Weight loss in adult male Wistar rats by Roux-en-Y gastric bypass is primarily explained by caloric intake reduction and presurgery body weight.

Diets varying in macronutrient composition, energy density, and/or palatability may cause differences in outcome of bariatric surgery. In the present study, rats feeding a healthy low-fat (LF) diet or an obesogenic high-fat/sucrose diet (HF/S) were either subjected to Roux-en-Y gastric bypass surgery (RYGB) or sham surgery, and weight loss trajectories and various energy balance parameters were assessed. Before RYGB, rats eating an HF/S (n = 14) diet increased body weight relative to rats eating an LF diet (n = 20; P < 0.01). After RYGB, absolute weight loss was larger in HF/S (n = 6) relative to LF feeding (n = 6) rats, and this was associated with reduced cumulative energy intake (EI; P < 0.05) and increased locomotor activity (LA; P < 0.05-0.001), finally leading to similar levels of reduced body fat content in HF/S and LF rats 3 wk after surgery. Regression analysis revealed that variation in RYGB-induced body weight loss was best explained by models including 1) postoperative cumulative EI and preoperative body weight (R2 = 0.87) and 2) postoperative cumulative EI and diet (R2 = 0.79), each without significant contribution of LA. Particularly rats on the LF diet became transiently more hypothermic and circadianally arrhythmic following RYGB (i.e., indicators of surgery-associated malaise) than HF/S feeding rats. Our data suggest that relative to feeding an LF diet, continued feeding an HF/S diet does not negatively impact recovery from RYGB surgery, yet it promotes RYGB-induced weight loss. The RYGB-induced weight loss is primarily explained by reduced cumulative EI and higher preoperative body weight, leading to comparably low levels of body fat content in HF/S and LF feeding rats.NEW & NOTEWORTHY Relative to feeding an LF diet, continued feeding an HF/S diet does not negatively impact recovery from RYGB surgery in rats. Relative to feeding an LF diet, continued feeding an HF/S diet promotes RYGB-induced weight loss. The RYGB-induced weight loss is primarily explained by reduced cumulative EI and higher preoperative body weight, leading to comparably low levels of body fat content in HF/S and LF feeding rats.

The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

Body weight lowering effect of glucose-dependent insulinotropic polypeptide and glucagon-like peptide receptor agonists is more efficient in RAMP1/3 KO than in WT mice.

The glucose-dependent insulinotropic polypeptide (GIPR) and glucagon-like peptide (GLP-1R) receptor agonists are insulin secretagogues that have long been shown to improve glycemic control and dual agonists have demonstrated successful weight loss in the clinic. GIPR and GLP-1R populations are located in the dorsal vagal complex where receptor activity-modifying proteins (RAMPs) are also present. According to recent literature, RAMPs not only regulate the signaling of the calcitonin receptor, but also that of other class B G-protein coupled receptors, including members of the glucagon receptor family such as GLP-1R and GIPR. The aim of this study was to investigate whether the absence of RAMP1 and RAMP3 interferes with the action of GIPR and GLP-1R agonists on body weight maintenance and glucose control. To this end, WT and RAMP 1/3 KO mice were fed a 45% high fat diet for 22 weeks and were injected daily with GLP-1R agonist (2 nmol/kg/d; NN0113-2220), GIPR agonist (30 nmol/kg/d; NN0441-0329) or both for 3 weeks. While the mono-agonists exerted little to no body weight lowering and anorectic effects in WT or RAMP1/3 KO mice, but at the given doses, when both compounds were administered together, they synergistically reduced body weight, with a greater effect observed in KO mice. Finally, GLP-1R and GIP/GLP-1R agonist treatment led to improved glucose tolerance, but the absence of RAMPs resulted in an improvement of the HOMA-IR score. These data suggest that RAMPs may play a crucial role in modulating the pharmacological actions of GLP-1 and GIP receptors.

Early Postoperative Exposure to High-Fat Diet Does Not Increase Long-Term Weight Loss or Fat Avoidance After Roux-en-Y Gastric Bypass in Rats.

