Growth differentiation factor 15 (GDF15) and semaglutide inhibit food intake and body weight through largely distinct, additive mechanisms.

AIMS: To evaluate whether the potent hypophagic and weight-suppressive effects of growth differentiation factor-15 (GDF15) and semaglutide combined would be a more efficacious antiobesity treatment than either treatment alone by examining whether the neural and behavioural mechanisms contributing to their anorectic effects were common or disparate. MATERIALS/METHODS: Three mechanisms were investigated to determine how GDF15 and semaglutide induce anorexia: the potentiation of the intake suppression by gastrointestinal satiation signals; the reduction in motivation to feed; and the induction of visceral malaise. We then compared the effects of short-term, combined GDF15 and semaglutide treatment on weight loss to the individual treatments. Rat pharmaco-behavioural experiments assessed whether GDF15 or semaglutide added to the satiating effects of orally gavaged food and exogenous cholecystokinin (CCK). A progressive ratio operant paradigm was used to examine whether GDF15 or semaglutide reduced feeding motivation. Pica behaviour (ie, kaolin intake) and conditioned affective food aversion testing were used to evaluate visceral malaise. Additionally, fibre photometry studies were conducted in agouti-related protein (AgRP)-Cre mice to examine whether GDF15 or semaglutide, alone or in combination with CCK, modulate calcium signalling in hypothalamic AgRP neurons. RESULTS: Semaglutide reduced food intake by amplifying the feeding-inhibitory effect of CCK or ingested food, inhibited the activity of AgRP neurons when combined with CCK, reduced feeding motivation and induced malaise. GDF15 induced visceral malaise but, strikingly, did not affect feeding motivation, the satiating effect of ingested food or CCK signal processing. Combined GDF15 and semaglutide treatment produced greater food intake and body weight suppression than did either treatment alone, without enhancing malaise. CONCLUSIONS: GDF15 and semaglutide reduce food intake and body weight through largely distinct processes that produce greater weight loss and feeding suppression when combined.

Liraglutide, leptin and their combined effects on feeding: additive intake reduction through common intracellular signalling mechanisms.

AIM: To investigate the behavioural and intracellular mechanisms by which the glucagon like peptide-1 (GLP-1) receptor agonist, liraglutide, and leptin in combination enhance the food intake inhibitory and weight loss effects of either treatment alone. METHODS: We examined the effects of liraglutide (a long-acting GLP-1 analogue) and leptin co-treatment, delivered in low or moderate doses subcutaneously (s.c.) or to the third ventricle, respectively, on cumulative intake, meal patterns and hypothalamic expression of intracellular signalling proteins [phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein tyrosine phosphatase-1B (PTP1B)] in lean rats. RESULTS: A low-dose combination of liraglutide (25 µg/kg) and leptin (0.75 µg) additively reduced cumulative food intake and body weight, a result mediated predominantly through a significant reduction in meal frequency that was not present with either drug alone. Liraglutide treatment alone also reduced meal size; an effect not enhanced with leptin co-administration. Moderate doses of liraglutide (75 µg/kg) and leptin (4 µg), examined separately, each reduced meal frequency, cumulative food intake and body weight; only liraglutide reduced meal size. In combination these doses did not further enhance the anorexigenic effects of either treatment alone. Ex vivo immunoblot analysis showed elevated pSTAT3 in the hypothalamic tissue after liraglutide-leptin co-treatment, an effect which was greater than that of leptin treatment alone. In addition, s.c. liraglutide reduced the expression of PTP1B (a negative regulator of leptin receptor signalling), revealing a potential mechanism for the enhanced pSTAT3 response after liraglutide-leptin co-administration. CONCLUSIONS: Collectively, these results show novel behavioural and molecular mechanisms underlying the additive reduction in food intake and body weight after liraglutide-leptin combination treatment.

Hindbrain leptin and glucagon-like-peptide-1 receptor signaling interact to suppress food intake in an additive manner.

