Immunochemical identification of the mammalian peptide hormone obestatin from tea plant (Camellia sinensis).

This study determines obestatin-like substances from the young shoots of the tea plant [Camellia sinensis (L.) O. Kuntze (Theaceae)]. Proteins were extracted from the vegetative tea leaves using the QB (Quick Buffer) buffer as an extraction buffer. Obestatin-like substances in tea extract were investigated using an indirect home-made enzyme-linked immunosorbent assay (ELISA). Human obestatin-like immunoreactive substances from tea extract were isolated and characterized by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) and immunoblotting techniques. Immunochemical results showed that there are strong human obestatin-like immunoreactive substances (0.048±0.0064ng/mg protein) in vegetative tea leaves. This finding was completely unexpected since this hormone was considered to be present solely in animals. Furthermore, a single obestatin-like immunoreactive protein band of 13kDa was identified by tricine-SDS-PAGE and Western blotting of extract of vegetative tea leaf proteins. Present investigation is the first report of presence of obestatin-like immunoreactive substances in plants. It is concluded that obestatin-like bioactive peptides derived from plants can affect gastrointestinal tract structures as endogenous obestatin does and hence play a role in appetite regulation and body weight gain.

Supplementation with eicosapentaenoic and docosahexaenoic acids in late gestation in ewes changes adipose tissue gene expression in the ewe and growth and plasma concentration of ghrelin in the offspring1.

Omega-3 long chain fatty acids have a positive impact on production. When consumed during late gestation, it might have fetal programming effects on the fetus, which will have lifelong impacts on development and production. The objectives of the present study were to evaluate the effect of increasing doses of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the diet of ewes in the last third of gestation on their body weight (BW), subcutaneous adipose tissue relative mRNA abundance of genes associated with adipose tissue metabolism, and growth performance and plasma metabolites and hormones of their offspring during the finishing phase. Ewes (n = 72) were blocked by BW and allotted to pens (8 per treatment) with 3 ewes per pen. Ewes were supplemented with an EPA and DHA source (Strata G113) at concentrations of 0, 1, or 2% of dry matter intake during the last 50 d of gestation. At lambing, all ewes were penned together and offered the same diet. After weaning at 60 d of age, lambs were blocked by BW and sex and fed for 56 d. All lambs were fed the same pellet diet (61.09% ground corn, 24.08% soy hulls, 11.09% soybean meal, 1.48% Ca salt of palm oil, and 2.26% mixed mineral vitamin), and were weighed every 14 d until the end of the trial. Blood samples were collected on the weight sampling days. Dry matter intake and refusals were weighed daily. Data were analyzed as a randomized complete block design with repeated measurements (SAS 9.4). Polynomial contrast (linear-L and quadratic-Q) was used for mean separation. There were no differences in ewe body condition score, milk production, milk fat, or milk protein, but there was a trend for increased (L, P = 0.06) lactose concentration, and also differences in DGAT1 (L, P = 0.04), Δ5-desaturase (Q, P = 0.06) and Δ6-desaturase (Q, P = 0.07), PPARα (Q, P = 0.03), ELOVL2 and 5 (Q, P < 0.07), FABP4 (Q, P = 0.04), FATP1 (Q, P = 0.06), leptin (Q, P = 0.02), and resistin (L, P = 0.05). Feeding pregnant ewes an increased amount of EPA and DHA in late gestation increased final BW (L, P = 0.01), ADG (L, P = 0.04; Q, P = 0.01), DMI (Q, P ≤ 0.01), plasma glucose concentration (L, P = 0.04), and trended to decrease ghrelin concentrations (L, P = 0.07) in offspring during the finishing period. Dam supplementation did not affect G:F, nor plasma NEFA concentration (P ≥ 0.53) of lambs. Therefore, increasing supplementation of EPA and DHA in pregnant ewes has an impact on offspring performance, increasing DMI, ADG, and BW.

Conjugated Linoleic Acid Supplementation under a High-Fat Diet Modulates Stomach Protein Expression and Intestinal Microbiota in Adult Mice.