Background: Bariatric surgery alters food preferences in rats and reportedly decreases desire to consume high-fat high-sugar food in humans. The aim of this study was to investigate whether early post-operative exposure to high-fat food could increase body weight loss after Roux-en-Y gastric bypass (RYGB) by triggering fat avoidance. Methods: Male Wistar rats underwent either RYGB (n = 15) or sham-operations (n = 16). Preoperatively a standardized 4-choice cafeteria diet [dietary options: low-fat/low-sugar (LFLS), low-fat/high-sugar (LFHS), high-fat/low-sugar (HFLS), high-fat/high-sugar (HFHS)] was offered. First, each option was available for 4 days, thereafter rats were offered the 4 options simultaneously for 3 days preoperatively. Post-surgery, 8 rats in the RYGB- and 8 in the sham-group were exposed to a high-fat content diet (Oatmeal + 30% lard, OM+L) for 10 days, while 7 RYGB rats and 8 sham-rats received OM alone. From the 11th postoperative day, the 4-choice cafeteria diet was reintroduced for 55-days. The intake of all available food items, macronutrients and body weight changes were monitored over 8 weeks. Main outcomes were long-term body-weight and daily change in relative caloric intake during the postoperative cafeteria period compared to the preoperative cafeteria. Results: During the first 12 days of postoperative cafeteria access, RYGB-rats exposed to OM+L had a higher mean caloric intake per day than RYGB rats exposed to OM alone (Δ10 kCal, P adj = 0.004), but this difference between the RYGB groups disappeared thereafter. Consequently, in the last 33 days of the postoperative cafeteria diet, the mean body weight of the RYGB+OM+L group was higher compared to RYGB+OM (Δ51 g, P adj < 0.001). RYGB rats, independently from the nutritional intervention, presented a progressive decrease in daily consumption of calories from fat and increased their daily energy intake mainly from non-sugar carbohydrates. No such differences were detected in sham-operated controls exposed to low- or high fat postoperative interventions. Conclusion: A progressive decrease in daily fat intake over time was observed after RYGB, independently from the nutritional intervention. This finding confirms that macronutrient preferences undergo progressive changes over time after RYGB and supports the role of ingestive adaptation and learning. Early postoperative exposure to high-fat food failed to accentuate fat avoidance and did not lead to superior weight loss in the long-term.

Mediators of Amylin Action in Metabolic Control.

Amylin (also called islet amyloid polypeptide (IAPP)) is a pancreatic beta-cell hormone that is co-secreted with insulin in response to nutrient stimuli. The last 35 years of intensive research have shown that amylin exerts important physiological effects on metabolic control. Most importantly, amylin is a physiological control of meal-ending satiation, and it limits the rate of gastric emptying and reduces the secretion of pancreatic glucagon, in particular in postprandial states. The physiological effects of amylin and its analogs are mediated by direct brain activation, with the caudal hindbrain playing the most prominent role. The clarification of the structure of amylin receptors, consisting of the calcitonin core receptor plus receptor-activity modifying proteins, aided in the development of amylin analogs with a broad pharmacological profile. The general interest in amylin physiology and pharmacology was boosted by the finding that amylin is a sensitizer to the catabolic actions of leptin. Today, amylin derived analogs are considered to be among the most promising approaches for the pharmacotherapy against obesity. At least in conjunction with insulin, amylin analogs are also considered important treatment options in diabetic patients, so that new drugs may soon be added to the only currently approved compound pramlintide (Symlin®). This review provides a brief summary of the physiology of amylin's mode of actions and its role in the control of the metabolism, in particular energy intake and glucose metabolism.

Roux-En-Y Gastric Bypass (RYGB) Surgery during High Liquid Sucrose Diet Leads to Gut Microbiota-Related Systematic Alterations.

Roux-en-Y gastric bypass (RYGB) surgery has been proven successful in weight loss and improvement of co-morbidities associated with obesity. Chronic complications such as malabsorption of micronutrients in up to 50% of patients underline the need for additional therapeutic approaches. We investigated systemic RYGB surgery effects in a liquid sucrose diet-induced rat obesity model. After consuming a diet supplemented with high liquid sucrose for eight weeks, rats underwent RYGB or control sham surgery. RYGB, sham pair-fed, and sham ad libitum-fed groups further continued on the diet after recovery. Notable alterations were revealed in microbiota composition, inflammatory markers, feces, liver, and plasma metabolites, as well as in brain neuronal activity post-surgery. Higher fecal 4-aminobutyrate (GABA) correlated with higher Bacteroidota and Enterococcus abundances in RYGB animals, pointing towards the altered enteric nervous system (ENS) and gut signaling. Favorable C-reactive protein (CRP), serine, glycine, and 3-hydroxybutyrate plasma profiles in RYGB rats were suggestive of reverted obesity risk. The impact of liquid sucrose diet and caloric restriction mainly manifested in fatty acid changes in the liver. Our multi-modal approach reveals complex systemic changes after RYGB surgery and points towards potential therapeutic targets in the gut-brain system to mimic the surgery mode of action.

