The long-acting amylin/calcitonin receptor agonist ZP5461 suppresses food intake and body weight in male rats.

The peptide hormone amylin reduces food intake and body weight and is an attractive candidate target for novel pharmacotherapies to treat obesity. However, the short half-life of native amylin and amylin analogs like pramlintide limits these compounds' potential utility in promoting sustained negative energy balance. Here, we evaluate the ability of the novel long-acting amylin/calcitonin receptor agonist ZP5461 to reduce feeding and body weight in rats, and also test the role of calcitonin receptors (CTRs) in the dorsal vagal complex (DVC) of the hindbrain in the energy balance effects of chronic ZP5461 administration. Acute dose-response studies indicate that systemic ZP5461 (0.5-3 nmol/kg) robustly suppresses energy intake and body weight gain in chow- and high-fat diet (HFD)-fed rats. When HFD-fed rats received chronic systemic administration of ZP5461 (1-2 nmol/kg), the compound initially produced reductions in energy intake and weight gain but failed to produce sustained suppression of intake and body weight. Using virally mediated knockdown of DVC CTRs, the ability of chronic systemic ZP5461 to promote early reductions in intake and body weight gain was determined to be mediated in part by activation of DVC CTRs, implicating the DVC as a central site of action for ZP5461. Future studies should address other dosing regimens of ZP5461 to determine whether an alternative dose/frequency of administration would produce more sustained body weight suppression.

Combined Amylin/GLP-1 pharmacotherapy to promote and sustain long-lasting weight loss.

A growing appreciation of the overlapping neuroendocrine mechanisms controlling energy balance has highlighted combination therapies as a promising strategy to enhance sustained weight loss. Here, we investigated whether amylin- and glucagon-like-peptide-1 (GLP-1)-based combination therapies produce greater food intake- and body weight-suppressive effects compared to monotherapies in both lean and diet-induced obese (DIO) rats. In chow-maintained rats, systemic amylin and GLP-1 combine to reduce meal size. Furthermore, the amylin and GLP-1 analogs salmon calcitonin (sCT) and liraglutide produce synergistic-like reductions in 24 hours energy intake and body weight. The administration of sCT with liraglutide also led to a significant enhancement in cFos-activation in the dorsal-vagal-complex (DVC) compared to mono-therapy, suggesting an activation of distinct, yet overlapping neural substrates in this critical energy balance hub. In DIO animals, long-term daily administration of this combination therapy, specifically in a stepwise manner, results in reduced energy intake and greater body weight loss over time when compared to chronic mono- and combined-treated groups, without affecting GLP-1 receptor, preproglucagon or amylin-receptor gene expression in the DVC.

Glucagon-Like Peptide-1 Receptor Signaling in the Lateral Dorsal Tegmental Nucleus Regulates Energy Balance.

The neurobiological substrates that mediate the anorectic effects of both endogenous glucagon-like peptide-1 (GLP-1) and exogenous GLP-1 receptor (GLP-1R) agonists are an active area of investigation. As the lateral dorsal tegmental nucleus (LDTg) expresses the GLP-1R and represents a potential neuroanatomical hub connecting the nucleus tractus solitarius (NTS), the major central source of GLP-1, with the other nuclei in the midbrain and forebrain, we tested the hypothesis that GLP-1R signaling in the LDTg controls food intake. Direct activation of LDTg GLP-1R suppresses food intake through a reduction in average meal size and independent of nausea/malaise. Immunohistochemical data show that GLP-1-producing neurons in the NTS project to the LDTg, providing anatomical evidence of endogenous central GLP-1 in the LDTg. Pharmacological blockade of LDTg GLP-1Rs with exendin-(9-39) dose-dependently increases food intake and attenuates the hypophagic effects of gastric distension. As GLP-1 mimetics are administered systemically in humans, we evaluated whether peripherally administered GLP-1R agonists access the LDTg to affect feeding. Immunohistochemical data show that a systemically administered fluorescent GLP-1R agonist accesses the LDTg and is juxtaposed with neurons. Additionally, blockade of LDTg GLP-1Rs attenuates the hypophagic effects of a systemic GLP-1R agonist. Together, these data indicate that LDTg GLP-1R signaling controls energy balance and underscores the role of the LDTg in integrating energy balance-relevant signals to modulate feeding.

Thioamide Substitution Selectively Modulates Proteolysis and Receptor Activity of Therapeutic Peptide Hormones.