The gastrointestinal tract constitutes a physiological interface integrating nutrient and microbiota-host metabolism. Conjugated linoleic acids (CLA) have been reported to contribute to decreased body weight and fat accretion. The modulation by dietary CLA of stomach proteins related to energy homeostasis or microbiota may be involved, although this has not been previously analysed. This is examined in the present study, which aims to underline the potential mechanisms of CLA which contribute to body weight regulation. Adult mice were fed either a normal fat (NF, 12% kJ content as fat) or a high-fat (HF, 43% kJ content as fat) diet. In the latter case, half of the animals received daily oral supplementation of CLA. Expression and content of stomach proteins and specific bacterial populations from caecum were analysed. CLA supplementation was associated with an increase in stomach protein expression, and exerted a prebiotic action on both Bacteroidetes/Prevotella and Akkermansia muciniphila. However, CLA supplementation was not able to override the negative effects of HF diet on Bifidobacterium spp., which was decreased in both HF and HF+CLA groups. Our data show that CLA are able to modulate stomach protein expression and exert a prebiotic effect on specific gut bacterial species.

[Assay of adiponectin, leptin, true insulin and ghrelin levels in preterm human milk, and its relationship with infants growth].

OBJECTIVE: To understand adiponectin, leptin, insulin and ghrelin levels in preterm colostrum and mature milk and their influence on the growth and development of the premature infant. METHOD: The study subjects were divided into two groups: preterm group and control group. Specimens of colostrum and mature milk on 42nd day after delivery were collected, the general situation of maternal and infants growth parameters at birth and at postnatal 42 days were recorded. Leptin, adiponectin, insulin and ghrelin levels in colustrum and mature milk were determined and compared. RESULT: A total of 128 mother-infant pairs were involved. There were 128 specimens of colostrums (80 from preterm group, 48 from control group) and 94 specimens of mature milk(50 from premature group, 44 from control group). The levels of colostrum, mature milk adiponectin, leptin, and insulin were not significantly different between the 2 groups; ghrelin levels in colostrum and mature milk of premature group were significantly lower than those in control group (P = 0.038), adiponectin and leptin levels in colostrum were higher than those of the mature milk (P < 0.05), colostrum ghrelin levels were lower than those of mature milk (P < 0.05). Adiponectin, leptin, and ghrelin showed no significant difference between different gestational age groups ( ≤ 34 weeks group vs. > 34 weeks group). True insulin level of mature milk in 34 weeks group was higher than that of > 34 weeks group (29.3 vs. 21.6 mU/L, P = 0.045); true insulin level in colostrums in ≤ 34 weeks group was lower than that in mature milk (21.7 vs. 29.3 mU/L, P = 0.000). Adiponectin levels in colostrum and 42 days weight gain were negatively correlated (r = -0.362, P = 0.025) . Insulin level in mature milk had a negative correlation with birth weight (r = -0.319, P = 0.029) . Ghrelin levels in colostrum and birth weight, length, head circumference, head circumference on 42(nd) day were positively correlated (r = 0.271,0.261,0.360, P < 0.05); weight, length at 42(nd) day and ghrelin levels showed borderline positive correlation (P = 0.050, 0.058). CONCLUSION: Many bioactive hormones in milk might participate in the regulation of suitable growth after birth. Premature birth affects hormone levels in breast milk. Breast feeding is very important in preterm infants.

Leptin and ghrelin levels in colostrum, milk and blood plasma of sows and pig neonates during the first week of lactation.

Radioimmunology was used to determine leptin and ghrelin levels in sow colostrum and milk in relation to those in sow and neonatal pig blood plasma and to the body weight of piglets during the first week of lactation. The highest concentration of leptin was found in colostrum on the second day of lactation (69.3 ± 6.3 ng/mL). Leptin concentrations in sow plasma were significantly lower than in colostrum/milk (2.19 ± 0.9 ng/mL, P = 0.7692) and were stable in the first 7 days of lactation. Total and active ghrelin concentrations in colostrum/milk were stable in the measured time points (6734 ± 261 pg/mL, P = 0.3397; 831 ± 242 pg/mL, P = 0.3988, respectively). Total ghrelin concentrations in sow plasma were lower than in colostrum/milk. These results indicate that pigs follow a unique species-specific pattern of leptin and ghrelin synthesis, release and existence, and that the mammary gland is an important source of leptin and ghrelin contained in colostrum/milk.

