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 β-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.

These novel findings highlight the potential clinical utility of B12-Ex4 conjugates as possible future T2DM therapeutics with reduced incidence of adverse effects.

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.

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 (IHC) and quantitative PCR 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 IHC and quantitative PCR 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 (AAV)-knockdown experiments indicate that LDTg amylin receptor signaling is physiologically and potentially pre-clinically relevant for energy balance control. Finally, IHC data indicate that LDTg amylin receptors are expressed on gamma-aminobutyric acid (GABA)-ergic neurons and behavioral results suggest that local GABA receptor signaling mediates the hypophagia following 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 GABA receptor signaling.

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.