Christina N. Boyle

Specific amino acids inhibit food intake via the area postrema or vagal afferents

•u2002 Proteins are more satiating than fats or lipids. Proteins are built by the 20 proteogenic amino acids. •u2002 Here, we identified l‐arginine, l‐lysine and l‐glutamic acid as the most potent anorectic amino acids in rats. •u2002 l‐Arginine and l‐glutamic acid require intact neurons in the area postrema to inhibit food intake, whereas l‐lysine requires intact afferent fibres of the vagus nerve. All three mediate their effect by the blood stream. •u2002 All three amino acids induce gastric distension by delaying gastric emptying and inducing secretion. However, the gastric phenotype does not mediate the anorectic response. •u2002 These results unravel amino acid‐specific mechanisms regulating digestion and eating behaviour and thereby contribute to the understanding of nutrient sensing in vivo.

Influence of high-fat feeding, diet-induced obesity, and hyperamylinemia on the sensitivity to acute amylin

Obesity results in the increased secretion of various hormones controlling food intake and body weight, such as leptin, and insulin; increased circulating levels of pancreatic amylin have also been described in obese humans and rodents. Because leptin-resistance is present in diet-induced obese (DIO) rats, and because hyperleptinemia seems necessary for the full development of leptin resistance, we tested whether amylin sensitivity is inversely correlated with adiposity, such that DIO reduces the anorectic action of acute amylin. We also determined if hyperamylinemia leads to a change in amylin sensitivity. In the first experiment, rats were chronically exposed to a high fat (HF; 60% fat) diet or fed standard chow for control. The anorectic response to amylin was tested on several occasions over a 14 week observation period. HF feeding led to the expected increase in body adiposity; the response to an acute amylin injection (5-50 μg/kg s.c.) was unaltered for 10 weeks of HF feeding. Even after 12 weeks on a HF diet, which clearly caused obesity, acute administration of amylin (5 μg/kg, s.c.) was still able to suppress food intake, although the suppression was not statistically significant. Further experiments using additional doses of amylin will be necessary to demonstrate possible amylin resistance after HF feeding or in DIO rats. In the second experiment, we tested more specifically whether hyperamylinemia that may result from HF feeding and subsequent obesity, reduces the sensitivity of the amylin signaling system. To avoid confounding factors, we chronically infused lean chow fed rats with amylin (5 or 10 μg/kg/day s.c.) to elevate their plasma amylin concentration to levels observed in obese rats (30-40 pM). In the absence of obesity, hyperamylinemia did not lead to a reduced sensitivity to acute amylin (5-20 μg/kg s.c.) injections; acute amylin reduced eating similarly in all groups of rats. Overall, we concluded that direct diet effects by short term exposure to HF appear to be of little importance for amylin sensitivity; further, long-term maintenance on a HF diet and the resulting obesity only slightly attenuated the anorectic response to acute amylin. Because we observed no marked changes in amylin sensitivity in lean, chow fed rats with induced hyperamylinemia, amylin receptor downregulation in chronic hyperamylinemia does not seem to occur.

Involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in amylin's eating inhibitory effect

Peripheral amylin inhibits eating via the area postrema (AP). Because amylin activates the extracellular-signal regulated kinase 1/2 (ERK) pathway in some tissues, and because ERK1/2 phosphorylation (pERK) leads to acute neuronal responses, we postulated that it may be involved in amylins eating inhibitory effect. Amylin-induced ERK phosphorylation (pERK) was investigated by immunohistochemistry in brain sections containing the AP. pERK-positive AP neurons were double-stained for the calcitonin 1a/b receptor, which is part of the functional amylin-receptor. AP sections were also phenotyped using dopamine-β-hydroxylase (DBH) as a marker of noradrenergic neurons. The effect of fourth ventricular administration of the ERK cascade blocker U0126 on amylins eating inhibitory action was tested in feeding trials. The number of pERK-positive neurons in the AP was highest ∼10-15 min after amylin treatment; the effect appeared to be dose-dependent (5-20 μg/kg amylin). A portion of pERK-positive neurons in the AP carried the amylin-receptor and 22% of the pERK-positive neurons were noradrenergic. Pretreatment of rats with U0126 decreased the number of pERK-positive neurons in the AP after amylin injection. U0126 also attenuated the ability of amylin to reduce eating, at least when the animals had been fasted 24 h prior to the feeding trial. Overall, our results suggest that amylin directly stimulates pERK in AP neurons in a time- and dose-dependent manner. Part of the AP neurons displaying pERK were noradrenergic. At least under fasting conditions, pERK was shown to be a necessary part in the signaling cascade mediating amylins anorectic effect.