Gut peptide profile and chemotherapy-associated dyspepsia syndrome in patients with breast cancer undergoing FEC60 chemotherapy.

AIM: The association of motilin, ghrelin, leptin, gastrin, pepsinogen (PG) I and II with cancer chemotherapy-associated dyspepsia syndrome (CADS) was investigated in 35 patients with breast cancer receiving first cycle of 5-fluorouracil, cyclophosphamide, epirubicin (FEC60) chemotherapy. PATIENTS AND METHODS: The onset of dyspeptic symptoms on days 3 and 10 after chemotherapy identified patients with and without CADS. Gastrointestinal symptoms were scored with the Gastrointestinal Symptom Scoring Rate (GSRS) questionnaire. Gastrointestinal peptides were evaluated by enzyme-linked immunosorbent assay. RESULTS: Twenty-one patients (60%) had CADS. The area under the curve (AUC) of ghrelin was higher, whereas that of PGI, PGII and motilin were lower in patients with CADS compared to those without. In patients with CADS, the AUC of PGI and PGII negatively correlated with the GSRS indigestion cluster. CONCLUSION: Impairment of gastrointestinal motility suggested by low motilin concentrations and mucosal damage mirrored by an increase of ghrelin seem to be involved in the onset of CADS in patients during chemotherapy for breast cancer.

Localization of ghrelin and its receptor in the reproductive tract of Holstein heifers.

The aim of this experiment was to localize the mRNA and protein of ghrelin and its active receptor, growth hormone secretagogue 1A (GHS-R1A), within the reproductive tract of dairy cattle. Ghrelin is an orexigenic hormone that has been identified as a potent regulator of energy homeostasis. Recent evidence suggests that ghrelin may also serve as a metabolic signal to the reproductive tract. Ghrelin and GHS-R1A have been identified in the reproductive tract of several species, including humans, mice, and rats. However, ghrelin and GHS-R1A expression have not been described within bovine reproductive tissues. Therefore, the ampulla, isthmus, uterine body, corpus luteum, and follicles were harvested from 3 Holstein heifers (15.91±0.07 mo of age) immediately following exsanguination. Duodenum and hypothalamus were collected as positive controls for ghrelin and GHS-R1A, respectively. Tissues were fixed in 10% formalin and embedded in paraffin for microscopy. Additional samples were stored at -80°C for detection of mRNA. Ghrelin and GHS-R1A mRNA and protein were observed in all tissue types within the reproductive tract of dairy heifers; however, expression appeared to be cell specific. Furthermore, ghrelin protein appeared to be localized to the cytoplasm, whereas GHS-R1A protein was found on the plasma membrane. Within the reproductive tissues, ghrelin mRNA and protein were most abundantly expressed in the ampulla of the oviduct. Concentrations of GHS-R1A were lower than those of ghrelin but differed between tissues. This is one of the first studies to provide molecular evidence for the presence of ghrelin and GHS-R1A within the entire reproductive tract. However, implications for fertility remain to be determined.

High-performance liquid chromatography analysis of hypothalamic ghrelin.

Ghrelin, first identified in the stomach, is a ligand of an orphan G-protein coupled receptor. Early studies indicated that the growth hormone secretagogue receptor (GHS-R; ghrelin receptor) is ubiquitously distributed in the brain. In addition, centrally administered ghrelin and ghrelin receptor agonist have effects on central neurons in many regions, including the hypothalamus, caudal brain stem, and spinal cord. These effects are due to ghrelin secreted from the brain, rather than from the stomach; ghrelin does not cross efficiently through the blood-brain barrier. Identification of ghrelin in the hypothalamus demonstrated that, as with stomach ghrelin, hypothalamic ghrelin also has two molecular forms, namely, octanoyl ghrelin and des-acyl ghrelin. Hypothalamic ghrelin plays diverse roles in processes including feeding regulation and thermoregulation. Thus, the analysis of hypothalamic ghrelin will provide new information about the action of ghrelin in the central nervous system. In this chapter, we outline high-performance liquid chromatography and real-time PCR analysis of hypothalamic ghrelin.