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism.

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.

Hypoglycemia attenuates acute amylin-induced reduction of food intake in male rats.

The ability of amylin to inhibit gastric emptying and glucagon secretion in rats is reduced under hypoglycemic conditions. These effects are considered part of a fail-safe mechanism that prevents amylin from further decreasing nutrient supply when blood glucose levels are low. Because these actions and amylin-induced satiation are mediated by the area postrema (AP), it is plausible that these phenomena are based on the co-sensitivity of AP neurons to amylin and glucose. Using hyperinsulinemic glucose clamps in unrestrained and freely-feeding rats, we investigated whether amylin's ability to inhibit food intake is also reduced by hypoglycemia (HYPO). Following an 18 h fast, rats were infused with insulin and glucose for 45 min to clamp blood glucose at baseline levels (between 90 and 100 mg/dL). HYPO (approximately 55 mg/dL) was induced between 45 and 60 min and then maintained for the remainder of the clamp. Rats were injected with amylin (20 µg/kg) or saline and offered normal chow at 85 min. Food intake was measured at 30 and 60 min after amylin. Control hyperinsulinemic/euglycemic (EU) rats were maintained at approximately 150 mg/dL (which is a physiological periprandial glucose level) before and after amylin injection. Terminal experiments tested the effect of amylin to induce the phosphorylation of ERK, a marker of amylin action in the AP, in EU and HYPO conditions. Amylin significantly reduced 30- and 60-min food intake in EU rats, but the effect at 60-min was attenuated in HYPO rats. Interestingly, glucose infusion rate had to be dramatically reduced at meal onset in saline-treated, but not in amylin-treated, EU or HYPO rats; this suggests that meal-related glucose appearance in the blood was inhibited by amylin under both EU and HYPO. Finally, amylin induced a similar pERK response in the AP in EU and HYPO rats. We conclude that amylin's action to decrease eating is blunted in hypoglycemia, and this effect seems to be downstream from amylin-induced pERK in AP neurons. These data allow us to extend the idea of a hypoglycemic brake on amylin's actions to its food intake-reducing effect, but also demonstrate that amylin can buffer meal-induced glucose appearance at EU and HYPO levels.

Influence of High Energy Diet and Polygenic Predisposition for Obesity on Postpartum Health in Rat Dams.

It is estimated that 30% of pregnant women worldwide are overweight or obese, leading to adverse health effects for both mother and child. Women with obesity during pregnancy are at higher risk for developing both metabolic and mental disorders, such as diabetes and depression. Numerous studies have used rodent models of maternal obesity to understand its consequences on the offspring, yet characterization of changes in the dams is rare, and most rodent models rely solely on a high fat diet to induce maternal obesity, without regarding genetic propensity for obesity. Here we present the influence of both peripartum high energy diet (HE) and obesity-proneness on maternal health using selectively bred diet-resistant (DR) and diet-induced obese (DIO) rat dams. Outbred Sprague-Dawley rats were challenged with HE diet prior to mating and bred according to their propensity to gain weight. The original outbred breeding dams (F0) were maintained on low-fat chow during pregnancy and lactation. By comparison, the F1 dams consuming HE diet during pregnancy and lactation displayed higher gestational body weight gain (P < 0.01), and HE diet caused increased meal size and reduced meal frequency (P < 0.001). Sensitivity to the hormone amylin was preserved during pregnancy, regardless of diet. After several rounds of selective breeding, DIO and DR dams from generation F3 were provided chow or HE during pregnancy and lactation and assessed for their postpartum physiology and behaviors. We observed strong diet and phenotype effects on gestational weight gain, with DIO-HE dams gaining 119% more weight than DR-chow (P < 0.001). A high-resolution analysis of maternal behaviors did not detect main effects of diet or phenotype, but a subset of DIO dams showed delayed nursing behavior (P < 0.05). In generation F6/F7 dams, effects on gestational weight gain persisted (P < 0.01), and we observed a main effect of phenotype during a sucrose preference test (P < 0.05), with DIO-chow dams showing lower sucrose preference than DR controls (P < 0.05). Both DIO and DR dams consuming HE diet had hepatic steatosis (P < 0.001) and exhibited reduced leptin sensitivity in the arcuate nucleus (P < 0.001). These data demonstrate that both diet and genetic obesity-proneness have consequences on maternal health.