Amylin-Induced Central IL-6 Production Enhances Ventromedial Hypothalamic Leptin Signaling

Amylin acts acutely via the area postrema to reduce food intake and body weight, but it also interacts with leptin over longer periods of time, possibly via the ventromedial hypothalamus (VMH), to increase leptin signaling and phosphorylation of STAT3. We postulated that amylin enhances VMH leptin signaling by inducing interleukin (IL)-6, which then interacts with its gp130 receptor to activate STAT3 signaling and gene transcription downstream of the leptin receptor. We found that components of the amylin receptor (RAMPs1–3, CTR1a,b) are expressed in cultured VMH astrocytes, neurons, and microglia, as well as in micropunches of arcuate and ventromedial hypothalamic nuclei (VMN). Amylin exposure for 5 days increased IL-6 mRNA expression in VMH explants and microglia by two- to threefold, respectively, as well as protein abundance in culture supernatants by five- and twofold, respectively. Amylin had no similar effects on cultured astrocytes or neurons. In rats, 5 days of amylin treatment decreased body weight gain and/or food intake and increased IL-6 mRNA expression in the VMN. Similar 5-day amylin treatment increased VMN leptin-induced phosphorylation of STAT3 expression in wild-type mice and rats infused with lateral ventricular IgG but not in IL-6 knockout mice or rats infused with ventricular IL-6 antibody. Lateral ventricular infusion of IL-6 antibody also prevented the amylin-induced decrease of body weight gain. These results show that amylin-induced VMH microglial IL-6 production is the likely mechanism by which amylin treatment interacts with VMH leptin signaling to increase its effect on weight loss.

Amylinergic control of food intake in lean and obese rodents

Obesity develops despite a complex and seemingly well orchestrated network that controls eating, energy expenditure and ultimately body weight; many of the involved signals are derived from the gastrointestinal tract. It is assumed that this network as an entity aims at maintaining body weight and body adiposity at a relatively constant level, but the control mechanisms seem to fail at least if an individual is chronically exposed to an oversupply of food. This article summarizes recent findings about the role of amylin in the control of eating in lean and obese rodents. The article gives some short background information about the well investigated adiposity and satiating signals leptin and cholecystokinin, respectively; this will provide the framework to discuss aspects of amylin physiology and pathophysiology in the control of eating in leanness and obesity. This discussion also involves the mechanisms mediating amylins eating inhibitory effect in the area postrema and the interactions between amylin and leptin. Further, we discuss the effect of high fat diets on amylin release and amylin action in lean and obese rats. The last part of this article raises the question whether amylin interacts with the reward system in the forebrain.

Interleukin-6 contributes to early fasting-induced free fatty acid mobilization in mice

Contracting muscle releases interleukin-6 (IL-6) enabling the metabolic switch from carbohydrate to fat utilization. Similarly, metabolism is switched during transition from fed to fasting state. Herein, we examined a putative role for IL-6 in the metabolic adaptation to normal fasting. In lean C57BL/6J mice, 6 h of food withdrawal increased gene transcription levels of IL-6 in skeletal muscle but not in white adipose tissue. Concomitantly, circulating IL-6 and free fatty acid (FFA) levels were significantly increased, whereas respiratory quotient (RQ) was reduced in 6-h fasted mice. In white adipose tissue, phosphorylation of hormone-sensitive lipase (HSL) was increased on fasting, indicating increased lipolysis. Intriguingly, fasting-induced increase in circulating IL-6 levels and parallel rise in FFA concentration were absent in obese and glucose-intolerant mice. A causative role for IL-6 in the physiological adaptation to fasting was further supported by the fact that fasting-induced increase in circulating FFA levels was significantly blunted in lean IL-6 knockout (KO) and lean C57BL/6J mice treated with neutralizing IL-6 antibody. Consistently, phosphorylation of HSL was significantly reduced in adipose tissue of IL-6-depleted mice. Hence, our findings suggest a novel role for IL-6 in energy supply during early fasting.

Amylin receptor components and the leptin receptor are co‐expressed in single rat area postrema neurons

Amylin is a pancreatic β‐cell hormone that acts as a satiating signal to inhibit food intake by binding to amylin receptors (AMYs) and activating a specific neuronal population in the area postrema (AP). AMYs are heterodimers that include a calcitonin receptor (CTR) subunit [CTR isoform a or b (CTRa or CTRb)] and a member of the receptor activity‐modifying proteins (RAMPs). Here, we used single‐cell quantitative polymerase chain reaction to assess co‐expression of AMY subunits in AP neurons from rats that were injected with amylin or vehicle. Because amylin interacts synergistically with the adipokine leptin to reduce body weight, we also assessed the co‐expression of AMY and the leptin receptor isoform b (LepRb) in amylin‐activated AP neurons. Single cells were collected from Wistar rats and from transgenic Fos‐GFP rats that express green fluorescent protein (GFP) under the control of the Fos promoter. We found that the mRNAs of CTRa, RAMP1, RAMP2 and RAMP3 were all co‐expressed in single AP neurons. Moreover, most of the CTRa+ cells co‐expressed more than one of the RAMPs. Amylin down‐regulated RAMP1 and RAMP3 but not CTR mRNAs in AMY+ neurons, suggesting a possible negative feedback mechanism of amylin at its own primary receptors. Interestingly, amylin up‐regulated RAMP2 mRNA. We also found that a high percentage of single cells that co‐expressed all components of a functional AMY expressed LepRb mRNA. Thus, single AP cells expressed both AMY and LepRb, which formed a population of first‐order neurons that presumably can be directly activated by amylin and, at least in part, also by leptin.