Peptide hormones are attractive as injectable therapeutics and imaging agents, but they often require extensive modification by mutagenesis and/or chemical synthesis to prevent rapid in vivo degradation. Alternatively, the single-atom, O-to-S modification of peptide backbone thioamidation has the potential to selectively perturb interactions with proteases while preserving interactions with other proteins, such as target receptors. Here, we use the validated diabetes therapeutic, glucagon-like peptide-1 (GLP-1), and the target of clinical investigation, gastric inhibitory polypeptide (GIP), as proof-of-principle peptides to demonstrate the value of thioamide substitution. In GLP-1 and GIP, a single thioamide near the scissile bond renders these peptides up to 750-fold more stable than the corresponding oxopeptides toward cleavage by dipeptidyl peptidase 4, the principal regulator of their in vivo stability. These stabilized analogues are nearly equipotent with their parent peptide in cyclic AMP activation assays, but the GLP-1 thiopeptides have much lower ß-arrestin potency, making them novel agonists with altered signaling bias. Initial tests show that a thioamide GLP-1 analogue is biologically active in rats, with an in vivo potency for glycemic control surpassing that of native GLP-1. Taken together, these experiments demonstrate the potential for thioamides to modulate specific protein interactions to increase proteolytic stability or tune activation of different signaling pathways.

Amylin receptor activation in the ventral tegmental area reduces motivated ingestive behavior.

Amylin is produced in the pancreas and the brain, and acts centrally to reduce feeding and body weight. Recent data show that amylin can act in the ventral tegmental area (VTA) to reduce palatable food intake and promote negative energy balance, but the behavioral mechanisms by which these effects occur are not fully understood. The ability of VTA amylin signaling to reduce intake of specific palatable macronutrients (fat or carbohydrate) was tested in rats in several paradigms, including one-bottle acceptance tests, two-bottle choice tests, and a free-choice diet. Data show that VTA amylin receptor activation with the amylin receptor agonist salmon calcitonin (sCT) preferentially and potently reduces intake of fat, with more variable suppression of sucrose intake. Intake of a non-nutritive sweetener is also decreased by intra-VTA administration of sCT. As several feeding-related signals that act in the mesolimbic system also impact motivated behaviors besides feeding, we tested the hypothesis that the suppressive effects of amylin signaling in the VTA extend to other motivationally relevant stimuli. Results show that intra-VTA sCT reduces water intake in response to central administration of the dipsogenic peptide angiotensin II, but has no effect on ad libitum water intake in the absence of food. Importantly, open field and social interaction studies show that VTA amylin signaling does not produce anxiety-like behaviors. Collectively, these findings reveal a novel ability of VTA amylin receptor activation to alter palatable macronutrient intake, and also demonstrate a broader role of VTA amylin signaling for the control of motivated ingestive behaviors beyond feeding.

Amylin Acts in the Lateral Dorsal Tegmental Nucleus to Regulate Energy Balance Through Gamma-Aminobutyric Acid Signaling.

BACKGROUND: The pancreatic- and brain-derived hormone amylin promotes negative energy balance and is receiving increasing attention as a promising obesity therapeutic. However, the neurobiological substrates mediating amylin's effects are not fully characterized. We postulated that amylin acts in the lateral dorsal tegmental nucleus (LDTg), an understudied neural processing hub for reward and homeostatic feeding signals. METHODS: We used immunohistochemical and quantitative polymerase chain reaction analyses to examine expression of the amylin receptor complex in rat LDTg tissue. Behavioral experiments were performed to examine the mechanisms underlying the hypophagic effects of amylin receptor activation in the LDTg. RESULTS: Immunohistochemical and quantitative polymerase chain reaction analyses show expression of the amylin receptor complex in the LDTg. Activation of LDTg amylin receptors by the agonist salmon calcitonin dose-dependently reduces body weight, food intake, and motivated feeding behaviors. Acute pharmacological studies and longer-term adeno-associated viral knockdown experiments indicate that LDTg amylin receptor signaling is physiologically and potentially preclinically relevant for energy balance control. Finally, immunohistochemical data indicate that LDTg amylin receptors are expressed on gamma-aminobutyric acidergic neurons, and behavioral results suggest that local gamma-aminobutyric acid receptor signaling mediates the hypophagia after LDTg amylin receptor activation. CONCLUSIONS: These findings identify the LDTg as a novel nucleus with therapeutic potential in mediating amylin's effects on energy balance through gamma-aminobutyric acid receptor signaling.

Hindbrain DPP-IV inhibition improves glycemic control and promotes negative energy balance.

The beneficial glycemic and food intake-suppressive effects of glucagon-like peptide-1 (GLP-1) have made this neuroendocrine system a leading target for pharmacological approaches to the treatment of diabetes and obesity. One strategy to increase the activity of endogenous GLP-1 is to prevent the rapid degradation of the hormone by the enzyme dipeptidyl peptidase-IV (DPP-IV). However, despite the expression of both DPP-IV and GLP-1 in the brain, and the clear importance of central GLP-1 receptor (GLP-1R) signaling for glycemic and energy balance control, the metabolic effects of central inhibition of DPP-IV activity are unclear. To test whether hindbrain DPP-IV inhibition suppresses blood glucose, feeding, and body weight gain, the effects of 4th intracerebroventricular (ICV) administration of the FDA-approved DPP-IV inhibitor sitagliptin were evaluated. Results indicate that hindbrain delivery of sitagliptin improves glycemic control in a GLP-1R-dependent manner, suggesting that this effect is due at least in part to increased endogenous brainstem GLP-1 activity after sitagliptin administration. Furthermore, 4th ICV injection of sitagliptin reduced 24h body weight gain and energy intake, with a selective suppression of high-fat diet, but not chow, intake. These data reveal a novel role for hindbrain GLP-1R activation in glycemic control and also demonstrate that DPP-IV inhibition in the caudal brainstem promotes negative energy balance.