Chronic social stress in mice alters energy status including higher glucose need but lower brain utilization.

Chronic stress leads to changes in energy status and is a major risk factor for depression, with common symptoms of reductions in body weight and effortful motivation for reward. Indeed, stress-induced disturbed energy status could be a major aetio-pathogenic factor for depression. Improved understanding of these putative inter-relationships requires animal model studies of effects of stress on both peripheral and central energy-status measures and determinants. Here we conducted a study in mice fed on a standard low-fat diet and exposed to either 15-day chronic social stress (CSS) or control handling (CON). Relative to CON mice, CSS mice had attenuated body weight maintenance/gain despite consuming the same amount of food and expending the same amount of energy at any given body weight. The low weight of CSS mice was associated with less white and brown adipose tissues, and with a high respiratory exchange ratio consistent with increased dependence on glucose as energy substrate. Basal plasma insulin was low in CSS mice and exogenous glucose challenge resulted in a relatively prolonged elevation of blood glucose. With regard to hunger and satiety hormones, respectively, CSS mice had higher levels of acylated ghrelin in plasma and of ghrelin receptor gene expression in ventromedial hypothalamus and lower levels of plasma leptin, relative to CON mice. However, whilst CSS mice displayed this constellation of peripheral changes consistent with increases in energy need and glucose utilization relative to CON mice, they also displayed attenuated uptake of [18F]FDG in brain tissue specifically. Reduced brain glucose utilization in CSS mice could contribute to the reduced effortful motivation for reward in the form of sweet-tasting food that we have reported previously for CSS mice. It will now be important to utilize this model to further understanding of the mechanisms via which chronic stress can increase energy need but decrease brain glucose utilization and how this relates to regional and cellular changes in neural circuits for reward processing relevant to depression.

Vaccination Against Amyloidogenic Aggregates in Pancreatic Islets Prevents Development of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic progressive disease characterized by insulin resistance and insufficient insulin secretion to maintain normoglycemia. The majority of T2DM patients bear amyloid deposits mainly composed of islet amyloid polypeptide (IAPP) in their pancreatic islets. These-originally ß-cell secretory products-extracellular aggregates are cytotoxic for insulin-producing ß-cells and are associated with ß-cell loss and inflammation in T2DM advanced stages. Due to the absence of T2DM preventive medicaments and the presence of only symptomatic drugs acting towards increasing hormone secretion and action, we aimed at establishing a novel disease-modifying therapy targeting the cytotoxic IAPP deposits in order to prevent the development of T2DM. We generated a vaccine based on virus-like particles (VLPs), devoid of genomic material, coupled to IAPP peptides inducing specific antibodies against aggregated, but not monomeric IAPP. Using a mouse model of islet amyloidosis, we demonstrate in vivo that our vaccine induced a potent antibody response against aggregated, but not soluble IAPP, strikingly preventing IAPP depositions, delaying onset of hyperglycemia and the induction of the associated pro-inflammatory cytokine Interleukin 1ß (IL-1ß). We offer the first cost-effective and safe disease-modifying approach targeting islet dysfunction in T2DM, preventing pathogenic aggregates without disturbing physiological IAPP function.

Amylin and Leptin interaction: Role During Pregnancy, Lactation and Neonatal Development.