The role of the area postrema in the anorectic effects of amylin and salmon calcitonin: behavioral and neuronal phenotyping

Amylin reduces meal size by activating noradrenergic neurons in the area postrema (AP). Neurons in the AP also mediate the eating‐inhibitory effects of salmon calcitonin (sCT), a potent amylin agonist, but the phenotypes of the neurons mediating its effect are unknown. Here we investigated whether sCT activates similar neuronal populations to amylin, and if its anorectic properties also depend on AP function. Male rats underwent AP lesion (APX) or sham surgery. Meal patterns were analysed under ad libitum and post‐deprivation conditions. The importance of the AP in mediating the anorectic action of sCT was examined in feeding experiments of dose–response effects of sCT in APX vs. sham rats. The effect of sCT to induce Fos expression was compared between surgery groups, and relative to amylin. The phenotype of Fos‐expressing neurons in the brainstem was examined by testing for the co‐expression of dopamine beta hydroxylase (DBH) or tryptophan hydroxylase (TPH). By measuring the apposition of vesicular glutamate transporter‐2 (VGLUT2)‐positive boutons, potential glutamatergic input to amylin‐ and sCT‐activated AP neurons was compared. Similar to amylin, an intact AP was necessary for sCT to reduce eating. Further, co‐expression between Fos activation and DBH after amylin or sCT did not differ markedly, while co‐localization of Fos and TPH was minor. Approximately 95% of neurons expressing Fos and DBH after amylin or sCT treatment were closely apposed to VGLUT2‐positive boutons. Our study suggests that the hindbrain pathways engaged by amylin and sCT share many similarities, including the mediation by AP neurons.

Early postnatal amylin treatment enhances hypothalamic leptin signaling and neural development in the selectively bred diet-induced obese rat

Selectively bred diet-induced obese (DIO) rats become obese on a high-fat diet and are leptin resistant before becoming obese. Compared with diet-resistant (DR) neonates, DIO neonates have impaired leptin-dependent arcuate (ARC) neuropeptide Y/agouti-related peptide (NPY/AgRP) and α-melanocyte-stimulating hormone (α-MSH; from proopiomelanocortin (POMC) neurons) axon outgrowth to the paraventricular nucleus (PVN). Using phosphorylation of STAT3 (pSTAT3) as a surrogate, we show that reduced DIO ARC leptin signaling develops by postnatal day 7 (P7) and is reduced within POMC but not NPY/AgRP neurons. Since amylin increases leptin signaling in adult rats, we treated DIO neonates with amylin during postnatal hypothalamic development and assessed leptin signaling, leptin-dependent ARC-PVN pathway development, and metabolic changes. DIO neonates treated with amylin from P0-6 and from P0-16 increased ARC leptin signaling and both AgRP and α-MSH ARC-PVN pathway development, but increased only POMC neuron number. Despite ARC-PVN pathway correction, P0-16 amylin-induced reductions in body weight did not persist beyond treatment cessation. Since amylin enhances adult DIO ARC signaling via an IL-6-dependent mechanism, we assessed ARC-PVN pathway competency in IL-6 knockout mice and found that the AgRP, but not the α-MSH, ARC-PVN pathway was reduced. These results suggest that both leptin and amylin are important neurotrophic factors for the postnatal development of the ARC-PVN pathway. Amylin might act as a direct neurotrophic factor in DIO rats to enhance both the number of POMC neurons and their α-MSH ARC-PVN pathway development. This suggests important and selective roles for amylin during ARC hypothalamic development.

The satiating hormone amylin enhances neurogenesis in the area postrema of adult rats

Objective Adult neurogenesis in the subgranular zone and subventricular zone is generally accepted, but its existence in other brain areas is still controversial. Circumventricular organs, such as the area postrema (AP) have recently been described as potential neurogenic niches in the adult brain. The AP is the major site of action of the satiating hormone amylin. Amylin has been shown to promote the formation of neuronal projections originating from the AP in neonatal rodents but the role of amylin in adult neurogenesis remains unknown. Methods To test this, we first performed an RNA-sequencing of the AP of adult rats acutely injected with either amylin (20 μg/kg), amylin plus the amylin receptor antagonist AC187 (500 μg/kg) or vehicle. Second, animals were subcutaneously equipped with minipumps releasing either amylin (50 μg/kg/day) or vehicle for 3 weeks to assess cell proliferation and differentiation with the 5′-bromo-2-deoxyuridine (BrdU) technique. Results Acute amylin injections affected genes involved in pathways and processes that control adult neurogenesis. Amylin consistently upregulated NeuroD1 transcript and protein in the adult AP, and this effect was blocked by the co-administration of AC187. Further, chronic amylin treatment increased the number of newly proliferated AP-cells and significantly promoted their differentiation into neurons rather than astrocytes. Conclusion Our findings revealed a novel role of the satiating hormone amylin in promoting neurogenesis in the AP of adult rats.