Amylin modulates the mesolimbic dopamine system to control energy balance.

Amylin acts in the CNS to reduce feeding and body weight. Recently, the ventral tegmental area (VTA), a mesolimbic nucleus important for food intake and reward, was identified as a site-of-action mediating the anorectic effects of amylin. However, the long-term physiological relevance and mechanisms mediating the intake-suppressive effects of VTA amylin receptor (AmyR) activation are unknown. Data show that the core component of the AmyR, the calcitonin receptor (CTR), is expressed on VTA dopamine (DA) neurons and that activation of VTA AmyRs reduces phasic DA in the nucleus accumbens core (NAcC). Suppression in NAcC DA mediates VTA amylin-induced hypophagia, as combined NAcC D1/D2 receptor agonists block the intake-suppressive effects of VTA AmyR activation. Knockdown of VTA CTR via adeno-associated virus short hairpin RNA resulted in hyperphagia and exacerbated body weight gain in rats maintained on high-fat diet. Collectively, these findings show that VTA AmyR signaling controls energy balance by modulating mesolimbic DA signaling.

Intraduodenal milk protein concentrate augments the glycemic and food intake suppressive effects of DPP-IV inhibition.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone released from intestinal L-cells in response to food entering into the gastrointestinal tract. GLP-1-based pharmaceuticals improve blood glucose regulation and may hold promise for obesity treatment, as GLP-1 drugs reduce food intake and body weight in humans and animals. In an effort to improve GLP-1 pharmacotherapies, we focused our attention on macronutrients that, when present in the gastrointestinal tract, may enhance GLP-1 secretion and improve glycemic regulation and food intake suppression when combined with systemic administration of sitagliptin, a pharmacological inhibitor of DPP-IV (enzyme responsible for GLP-1 degradation). In particular, previous data suggest that specific macronutrient constituents found in dairy foods may act as potent secretagogues for GLP-1 and therefore may potentially serve as an adjunct dietary therapy in combination with sitagliptin. To directly test this hypothesis, rats received intraperitoneal injections of sitagliptin (6 mg/kg) or saline vehicle followed by intraduodenal infusions of either milk protein concentrate (MPC; 80/20% casein/whey; 4 kcal), soy protein (nondairy control infusate; 4 kcal), or 0.9% NaCl. Food intake was assessed 30 min postinfusion. In separate studies, regulation of blood glucose was examined via a 2-h oral glucose tolerance test (2 g/kg) following identical sitagliptin treatment and intraduodenal nutrient infusions. Collectively, results show that intraduodenal MPC, but not soy protein, significantly enhances both the food intake suppression and improved control of blood glucose produced by sitagliptin. These data support the hypothesis that dietary intake of dairy protein may be beneficial as an adjunct behavioral therapy to enhance the glycemic and food intake suppressive effects of GLP-1-based pharmacotherapies.

Hindbrain GLP-1 receptor-mediated suppression of food intake requires a PI3K-dependent decrease in phosphorylation of membrane-bound Akt.

Glucagon-like peptide-1 (GLP-1) receptors (GLP-1R) expressed in the nucleus tractus solitarius (NTS) are physiologically required for the control of feeding. Recently, NTS GLP-1R-mediated suppression of feeding was shown to occur via a rapid PKA-induced suppression of AMPK and activation of MAPK signaling. Unknown are the additional intracellular signaling pathways that account for the long-term hypophagic effects of GLP-1R activation. Because cAMP/PKA activity can promote PI3K/PIP3-dependent translocation of Akt to the plasma membrane, we hypothesize that hindbrain GLP-1R-mediated control of feeding involves a PI3K-Akt-dependent pathway. Importantly, the novel evidence presented here challenges the dogmatic view that PI3K phosphorylation results in an obligatory activation of Akt and instead supports a growing body of literature showing that activation of cAMP/PKA can inhibit Akt phosphorylation at the plasma membrane. Behavioral data show that inhibition of hindbrain PI3K activity by a fourth icv administration of LY-294002 (3.07 µg) attenuated the food intake- and body weight-suppressive effects of a fourth icv administration of the GLP-1R agonist exendin-4 (0.3 µg) in rats. Hindbrain administration of triciribine (10 µg), an inhibitor of PIP3-dependent translocation of Akt to the cell membrane, also attenuated the intake-suppressive effects of a fourth icv injection of exendin-4. Immunoblot analyses of ex vivo NTS tissue lysates and in vitro GLP-1R-expressing neurons (GT1-7) support the behavioral findings and show that GLP-1R activation decreases phosphorylation of Akt in a time-dependent fashion. Current data reveal the requirement of PI3K activation, PIP3-dependent translocation of Akt to the plasma membrane, and suppression in phosphorylation of membrane-bound Akt to mediate the food intake-suppressive effects of hindbrain GLP-1R activation.