BACKGROUND: The physiological control of feeding behavior involves modulation of the intake inhibitory effects of gastrointestinal satiation signaling via endogenous hindbrain leptin receptor (LepR) and glucagon-like-peptide-1 receptor (GLP-1R) activation. DESIGN AND RESULTS: Using a variety of dose-combinations of hindbrain delivered (4th intracerebroventricular; i.c.v.) leptin and the GLP-1R agonist exendin-4, experiments demonstrate that hindbrain LepR and GLP-1R signaling interact to control food intake and body weight in an additive manner. In addition, the maximum intake suppressive response that could be achieved by 4th i.c.v. leptin alone in non-obese rats (∼33%) was shown to be further suppressed when exendin-4 was co-administered. Importantly, it was determined that the interaction between hindbrain LepR signaling and GLP-1R signaling is relevant to endogenous food intake control, as hindbrain GLP-1R blockade by the selective antagonist exendin-(9-39) attenuated the intake inhibitory effects of hindbrain leptin delivery. CONCLUSIONS: Collectively, the findings reported here show that hindbrain LepR and GLP-1R activation interact in at least an additive manner to control food intake and body weight. As evidence is accumulating that combination pharmacotherapies offer greater sustained food intake and body weight suppression in obese individuals when compared with mono-drug therapies or lifestyle modifications alone, these findings highlight the need for further examination of combined central nervous system GLP-1R and LepR signaling as a potential drug target for obesity treatment.

The nucleus tractus solitarius: a portal for visceral afferent signal processing, energy status assessment and integration of their combined effects on food intake.

For humans and animal models alike there is general agreement that the central nervous system processing of gastrointestinal (GI) signals arising from ingested food provides the principal determinant of the size of meals and their frequency. Despite this, relatively few studies are aimed at delineating the brain circuits, neurochemical pathways and intracellular signals that mediate GI-stimulation-induced intake inhibition. Two additional motivations to pursue these circuits and signals have recently arisen. First, the success of gastric-bypass surgery in obesity treatment is highlighting roles for GI signals such as glucagon-like peptide-1 (GLP-1) in intake and energy balance control. Second, accumulating data suggest that the intake-reducing effects of leptin may be mediated through an amplification of the intake-inhibitory effects of GI signals. Experiments reviewed show that: (1) the intake-suppressive effects of a peripherally administered GLP-1 receptor agonist is mediated by caudal brainstem neurons and that forebrain-hypothalamic neural processing is not necessary for this effect; (2) a population of medial nucleus tractus solitarius (NTS) neurons that are responsive to gastric distention is also driven by leptin; (3) caudal brainstem-targeted leptin amplifies the food-intake-inhibitory effects of gastric distention and intestinal nutrient stimulation; (4) adenosine monophosphate-activated protein kinase (AMPK) activity in NTS-enriched brain lysates is elevated by food deprivation and reduced by refeeding and (5) the intake-suppressive effect of hindbrain-directed leptin is reversed by elevating hindbrain AMPK activity. Overall, data support the view that the NTS and circuits within the hindbrain mediate the intake inhibition of GI signals, and that the effects of leptin on food intake result from the amplification of GI signal processing.

Chronically decerebrate rats demonstrate satiation but not bait shyness.

Taste substances applied to the oral cavity result in either ingestion or rejection, each with a characteristic muscular response pattern. These responses are the same in decerebrate and intact rats; the caudal brainstem appears to be the neural substrate of ingestion and rejection responses. The experiment determined whether decerebrates can alter these discriminative responses as a function of food deprivation or toxicosis. Food-deprived decerebrate rats, like intact ones, ingested a taste substance they had rejected when sated. However, these same decerebrates, in contrast to controls, neither rejected nor decreased ingestive reactions to a novel taste after that taste had been repeatedly paired with lithium chloride-induced illness. Although the forebrain may be important for integrating ingestion, some aspects of this control seem to be represented in caudal brain areas.

The forebrain is not essential for sympathoadrenal hyperglycemic response to glucoprivation.

The reduction of glycolysis by hypoglycemia or the glucose analog 2-deoxy-D-glucose (2DG) stimulates compensatory sympathetic alterations of metabolism. Considerable attention has been focused on the hypothalamus as the probable locus of requisite metabolic signal detection. We report, however, that unanesthetized chronically decerebrate rats are capable of exhibiting sympathoadrenal hyperglycemia in response to the metabolic challenge presented by 2DG. This findings demonstrates that the forebrain is not necessary for glucoprivic stimulation of this reflex. Since cervical cord transection has been shown to eliminate hyperglycemia induced by 2DG, we conclude that the caudal brainstem contains an essential part of the neural mechanism which both detects metabolic need and ameliorates that need through the release of stored fuels.

Insulin elicits ingestion in decerebrate rats.

