

The aim of this study was to investigate plasma IL-6, TNF-α, leptin and ghrelin concentrations during high-volume training. Eight trained male rowers participated. Fasting blood was sampled before (T1) and after (T2) increased training volume and after recovery period (T3). Two-hour rowing was performed at T1, T2 and at T3 with blood samples before, POST and POST 30′. Decrease in fasting leptin was observed at T2 (from 1.31 (0.53) to 0.93 (0.27) ng ml⁻¹; P < 0.05). Leptin was also significantly decreased at POST and POST 30 exercise compared to PRE test at T2. At T2 POST 30′ leptin was significantly lower compared to corresponding value at T1. There were no significant post-exercise changes in ghrelin at T2 compared to T1 and T3. TNF-α was significantly increased POST exercise only at T2. In conclusion, high-volume training causes alterations of post exercise leptin and TNF-α, while increases in ghrelin are down regulated.     

Gastric capacity, test meal intake, and appetitive hormones in binge eating disorder

Binge eating disorder (BED), characterized by ingestion of very large meals without purging afterwards, is found in a subset of obese individuals. We showed previously that stomach capacity is greater in obese than in lean subjects, and in this study, we investigated capacity in obese individuals with BED. We also determined ad-libitum intake of a test meal until extremely full. Furthermore, we measured various appetitive hormones (insulin, leptin, glucagon, CCK, ghrelin) and glucose before a fixed meal and for 120 min afterwards. An acetaminophen tracer was used to assess gastric emptying rate. We compared three groups of overweight women: 11 BED, 13 BE (subthreshold BED), and 13 non-binge-eating normals. The BED individuals had the largest stomach capacity as assessed by either maximum volume tolerated (P=.05) or by gastric compliance to pressure (P=.02) using an intragastric balloon. Although test meal intake did not differ between groups, it correlated (P=.03) with gastric capacity. The BED group showed a tendency (P=.06) to have greater area under the curve (AUC) and had higher values at 5 and 60 min (P<.05) for insulin compared to normals. Moreover, the BED subjects had lower ghrelin baselines premeal, and lower AUC for ghrelin, which then declined less postmeal than for the normals (P<.05). None of the other blood values differed, including glucose, leptin glucagon, and CCK, as well as acetaminophen, reflecting gastric emptying. The lower ghrelin in BED, although contrary to what was expected, is consistent with lower ghrelin in obesity, and suggests down-regulation of ghrelin by overeating. The lack of differences in CCK is consistent with the lack of differences in gastric emptying rate, given that CCK is released when nutrients reach the intestine. The results show that BED subjects have a large gastric capacity as well as abnormalities in meal-related ghrelin and insulin patterns that may be factors in binge eating.     

Diurnal variation in circulating leptin is dependent on gender, food intake and circulating insulin in mice

Leptin is an adipocyte hormone involved in the regulation of energy homeostasis. Its circulating levels show a diurnal rhythm with a nocturnal peak. We examined the influences of gender, feeding state, and plasma insulin and glucose on the diurnal rhythm in normal mice. Plasma was sampled at 4-h interval for 24 h in female (n=80) and male (n=80) mice, which were freely fed or fasted. In both genders, plasma leptin displayed a diurnal rhythm with a nadir at 8 or 10 AM and a nocturnal peak at 10 PM to 2 AM. The nocturnal increase in leptin was higher in females (+160+/-18%) than in males (61+/- 16%; P<0.001), completely abolished by fasting, and correlated significantly to the diurnal variation in plasma insulin both in females (r=0.44, P=0.003) and males (r=0.46, P<0.001). Baseline plasma leptin in non-fasted animals were not different between the genders, whereas during fasting, the reduction in leptin was more pronounced in males than in females, resulting in a higher plasma leptin after fasting in females. Plasma insulin was higher in males under non-fasted conditions (P=0.003), but not significantly different between genders in fasted animals. In conclusion, plasma leptin displays a nocturnal increase in mice, which is more pronounced in female mice than in male mice, is completely abolished by fasting and correlates to the diurnal variation in circulating insulin. It is suggested that the nocturnal rise in leptin shows gender dependency and is caused by the increase in plasma insulin caused by food intake.     

Diurnal rhythm disorder of behavioral activity in SAMP1 mice is partially normalized by spontaneous wheel running

We examined the diurnal rhythms of food and water intake, spontaneous wheel running (SWR), and spontaneous motor activity (SMA) in the SAMP1 strain, a mouse model of accelerated senescence. Without SWR exercise, food, and water intake in the SAMP1 mice was significantly higher during the light (L)-phase of the light-darkness (LD) cycle than in the control SAMR1 strain. Additionally, SWR and SMA activity rhythms were split in SAMP1 mice, as demonstrated by the appearance of a secondary peak starting from the end of the dark (D)-phase. SWR exercise significantly increased the percentages of nocturnal food and water intake and SMA in the SAMP1 mice, although food and water intake did not reach the level of control SAMR1 mice. Thus, the disordered diurnal rhythms in SAMP1 mice can be normalized, even if only partially, by SWR exercise.     

