Suppressive effect on food intake of a potato extract (Potein®) involving cholecystokinin release in rats

We have recently reported that oral gavage of a potato extract (Potein?) suppressed the food intake in rats. The satiating effect of the potato extract was compared in the present study to other protein sources, and the involvement of endogenous cholecystokinin (CCK) secretion was examined. Food consumption was measured in 18-h fasted rats after oral gavage of the potato extract or other protein sources. The CCK-releasing activity of the potato extract was then examined in anesthetized rats with a portal cannula. Oral gavage of the potato extract reduced the food intake in the rats, the effect being greater than with casein and a soybean β-conglycinin hydrolysate. The suppressive effect on appetite of the potato extract was attenuated by treating with a CCK-receptor antagonist (devazepide). The portal CCK concentration was increased after a duodenal administration of the potato extract to anesthetized rats. These results indicate that the potato extract suppressed the food intake in rats through CCK secretion.     

Dose-Dependent Effects of a Soluble Dietary Fibre (Pectin) on Food Intake,Adiposity, Gut Hypertrophy and Gut Satiety Hormone Secretion in Rats

Soluble fermentable dietary fibre elicits gut adaptations, increases satiety and potentially offers a natural sustainable means of body weight regulation. Here we aimed to quantify physiological responses to graded intakes of a specific dietary fibre (pectin) in an animal model. Four isocaloric semi-purified diets containing 0, 3.3%, 6.7% or 10% w/w apple pectin were offered ad libitum for 8 or 28 days to young adult male rats (n = 8/group). Measurements were made of voluntary food intake, body weight, initial and final body composition by magnetic resonance imaging, final gut regional weights and histology, and final plasma satiety hormone concentrations. In both 8- and 28-day cohorts, dietary pectin inclusion rate was negatively correlated with food intake, body weight gain and the change in body fat mass, with no effect on lean mass gain. In both cohorts, pectin had no effect on stomach weight but pectin inclusion rate was positively correlated with weights and lengths of small intestine and caecum, jejunum villus height and crypt depth, ileum crypt depth, and plasma total glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) concentrations, and at 8 days was correlated with weight and length of colon and with caecal mucosal depth. Therefore, the gut’s morphological and endocrine adaptations were dose-dependent, occurred within 8 days and were largely sustained for 28 days during continued dietary intervention. Increasing amounts of the soluble fermentable fibre pectin in the diet proportionately decreased food intake, body weight gain and body fat content, associated with proportionately increased satiety hormones GLP-1 and PYY and intestinal hypertrophy, supporting a role for soluble dietary fibre-induced satiety in healthy body weight regulation.     

Soybean β-Conglycinin Bromelain Hydrolysate Stimulates Cholecystokinin Secretion by Enteroendocrine STC-1 Cells to Suppress the Appetite of Rats under Meal-Feeding Conditions

A peptic digest of soybean β-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. β-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of β-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.     

Soybean β-conglycinin bromelain hydrolysate stimulates cholecystokinin secretion by enteroendocrine STC-1 cells to suppress the appetite of rats under meal-feeding conditions

A peptic digest of soybean β-conglycinin (BconP) suppresses the appetite in rats through cholecystokinin (CCK) secretion by enteroendocrine cells. We investigate in this study more appetite-suppressing hydrolysates. β-Conglycinin hydrolyzed with food-processing proteases thermolysin (BconT), bromelain (BconB), chymotrypsin, protease S, and protease M was examined for CCK-secreting activity in a CCK-producing cell line for comparison with BconP. The potent CCK-releasing hydrolysates were then tested for their suppression of the food intake by rats. BconB, BconT, and BconP stimulated high CCK secretion, with the highest by BconB. Orogastric preloading by BconB, but not by BconT, suppressed the 60-min food intake. A meal-feeding trial twice a day in the morning (a.m.) and evening (p.m.) for 10 d showed that BconB preloading before every meal attenuated the p.m. meal size, but not that a.m., resulting in an overall reduction of the daily meal size. These results demonstrate that the bromelain hydrolysate of β-conglycinin having potent CCK-releasing activity suppressed the appetite of rats under meal-feeding conditions.     

