Insulin blunts the response of glucose-excited neurons in the ventrolateral-ventromedial hypothalamic nucleus to decreased glucose

Insulin signaling is dysfunctional in obesity and diabetes. Moreover, central glucose-sensing mechanisms are impaired in these diseases. This is associated with abnormalities in hypothalamic glucose-sensing neurons. Glucose-sensing neurons reside in key areas of the brain involved in glucose and energy homeostasis, such as the ventromedial hypothalamus (VMH). Our results indicate that insulin opens the KATP channel on VMH GE neurons in 5, 2.5, and 0.1 mM glucose. Furthermore, insulin reduced the sensitivity of VMH GE neurons to a decrease in extracellular glucose level from 2.5 to 0.1 mM. This change in the glucose sensitivity in the presence of insulin was reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (10 nM) but not by the mitogen-activated kinase (MAPK) inhibitor PD-98059 (PD; 50 μM). Finally, neither the AMPK inhibitor compound C nor the AMPK activator AICAR altered the activity of VMH GE neurons. These data suggest that insulin attenuates the ability of VMH GE neurons to sense decreased glucose via the PI3K signaling pathway. Furthermore, these data are consistent with the role of insulin as a satiety factor. That is, in the presence of insulin, glucose levels must decline further before GE neurons respond. Thus, the set point for detection of glucose deficit and initiation of compensatory mechanisms would be lowered

Media 1: Dual-mode laparoscopic fluorescence image-guided surgery using a single camera

Originally published in Biomedical Optics Express on 01 August 2012 (boe-3-8-1880

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging

<div><p>The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.</p></div

Kinetics and dosing in mice following IV administration of GE3126 formulated with 80% distilled/deionized water, 10% 2- HPβCD, and 10% propylene glycol.

<p>Imaging was performed using a commercial fluorescence stereomicroscope with multispectral detection. Excitation was achieved using a filter centered at 406 nm with a 15 nm bandwidth. Numerical data represented the area under the curve for emission wavelengths ranging from 550 nm to 720 nm acquired in sciatic nerves, adjacent muscle and adipose tissue, with n = 3 mice per group. (A) For kinetics, each mouse was given 16 mg/kg of GE3126 and imaging was performed at 0.5, 1, 2, 3, or 4 h post-injection. (B) In the dosing study, mice were given 1.6, 3.3, 6.6., 10.0, 13.3, 16.6, 20.0, 23.3, or 46.6 mg/kg of GE3126 and imaging was performed 1 h post-injection. Control mice were given a single injection of IV formulation (vehicle only) and imaged to determine background fluorescence. (C) Representative fluorescence multispectral image of a mouse injected with 16.6 mg/kg of GE3126. The sciatic nerve (reddish-orange) is indicated by an arrow. Note fluorescence labeling of adjacent adipose tissue in green. The control animal tissue appeared dark. Scale bar ~0.5 mm. (D) Emission spectra of nerve, muscle, and adipose tissue are shown to illustrate spectral separations between tissue types in both GE3126-treated mouse and control mouse.</p

Dual-mode laparoscopic imaging in the porcine model.

<p>A dose of 0.74 mg/kg GE3126 was injected into the pig. An incision was made into the left brachial plexus and video was recorded just prior to GE3126 injection and at defined time points up to 100 min post-injection. (A) Individual frames were extracted and the fluorescence intensities of nerve, adjacent muscle and adipose tissue were plotted over time. (B) Representative white light and fluorescence images extracted from the video show the brachial plexus (top) and the retroperitoneal region (bottom) at ~80 min post-injection. Nerves are shown with white arrows. (C) Fluorescence microscopy images of a pig nerve tissue section taken prior to injection of GE3126, and at ~90 min post-injection. Scale bar in B ~1 mm; scale bar in C = 20 μm.</p

Optical properties of GE3126 in different solvents.

<p>Ɛ = molar extinction coefficient; Abs Max = absorbance maximum wavelength; Em Max = emission maximum wavelength; QY = quantum yield.</p><p>Optical properties of GE3126 in different solvents.</p

<i>In vitro</i> properties of the fluorophores.

<p>^a Measured maximal solubility when dissolved in 58.5% distilled water, 30% 2- HPβCD, 10% propylene glycol, 1% polyethylene glycol-300, and 0.5% DMSO</p><p>^b From Reference [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130276#pone.0130276.ref031" target="_blank">31</a>]</p><p><i>In vitro</i> properties of the fluorophores.</p

Dual-mode laparoscopic imaging in a porcine model and corresponding histology confirmation.

<p>(A) White light imaging (top left) showing a vessel, marked “a”, and a harder-to-visualize nerve, in the vena cava, marked “b”. The corresponding fluorescence image (top right) shows the fluorescently labeled nerve “b” more clearly than white light imaging. (B) H&E staining of tissue sections of “a” and “b” confirmed the identity of the non-labeled blood vessel (left) and labeled nerve (right). Scale bar in A~ 1 mm; scale bar in B = 100 μm.</p