Amylin is co-secreted with insulin by pancreatic ß-cells in response to a meal and produced by neurons in discrete hypothalamic brain areas. Leptin is proportionally secreted by the adipose tissue. Both hormones control food intake and energy homeostasis post-weaning in rodents. While amylin's main site of action is located in the area postrema (AP) and leptin's is located in the mediobasal hypothalamus, both hormones can also influence the other's signaling pathway; amylin has been shown enhance hypothalamic leptin signaling, and amylin signaling in the AP may rely on functional leptin receptors to modulate its effects. These two hormones also play major roles during other life periods. During pregnancy, leptin levels rise as a result of an increase in fat depot resulting in gestational leptin-resistance to prepare the maternal body for the metabolic needs during fetal development. The role of amylin is far less studied during pregnancy and lactation, though amylin levels seem to be elevated during pregnancy relative to insulin. Whether amylin and leptin interact during pregnancy and lactation remains to be assessed. Lastly, during brain development, amylin and leptin are major regulators of cell birth during embryogenesis and act as neurotrophic factors in the neonatal period. This review will highlight the role of amylin and leptin, and their possible interaction, during these dynamic time periods of pregnancy, lactation, and early development.

RAMP1 and RAMP3 Differentially Control Amylin's Effects on Food Intake, Glucose and Energy Balance in Male and Female Mice.

Amylin is a pancreatic peptide, which acts as a key controller of food intake and energy balance and predominately binds to three receptors (AMY 1-3). AMY 1-3 are composed of a calcitonin core receptor (CTR) and associated receptor-activity modifying proteins (RAMPs) 1-3. Using RAMP1, RAMP3 and RAMP1/3 global KO mice, this study aimed to determine whether the absence of one or two RAMP subunits affects food intake, glucose homeostasis and metabolism. Of all the RAMP-deficient mice, only high-fat diet fed RAMP1/3 KO mice had increased body weight. Chow-fed RAMP3 KO and high-fat diet fed 1/3 KO male mice were glucose intolerant. Fat depots were increased in RAMP1 KO male mice. No difference in energy expenditure was observed but the respiratory exchange ratio (RER) was elevated in RAMP1/3 KO. RAMP1 and 1/3 KO male mice displayed an increase in intermeal interval (IMI) and meal duration, whereas IMI was decreased in RAMP3 KO male and female mice. WT and RAMP1, RAMP3, and RAMP1/3 KO male and female littermates were then assessed for their food intake response to an acute intraperitoneal injection of amylin or its receptor agonist, salmon calcitonin (sCT). RAMP1/3 KO were insensitive to both, while RAMP3 KO were responsive to sCT only and RAMP1 KO to amylin only. While female mice generally weighed less than male mice, only RAMP1 KO showed a clear sex difference in meal pattern and food intake tests. Lastly, a decrease in CTR fibers did not consistently correlate with a decrease in amylin- induced c-Fos expression in the area postrema (AP). Ultimately, the results from this study provide evidence for a role of RAMP1 in mediation of fat utilization and a role for RAMP3 in glucose homeostasis and amylin's anorectic effect.

Body weight-dependent and independent improvement in lipid metabolism after Roux-en-Y gastric bypass in ApoE*3Leiden.CETP mice.

BACKGROUND/OBJECTIVES: The incidence of obesity and metabolic syndrome (MetS) has rapidly increased worldwide. Roux-en-Y gastric bypass (RYGB) achieves long-term weight loss and improves MetS-associated comorbidities. Using a mouse model with a humanized lipoprotein metabolism, we elucidated whether improvements in lipid and glucose metabolism after RYGB surgery are body weight loss-dependent or not. SUBJECTS/METHODS: Male ApoE*3Leiden.CETP (ApoE3L.CETP) mice fed Western type diet for 6 weeks underwent RYGB or Sham surgery. Sham groups were either fed ad libitum or were body weight-matched (BWm) to the RYGB mice to discriminate surgical effects from body weight loss-associated effects. Before and after surgery, plasma was collected to assess the metabolic profile, and glucose tolerance and insulin sensitivity were tested. Twenty days after surgery, mice were sacrificed, and liver was collected to assess metabolic, histological and global gene expression changes after surgery. RESULTS: RYGB induced a marked reduction in body weight, which was also achieved by severe food restriction in BWm mice, and total fat mass compared to Sham ad libitum mice (Sham AL). Total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) and ceramide were strongly reduced 20 days after surgery in RYGB compared to BWm mice. Glucose tolerance and insulin sensitivity improved 13 days after surgery similarly in RYGB and BWm mice. Liver histology confirmed lipid reduction in RYGB and BWm mice while the transcriptomics data indicated altered genes expression in lipid metabolism. CONCLUSIONS: RYGB surgery improves glucose metabolism and greatly ameliorates lipid metabolism in part in a body weight-dependent manner. Given that ApoE3L.CETP mice were extensively studied to describe the MetS, and given that RYGB improved ceramide after surgery, our data confirmed the usefulness of ApoE3L.CETP mice after RYGB in deciphering the metabolic improvements to treat the MetS.

