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Thomas Tai-Seale, Dr.P.H. M.M.S.,M.P.H.,M.A.
School of Rural Public Health
Texas A&M Health Science Center
is the inability of your muscle, fat, and liver cells to use insulin properly.
How prevalent is IR?
• Well, as the tests are not routine, no
one seems to know for the general
population, though the figure 25% is
• Among healthy (non-diabetic) 1st
degree relatives of Type 2 diabetics,
a good estimate is about 40%.
Volek A and Ronn W, 1999. Experimental and Clinical Endocrinology and Diabetes 107,
Here’s the relationship of blood sugar
levels and mortality
N= 25,364 > • Impaired fasting glucose is
blood glucose levels
30 years old between 100-125 mg/dl.
• ADA criteria for diabetes is
>125 mg/dl.- this includes
many who don’t know they
DECODE Study Group Lancet 1999, 354: 617-621.
So, let’s start at the beginning – where we’d like
to stop this disease progression - and study
(and to a much lesser degree in fat)
Here’s a muscle cell in red. Note, it has a “lock.”
The lock is the insulin receptor.
(or some amino acids) Insulin receptor
after a meal and makes
insulin in response. Insulin
Insulin is carried in the blood
to the lock on the cell surface.
This begins the process to bring a
glucose transporting door to the cell
surface and open it.
Sugar enters and the muscle then uses it
for fuel or stores it as glycogen.
Technical mumbo jumbo: Insulin binding causes glucose transporters (GLUT4)
stored in vesicles inside the cell to be slowly released (translocated) to the surface
where they allow glucose in by diffusion. In the cell, glucose binds to and inhibits
glycogen phosphorylase (the enzyme which breaks down glycogen). Within an hour of
insulin removal, GLUT4 are largely restored to the cytoplasm by endocytosis in what
are called “clatharin-coated pits.” For FFA insulin effects see Newgard & McGarry 1995,
Ann Review Biochem 64,689-719 and McGarry 2002 Diabetes 51:7-18. 7
Note: it’s not only
malfunctioning But, what would happen
receptors on muscle to the blood sugar level if
that causes blood some of the locks were
sugar to rise, the
liver also produces damaged, as sugar keeps
excess glucose in IR.coming in? Insulin receptors
That’s right, blood sugar would rise as long as
sugar has trouble getting into the cell.
And what would happen
to the insulin level?
It too would rise in response to persistent sugar.
In Phase 1 (a.k.a. early) IR, the extra
insulin would open some doors, so Pancreas
blood sugar and insulin would fall, and you
wouldn’t’ test hyperglycemic or hyperinsulinemic even
though some IR receptors are not working.
Sad note: If you have relatives who are diabetic (like me),
chances are good that your insulin receptors are not working
well, even if you don’t test positive for hyperglycemia.
Pratipanawatr W, Pratipanawatr T, Cusi K, Berria R, Adams JM, Jenkinson CP, Maezono K, DeFronzo RA, Mandarino LJ, 2001
Skeletal muscle insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes is associated with
decreased insulin stimulated, insulin receptor substrate-1 tyrosine phosphorylation Diabetes 50, 2572-2578. Muscle cell 8
In Phase 2 IR, there is increased
insulin resistance, i.e. more
locks are damaged, so blood
sugar builds up
and your pancreas responds
by making more insulin. Insulin
The load of sugar,
however, is too great,
and the pancreas can’t
produce enough insulin
to reduce it.
You now test positive for BOTH
Impaired Glucose Tolerance (glucose
levels of 140 to 199 mg per dL (7.8 to 11.0
mmol) two-hours after you’ve had 75-g
oral glucose) AND hyperinsulinemia.
Increased IR in IGT - see: Tripathy D, Carlsson M, Almgren P, Isomaa B, Taskinen M-R, Tuomi T,
Groop LC, 2000 Insulin secretion and insulin sensitivity in relation to glucose-tolerance. Lessons
from the Botnia Study. Diabetes 49 975-980. Muscle cell 9
I’m sick of
…the pancreas crazy lock
gets tired of Insulin
having to make
so much insulin.