Insulin administered to rats reliably elicits ingestion of food. To determine whether the neural mechanisms sufficient to control insulin-elicited ingestion are located in or caudal to the forebrain, decerebrate rats were treated with insulin and ingestive responses were measured. Insulin treatment produced hypoglycemia that was comparable, in magnitude and duration, in control and decerebrate rats. Decerebrate and control rats ingested significantly more sucrose solution while hypoglycemic than while normoglycemic. In contrast, insulin did not augment the water consumption of either group. These data indicate that neural systems caudal to the forebrain are sufficient to control ingestive consummatory behavior through the integration of metabolic signals generated by insulin treatment and taste afferent input from the oropharynx.

Glucagon-like peptide 1 (GLP-1).

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.

The hindbrain is a site of energy balance action for prolactin-releasing peptide: feeding and thermic effects from GPR10 stimulation of the nucleus tractus solitarius/area postrema.

PURPOSE: Prolactin-releasing peptide (PrRP) is a neuropeptide that suppresses food intake and increases body temperature when delivered to the forebrain ventricularly or parenchymally. However, PrRP's receptor GPR10 is widely distributed throughout the brain with particularly high levels found in the dorsomedial hindbrain. Thus, we hypothesized that hindbrain-directed PrRP administration would affect energy balance and motivated feeding behavior. METHODS: To address this hypothesis, a range of behavioral and physiologic variables were measured in Sprague-Dawley rats that received PrRP delivered to the fourth ventricle (4V) or the nucleus of the solitary tract (NTS) at the level of the area postrema (AP). RESULTS: 4V PrRP delivery decreased chow intake and body weight, in part, through decreasing meal size in ad libitum maintained rats tested at dark onset. PrRP inhibited feeding when delivered to the nucleus tractus solitarius (NTS), but not to more ventral hindbrain structures. In addition, 4V as well as direct NTS administration of PrRP increased core temperature. By contrast, 4V PrRP did not reduce ad libitum intake of highly palatable food or the motivation to work for or seek palatable foods. CONCLUSIONS: The dorsomedial hindbrain and NTS/AP, in particular, are sites of action in PrRP/GPR10-mediated control of chow intake, core temperature, and body weight.

Overfeeding-induced weight gain suppresses plasma ghrelin levels in rats.

The elevation of plasma ghrelin associated with weight loss has been taken as evidence of a role for ghrelin in the adaptive response to body weight change. However, there has been no clear experimental evidence that circulating ghrelin is suppressed by weight gain. We investigate this issue using a model of involuntary (intra-gastric gavage) overfeeding-induced obesity. Rats were first maintained at normal body weight with 4 daily tube-feedings of liquid diet (2.11 kcal/ml), each delivered at a volume of 9 ml. Gavage volume was then increased to 13 ml/feeding for 2 weeks, during which rats gained 25% of their initial body weight. Fasting plasma ghrelin levels and the response to 9- and 13-ml intra-gastric load sizes were measured during the weight-stable and overfed conditions. We found that: 1) weight gain decreased circulating ghrelin levels; 2) this response could not be attributed to additional food in the gastrointestinal tract; 3) the ghrelin response to nutrient loads was diminished in the obese vs normal-weight conditions. Having discounted diet composition and differences in gastric contents at the time of blood sampling, the decrease in ghrelin levels with overfeeding can be unambiguously attributed to physiological correlates of weight gain.

Comparison of immunocytochemical estrogen receptor assay, estrogen receptor enzyme immunoassay, and radioligand-labeled estrogen receptor assay in human breast cancer and uterine tissue.

Determination of estrogen receptor content in 82 breast cancer specimens with immunocytochemical estrogen receptor assay (ER-EIA) (Abbott) was compared with our routinely used binding assay using 125I-estradiol as radioligand with Scatchard plot analysis of the binding data. Although the estrogen receptor content measured with the ER-EIA was approximately 2-fold higher compared with the binding assay, the immunochemical method proved to be a useful alternative for estrogen receptor determination. Furthermore, it is possible to detect estrogen receptors in FPLC Superose 12 (size exclusion column) eluates or in the fractions obtained after sucrose density centrifugation using the ER-EIA. Forty breast cancer samples were analyzed utilizing the immunocytochemical technique (ER-ICA) for visualization of the estrogen receptor content in frozen tumor tissues in relationship to the quantitative results obtained with the ER-EIA assay. Specific staining for estrogen receptor was confined only to the cell nucleus, was distributed irregularly among the tumor cells, and was variable in intensity. The staining intensity and the percentage of positively stained cells increased with increasing level of cytosolic estrogen receptor. In 27 of 40 cases the immunocytochemical results correlated well with the ER-EIA assay. Nine cases were ER-ICA negative with positive ER-EIA, and four were ER-ICA positive with negative ER-EIA.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Ingestive taste reactivity as licking behavior.