Breastmilk ghrelin, leptin, and fat levels changing foremilk to hindmilk: is that important for self-control of feeding?

The aim of this study was to evaluate the changes in the ghrelin, leptin, and fat levels in the foremilk and hindmilk and the possible relationship between these levels with the age and growth of term healthy infants. Sixty-two babies were subdivided (according to their nutrition) into breastfed (BF), formula-fed (FF), and BF plus FF (BF + FF) groups. The total and active ghrelin and tryglyceride levels and the total cholesterol levels in the foremilk and hindmilk were studied at the first and second visits (mean of the second and fifth months, respectively). At both visits, the total and active ghrelin and the total cholesterol levels were lower in the hindmilk than in the foremilk. However, the triglyceride levels were higher in the hindmilk than in the foremilk (p < 0.001). The leptin levels were also higher in the hindmilk, but this difference was not statistically significant. At the second visit, the mean total foremilk ghrelin (p < 0.01), leptin (p < 0.05), tryglyceride (p < 0.001), and cholesterol (p < 0.01) levels in the BF group were decreased compared with the levels at the first visit, whereas the active ghrelin levels increased (p < 0.001). At the second visit, we observed a 3.5% increase in the body mass index in BF infants, a 14.6% increase in FF infants, and an 11.8% increase in BF + FF infants (p < 0.01). The foremilk leptin levels were lower in the BF + FF group than in the BF group at both visits. In conclusion, at the first and second visits, the decreased ghrelin and increased tryglyceride and leptin levels in the hindmilk might be associated with the important role of self-control when feeding BF infants. The stable content of formulas might be associated with a lack of self-control during feeding and increased nutrition. Changing the breast milk ghrelin, leptin, and fat levels between the foremilk and hindmilk and between the first and second visits might explain the differences in the weight gain patterns of BF and FF infants.

Macronutrients act directly on the stomach to regulate gastric ghrelin release.

BACKGROUND: Ghrelin is a gastric secreted hormone deeply implicated in meal initiation and body weight regulation. This peptide is a peripheral orexigenic hormone with a nutritional status-dependent regulation showing a pre-pandrial rise and post-prandial fall pattern. A wide variety of studies have tested the effect of meal different nutrient composition over stomach mucosa ghrelin content and plasmatic ghrelin levels; nevertheless, few and non-conclusive data exist about the direct action of macronutrients on the stomach in order to regulate ghrelin secretion. The recent identification of taste receptors or chemoreceptors in the stomach mucosa would reinforce this paradigm. AIMS: To investigate the individual effect of different macronutrients (l-glutamine, lipids, and glucose) over gastric ghrelin secretion by using an in vitro gastric explants model. RESULTS: L-glutamine and intralipid emulsion act locally in the stomach decreasing ghrelin secretion, while no effect was found after glucose exposure. CONCLUSIONS: These results show for the first time that macronutrients, and specially amino acids and lipids, act directly in the stomach in order to regulate gastric ghrelin release. Consequently, the chemosensory capacity of the stomach, until now restricted to the oral cavity or intestine, is demonstrated.

Selective expression of TLQP-21 and other VGF peptides in gastric neuroendocrine cells and modulation by feeding.