In progressing from IGT to T2DM, IR does not change, but the pancreas wears out: Reaven GM, Holenbach CB,
Chen YDI, 1989. Relationship between glucose tolerance, insulin secretion, and insulin action in non-obese
individuals with varying degrees of glucose tolerance. Diabetologica 32:52-55. Bogardus C, Lillioja S, Howard
BV, Reaven G, Mott D, 1984. Relationship between insulin secretion, insulin action, and fasting plasma glucose
concentration in nondiabetic and noninsulin dependent diabetic subjects. J. Clinical Investigations, 74:1238-1246. Muscle cell 11
You’d no longer be
and you’d have to start
buying your insulin at
the drug store.
You’d also begin to have to deal with the
bad effects of all that excess sugar.
Muscle cell 12
So, IR (and thus diabetes) is a disease about
broken insulin receptors (locks) that govern
the inflow of glucose and fats (through
doors) into muscle cells - and by a similar
mechanism, but to a much lesser extent, into
fat cells. It is also a disease about defective
pancreatic beta cells.
Note: It is still unknown if insulin resistance (broken
locks) or defective insulin secretion (broken keys) is the
primary defect leading to Type 2 diabetes, but both are
present in early stages. –see Williams 11th ed, pp. 6-7 of
the section on pathogenesis.
Please note that in addition to
removing sugar from the blood,
insulin also clears Free Fatty Acids
(a.k.a. non-esterified fatty acids, NEFA)
from plasma – especially after a meal.
Bonen et al, 2004. Regulation of fatty acid transport by fatty acid translocase/CD36. Proc Nutr Soc 63: 245–249.
Miles et al. 2003 Nocturnal and postprandial free fatty acid kinetics in normal and type 2 diabetic subjects: effects
of insulin sensitization therapy. Diabetes 52: 675–681, 2003.
The main mechanism by which insulin does this
is blocking the appearance of Free Fatty Acids
(FFA) into the blood - by blocking lipolysis.
Secondarily, it also removes FFA from the blood.
Carpentier et al 2007. Am J Physiol Endocrinol Metab 292: E693–E701.
Is Insulin Resistance Bad
• Consider what it
would do in times of
• Insulin resistance
would cause more
glucose to be
available to the
muscles could run
• Thus, it was
adaptive (Landsberg 2006,
Clinical and Experimental Pharmacology and
Physiology 33: 863-867.)
Of course, that was before television!
• As you’ve seen, insulin is
secreted in response to
• It is also released in response
to certain free amino acids –
though this is not as well
• Initially, these were identified
as arginine, leucine, and
phenylalanine (Floyd et al 1966 J Clin
Invest 45, 1487-1502; Floyd et al 1970
Diabetes 19, 102-108.)
• More recent studies find that
arginine, leucine, isoleucine, and
alanine are particularly potent at
stimulating beta cells (Bolea et al, 1997.
Pflugers Arch 433:699-704.)
• A 2006 review indicates that
arginine, leucine, and alanine,
stimulate insulin release (Newsholme
et al Diabetes 55 Sup 2: S39-s47).
• One amino acid, homocystein, inhibits
• Glutamine can only stimulate insulin release
in the presence of glucose.
• The ability of glutamate to stimulate insulin
release is controversial.
• If, however, you give a
protein or amino acid source
AND a glucose source – the
insulin secreting capacity of
beta cells INCREASES! (Calbet &
Maclean, 2002. J Nutr 132:2174-2182.)
• As glucose levels drop as
insulin rises – until late-stage
diabetes – it may be possible
to delay the onset of diabetes
by ingestion of specific
amino acids with meals (Van
Loon et al 2003, Diabetes Care 26(3) 625-630).
• This mixture would also
stimulate protein synthesis
and inhibit the breakdown of
protein seen in diabetes (Van
Loon et al 2003, Diabetes Care 26(3) 625-630).
What about fatty acids and insulin
• Up until recently, fatty acids
have been thought not to cause
insulin release, but to amplify
the effect of glucose – if present
– on insulin release. Warnotte et al 1994
Diabetes 43: 703-711. Parker et al 2003 Metabolism 52:1367-1371.
• This view may be changing.
The question we will consider next is:
How do the keys and locks get broken?
Well, sometimes, it’s
Here, I’ll show you…
First, let’s study the normal insulin
response to sugar.
The figure to the left shows
what happens to insulin when
glucose is infused – enough to
maintain blood glucose levels
two to three times the fasting
level for an hour.
Almost immediately after the
glucose infusion begins, plasma
insulin levels increase
This initial increase is due to
secretion of preformed insulin,
which in a few minutes is
The secondary rise in insulin
reflects the considerable
amount of newly synthesized
insulin that is released after
about 15 minutes.