Effect of a protein preload on food intake and satiety feelings in response to duodenal fat perfusions in healthy male subjects

The control of food intake and satiety requires a coordinated interplay. Oral protein and duodenal fat inhibit food intake and induce satiety, but their interactive potential is unclear. Our aim was therefore to investigate the interactions between an oral protein preload and intraduodenal (ID) fat on food intake and satiety feelings. Twenty healthy male volunteers were studied in a randomized, double-blind, four-period crossover design. On each study day, subjects underwent one of the following treatments: 1) water preload plus ID saline perfusion, 2) water preload plus ID fat perfusion, 3) protein preload plus ID saline perfusion, or 4) protein preload plus ID fat perfusion. Subjects were free to eat and drink as much as they wished. An oral protein preload significantly reduced caloric intake (19%, P < 0.01). Simultaneous administration of an oral protein preload and ID fat did not result in a positive synergistic effect with respect to caloric consumption, rejecting the initial hypothesis that the two nutrients exert a positive synergistic effect on food intake. An oral protein preload but not ID fat altered the feelings of hunger and fullness. These data indicate that the satiety effect of an oral protein preload is not amplified by ID fat; indeed, the effect of a protein preload does not seem to be mediated by cholecystokinin, glucagon-like peptide-1, or peptide YY. Much more information is necessary to understand the basic physiological mechanisms that control food intake and satiety.     

Glucagon-like peptide-1 (GLP-1) increases in plasma and colon tissue prior to estrus and circulating levels change with increasing age in reproductively competent Wistar rats

There is a well-documented association between cyclic changes to food intake and the changing ovarian hormone levels of the reproductive cycle in female mammals. Limited research on appetite-controlling gastrointestinal peptides has taken place in females, simply because regular reproductive changes in steroid hormones present additional experimental factors to account for. This study focussed directly on the roles that gastrointestinal-secreted peptides may have in these reported, naturally occurring, changes to food intake during the rodent estrous cycle and aimed to determine whether peripheral changes occurred in the anorexigenic (appetite-reducing) hormones peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) in female Wistar rats (32–44 weeks of age). Total forms of each peptide were measured in matched fed and fasted plasma and descending colon tissue samples for each animal during the dark (feeding) phase. PYY concentrations did not significantly change between defined cycle stages, in either plasma or tissue samples. GLP-1 concentrations in fed plasma and descending colon tissue were significantly increased during proestrus, just prior to a significant reduction in fasted stomach contents at estrus, suggesting increased satiety and reduced food intake at this stage of the cycle. Increased proestrus GLP-1 concentrations could contribute to the reported reduction in food intake during estrus and may also have biological importance in providing the optimal nutritional and metabolic environment for gametes at the potential point of conception. Additional analysis of the findings demonstrated significant interactions of ovarian cycle stage and fed/fasted status with age on GLP-1, but not PYY plasma concentrations. Slightly older females had reduced fed plasma GLP-1 suggesting that a relaxation of regulatory control of this incretin hormone may also take place with increasing age in reproductively competent females.     

Critical role for peptide YY in protein-mediated satiation and body-weight regulation

Dietary protein enhances satiety and promotes weight loss, but the mechanisms by which appetite is affected remain unclear. We investigated the role of gut hormones, key regulators of ingestive behavior, in mediating the satiating effects of different macronutrients. In normal-weight and obese human subjects, high-protein intake induced the greatest release of the anorectic hormone peptide YY (PYY) and the most pronounced satiety. Long-term augmentation of dietary protein in mice increased plasma PYY levels, decreased food intake, and reduced adiposity. To directly determine the role of PYY in mediating the satiating effects of protein, we generated Pyy null mice, which were selectively resistant to the satiating and weight-reducing effects of protein and developed marked obesity that was reversed by exogenous PYY treatment. Our findings suggest that modulating the release of endogenous satiety factors, such as PYY, through alteration of specific diet constituents could provide a rational therapy for obesity.     

Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.     

Pork peptone stimulates cholecystokinin secretion from enteroendocrine cells and suppresses appetite in rats

We found that soybean beta-conglycinin peptone (BconP) suppresses food intake through cholecystokinin (CCK) release from enteroendocrine cells in association with binding of the peptone to rat small intestinal brush border membrane (BBM). The aim of the present study was to find new appetite suppressing peptides. Peptones from chicken, pork, beef, beef liver, and egg white were examined for activities to bind with rat BBM, CCK-release from enteroendocrine cell line STC-1, and induce satiety in rats. Chicken and pork peptone (ChickP and PorkP) bound to BBM with highest ability as evaluated with a surface plasmon biosensor. PorkP and ChickP released CCK in higher amounts than BconP from STC-1 cells dose-dependently, with highest stimulation by PorkP. An orogastric preload of PorkP, but not ChickP, suppressed food intake similarly to BconP, dose-dependently. These results suggest that PorkP interacts directly with the small intestinal CCK cells to release CCK, and that it suppresses appetite in rats.     