Rodent models of leptin receptor deficiency are less sensitive to amylin.

The pancreatic hormone amylin is released from beta cells following nutrient ingestion and contributes to the control of body weight and glucose homeostasis. Amylin reduces food intake by activating neurons in the area postrema (AP). Amylin was also shown to synergize with the adipokine leptin, with combination therapy producing greater weight loss and food intake reduction than either hormone alone. Although amylin and leptin were initially thought to interact downstream of the AP in the hypothalamus, recent findings show that the two hormones can act on the same AP neurons, suggesting a more direct relationship. The objective of this study was to determine whether amylin action depends on functional leptin signaling. We tested the ability of amylin to induce satiation and to activate its primary target neurons in the AP in two rodent models of LepR deficiency, the db/db mouse and the Zucker diabetic fatty (ZDF) rat. When compared with wild-type (WT) mice, db/db mice exhibited reduced amylin-induced satiation, reduced amylin-induced Fos in the AP, and a lower expression of calcitonin receptor (CTR) protein, the core component of all amylin receptors. ZDF rats also showed no reduction in food intake following amylin treatment; however, unlike the db/db mice, levels of amylin-induced Fos and CTR in the AP were no different than WT rats. Our results suggest that LepR expression is required for the full anorexic effect of amylin; however, the neuronal activation in the AP seems to depend on the type of LepR mutation.

Sensitive quantification of the somatostatin analog AP102 in plasma by ultra-high pressure liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study in rats.

AP102 is a di-iodinated octapeptide somatostatin agonist (SSA) designed to treat acromegaly and neuroendocrine tumors. A sensitive and selective method was validated for the quantification of AP102 in plasma following the European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. Sample preparation was performed using solid-phase extraction microplates. Chromatographic separation was achieved on an ultra-high pressure liquid chromatography (UHPLC) C18 column in 6.0 minutes. The compounds were quantified using multiple reaction monitoring on a tandem quadrupole mass spectrometer with 13 C,15 N-labeled AP102 as internal standard. Calibration ranged from 50 to 10000 pg/mL. The lower limit of quantification (LLOQ) was measured at 20 pg/mL, and robust analytical performances were obtained with trueness at 99.2%-100.0%, intra-assay imprecision at 2.5%-4.4%, and inter-assay imprecision at 8.9%-9.7%. The accuracy profiles (total error) built on the 3 concentrations levels showed accuracy within the 70%-130% range. AP102 is remarkably stable since no proteolytic fragments were detected on plasma samples analyzed by Orbitrap-MS. Pharmacokinetic studies were conducted in rats, after single dose (1, 3, and 10 µg/kg, sc) and continuous subcutaneous administration (osmotic minipumps for 28 days, 3.0 or 10.0 µg/kg/h). AP102 showed a rapid absorption by the subcutaneous route (Tmax : 15-30 minutes) and a fast elimination (t1/2 : 33-86 minutes). The PK profile of AP102 exhibited a mean clearance of 1.67 L/h and a mean distribution volume at steady state of 7.16 L/kg, about 10-fold higher than those observed with other SSA or non- and mono-iodinated AP102. LogD7.4 determination confirmed the lipophilic properties of AP102 that might influence its distribution in tissues.

Amylin Selectively Signals Onto POMC Neurons in the Arcuate Nucleus of the Hypothalamus.

