Mechanisms of Insulin Action 
Perry J. Blackshear, M.D., D.Phil. — Investigator 
Dr. Blackshear is also Professor of Medicine and Assistant Professor of Biochemistry at Duke University 
Medical Center. He received his D.Phil, degree in biochemistry from Trinity College, Oxford University, 
and his M.D. degree from Harvard Medical School. Before moving to Duke University, he was Assistant 
Professor of Medicine at Harvard Medical School. Dr. Blackshear has received the Young Investigator 
Award for Clinical Research from the American Federation for Clinical Research. 
OUR laboratory is mainly interested in the mo- 
lecular mechanisms of action of insulin and 
polypeptide growth factors. The studies involv- 
ing insulin action are particularly relevant to 
common clinical disorders of insulin resistance, 
such as type II (adult-onset) diabetes and obesity, 
in which the locus of the resistance is thought to 
be at a "postreceptor" step within the cells of 
muscle, liver, and adipose tissue. Our work is 
aimed at understanding the biochemical steps in- 
volved in insulin action in these tissues, with the 
ultimate hope of identifying the abnormal steps 
in these insulin-resistant states and possibly using 
this knowledge to develop novel drugs aimed at 
correcting the abnormalities. 
Another major area of interest is the molecular 
steps involved in mediating the effects of a wide 
variety of hormones, neurotransmitters, and 
drugs on their target cells. The common denomi- 
nator of these agents is that the mechanism of 
action involves the stimulated breakdown of cer- 
tain membrane lipid compounds, leading to the 
generation of intracellular lipid mediators known 
as diacylglycerols. These in turn can activate an 
important cellular enzyme, protein kinase C (PKC). 
Over the past several years, a large amount of 
information has accumulated about the way in 
which hormones stimulate the breakdown of 
these membrane lipids and about the molecular 
biology and biochemistry of the PKC family of 
enzymes. However, almost nothing is yet known 
about the proteins that this kinase phosphorylates 
in cells and tissues, or about the involvement of 
these phosphorylated proteins in mediating a vari- 
ety of cellular effects. To understand the role of 
these PKC substrates in the cell is a major goal of 
our laboratory. Within the past year we have 
made several advances in each of these areas. 
With regard to signal transduction, we are in- 
vestigating the molecular nature of a family of 
PKC substrates known by the acronym MARCKS 
(myristoylated alanine-rich C-kinase substrates). 
These are widely distributed cellular proteins 
that are phosphorylated within seconds of PKC 
activation in intact cells, and it is presumed that 
this phosphorylation serves some function in me- 
diating the effects of the activated kinase. Our 
group has established that these proteins are ex- 
cellent substrates for PKC, and that phosphoryla- 
tion leads to changes in their properties that may 
influence their behavior in the cell. For example, 
phosphorylation of the protein disrupts its associ- 
ation with model cellular membranes, perhaps 
allowing for phosphorylation-dependent changes 
in the protein's intracellular location. 
Similarly, phosphorylation of the protein 
disrupts its ability to form a tight complex with 
calmodulin, the ubiquitous calcium-dependent 
regulator of a wide variety of cellular enzymes. 
We postulate that such disruption leads to an in- 
crease in the cellular content of free calmodulin, 
which might then lead to concomitant activation 
of calmodulin-sensitive enzymes. In this way 
phosphorylation of the MARCKS protein and its 
relatives by PKC might lead to increases in the 
activity of calcium/calmodulin-dependent pro- 
cesses, a synergistic relationship that has long 
been postulated but not proved. 
The overall goal of these studies is to try to 
determine the role that the MARCKS protein fam- 
ily plays, if any, in mediating the actions of PKC 
in various tissues. One important series of studies 
now under way involves attempts to render cells 
and intact animals deficient in these proteins, us- 
ing the techniques of antibody microinjection, 
antisense RNA expression, and ultimately gene 
disruption. It is hoped that within the next year 
or two, such deficient cells and animals will be 
available to study the potential role of this family 
of proteins in development, particularly of the 
nervous system, in which the MARCKS proteins 
and their relatives are highly expressed. 
Considerable progress has also been made in 
the studies of the molecular mechanisms of insu- 
lin action. In one group of studies involving two 
genes whose transcription is rapidly stimulated 
by insulin, we have made progress in identifying 
regions of the DNA that are implicated in the in- 
sulin reaction. We have also identified proteins 
that bind to these regions and have demonstrated 
that they are modified in some way by rapid insu- 
lin exposure of the cells. Current studies in the 
laboratory are attempting to clone and sequence 
43 