Individual subtypes of enteroendocrine cells in the mouse small intestine exhibit unique patterns of inositol 1,4,5-trisphosphate receptor expression.

Enteroendocrine cells are a complex population of intestinal epithelial cells whose hormones play critical roles in regulating gastrointestinal and whole-animal physiology. There are many subpopulations of enteroendocrine cells based on the major hormone(s) produced by individual cells. Intracellular calcium plays a critical role in regulating hormone release. Inositol 1,4,5-trisphophate (IP3) receptors regulate calcium mobilization from endoplasmic reticulum-derived calcium stores in many endocrine and excitatory cells and are expressed in the intestine. However, the specific subtypes of enteroendocrine cells that express these receptors have not been reported. Immunohistochemical (IHC) studies revealed that enteroendocrine cells did not express detectable levels of type 2 IP3 receptors, whereas nearly all enteroendocrine cells that produced chromogranin A and/or serotonin expressed type 1 and type 3 IP3 receptors. Conversely, enteroendocrine cells that produced glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, cholecystokinin, or somatostatin did not express detectable levels of any IP3 receptors. Subsets of enteroendocrine cells that produced substance P or secretin expressed type 1 (33% or 18%, respectively) and type 3 (10% or 62%, respectively) IP3 receptors. Thus, different subtypes of enteroendocrine cells, as well as individual cells that express a particular hormone, exhibit remarkable heterogeneity in the molecular machineries that regulate hormone release in vivo. These results suggest that therapeutic agents can be developed that could potentially inhibit or promote secretion of hormones from specific subtypes of enteroendocrine cells.     

Mu-opioid receptors and dietary protein stimulate a gut-brain neural circuitry limiting food intake

Intestinal gluconeogenesis is involved in the control of food intake. We show that mu-opioid receptors (MORs) present in nerves in the portal vein walls respond to peptides to regulate a gut-brain neural circuit that controls intestinal gluconeogenesis and satiety. In vitro, peptides and protein digests behave as MOR antagonists in competition experiments. In vivo, they stimulate MOR-dependent induction of intestinal gluconeogenesis via activation of brain areas receiving inputs from gastrointestinal ascending nerves. MOR-knockout mice do not carry out intestinal gluconeogenesis in response to peptides and are insensitive to the satiety effect induced by protein-enriched diets. Portal infusions of MOR modulators have no effect on food intake in mice deficient for intestinal gluconeogenesis. Thus, the regulation of portal MORs by peptides triggering signals to and from the brain to induce intestinal gluconeogenesis are links in the satiety phenomenon associated with alimentary protein assimilation.     

Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin

Intracerebroventricular (ICV) administration of Neuromedin U (NMU), a hypothalamic neuropeptide, or leptin, an adipostat hormone released from adipose tissue, reduces food intake and increases energy expenditure. Leptin stimulates the release of NMU in vitro, and NMU expression is reduced in models of low or absent leptin. We investigated the role of NMU in mediating leptin-induced satiety. ICV administration of anti-NMU immunoglobulin G (IgG) (5 nmol) to satiated rats significantly increased food intake 4 h after injection, an effect seen for 相似文献   

CCK-resistance in Zucker obese versus lean rats

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 μg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20–100 μg/kg) in lean versus obese rats. In lean animals the satiety effects of the “near physiological” 4 μg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of ¹²⁵I- labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of “near physiological” doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.     

Ileal interposition attenuates the satiety responses evoked by cholecystokinin-8 and -33

One of the possible mechanisms by which the weight-reducing surgical procedure ileal interposition (II) works is by increasing circulating levels of lower gut peptides that reduce food intake, such as glucagon like peptide-1 and peptide YY. However, since this surgery involves both lower and upper gut segments, we tested the hypothesis that II alters the satiety responses evoked by the classic upper gut peptide cholecystokinin (CCK). To test this hypothesis, we determined meal size (MS), intermeal interval (IMI) and satiety ratio (SR) evoked by CCK-8 and -33 (0, 1, 3, 5 nmol/kg, i.p.) in two groups of rats, II and sham-operated. CCK-8 and -33 reduced MS more in the sham group than in the II group; CCK-33 prolonged IMI in the sham group and increased SR in both groups. Reduction of cumulative food intake by CCK-8 in II rats was blocked by devazepide, a CCK₁ receptor antagonist. In addition, as previously reported, we found that II resulted in a slight reduction in body weight compared to sham-operated rats. Based on these observations, we conclude that ileal interposition attenuates the satiety responses of CCK. Therefore, it is unlikely that this peptide plays a significant role in reduction of body weight by this surgery.     