Although vgf gene knockout mice are hypermetabolic, administration of the VGF peptide TLQP-21 itself increased energy consumption. Agonist-antagonist roles are thus suggested for different VGF peptides, and the definition of their tissue heterogeneity is mandatory. We studied the rat stomach using antisera to C- or N-terminal sequences of known or predicted VGF peptides in immunohistochemistry and ELISA. TLQP (rat VGF(556-565)) peptide/s were most abundant (162±11 pmol/g, mean±s.e.m.) and were brightly immunostained in enterochromaffin-like (ECL) cells and somatostatin cells. A peptide co-eluting with TLQP-21 was revealed in HPLC of gastric and hypothalamic extracts, while the extended TLQP-62 form was restricted to the hypothalamus. Novel PGH (rat VGF(422-430)) peptide/s were revealed in ghrelin cells, mostly corresponding to low MW forms (0.8-1.5  kDa), while VGF C-terminus peptides were confined to neurons. VGF mRNA was present in the above gastric endocrine cell types, and was prominent in chief cells, in parallel with low-intensity staining for further cleaved products from the C-terminal region of VGF (HVLL peptides: VGF(605-614)). In swine stomach, a comparable profile of VGF peptides was revealed by immunohistochemistry. When fed and fasted rats were studied, a clear-cut, selective decrease on fasting was observed for TLQP peptides only (162±11 vs 74±5.3  pmol/g, fed versus fasted rats, mean±s.e.m., P<0.00001). In conclusion, specific VGF peptides appear to be widely represented in different gastric endocrine and other mucosal cell populations. The selective modulation of TLQP peptides suggests their involvement in peripheral neuro-endocrine mechanisms related to feeding responses and/or ECL cell regulation.

[Relationship between Ghrelin and growth hormone secretagogue receptor expression and catch-up growth in rats with intrauterine growth restriction].

OBJECTIVE: To study the relationship between Ghrelin and growth hormone secretagogue receptor (GHSR) expression and the catch-up growth in rats with intrauterine growth restriction (IUGR). METHODS: The rat model of IUGR was established by food restriction during pregnancy. The small for gestational age (SGA) and appropriate for gestational age (AGA) rat pups from the pregnant rats were used as the experimental group. The AGA rat pups from the pregnant rats without food restriction served as the control group. The samples from the stomach fundus and hypothalamus were taken postnatal days 0, 20 and 40. Ghrelin mRNA and GHSR mRNA expression were determined by real-time fluorescence quantitative PCR (real-time FQ-PCR). Ghrelin protein and GHSR protein expression were examined by immunohistochemistry (IHC). RESULTS: At postnatal day 0, both Gherlin mRNA and protein levels in the stomach fundus were significantly higher, while GHSR mRNA expression in the hypothalamus were significantly lower in SGA rats from food restriction group than those in AGA rats from restriction and control groups. At postnatal day 20, the ghrelin protein expression in the stomach of fundus, and GHSR mRNA and protein expression in the hypothalamus in SGA catch-up rats were significantly higher than those in SGA non-catch-up growth rats and AGA rats from the control group. At postnatal day 40, there were no significant differences among SGA catch-up growth rats, SGA non-catch-up growth rats and normal AGA rats. CONCLUSIONS: Ghrelin-GHSR might be involved in the physiological regulation and pathological process in IUGR rats. It is also possibly involved in the regulation of catch-up growth in the early life of SGA rats.

Ghrelin and adiponectin levels in colostrum, cord blood and maternal serum.

BACKGROUND: Ghrelin and adiponectin, which are considered to take part in the regulation of energy metabolism, have been found in breast milk and cord blood. The aims of this study were to determine ghrelin and adiponectin levels in colostrum, cord blood and maternal serum and to investigate the correlations between colostrum and cord blood levels of these peptides and the anthropometry of newborn infants and their mothers. METHODS: Total ghrelin (TGHR), free ghrelin (FGHR) and adiponectin levels were studied in colostrum and the serum samples of 25 healthy lactating women and the cord blood of their healthy full-term infants. RESULTS: No significant differences could be found among TGHR and adiponectin levels in colostrum, cord blood and maternal serum. The median FGHR level in colostrum was significantly higher than that of maternal serum and cord blood. The colostrum TGHR was negatively correlated with body mass index (BMI) and weight of the infants at birth. TGHR and FGHR levels in colostrum were found to be positively correlated with those of maternal TGHR and FGHR concentrations, respectively. Adiponectin levels in colostrum were not correlated with BMI or birthweight of the infants or BMI of the mothers. CONCLUSION: These findings suggest that the source of ghrelin in breast milk is probably both breast tissue itself and the serum of the mother. Ghrelin in colostrum seems to be related to the anthropometry of infants even at birth, unlike adiponectin.