Clearly, elevated glucose not
only simulates insulin secretion,
but also transcription of the
insulin gene and translation of
N Insulin levels
a These first 5 graphs show the insulin levels (from a OGTT) across a 20 year time series.
t Those without genetic risk for diabetes are graphed in yellow. The pattern is steady.
Those with a family history for type 2 diabetes are in orange/red.
The pancreas begins to lose tha ability to make insulin after the third graph.
The next 5 graphs are matched glucose levels (mmol/l) across the same time, for those
t without genetic risk (yellow) and with risk (orange/red).
The horizontal white line is the cut-point for diabetes.
h Note: even at the start of the 20 year study (furthest left), those who are at risk have
i elevated insulin levels, but they won’t be diagnosed with diabetes for a long time!
s Pathophysiology of Insulin Resistance James R. Gavin III, MD, PhD. http://www.medscape.com/viewarticle/442813_9
Here’s more evidence of genetic cause
Relatives of diabetics often have
IR – even if not obese1 and even
if not hyperglycemic.2
1. Warram JH, Martin BC, Krowelski AS, et al. Slow glucose removal rate and hyperinsulinemia
precede the development of type II diabetes in the offspring of diabetic parents. Ann Intern Med
2. Pratipanawatr W, Pratipanawatr T, Cusi k, Berria R, Adams JM, Jenkinson CP, Maezono K,
DeFronzo RA, Mandarino LJ, 2001. Skeletal muscle insulin resistance in normoglycemic subjects with
a strong family history of type 2 diabetes is associated with decreased insulin-stimulated insulin
receptor substrate-1 tyrosine phosphorylation. Diabetes 50, 2572-2578.
Twins often both have IR.
Lehtovirta M, Kaprio J, Forsblom C, et al. Insulin sensitivity and insulin secretion in
monozygotic and dizygotic twins. Diabetologia 2000; 43:285–293.
Some ethnic groups have insulin
resistance, e.g. Pima Indians
So, if you’ve got it, it may not be all your fault.
Any of the following single gene defects
will cause diabetes:
• A defect in the key (i.e. the insulin molecule) or in
the beta cell insulin secreting mechanism.
• For example, defective proinsulin or insulin genes, genes that code
mitochondrial enzymes in beta cells needed to produce ATP to
depolarize the beta cell and cause insulin release, and defects in
several other beta cell genes (e.g. for glucokinase needed to
provide G-6-P for mitochondria and thus precursor for cell
depolization and insulin release) that give rise to maturity-onset –
(manifesting before age 25) diabetes of the young (MODY).
• Defects in the lock (i.e. the insulin receptor)
• Reduced manufacture of lock. Class 1 diabetes
• Poor transport of lock to cell surface. Class 2 diabetes
• Dysfunctional lock – key won’t fit. Class 3 diabetes
• Poor lock functioning (signaling thru tyrosine kinase). Class 4
• Increased breakdown and recycling of lock. Class 5
We see defective insulin receptors in
Monogenic causes of IR and
Type A Insulin Resistance
diabetes however are rare!
Reviewed in Williams Textbook of Endocrinology 10th
ed pp 1430-1432. & 11th ed. Rabson-Mendenhall Syndrome 24
A number of enzymes are probably involved in the more
common types of type 2 diabetes
• One of the most promising under study is Calpain 10.
• Calpain, discovered in 1976, is an intracellular enzyme
that cleaves proteins containing cysteine (an amino
acid containing sulfur). Its name comes from its
similarity to two other enzymes: calmodulin and
papain. Like calmodulin, calpain requires calcium to
• If calpain 10 is inhibited, the result is insulin
resistance and impaired insulin secretion in response
Zhou, Y-P, et al. Calpain inhibitors impair insulin secretion after 48-hours: a model
for beta-cell dysfunction in type 2 diabetes? Diabetes 2000. 49:A80
Seamus, K, et al. Calpain-sensitive pathways in insulin secretion and action: a
pathophysiological basis for type 2 diabetes? Diabetes 2000. 49:A62.
We too may cause IR …
If we look like this.
Did you know that it’s not obesity per se that’s
related to IR.
It’s abdominal size.
The bigger in the belly you are, the less you can use insulin.