Intraportal administration of DPP-IV inhibitor regulates insulin secretion and food intake mediated by the hepatic vagal afferent nerve in rats

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and suppresses food intake. Recent studies indicate that the hepatic vagal afferent nerve is involved in this response. Dipeptidyl peptidase-IV (DPP-IV) inhibitor extends the half-life of endogenous GLP-1 by preventing its degradation. This study aimed to determine whether DPP-IV inhibitor-induced elevation of portal GLP-1 levels affect insulin secretion and feeding behavior via the vagal afferent nerve and hypothalamus. The effect of DPP-IV inhibitor infusion into the portal vein or peritoneum on portal and peripheral GLP-1 levels, food intake, and plasma insulin and glucose was examined in sham-operated and vagotomized male Sprague-Dawley rats. Analyses of neuronal histamine turnover and immunohistochemistry were used to identify the CNS pathway that mediated the response. Intraportal administration of the DPP-IV inhibitor significantly increased portal (but not peripheral) GLP-1 levels, increased insulin levels, and decreased glucose levels. The DPP-IV inhibitor suppressed 1- and 12- but not 24-h cumulative food intake. Intraportal infusion of the DPP-IV inhibitor increased hypothalamic neuronal histamine turnover and increased c-fos expression in several areas of the brain. These responses were blocked by vagotomy. Our results indicate that DPP-IV inhibitor-induced changes in portal but not systemic GLP-1 levels affect insulin secretion and food intake. Furthermore, our findings suggest that a neuronal pathway that includes the hepatic vagal afferent nerve and hypothalamic neuronal histamine plays an important role in the pharmacological actions of DPP-IV inhibitor.     

Effect of intraperitoneal CCK-8 on food intake and brain orexin-A after 48 h of fasting in the rat

We investigated the interactions of the peripheral satiety peptide cholecystokinin and the brain orexin-A system in the control of food intake. The effect of an intraperitoneal (i.p.) injection of sulfated cholecystokinin octapeptide (in this article called CCK) (5 microg/kg, 4.4 nmol/kg) or of phosphate-buffered saline (PBS, vehicle control) on 48 h fasting-induced feeding and on orexin-A peptide content was analyzed in diverse brain regions innervated by orexin neurons and involved in the control of food intake. Administration of CCK after a 48 h fast reduced fasting-induced hyperphagia (P<0.05). I.p. CCK increased the orexin-A content in the posterior brainstem of 48 h fasted rats by 35% (P<0.05). Fed animals receiving CCK had 48% higher orexin-A levels in the posterior brainstem than fasted rats (P<0.05). In the lateral hypothalamus, fasting decreased orexin-A levels by 50% as compared to fed rats (P<0.05). In the septal nuclei, the combination of fasting and CCK administration reduced orexin-A contents compared to fed PBS and CCK animals by 13% and 17%, respectively (P<0.05). These results suggest a convergence of pathways activated by peripheral CCK and by fasting on the level of orexin-A released in the posterior brainstem and provide evidence for a novel interaction between peripheral satiety signaling and a brain orexigen in the control of food intake.     

The endogenous CCK mediation of electroacupuncture stimulation-induced satiety in rats

A major satiety hormone, cholecystokinin (CCK) is well known to be released by electroacupuncture (EA) stimulation at certain body sites which elicits profound psychophysiological responses. Previous clinical and animal studies have shown that EA stimulation reduces food intake and body weight in both normal and obese subjects. The aim of the present study was to elucidate the satiety effect of EA stimulation and its mechanism related to CCK in rats. Here we show that EA stimulation at "Zusanli" (ST36) acupoint significantly reduced 30-min and 60-min food intake in 48-h fasted Sprague-Dawley rats, and such effect was reversed by a lorglumide (CCK-1 receptor antagonist, 10mg/kg, i.p.) pretreatment. The ST36 EA stimulation-induced satiety was not observed in CCK-1 receptor knockout, Otsuka Long-Evans Tokushima Fatty rats, but in their controls, Long-Evans Tokushima Otsuka rats. Subdiaphragmatic vagotomy also blocked the satiety effect of ST36 EA stimulation in Sprague-Dawley rats. These results suggest that ST36 EA stimulation elicits satiety in rats and this is mediated by the endogenous CCK signaling pathway.     

