Dr. Krishana Sankar

Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells

by Sankar KS, Green BJ, Crocker AR, Verity JE, Altamentova SM, Rocheleau JV.

Published by PLoS ONE

September 22, 2011

Abstract
Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC). This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and -connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca(2+)-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(P)H response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.

URL: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024904

LKB1 couples glucose metabolism to insulin secretion in mice

by Accalia FuKarine RobitailleBrandon FaubertCourtney ReeksXiao-Qing DaiAlexandre B. HardyKrishana S. SankarSvetlana OgrelOsama Y. Al-DirbashiJonathan V. RocheleauMichael B. WheelerPatrick E. MacDonaldRussell JonesRobert A. Screaton

Published by Diabetologia

July 1, 2015

Abstract
AIMS/HYPOTHESIS:
Precise regulation of insulin secretion by the pancreatic beta cell is essential for the maintenance of glucose homeostasis. Insulin secretory activity is initiated by the stepwise breakdown of ambient glucose to increase cellular ATP via glycolysis and mitochondrial respiration. Knockout of Lkb1, the gene encoding liver kinase B1 (LKB1) from the beta cell in mice enhances insulin secretory activity by an undefined mechanism. Here, we sought to determine the molecular basis for how deletion of Lkb1 promotes insulin secretion.

METHODS:
To explore the role of LKB1 on individual steps in the insulin secretion pathway, we used mitochondrial functional analyses, electrophysiology and metabolic tracing coupled with by gas chromatography and mass spectrometry.

RESULTS:
Beta cells lacking LKB1 surprisingly display impaired mitochondrial metabolism and lower ATP levels following glucose stimulation, yet compensate for this by upregulating both uptake and synthesis of glutamine, leading to increased production of citrate. Furthermore, under low glucose conditions, Lkb1(-/-) beta cells fail to inhibit acetyl-CoA carboxylase 1 (ACC1), the rate-limiting enzyme in lipid synthesis, and consequently accumulate NEFA and display increased membrane excitability.

CONCLUSIONS/INTERPRETATION:
Taken together, our data show that LKB1 plays a critical role in coupling glucose metabolism to insulin secretion, and factors in addition to ATP act as coupling intermediates between feeding cues and secretion. Our data suggest that beta cells lacking LKB1 could be used as a system to identify additional molecular events that connect metabolism to cellular excitation in the insulin secretion pathway.

URL: https://link.springer.com/article/10.1007%2Fs00125-015-3579-7

Allosteric modulation in monomers and oligomers of a G protein-coupled receptor

by Rabindra V Shivnaraine, Brendan Kelly, Krishana S Sankar, Dar'ya S Redka, Yi Rang Han, Fei Huang, Gwendolynne Elmslie, Daniel Pinto, Yuchong Li, Jonathan V Rocheleau, Claudiu C Gradinaru Is a corresponding author , John Ellis Is a corresponding author , James W Wells

Published by eLIFE

May 6, 2016

Abstract
The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.

URL: https://elifesciences.org/articles/11685

Single-Molecule Analysis of the Supramolecular Organization of the M2 Muscarinic Receptor and the Gαi1 Protein.

by Rabindra V. Shivnaraine, Dennis D. Fernandes, Huiqiao Ji, Yuchong Li, Brendan Kelly, Zhenfu Zhang, Yi Rang Han, Fei Huang, Krishana S. Sankar, David N. Dubins, Jonathan V. Rocheleau, James W. Wells, Claudiu C. Gradinaru

Published by Journal of the American Chemical Society

September 24, 2016

Abstract
G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes. The method was calibrated with multiplexed controls comprising 1-4 copies of fused eGFP. The photobleaching patterns of eGFP-M2R were indicative of a tetramer and unaffected by muscarinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected by GTPγS. A complex of M2R and Gi1 was tetrameric in both, and activation by a full agonist plus GTPγS reduced the oligomeric size of Gi1 without affecting that of the receptor. A similar reduction was observed upon activation of eGFP-Gαi1 by the receptor-mimic mastoparan plus GTPγS, and constitutively active eGFP-Gαi1 was predominantly dimeric. The oligomeric nature of Gi1 in live CHO cells was demonstrated by means of Förster resonance energy transfer and dual-color fluorescence correlation spectroscopy in studies with eGFP- and mCherry-labeled Gαi1; stochastic FRET was ruled out by means of non-interacting pairs. These results suggest that the complex between M2R and holo-Gi1 is an octamer comprising four copies of each, and that activation is accompanied by a decrease in the oligomeric size of Gi1. The structural feasibility of such a complex was demonstrated in molecular dynamics simulations.

URL: https://pubs.acs.org/doi/10.1021/jacs.6b04032