Effect of salsalate on lipid induced insulin resistance

Insulin resistance is a primary factor causally linked to the pathogenesis of
type 2 diabetes. Skeletal muscle accounts for a substantial part of the total
body mass, and is responsible for ~80% of insulin-stimulated glucose uptake.
There is evidence that accumulation of fat in non-adipose tissues, like heart,
liver and muscle contributes to development of insulin resistance in
non-trained individuals. Thus, intramyocellular lipids (IMCL) are tightly
correlated with the severity of insulin resistance and it has been shown that
intracellular fatty acid metabolites, like diacylglycerol (DAG), long-chain
fatty acyl CoA (LCFACoA) and ceramides, may cause insulin resistance by
activation of the serine/threonine kinase PKC, which impedes insulin signaling
at the level of insulin receptor substrate-1 (IRS1). Next to PKC-induced
inhibition of insulin signaling, intracellular fatty acid metabolites can
activate the inflammatory I*B/NF*B pathway, which is also associated with the
development of insulin resistance. Under resting conditions cytosolic NF*B is
bound to its inhibitor protein subunit, I*B. Upon activation of I*B kinase
(IKK) I*B gets phosphorylated and degrades, disinhibiting NF*B. Than NF*B
translocates to the nucleus where it binds to its target genes and increases
the expression of chemokines, proinflammatory cytokines and inflammatory
enzymes. which in a feedforward loop also activates NF*B, leading to a
progressive inflammatory responses, and a deterioration of insulin resistance.
Interestingly, chronic low-grade inflammation is also associated with insulin
resistance and the use of high-doses non-steroidal anti inflammatory agents
like Aspirin (acetylsalicylic acid) or salsalate (a non-acetylated dimer of
salicylic acid which, in contrast to Aspirin, does not affect COX activity and
therefore bleeding time) can improve glycemic control as indicated by decreased
fasting glucose levels, a decrease of glycosuria and an increase in insulin
secretion. The mechanism underlying the insulin sensitizing effect of
acetylsalicylic acid is not yet known, although recent animal research
implicates a role for IKK: after inhibition of IKK, the I*B/NF*B pathway
remained inactivated and insulin resistance improved. In contrast,
overexpression of IKK led to activation of this pathway and resulted in an
increase in insulin resistance.

Using the model of lipid-induced insulin resistance we will study the
hypothesis that pre-treatment with salsalate (salicylsalicylic acid) blunts the
development of lipid-induced insulin resistant in an I*B/NF*B dependent manner
in healthy human subjects. Using this approach we will be the first to examine
the effects of an anti-inflammatory agent (salsalate) on acute low-grade
inflammation in muscle upon acute lipid-induced insulin resistance and to
identify targets for early intervention towards the development of type 2
diabetes.

As such, we would like to get more insight in the pathogenesis of insulin
resistance which would lead to a better understanding of type 2 diabetes
mellitus and to the prevention and treatment of this disease.

Subjects will be invited for a screening visit which will include measurement
of body composition and a maximal aerobic exercise test. Subsequently, all
subjects will undergo two hyperinsulinemic euglycemic clamps with simultaneous
infusion of LCT separated by at least 1 week and in a random order. In one of
these trials, subjects receive in the 4 preceding days of the clamp 4 g
salsalate a day. In the other trial, salsalate will be replaced by placebo
tablets. In addition to these two hyperinsulinemic euglycemic clamps with
simultaneous infusion of LCT, a third hyperinsulinemic euglycemic clamp will be
performed without simultaneous infusion of LCT and will serve as a control
trial.

Disalcid is insoluble in acid gastric fluids but readily soluble in the small
intestine where it is partially hydrolyzed to two molecules of salicylic acid.
In contrast to acetylsalicylic acid (better known as aspirin), the use of
Disalcid is much safer: acetylsalicylic acid inhibits the cyclooxygenase (COX)
activity, resulting in decreased synthesis of prostaglandin, leukotriene and
thromboxane precursors such as the ubiquitous enzyme which catalyzes the
initial step in the synthesis of prostanoids. Low stomach prostanoid levels
strongly increases the risk of ulceration, internal bleeding and perforation of
the stomach. Therefore, salsalate will be used in this study.

In this study, the effect of salsalate on insulin resistance will be
investigated. All subjects will undergo three hyperinsulinemic euglycemic
clamps. Two hyperinsulinemic euglycemic clamps will be with simultaneous
infusion of lipids, separated by at least 1 week and in a random order. In one
of these trials, subjects receive in the 4 preceding days of the clamp 4 g
salsalate a day. In the other trial, salsalate will be replaced by placebo
tablets. In addition to these two hyperinsulinemic euglycemic clamps with
simultaneous infusion of lipids, a third hyperinsulinemic euglycemic clamp will
be performed after the intake of placebo tablets, but without simultaneous
infusion of lipids. This trial will serve as a control trial.

Blood samples, infusions and muscle biopsies might cause bruises. Infections or
continued bleeding are exceptional. The subjects will be instructed to refrain
from heavy physical labour and not to remove the pressure bandage for at least
24 hours after the biopsy. Biopsies will be taken by a skilled medical doctor.

Hyperinsulinemic euglycemic clamping is a procedure we perform routinely in our
laboratory without notable complications. In rare occasions subjects exhibit
symptoms of hypoglycaemia (even if their blood glucose levels are still above 3
mmol). In these occasions insulin infusion will be terminated until
normoglycemia is achieved. After successfully performing the clamp blood
glucose values will be monitored for an additional 30 minutes with glucose
infusion stand-by if glucose levels happen to drop. Solid food will be provided
directly after finalising the clamp to avoid the experience of hypoglycaemia.

Infusion of Intralipid is routinely used in clinical settings. Adverse effects
are rare but nausea, vomiting, diarrhoea, shivering and fever might occur
during long term administration of intralipid. Given the sort time span of
infusion in the present study (less than 6 hours) we anticipate no such adverse
effects. If they happen to occur anyway, the infusion will be terminated
promptly.

Salsalate is a drug which in general is well tolerated without major risks. The
following reversible adverse experiences characteristic of salicylates were
most commonly reported with Disalcid, listed in descending order of frequency:
tinnitus, nausea, hearing impairment, rash, and vertigo. Although
cause-and-effect relationships have not been established, spontaneous reports
over a ten-year period have included the following additional medically
significant adverse experiences: abdominal pain, abnormal hepatic function,
anaphylactic shock, angioedema, bronchospasm, decreased creatinine dearance,
diarrhea, gastro intestinal bleeding, hepatitis, hypotension, nephritis and
urticaria. The usual dosage is 3000 mg daily, divided over three dosages.
However, studies by the group of Shoelson revealed that a dose of 4000 mg was
required for insulin sensitizing effects. Therefore, the dosage of salsalate
intake in this investigation will be 4000 milligrams daily. Previous
investigations showed no additional side effects after the intake of 4000 mg of
salsalate/day.

As drug interaction of Disalcid is recognized, use of any medication is an
exclusion criterion. Given the fact that Disalcid has additive effects to
aspirin and other salicylate derivates, special care will be taken to exclude
the use of any NSAID during the study. Furthermore, the intake of salsalate
should be avoided in following conditions: before (dental) surgery, anaemia,
bleeding problems, ulcers, asthma, kidney or liver disease, gout, Hodgkin*s
disease, nasal polyps flu symptoms, chickenpox or when a subject is allergic to
salsalate, aspirin or other medications for arthritis, pain, or any other drugs.
Finally, it is not allowed to take aspirin, or drink alcohol while taking
salsalate.