Comparison of crowding and food restriction effects on growth, body weight gain and endocrine status in the rat

The present work compares the effects of post-weaning crowding and those of food restriction on growth, body weight gain and the levels of several hormones, in male Sprague-Dawley rats. Crowding resulted in reduced food intake and diminished body weight gain. Rats daily receiving the same amount of food as that eaten by crowded rats (food-restricted group) showed similar body weight gain as crowded rats, but higher growth rate. Neither crowding nor food restriction altered the pituitary-adrenal axis. In contrast, both treatments decreased serum insulin, growth hormone (GH), somatomedin C (Sm-C) and thyroid-stimulating hormone (TSH) as compared to control rats. The reduction of GH and Sm-C levels was similar in crowded and food-restricted rats but that of TSH was higher in food-restricted, rather than in crowded rats. The present data indicate that the effects of crowding could only be partially explained by the concomittant reduction of food intake and that the serum levels of the hormones studied could not explain the differences between the crowded and food-restricted rats with regard to growth.     

The anorectic effect of fenfluramine is influenced by sex and stage of the estrous cycle in rats

The controls of food intake differ in male and female rats. Daily food intake is typically greater in male rats, relative to female rats, and a decrease in food intake, coincident with the estrous stage of the ovarian reproductive cycle, is well documented in female rats. This estrous-related decrease in food intake has been attributed to a transient increase in the female rat's sensitivity to satiety signals generated during feeding bouts. Here, we investigated whether sex or stage of the estrous cycle modulate the satiety signal generated by fenfluramine, a potent serotonin (5-HT) releasing agent. To examine this hypothesis, food intake was monitored in male, diestrous female, and estrous female rats after intraperitoneal injections of 0, 0.25, and 1.0 mg/kg D-fenfluramine. The lower dose of fenfluramine decreased food intake only in diestrous and estrous females, suggesting that the minimally effective anorectic dose of fenfluramine is lower in female rats, relative to male rats. Although the larger dose of fenfluramine decreased food intake in both sexes, the duration of anorexia was greater in diestrous and estrous female rats, relative to male rats. Moreover, the magnitude of the anorectic effect of the larger dose of fenfluramine was greatest in estrous rats, intermediate in diestrous rats, and least in male rats. Thus our findings indicate that the anorectic effect of fenfluramine is modulated by gonadal hormone status.     

Regulation of Resistin by Gonadal,Thyroid Hormone,and Nutritional Status

Objective: Resistin was recently identified as a hormone secreted by adipocytes that is under hormonal and nutritional control. This hormone has been suggested to be the link between obesity and type 2 diabetes. The aim of this study was to assess the influence of gender, gonadal status, thyroid hormones, pregnancy, and food restriction on resistin mRNA levels in adipose tissue of rats. Research Methods and Procedures: We have determined resistin mRNA expression by Northern blot analysis in all experimental sets. Results: Resistin mRNA expression is influenced by age, with the highest hormone levels existing at 45 days after birth and decreasing thereafter. Resistin mRNA expression is higher in men than in women. Moreover, we studied the effect of orchidectomy and ovariectomy in rats of different ages and showed that gonadal hormones increase adipose tissue resistin mRNA expression in male rats. Resistin is also regulated by thyroid hormones; it is severely decreased in hyperthyroid rats. Our results clearly show that chronic food restriction (30% of ad libitum food intake) led to a decrease in adipose tissue mRNA levels in normal cycling female rats and pregnant rats. In pregnancy, resistin mRNA levels were enhanced particularly at midgestation. Discussion: Our observations indicate that resistin is influenced by gender, gonadal status, thyroid hormones, and pregnancy. These findings suggest that resistin could explain the decreased insulin sensitivity during puberty and could be the link between sex steroids and insulin sensitivity. Moreover, resistin could mediate the effect of thyroid hormones on insulin resistance and the state of insulin resistance present during pregnancy.