Project Summary
Pancreatic beta cells (ß-cells) produce and secrete insulin in response to acute elevations in blood glucose.
Diminished ß-cell insulin production and secretion deregulate whole-body glucose homeostasis and are
hallmarks of Type 2 Diabetes (T2D). Endoplasmic Reticulum (ER) stress and Unfolded Protein Response
(UPR) activation are required steps in ß-cell dysfunction. Activating Transcription Factor 6 alpha (ATF6a) is
a UPR-effector protein that helps increase cellular tolerance to stress by inducing expression of genes
involved in ER function. Based on extensive literature documenting the beneficial role of ATF6a, we
hypothesized in vivo ß-cell ATF6a activation would protect mice against diabetes. Using destabilized domain
technology provided by Luke Wiseman’s lab at the Scripps Institute, the Alonso lab generated knock-in mice
(C57BL/6) expressing the N-terminal fragment of human ATF6a fused to a destabilized variant of E. coli
Dihydrofolate Reductase (DHFR-ATF6a). Upon constitutive expression, the destabilized DHFR domain
directs DHFR-ATF6a towards proteasomal degradation until exposure to the pharmacologic chaperone,
Trimethoprim (TMP), which stabilizes DHFR and turns on DHFR-ATF6a transcriptional activity. These mice
allow cell-type-specific (via Cre recombinase) and temporal (via TMP) activation of ATF6a in ß-cells.
Surprisingly, contrary to our hypothesis in vivo ß-cell DHFR-ATF6a activation caused marked glucose
intolerance during an intraperitoneal (i.p.) glucose challenge. There was no loss of insulin responsiveness
and ß-cell mass remained intact, suggesting ß-cell DHFR-ATF6a activation causes insulin insufficiency.
Indeed, our preliminary data indicate loss of glucose stimulated insulin secretion in vivo and a reduced islet
insulin content. Intriguingly, loss of insulin secretion preceded reduced islet insulin content, suggesting ß-cell
DHFR-ATF6a activation impacts these processes by distinct molecular mechanisms. In Aim1, I propose to
examine the impact of in vivo ß-cell DHFR-ATF6a activation on insulin production; specifically, proinsulin
production, proinsulin processing, and insulin granule maturation using transmission electron microscopy. In
Aim 2, I propose to examine the impact of in vivo ß-cell DHFR-ATF6a activation on the insulin secretion
pathway; specifically, glucose uptake, glucose metabolism, ATP production, cAMP signaling, ATP-sensitive
potassium channel closure, calcium induced insulin release, and cortical actin remodeling, using the gold-
standard islet perifusion assay. These aims will contribute valuable knowledge for developing therapies that
help preserve and/or rescue ß-cell function in T2D. ATF6a is a key component of the chronic stress response
that is thought to contribute to ß-cell dysfunction. Understanding the impact of continuous, inappropriate ß-
cell ATF6a signaling may be useful for uncovering novel molecular mechanisms that explain one part of the
damaging effect of chronic stress that precedes diminished ß-cell insulin production and secretion in T2D.