A, From Fujimoto WY, Bergstrom RW, Boyko EJ, et al. Obesity Res 1995; Suppl 2:1795–1863; B, from Kahn SE, Prigeon
RL, McCulloch DK, et al. Quantification of the relationship between insulin sensitivity and beta-cell function in human
subjects: evidence for a hyperbolic function. Diabetes 1993; 42:1663–1672.) 27
It’s worse to be
an apple than a pear
Apple-shaped people have more intra-abdominal
fat than pear-shaped folk. Look…
Most fat, about
just under the
Visceral fat is
the fat around
On average, it’s
only about 10%
of body fat.
Two people of
the same weight,
can have very
and types of fat.
Why is central
obesity worse than
It leaks more fat!
and elevated free fatty acids predicts the progression to diabetes.
TECHY STUFF: 1. Central fat has more adrenergic receptors and when stimulated by
epinephrine, hormone sensitive lipase is activated which breaks down fat releasing it
to the blood stream. (See: Arner P, Hellstrom L, Wahrenberg H, Bronnegard M. Beta-adrenoceptor
expression in human fat cells from different regions. J Clin Invest 1990; 86:1595–1600. Nicklas BJ, Rogus EM,
Colman EG, Goldberg AP. Visceral adiposity, increased adipocyte lipolysis, and metabolic dysfunction in obese
postmenopausal women. Am J Physiol 1996; 270:E72–E78. )
2. Central fat is also resistant to insulin’s ability to inhibit lipolysis.
Note: 80% of diabetics are overweight with visceral obesity and thus have higher day-long
elevations of FFA. (See: Reaven GM, Hollenback C, Jeng C-Y, Wu MS, Chen Y-DI, 1988. Measurement of plasma glucose,
free fatty acid, lactate, and insulin for 24 hours in patients with NIDDM. Diabetes, 37, 1020-1024.)
3. Part of this is because of an increase in fat mass (Jensen MD, Haymond MW, Rizza RA, Cryer PE, Miles JM,
1989. Influence of body fat distribution on free fatty acid metabolism in obesity. J Clin Invest. 83,1168-1173) 30
Note: Most fats in the blood (99.9%) are bound to albumin.
Only a tiny amount are free (unbound). The levels of "free fatty
acid" in the blood are limited by the number of albumin
binding sites available.
Where do the free fatty acids we find in plasma come from?
As I said, much of Another source of
the free fatty acids
plasma free fatty
in blood plasma
originate from the acids are the
membranes of cells,
(TAG) stored in fat
whose fat is released
cells which are into blood by the
regularly broken enzyme
down by lipolysis.
By the way, lipolysis from TAG favors
unsaturated and short chained fatty acids.
The most mobile is eicosapentenoic acid
(C20:5n-3) and arachadonic acid (C20:4n-6).
Diet is not an immediate source of FFA in blood, rather diet supplies
the fat found in fat cells and the phospholipid membrane.
Leaf, 2001 Circulation 104, 744-745.
As there is very, very little
FFA in blood
• Only micrograms per liter – as FFA don’t like the
aqueous blood environment.
• By contrast there are grams of bound fat per liter
–usually expressed as mg/dl of blood.
• Measuring FFA is not a common practice.
Leaf, 2001 Circulation 104, 744-745.
There are four ways more FFA can
get into circulation.
1. If there is more fat mass over
which lipolysis can occur.
2. If subjects are stressed,
norepinephrine triggers a
sequence that activates
hormone sensitive lipase
(HSL) in fat cells
to break down triglycerides (TG).
3. If there’s less insulin or resistance
to insulin – as insulin has an
antilipolytic effect on the same
process. (Salaranta and Groop 1996:
Diabetes Metabolism Review 12:15-36.) From Holm 2003, Biochemical Society
. Transactions Volume 31, part 6.
4. If there is decreased uptake or
oxidation of FFA (Colberg et al 1995, J
Clin Investigation 95:1846-1853)
Now, the trouble is: Obese people suffer all four of these conditions. 34
Yes! Insulin resistance can be
induced in young healthy people
without diabetes in a matter of hours,
by simply exposing them to IV lipid
solutions (e.g. a 10% safflower oil
and 10% soy bean oil emulsion) while
keeping glucose and insulin levels
What happens is that lipid replaces
carbohydrate as fuel within a few
hours. FFA builds up in muscle,
glucose is not oxidized, and
glycogen synthesis is dramatically
reduced, (Boden et al, 1991 J. Clin.
Invest. 88:960-966. Roden et al, 1996.
J. Clin. Invest 97:2859-2865.)
So how does increased FFA
cause insulin resistance?