The intestinal lymph fistula model--a novel approach to study ghrelin secretion.

The orexigenic hormone ghrelin is secreted from the stomach and has been implicated in the regulation of energy and glucose homeostasis. We hypothesized that ghrelin, like other gastrointestinal (GI) hormones, is present in intestinal lymph, and sampling this compartment would provide advantages for studying ghrelin secretion in rodents. Blood and lymph were sampled from catheters in the jugular vein and mesenteric lymph duct before and after intraduodenal (ID) administration of isocaloric Ensure, dextrin, or Liposyn meals or an equal volume of saline in conscious Sprague-Dawley rats. Total ghrelin levels were measured using an established radioimmunoassay. Acyl and des-acyl ghrelin were measured using two-site ELISA. Fasting ghrelin levels in lymph were significantly higher than in plasma (means +/- SE: 3,307.9 +/- 272.9 vs. 2,127.1 +/- 115.0 pg/ml, P = 0.004). Postingestive acyl and des-acyl ghrelin levels were also significantly higher, whereas the ratio of acyl:des-acyl ghrelin was similar in lymph and plasma (0.91 +/- 0.28 vs. 1.20 +/- 0.36, P = 0.76). The principle enzymes responsible for deacylation of ghrelin were lower in lymph than in plasma. Following ID Ensure, maximum ghrelin suppression occurred at 2 h in lymph compared with at 1 h in plasma. The return of suppressed ghrelin levels to baseline was also delayed in lymph. Similarly, dextrin also induced significant suppression of ghrelin (two-way ANOVA: P = 0.02), whereas Liposyn did not (P = 0.32). On the basis of these findings, it appears that intestinal lymph, which includes drainage from the interstitium of the GI mucosa, is enriched in ghrelin. Despite reduced deacylating activity in lymph, there is not a disproportionate amount of acyl ghrelin in this pool. The postprandial dynamics of ghrelin are slower in lymph than plasma, but the magnitude of change is greater. Assessing ghrelin levels in the lymph may be advantageous for studying its secretion and concentrations in the gastric mucosa.

Presence of obestatin in breast milk: relationship among obestatin, ghrelin, and leptin in lactating women.

OBJECTIVE: The peptide hormones ghrelin and leptin have been found in blood and breast milk. This study was undertaken to investigate whether breast milk also contains obestatin, which is derived from the same gene as ghrelin but has opposite actions, and to characterize the relations among serum and milk ghrelin, obestatin, and leptin levels in lactating mothers. METHODS: Venous blood, colostrum, and mature milk were obtained from healthy lactating women (n = 31) just before suckling. The ghrelin and obestatin concentrations were determined by radioimmunoassay. Leptin levels were measured by enzyme-amplified sensitivity immunoassay. RESULTS: Obestatin levels in colostrum (538.9 pg/mL) and mature milk (528.5 pg/mL) were more than twice the corresponding blood levels (270.3 and 289.4 pg/mL, respectively). In contrast, leptin levels in colostrum (2.01 ng/mL) and mature milk (2.04 ng/mL) were more than five-fold lower than the corresponding blood levels (11.54 ng/mL). There was no correlation between breast milk ghrelin levels and leptin (r = -0.18, P > 0.05). However, there was a positive correlation between leptin levels in breast milk and blood (r = 0.369, P < 0.05). CONCLUSION: The origin of milk obestatin is not currently known, but it comes from the blood or breast and may drain through the mammary glands into the milk. Ghrelin, obestatin, and leptin in the milk may directly affect appetite and their levels may be related to the regulation of energy balance and the pathogenesis of obesity.

Influence of short- and long-term treadmill exercises on levels of ghrelin, obestatin and NPY in plasma and brain extraction of obese rats.