In ingestive taste reactivity analysis, the rhythmic oral motor responses observed during intraoral infusion of fluids normally ingested by rats are categorized and counted. These rhythmic movements can be likened to spout-licking in several respects. Both are emitted in the same frequency range (5-8 Hz), organized in a burst/pause pattern, and serve the function of intraoral transport of fluid into position for swallowing. The parallel suggests that a temporal pattern analysis, based on the spout-licking literature, can be fruitfully applied to the rhythmic movements that attend intraoral infusion. We provide a demonstration of such an analysis using an electromyographic (EMG) recording-based method for automated event detection. Eight rats received a 37.5% glucose solution (1.0 ml/min) in a series of 120 s infusion trials (45 s intertrial intervals) that was extended until the fluid was rejected. Movement counts declined 19.1% from the first to the last complete trial. Parameters derived from the pattern analysis (number of bursts, mean burst duration, pause durations, coefficient of variation for the distribution of within-burst intermovement intervals) were affected to a greater extent. The results indicate the potential value of temporal pattern analysis for various applications of the taste reactivity paradigm.

The role of the dorsal vagal complex and the vagus nerve in feeding effects of melanocortin-3/4 receptor stimulation.

Fourth intracerebroventricular (4th-icv) administration of the melanocortin-3/4 receptor (MC3/4-R) agonist, MTII, reduces food intake; the antagonist, SHU9119, increases feeding. The dorsal motor nucleus of the vagus nerve (DMX) contains the highest density of MC4-R messenger RNA in the brain. To explore the possibility that the DMX contributes to 4th-icv MC4-R effects, we delivered doses of MTII and SHU9119 that are subthreshold for ventricular response unilaterally through a cannula centered above the DMX. MTII markedly suppressed 2-h (50%), 4-h (50%), and 24-h (33%) intake. Feeding was significantly increased 4 h (50%) and 24 h (20%) after SHU9119 injections. These results suggest that receptors in the DMX, or the dorsal vagal complex more generally, underlie effects obtained with 4th-icv administration of these ligands. We investigated possible vagal mediation of 4th-icv MTII effects by giving the agonist to rats with subdiaphragmatic vagotomy. MTII suppressed 2-, 4-, and 24-h liquid diet intake (approximately 80%) to the same extent in vagotomized and surgical control rats. We conclude that stimulation or antagonism of MC3/4-Rs in the dorsal vagal complex yields effects on food intake that do not require an intact vagus nerve.

Brainstem melanocortin 3/4 receptor stimulation increases uncoupling protein gene expression in brown fat.

Central administration of melanocortin 3 and 4 receptor (MC3/4-R) agonists increases energy expenditure, with the hypothalamus commonly held as the primary site of action. It is also clear, however, that MC4-R are expressed in caudal brainstem structures of relevance to the control of energy expenditure. Three experiments investigated whether hindbrain MC-R contribute to the energy expenditure effects of central MC3/4-R agonist treatments; in each, we examined the effect of fourth intracerebroventricular (i.c.v.) administration of a MC3/4-R agonist, MTII (three injections, each separated by 12 h), on uncoupling protein 1 (UCP-1) gene expression in brown adipose tissue (BAT). First, we compared the effects of fourth and third i.c.v. administration of MTII and found that the hindbrain and forebrain treatments were equally effective at elevating UCP-1 mRNA expression in BAT compared with the respective vehicle-treated group results. A second experiment demonstrated that the fourth i.c.v. MTII-induced rise in UCP-1 expression was mediated by sympathetic outflow to BAT by showing that this response was abolished by surgical denervation of BAT. In the third experiment, we showed that chronic decerebrate rats, like their neurologically intact controls, elevated UCP-1 mRNA expression in response to fourth i.c.v. MTII administration. Taken together, the results indicate that: 1) there is an independent caudal brainstem MC3/4-R trigger for a sympathetically stimulated elevation in BAT UCP-1 gene expression, and 2) the MTII-induced rise in UCP-1 expression can be mediated by circuitry intrinsic to the caudal brainstem and spinal cord.

Effects of postnatal estradiol and progesterone replacement in extremely preterm infants.