Adapted from discussion in Williams 10th & 11th ed. Textbook of Endrocrinology and other referenced sources
• Well, the first hypothesis – which is
partially correct – is called the
Randle Hypothesis(Randle et al, 1963. Lancet 1: 785-789).
• It says that If tissue energy needs are
being met by burning fat, muscle
cells will not need glucose and will
move to decrease its uptake. Thus,
glucose will build up.
• Here’s the best current theory…
Free fatty acids are of different
types and shapes
Sometimes we Sometimes like this.
symbolize them like
this. And other ways too…
We need something simpler for this
presentation, let’s use just one
shape and call it…
The “free” fat that “leaks” from belly fat
is delivered to muscle cells in our blood.
We’ll start simply with one fat molecule.
With excess plasma
FFA, the fat is stored
in muscle (and liver)
as triglycerides which
are in a state of
constant turn over in
the cell back to FFA
(Goodpaster et al 2000 Am J
Clin Nutrition 71:885-892. and
Bays J Clin Endocrinology and Nucleus
Met 2004 89(2) 463-478).
Transfer of the free fatty acid (FFA) into the cell is facilitated by fatty acid
binding protein –plasma membrane (FABP-pm). Other enzymes may also be
involved, like fatty acid translocase and FA transport protein. 41
Once in the cell, small chain fats diffuse into the
mitochondria, but those over 10 carbons (which is
most) must be taken up by two enzymes on the
outer surface of the mitochondria that are
throughout the cell
The first mitochondrial enzyme is called Acyl-CoA synthetase (or fatty acyl-CoA synthetase) and it’s also found on
endoplasmic reticulum. The product of FFA and acyl-CoA synthase is fatty acyl-Co-A. This is then taken up by a
second enzyme on the outer mitochondrial surface that requires carnitine to work. It’s called Carnitine Palmitoyl
Transferase (CPT-1). The result is acyl-carnitine, which a second CPT enzyme (CPT-2) in the inner mitochondrial
membrane converts back to acyl-CoA, recovering the carnitine. The fatty acyl –coA is now inside the
mitochondria and can proceed to Beta oxidation. The reaction is at Appendix 1. More information follows...
Now, the mitochondria is amazing
It’s the boiler-room of the cell 43
Look, here’s one inside a cell
sugars move Beta
into the cell. Oxida-
digested to Acid
acetyl coA in Cycle
Then fat and
mitochondria. Sugars are broken down through glycolysis in the cytoplasm to acetyl-Co-A.
Short and medium chain FA diffuse across the mitochondrial membrane, but those longer than
C10 must be transported by carnitine palmitoyltransferase I (CPT-1) which resides on the outer
mitochondrial membrane –see diagram at Appendix 1 .
On the inner mitochondrial membrane fats are broken down by CPT II and a complex of enzymes
which vary depending on chain length. 44
In the Fats
fats are digested
in the beta Beta
If you pay me a Acid
dollar, I’ll show Cycle
you the cycle.
The product of beta oxidation is acetyl CoA (a 2 carbon unit – attached to CoA), the
same product of glycolysis in the cytosol.
Acetyl CoA from both sources then enters the citric acid cycle Pay here to see.
The citric acid cycle is also known as the tricarboxylic acid (TCA) or Krebs cycle
Thus, both fats and sugars are fuel for the citric acid cycle which
eventually makes energy (ATP molecules) for the body - some of
which were used to transport long-chain fatty acids into the
mitochondria. (Acyl-CoA synthetase requires ATP to make acyl CoA.)
But what happens if you
have too much fat and sugar
enter the citric acid cycle?
The citric acid cycle starts
Product Product But some steps happen
faster than others, so some
Citric Acid Cycle products build up at the
One build-up product of
particular note is “product 1”
5 4 which is called “citrate.”
Appendix 3: The CAC chemical names
Once made, citrate
can flow back into
and through a
series of steps,
shut off the
enzyme, CPT-1 Citric
(Remember that?) Acid
So that long-
chained fats can no
longer get into the
Now that would make sense. It would slow down the fuel supply
so things can work at normal pace.