This study aims to clarify the effects of exercise on levels of appetite regulatory hormones in plasma and hypothalamus of obese rats. Diet-induced obese rats undergo short- (40 min) and long-term (40 min, 5 days/week for 8 weeks) exercises. The rats ran at a speed of 20 m/min on a 5 degrees slope treadmill. Rats undergoing short-term exercise were divided into C, E0, E1, E3, E12, and E24. Rats undergoing long-term exercise (LE) were compared to long-term control (LC). Concentrations of ghrelin, obestatin, and neuropeptide Y (NPY) were measured using radio immuno-assay. Expression of ghrelin receptor (GHSR-1a), putative obestatin receptor (GPR-39), and NPY in the hypothalamus was measured by quantitative RT-PCR. After short-term exercise, the plasma concentrations of ghrelin and obestatin were not changed, but NPY decreased. Ghrelin and obestatin in the hypothalamus decreased, and recovered 12 until 24 h. NPY increased and recovered after 24 h. Expression of GHSR-1a and NPY was not changed and GPR-39 was not observed. In LE, these changes are different in plasma and hypothalamus. It would be concluded appetite and body weight of obese rats are decreased by exercise through reduced level of ghrelin in the hypothalamus. Obestatin seems to have no effect in exercise-induced change in appetite.

Milk and blood ghrelin level in diabetics.

OBJECTIVE: Besides its presence in various tissues, ghrelin has recently been shown to be present in blood and breast milk. No previous studies, however, have evaluated the level of this hormone under the condition of pregestational and gestational diabetes mellitus (P-GDM and GDM, respectively). This study was undertaken to show whether a relation exists between serum and milk ghrelin levels in lactating mothers with and without diabetes. METHODS: Venous blood was obtained from four groups of women (age range 22-37 y): GDM lactating (n = 12), P-GM lactating (n = 3), healthy non-diabetic lactating (n = 14), and healthy non-lactating (n = 14). Colostrum and mature milk samples were collected just before suckling. The ghrelin level was determined by radioimmunoassay and high-performance liquid chromatography. RESULTS: Radioimmunoassay results showed that women with GDM and P-GDM had greater than two-fold lower colostrum and serum levels of ghrelin than did lactating women with no GDM at 2 d after parturition. The GDM and non-diabetic groups at 15 d after delivery, however, showed similar levels of ghrelin in mature milk and serum. High-performance liquid chromatographic results indicated that in serum the deacylated form of ghrelin was 18-fold higher than the acylated form. Furthermore, in milk the acylated form of ghrelin was 24-fold that of the active form. CONCLUSION: These results indicate that mothers with GDM have a substantial (greater than two-fold) decrease in their serum and colostral ghrelin levels. This is, however, a temporary effect lasting only up to early postparturition (2 d after delivery). This peptide hormone restores to completely normal levels at day 15 of parturition, but not P-GDM. The significance of these results in terms of the health of the mother and her newborn, however, has yet to be determined.

Maternal adaptations to pregnancy in spontaneously hypertensive rats: leptin and ghrelin evaluation.

Leptin and/or ghrelin, initially thought to be considered messengers of energy metabolism, are now considered to play a role in normal and complicated pregnancy. In this study, pregnant, spontaneously hypertensive rats (SHR) have been used to evaluate, for the first time, the modification of leptin and ghrelin both at serum and tissue levels. In SHR, we evaluate plasma leptin level and tissue protein expression in both placenta and adipose tissue at the end of gestation (day 20) versus normotensive Wistar-Kyoto (WKY) animals. The expression of functional leptin receptor (Ob-Rb) in peripheral tissues and in the hypothalamus was evaluated. Moreover, we measured plasma ghrelin level and its mRNA expression in the stomach and placenta. SHR strain presented significantly lower plasma leptin levels when compared with those found in pregnant or not WKY controls. Interestingly, in the placenta, leptin gene expression was higher in SHR than normotensive WKY. Moreover, we demonstrated a resistance to the effects of leptin via 'downregulation' of hypothalamic receptors in pregnant SHR. Conversely, SHR presented significantly higher ghrelin plasma levels when compared with those found in pregnant or not WKY. However, we observed that ghrelin level in the stomach of SHR did not change during pregnancy, and on the opposite, mRNA ghrelin in the placenta of SHR was lower than that of normotensive rats, suggesting a different production of this hormone in the fetal-placental unit. These data gain further insight into metabolic hormone modifications observed in a model of pre-existing hypertension associated with pregnancy.