PMC

Systemically-delivered Cy5-B12-Ex4 is colocalized with insulin in the pancreas. The representative still images from three-dimensional rotational videos () demonstrate that Cy5-B12-Ex4 (yellow) is colocalized with insulin (red) in pancreatic beta cells. Sections were counterstained with DAPI (blue). Videos and corresponding representative images were taken from a z-stack (2μm step size) at 40× (A; ) and from a z-stack (1μm step size) at 40× with 4-5× optical zoom (B; ).

Systemically-delivered fluorescently labeled Ex-4 (Flex) highly penetrates within the PVN, whereas Cy5-B12 and Cy5-B12-Ex4 do not. Images were acquired at 10-20× (A,C,E,G) or 63× (with 2-3× optical zoom) (B,D,F,H) magnifications. Brains were processed for immunohistochemistry to label Flex, Equimolar-Flex, Cy5-B12 and Cy5-B12-Ex4 (yellow), astrocytes (GFAP; green) and neurons (NeuN; red). Sections were counterstained using DAPI (blue) to visualize cell nuclei. (B) Flex and (D) equimolar-Flex immunoreactivity is readily visualized in neurons in the PVN. (F) Cy5-B12 and (H) Cy5-B12-Ex4 are not present either in neurons or in astrocytes within the PVN. 3V, third ventricle; PVN paraventricular hypothalamic nucleus.

Systemically-delivered fluorescently labeled Ex-4 (Flex) highly penetrates within the DVC, whereas Cy5-B12 and Cy5-B12-Ex4 do not. Images were acquired at 10-20× (A,C,E,G) or 63× (with 2-3× optical zoom) (B,D,F,H) magnifications. Brains were processed for immunohistochemistry to label Flex, Equimolar-Flex, Cy5-B12 and Cy5-B12-Ex4 (yellow), astrocytes (GFAP; green) and neurons (NeuN; red). Sections were counterstained using DAPI (blue) to visualize cell nuclei. (B) Flex and (D) equimolar-Flex immunoreactivity is readily visualized in neurons and astrocytes in the DVC. (F) Cy5-B12 and (H) Cy5-B12-Ex4 are not present either in neurons or in astrocytes within the DVC. AP, area postrema; CC, central canal; DVC, dorsal vagal complex; NTS, nucleus tractus solitarius.

Systemic administration of B12-Ex4 or Ex4 suppresses blood glucose in mice. In an intraperitoneal glucose tolerance test, Ex4 (5μg/kg) and B12-Ex4 (dose equimolar to Ex4) suppressed blood glucose levels prior to (t=0min) and after (t=20, 40, 60, 120min) IP glucose administration (A); vehicle versus B12-Ex4: ** p<0.01, *** p<0.001; vehicle versus Ex4: ^### p<0.001; B12-Ex4 versus Ex4: ^§§ p<0.01. Area under the curve analyses from 0-120min (i.e., post-glucose load) show that B12-Ex4 (blue) and Ex4 (red) reduce AUC compared to saline vehicle (white) (B); * p<0.05, *** p<0.001. Data are mean ± SEM.

(A) Synthetic scheme for Cy5-B12, Cy5 alkyne was “clicked” onto a B12-azide conjugate. (B) RP-HPLC of Cy5-B12 showing purity ≥ 98% and LC-MS showing 1031[M+2H]⁺², 1042[M+Na+2H]⁺², and 1050 [M+K+2H]⁺², consistent with the conjugate. (C) Excitation and emission spectra of Cy5-B12 at 645 and 682nm, respectively. (D) Human recombinant TCII binding of B12-Ex4 and cyano-B12 with a K_d of 0.75 and 0.98nM, respectively. (E) Synthetic scheme for Cy5-B12-Ex4, Cy5-NHS ester was conjugated to Ex4’s lysine 26 and N-terminal. (F) RP-HPLC of Cy5-B12-Ex4 showing purity ≥ 91% and LC-MS showing m/z = 1728 [M+4H]⁺⁴, consistent with conjugate containing two molecules of Cy5 per B12-Ex4 component. (G) Excitation and emission spectra of Cy5-B12-Ex4 at 648 and 670 nm, respectively. (H) Cy5-B12-Ex4 agonism at the GLP-1 receptor reported using the FRET reporter H188; EC₅₀ = 13nM. Note that B12-Ex4 agonism at the GLP-1 receptor was previously reported (68 pM).

B12-Ex4 potently suppresses blood glucose in an oral glucose tolerance test but has minimal effects on energy balance control. Food intake and body weight change were measured after SC administration of B12-Ex4. Only the highest dose of drug, 20μg/kg, produced any reduction in feeding (A). No changes in 24h body weight gain were observed as a result of B12-Ex4 administration (B). In a separate experiment, SC injection of B12-Ex4 (0 μg/kg indicated by white circles, 5μg/kg by lighter blue circles, 20μg/kg by darker blue circles) reduced blood glucose in an oral glucose tolerance test from 20-40min after injection (C). *, significantly different from vehicle (p<0.05); &, significantly different from 5 μg/kg (p<0.05). Key in (A) also applies to (B). Data are mean ± SEM.

Systemic administration of Ex4 produces a different profile of metabolic effects than B12-Ex4. In contrast to the potent suppression of blood glucose produced by B12-Ex4, SC injection of Ex4 (0μg/kg indicated by white squares, 5μg/kg by lighter red squares, 20μg/kg by darker red squares) produced a robust hyperglycemic response (A). Food intake (B) and body weight gain (C) were suppressed by SC Ex4. To compare the induction of nausea/malaise by Ex4 with that potentially produced by B12-Ex4, rats were tested for expression of a conditioned taste avoidance (CTA) of a flavor paired with IP injection of B12-Ex4 (5μg/kg), Ex4 (5μg/kg), or LiCl as a positive control (0.15M). The percent acceptance of the drug-paired flavor is shown as a box-and-whiskers plot in (D). Both Ex4 (individual responses represented by black circles, overall group response represented in light gray box) and LiCl (individual responses represented by white circles, overall group response represented in white box) produce avoidance of the drug-paired flavor, as indicated by a reduced acceptance of the flavor. These effects are significantly different from acceptance of the drug-paired flavor in B12-Ex4-treated animals (individual responses represented by white squares, overall group response represented in dark gray box). *, significantly different from vehicle (p<0.05); &, significantly different from all other groups (p<0.05). Key under (B) and (C) applies to both panels. Data in (A-C) are mean ± SEM.