Citrate activates the enzyme acetyl CoA carboxylase, which catalyzes the conversion of acetyl CoA to malonyly-CoA. Malonyl CoA is
a potent inhibitor of CPT-1. See the reaction at Appendix 4. Bavenholm PN, Pigon J, Saha AK, et al. Fatty acid oxidation and the
regulation of malonyl-CoA in human muscle. Diabetes 2000; 49:1078–1083. Ruderman NB, Saha AK, Vavvas D, Witters LA. Malonyl-
CoA, fuel sensing, and insulin resistance. Am J Physiol 1999; 276:E1–E18. 48
The trouble is
that now fat Glycerol
starts to build Glycerol
up in the muscle
speeding up the
process of forming
intracellular TAG. Citric
The accumulation of TAG in the muscle
may not by itself be harmful (Boden and
Laakso 2004. Diabetes Care 27(9) 2253-987) . Rather, it
is probably one of the intermediary
products on the way to forming TAG
that causes problems, it is called diacyl
And it is often inserted into the cell membrane. 49
It may help to recall that the cell membrane
actually looks more like this
So it’s easy for diacyl glycerol (DAG) to slide in.
But excess DAG is not good!
DAG activates one of the forms of the enzyme Protein Kinase C – Calcium and DAG
dependent activation are shown below
The enzyme isoform is membrane-bound Protein Kinase C theta (PKC ), one of at least 12 forms of PKC. A protein kinase is an
enzyme that transfers a phosphate group from a donor molecule (usually ATP) to an amino acid residue of a protein. Most
protein kinases can only phosphorylate one kind of amino acid. PKC phosphorylates two: serine and threonine.
Phosphorylation can activate or inhibit an enzyme. PKC activation occurs with binding of diacylglycerol (DAG), often in the
presence of calcium (released from the sacroplasmic reticulum by inositol triphosphate –a sugar molecule) - though PKC theta
does not require it - resulting in translocation of the PKC-DAG complex to the cell membrane where it is active and activates
other signaling molecules. The whole reaction can be seen at Appendix 5. The exact mechanism whereby fat activates PKC is
not known; it may not be through citrate. The effect of activating PKC is a reduction in insulin receptor substrate-1 (IRS-1)
and phosphatidylionositol-3, required in translocating glucose transporter to the cell surface. The result is hyperglycemia. Good
review at Boden G and Shulman GI, 2002. Free fatty acids in obesity and type 2 diabetes: defining their role in the development
of insulin resistance and β-cell dysfunction. European Journal of Clinical Investigation (2002) 32 (Suppl. 3), 14–23.
Once activated, PKC phosphorylates the
MAPK protein and can also (next slide)
phosphorylate the insulin receptor
Here’s what’s going on at the insulin receptor
• Normally, insulin binds to the
• This activates the beta-subunits,
which become autophosphorylated
at tyrosine residues. (Thus the beta
unit is a tyrosine kinase.)
• Seven intracellular tyrosines become
autophosphorylated in response to
• This causes a 200-fold increase in
• However, if the Insulin Receptor is
phosphorylated by Protein Kinase C
–induced by excess fats -
phosphorylation occurs at a serine
or threonine amino acid and insulin
action is inhibited.
• The result is less IRS-1 and
phosphatidylionositol-3, and thus
ultimately fewer GLUT 4s are
transported to the surface..
Here’s a little more detail of what happens
below the insulin receptor.
Insulin binding to receptor can either stimulate
the PI(3)K or the MAP kinase pathway.
But if excess blood fat (DAG) stimulates PKC then both the insulin receptor is
inhibited and the MAP kinase pathway is favored over the PI(3)K pathway and its
products, like glucose transporters.
So glucose builds up
Here’s the fats (NEFA) coming into
in the blood.
or building up in the cell.
PKC alters They are first A simplified
version of the
the insulin turned into fatty
it. DAG. whole process
It also inhibits is shown here
IRS-1 and as a From: Hulver MW and Dohm
consequence, GL, 2004. The molecular
GLUT 4, “the mechanism linking muscle fat
cell door” for insulin resistance. Proceedings
glucose, is not Both DAG and of the Nutrition Society (2004),
Though it’s not
made and fatty acyl co-A shown, FFA 63, 375–380.
transported. To can activate
oxidative stress Note that increased NEFA leads
Protein Kinase C. and the to increased fatty acyl-CoA and
resulting also leads to increased
reactive ceramide (a fat derived from cell
membrane – see Appendix 6
here for more information.)