Metabolic responses to intracerebroventricular leptin and restricted feeding

Leptin is a hormone secreted by adipocytes, which plays an important role in the control of food intake and metabolic processes. In the current study, a dose-dependent relationship was shown between a bolus intracerebroventricular rat recombinant leptin administration and reductions in food intake and body weight in Sprague-Dawley rats. During the 24 h postinjection period, food intake was decreased by 24, 26, and 52% with 0.625, 2.5, and 10 microg of leptin, respectively. Body weight was reduced by 2, 3, and 5% at 24 h after leptin administration at the doses of 0.156, 2.5, and 10 microg, respectively. Furthermore, indirect calorimetry demonstrated that five daily i.c.v. injections of leptin resulted in an increase in heat production per unit of metabolic body size and fat oxidation by approximately 10 and 48%, respectively. In contrast, food-restricted rats that consumed the equivalent amount of food as leptin-treated rats for 5 days decreased their energy expenditure by 10%. Food restriction was found to decrease respiratory quotient in a similar pattern as the leptin administration. When ad lib feeding was resumed, food-restricted rats quickly recovered their normal food intakes, body weights, and metabolism. Conversely, leptin treatment has prolonged effects on body weight resulting from different metabolic responses than food restriction. Leptin not only suppresses food intake, but also enhances energy expenditure to reduce fat depots.     

Modulation of the satiating effect of amylin by central ghrelin, leptin and insulin

Amylin is a pancreatic hormone that is considered to be a satiating signal acting on neurons of the area postrema (AP) in the hindbrain. The adiposity signals leptin and insulin act in the hypothalamus to influence feeding. They also enhance the hindbrain's responsivity to satiating signals, e.g. cholecystokinin (CCK). The orexigenic hormone ghrelin is thought to use the same hypothalamic pathways as leptin and insulin, with opposite actions on feeding behaviour. In fact, CCK and ghrelin also seem to interact in the control of feeding. Because CCK's anorectic effect depends on endogenous amylin, the aim of this study was therefore to evaluate a possible functional interaction between amylin and these hormones on short-term food intake in rats. The experiments were performed with male Wistar rats. Intracerebroventricular injection (i3vt) of an orexigenic dose of ghrelin (5 ng/5 microl) reduced but did not completely reverse the intraperitoneal amylin (5 microg/kg)-induced inhibition of food intake. In comparison, administration of a sub-threshold dose of ghrelin (3 ng/5 microl) did not affect the anorexigenic action of peripheral amylin. Leptin administered into the third ventricle (i3vt; 3.5 microg/5 microl) and intraperitoneal amylin (5 microg/kg) synergistically reduced food intake in chow-fed rats. I3vt insulin, administered at a sub-threshold dose (0.5 mU/5 microl), significantly enhanced the response to peripheral amylin. These results indicate that the lipostatic signals leptin and insulin may synergize with amylin to reduce food intake. In contrast, under the conditions tested, the orexigenic hormone ghrelin does not seem to influence the feeding response to peripheral amylin.     

Modulation of vagal afferent excitation and reduction of food intake by leptin and cholecystokinin

The gut-peptide, cholecystokinin (CCK), reduces food intake by acting at CCK-1 receptors on vagal afferent neurons, whereas the feeding effects of the adipokine hormone, leptin, are associated primarily with its action on receptors (ObRb) in the hypothalamus. Recently, however, ObRb mRNA has been reported in vagal afferent neurons, some of which also express CCK-1 receptor, suggesting that leptin, alone or in cooperation with CCK, might activate vagal afferent neurons, and influence food intake via a vagal route. To evaluate these possibilities we have been examining the cellular and behavioral effects of leptin and CCK on vagal afferent neurons. In cultured vagal afferent neurons leptin and CCK evoked short latency, transient depolarizations, often leading to action potentials, and increases in cytosolic calcium. There was a much higher prevalence of CCK and leptin sensitivity amongst cultured vagal afferent neurons that innervate stomach or duodenum than there was in the overall vagal afferent population. Furthermore, almost all leptin-responsive gastric and duodenal vagal afferents also were sensitive to CCK. Leptin, infused into the upper GI tract arterial supply, reduced meal size, and enhanced satiation evoked by CCK. These results indicate that vagal afferent neurons are activated by leptin, and that this activation is likely to participate in meal termination, perhaps by enhancing vagal sensitivity to CCK. Our findings are consistent with the view that leptin and CCK exert their influence on food intake by accessing multiple neural systems (viscerosensory, motivational, affective and motor) at multiple points along the neuroaxis.     

Circadian variation of food intake and digestive tract contents in the rat

The variation of food intake and digestive tract contents over a period of 24 hours was measured in the free feeding rat kept under a 12:12 hr light:dark cycle (lights on at 0700 hr). After a cessation between 0700 and 1000 hr, the rate of food intake increased progressively during daytime followed by a marked increase and a plateau after lights went off. In the dark, dry matter in the stomach was almost ten times the levels of the light phase. In the major part of the high feeding period, the rates of food intake and gastric emptying equilibrated. Dry matter content of the intestine also followed circadian variations that were specific for each segment. The time-to-time content of the mid-gut segment was closely related to gastric emptying. The extent of dry matter absorption in the proximal intestine decreased continuously from the time lights went on and reached a minimum at 22 hr. Absorption in the distal segments was much less subjected to variation over 24 hours. In the free feeding situation, the changes induced by the light cycle on the rhythm of food intake were accompanied by marked modifications of digestive tract contents including those of the intestine. Data indicate a close relationship between gastric function and the middle portion of the intestine.