NAD(P)H autofluorescence

hello Matthew,

We are measuring NAD(P)H - and in addition riboflavin endogenous
fluorescence FAD/FMN - for 20 years now in our lab, using FACS.  Our system
generates quantitative data, similar to the data obtained by Piston, in that
we get a doubling of endogenous blue (NAD(P)H) fluorescence over 0-20 mM
glucose range in pancreatic beta cells. NAD(P)H changes are dynamic, and can
be modulated by nutrients and classical inhibitors of metabolism. Keep in
mind, however, that the cells we are studying are really tuned to shifts in
glucose concentrations, and thus metabolically highly active. I would advise
you to play with some mitochondrial ETC inhibitors such as rotenone or
cyanide when setting up your system. I include some abstracts demonstrating
our method. Don't hesitate if you have further questions.


Geert Martens, M.D.

Diabetes Research Center
Brussels Free University - VUB
Laarbeeklaan 103
1090 Brussel
Belgium
tel 32-2 - 477 4557
fax 32-2 - 477 4545

Biochem Biophys Res Commun. 1983 Jul 29;114(2):835-42.	   Related Articles,
Links 

Autofluorescence-activated cell sorting of pancreatic islet cells:
purification of insulin-containing B-cells according to glucose-induced
changes in cellular redox state.
Van De Winkel M, Pipeleers D.
Autofluorescence-activated cell sorting can be employed for the
subfractionation of insulin-containing islet B-cells according to their
responsiveness to their physiologic stimulus, glucose. The method utilizes a
flow cytometric detection of the rapid variations in endogenous NAD (P) H -
and FAD - fluorescence after exposure to 20 mM glucose. Under these
conditions, a two-fold increase in NAD (P) H and a 40% decrease in FAD was
observed in more than 75% of B-cells isolated from fed normal rats. The
technique makes it possible to study the metabolic behaviour of the B-cell
population in (physio)pathological conditions of impaired glucose-induced
insulin release; the availability of functionally homogenous B-cell
preparations facilitates studies on stimulus-secretion coupling. In view of
the universal role of the cellular metabolic redox state in cell regulation,
it is suggested that similar techniques can be developed for the metabolic
analysis of other cell types and for their purification according to their
responsiveness to specific stimuli.

J Clin Invest. 1992 Jan;89(1):117-25.	  Related Articles, Links

Differences in glucose recognition by individual rat pancreatic B cells are
associated with intercellular differences in glucose-induced biosynthetic
activity.
Kiekens R, In 't Veld P, Mahler T, Schuit F, Van De Winkel M, Pipeleers D.
Department of Metabolism and Endocrinology, Vrije Universiteit Brussel,
Belgium.
In vitro incubated rat islet B cells differ in their individual rates of
protein synthesis. The number of cells in biosynthetic activity increases
with the glucose concentration. Flow cytometric monitoring of the cellular
redox states indicated that islet B cells differ in their individual
metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H
fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM,
and for 70% at 20 mM. The functional significance of this metabolic
heterogeneity was assessed by comparing protein synthesis in metabolically
responsive and unresponsive subpopulations, shortly after their separation
by autofluorescence-activated cell sorting. The glucose-sensitive
subpopulation exhibited four- to fivefold higher rates of insulin synthesis
during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic
activity was mainly caused by recruitment of cells into active synthesis
and, to a lesser extent, by higher biosynthetic activity per recruited cell.
Cells from the glucose-sensitive subpopulation were larger, and presented a
threefold higher density of a pale secretory vesicle subtype, which is
thought to contain unprocessed proinsulin. It is concluded that
intercellular differences in metabolic responsiveness result in functional
heterogeneity of the pancreatic B cell population.