The fetus is supplied from the placenta with estradiol (E2) and progesterone (P) in increasing amounts during gestation. After delivery of a premature infant, placental supply is disrupted, resulting in a rapid decrease in E2 and P. Replacement of these placental hormones may restore intrauterine conditions and may be beneficial for bone mineral accretion, clinical course, and outcome. Thirty female infants with a median gestational age of 26.6 weeks (between 24.1-28.7) and a birth weight of 675 g (370-990) were randomized to receive E2 and P replacement, aiming to maintain plasma levels equaling the intrauterine levels, or no replacement. The E2 and P replacement was started iv and was followed by transepidermal administration for a total duration of 6 weeks. Repeated measurements included plasma levels of E2, P, FSH, and LH; uterine volume; calcium and phosphorus in spot urine specimens; and bone mineral accretion by single photon absorption densitometry. Further, the incidence of chronic lung disease and various clinical outcome data were recorded. The plasma levels of E2 and P were within the intrauterine range with median replacements of 2.30 mg/kg x day E2 (1.13-6.23) and 21.20 mg/kg x day P (11.23-27.36), iv. Three- and 6-fold higher doses of E2 and P were needed via the transepidermal route. The uterine volumes increased, and FSH and LH as indicators for biological effectiveness were significantly lowered with replacement. The bone mineral accretion rates tended to be higher, and the incidence of chronic lung disease tended to be lower (0% vs. 29%; P = 0.097). E2 and P replacement via iv and transepidermal routes is capable of maintaining plasma levels as high as those in utero with biological effectiveness. Trends toward improved postnatal bone mineral accretion and less chronic lung disease were found with the hormone replacement. Further and more extensive studies are warranted to address the role of this new approach in the care of extremely premature infants.

Lack of binding of gestodene to estrogen receptor in human breast cancer tissue.

Competition studies with progesterone and estradiol receptors of human myometrial tissue as well as of mammary cancer tissue showed that gestodene bound with high affinity to the progesterone receptor, as did other synthetic and natural progestogens. However, gestodene did not bind to the estradiol receptor. The relative binding affinities of all tested synthetic and natural ligands showed no organ-specific differences and no differences between neoplastically transformed and normal tissues.

Detection of mammaglobin expressing cells in blood of breast cancer patients.

Expression of human mammaglobin (hMAM) was published to be exclusively expressed in mammary tissue, in solid tumors, axillary lymph nodes and disseminated cancer cells in blood of breast cancer patients. A quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) test was applied to investigate hMAM expression in blood of breast cancer patients. Mammaglobin mRNA expression was found not only in breast cancer cell lines but also in cell lines of other cancer origin. In our patient cohort hMAM expression in 11/98 (11%) samples of breast cancer and 3/12 (25%) ovarian cancer patients could be detected. hMAM mRNA expression as a candidate marker for the detection of disseminated cancer cells in blood of breast cancer patients showed low sensitivity and reduced tissue specificity. A prognostic significance of hMAM expression could not be demonstrated.

The tumour-inhibiting potential of the progesterone antagonist Onapristone in the human mammary carcinoma T61 in nude mice.

The progesterone antagonist Onapristone proved to possess strong tumour-inhibiting activity in a panel of experimental mammary carcinomas. Its underlying mechanism of action is due to a progesterone-receptor-mediated induction of terminal differentiation and a specific blockade of the cell cycle and is also present in the absence of progesterone as was shown in the MXT mammary tumour. To prove this further, the tumour-inhibiting activity of Onapristone was investigated in the human postmenopausal T61 mammary tumour implanted in castrated male nude mice. Whereas Onapristone given alone had no effect on growth of established tumours, after stimulation of the relatively low progesterone receptor content of this tumour line with an oestrogen, Onapristone significantly inhibited tumour growth. Thus, we suggest that Onapristone exerts its antitumour action via progesterone receptors. As there is no endogenous progesterone in these mice, the tumour-inhibiting activity of Onapristone is not primarily due to a classical antihormonal effect.

Alternating ingestive and aversive consummatory responses suggest a two-dimensional analysis of palatability in rats.

The hedonic response to a taste is typically regarded to be the product of a central integration of gustatory afferent information, which ends in a single decision about the nature and intensity of the response to be given. This hedonic response is often characterized as a point lying along a single dimension of palatability, stretching from strongly positive to strongly negative. The present analysis of species-specific consummatory responses suggests that the final response is not made on the basis of a single central analysis of taste information but rather is the result of a competition between two separate systems that are activated by tastes. A single oral infusion of a taste solution may elicit rapid alternation between ingestive and aversive consummatory responses. Such alternation is better interpreted as due to a simultaneous activation of two palatability dimensions than as a reflection of neutral palatability. When increases in the magnitude of aversive responses are produced by taste mixtures, there is not necessarily a reciprocal decrease in ingestive responses. This asymmetry supports the hypothesis of independent palatability dimensions.