Amylin phosphorylates ERK (p-ERK) in the area postrema to reduce eating and synergizes with leptin to phosphorylate STAT3 in the arcuate (ARC) and ventromedial (VMN) hypothalamic nuclei to reduce food intake and body weight. The current studies assessed potential amylin and amylin-leptin ARC/VMN interactions on ERK signaling and their roles in postnatal hypothalamic pathway development. In amylin knockout mice, the density of agouti-related protein (AgRP)-immunoreactive (IR) fibers in the hypothalamic paraventricular nucleus (PVN) was increased, while the density of α-melanocyte-stimulating hormone (αMSH) fibers was decreased. In mice deficient of the amylin receptor components RAMP1/3, both AgRP and αMSH-IR fiber densities were decreased, while only αMSH-IR fiber density was decreased in rats injected neonatally in the ARC/VMN with an adeno-associated virus short hairpin RNA against the amylin core receptor. Amylin induced p-ERK in ARC neurons, 60% of which was present in POMC-expressing neurons, with none in NPY neurons. An amylin-leptin interaction was shown by an additive effect on ARC ERK signaling in neonatal rats and a 44% decrease in amylin-induced p-ERK in the ARC of leptin receptor-deficient and of ob/ob mice. Together, these results suggest that amylin directly acts, through a p-ERK-mediated process, on POMC neurons to enhance ARC-PVN αMSH pathway development.

Involvement of Amylin and Leptin in the Development of Projections from the Area Postrema to the Nucleus of the Solitary Tract.

The area postrema (AP) and the nucleus of the solitary tract (NTS) are important hindbrain centers involved in the control of energy homeostasis. The AP mediates the anorectic action and the inhibitory effect on gastric emptying induced by the pancreatic hormone amylin. Amylin's target cells in the AP project to the NTS, an integrative relay center for enteroceptive signals. Perinatal hormonal and metabolic factors influence brain development. A postnatal surge of the adipocyte-derived hormone leptin represents a developmental signal for the maturation of projections between hypothalamic nuclei controlling energy balance. Amylin appears to promote neurogenesis in the AP in adult rats. Here, we examined whether amylin and leptin are required for the development of projections from the AP to the NTS in postnatal and adult mice by conducting neuronal tracing studies with DiI in amylin- (IAPP-/-) and leptin-deficient (ob/ob) mice. Compared to wild-type littermates, postnatal (P10) and adult (P60) IAPP-/- mice showed a significantly reduced density of AP-NTS projections. While AP projections were also reduced in postnatal (P14) ob/ob mice, AP-NTS fiber density did not differ between adult ob/ob and wild-type animals. Our findings suggest a crucial function of amylin for the maturation of neuronal brainstem pathways controlling energy balance and gastrointestinal function. The impaired postnatal development of neuronal AP-NTS projections in ob/ob mice appears to be compensated in this experimental model during later brain maturation. It remains to be elucidated whether an amylin- and leptin-dependent modulation in neuronal development translates into altered AP/NTS-mediated functions.

Effect of AP102, a subtype 2 and 5 specific somatostatin analog, on glucose metabolism in rats.

PURPOSE: Somatostatin analogs are widely used to treat conditions associated with hormonal hypersecretion such as acromegaly and metastatic neuroendocrine tumors. First generation somatostatin analogs, such as octreotide and lanreotide, have high affinity for somatostatin receptor subtype 2 (SSTR2), but have incomplete efficacy in many patients. Pasireotide targets multiple SSTRs, having the highest affinity for SSTR5, but causes hyperglycemia and diabetes mellitus in preclinical and clinical studies. AP102 is a new somatostatin analogs with high affinity at both SSTR2 and SSTR5. We aimed to characterize the effects of AP102 vs. pasireotide on random and dynamic glucose levels, glucoregulatory hormone concentrations and growth axis measures in healthy Sprague-Dawley rats. METHODS: Three doses of each compound were evaluated under acute conditions (1, 10, and 30 µg/kg s.c.), and two doses during a chronic (4-week) infusion (3 and 10 µg/kg/h s.c.). RESULTS: Neither acute nor chronic AP102 administration altered blood glucose concentrations or dynamic responses following an intraperitoneal glucose tolerance test. In contrast, acute and chronic pasireotide dosing increased random and post-intraperitoneal glucose tolerance test blood glucose measures, compared to vehicle-treated controls. Both AP102 and pasireotide acutely suppressed growth hormone levels, although insulin-like growth factor-1 and somatic growth was suppressed to a greater extent with pasireotide. CONCLUSIONS: AP102 is a new dual SSTR2/SSTR5-specific somatostatin analog that acutely reduces growth hormone but does not cause hyperglycemia during acute or chronic administration in a healthy rat model. Further studies in diabetic animals and in humans are necessary to determine the potential utility of AP102 in the clinical setting.