Boden and which has a negative effect on
Laakso, 2004) Akt and thus a negatiove affect
The unsimplified process is a bit overwhelming..see on GLUT 4 translocation. Here’
I’ll show you…
Fig. 3. Some of the cellular mechanisms that link intramyocellular lipid accumulation with insulin
resistance. DAG, diacylglycerols; TAG, triacylglycerols; IRS-1, insulin receptor substrate 1; PI3K,
phosphatidylinositol 3-kinase; PDK, phosphatidylinositol-dependent kinase; akt/PKB, protein kinase B;
PKC, protein kinase C; aPKC, atypical protein kinase C; PPase, protein phosphatase; CPT-1, carnitine
palmitoyltransferase 1; (+), activation; (–), inhibition.
And that’s the amazing and perhaps true story of how having
too much visceral fat may cause
Insulin Resistance and diabetes!!!! in muscle.
There is, of course, debate over the visceral
obesity causes IR hypothesis. Miles and Jensen
(2005) for example thinks subcutaneous fat is
the main contributor to FFA and IR - see
Diabetes Care 28(9) 2326-2328.
But it’s probably not intramuscular FFA that is the problem
- per se. For example:
Endurance trained athletes have high intramuscular TAG, but
don’t have IR. (Goodpaster et al 2001 J Clin Endocrin Metab
SO, IR SEEMS TO OCCUR WHEN THE
INTRAMUSCULAR FAT ISN’T OXIDIZED
And that leads to another hypothesis…Maybe the cause
of IR is some defect in mitochondrial functioning? (see
Schrauwen’s 2007 review in J Clin Endocrin and Metab
It could be too few mitochondria
• As you’ve seen, the number of mitochondria would be
important in energy balance. The more mitochondria the
more fats and sugars you could burn so they wouldn’t
build up to activate PKC.
• People with Insulin Resistance have fewer mitochondria,
Type 1 fibers are light, Type 2 are dark in and have more Type 2 muscle fibers than Type 1 muscle
this stained slide. fibers in relation to normal subjects (Diabetes 2005 54:8-14.)
TYPE 1 TYPE 2
Muscle Muscle Type 2 muscle fibers have fewer mitochondria and favor
Contraction Slow Fast
glycolysis as opposed to TCA cycle oxidation.
• A number of proteins regulate the number of
Color Red White
Oxidation High Low
• Peroxisome proliferator-activated receptor (PPAR)
Glycolysis Low High
gamma– coactivator 1a and 1b - say that three times fast!
Mitochondria Abundant Sparse - is one that is currently being researched. The name is
often shortened to PGC-1a and PGC-1b in the literature.
Light Dark PPAR gamma, by the way, is a receptor on the cell nucleus of a variety of tissues
(heart, muscle, colon, kidney, pancreas, and spleen , fat cells, and macrophages)
• Trouble is, we don’t know if the number of mitochondria
NADH-TR Dark Light (and their promoters) is a cause or effect of IR. (Note:
SDH Dark Light insulin stimulation up-regulates mitochondria.) In
COX Dark Light addition, muscle types may be inherited or acquired -
Glycogen Scant Abundant exercise changes muscle type patterns.
It could be dysfunctional
Subjects with IR They also have
or diabetes have reduced amounts of
a reduced a inner mitochondrial
number of genes membrane protein
responsive to (uncoupling protein-
PGC-1a that 3, UCP-3) that
code for oxidative moves fatty acids
metabolism. from the
(Mootha et al 2003 Nat mitochondria to the
Genet 34:267-273. Patti cytosol to protect the
et al, 2003, Proc Nat’l
Acad Sci USA 100:8466- mitochondria from
8471.). accumulation of
NEFA. (Schrauwen et al The
FASEB Journal. 2001;15:2497-
2502.) and Schrauwen and
Hesselink, 2004. Diabetes
• But are these causes of IR or the effects of FFA/IR?
(Roden 2005 Int J Obes (London) 29 (Suppl 2) S111-S115.) 62
Mitochondrial maladies may be behind the often reported
tiredness diabetics suffer.
YOU’D BE TIRED TOO IF YOUR
MITOCHONDRIA WEREN’T WORKING RIGHT!
The most simple etiological explanations
for IR is simply too much sugar
(hyperglycemia) or too much insulin.
• Decrease sugar intake and you decrease IR and diabetes.
This has been proven among Pima Indians.
• High levels of glucose (200 mg/dL and above) causes
defective action of insulin in skeletal muscle (which fails
to take up glucose) and liver (which overproduces
glucose). Thus excess glucose causes even greater
glucose. (Sheenan, 2008. New Mechanism of Glucose
• If you take normal subjects and subject them to high
insulin levels for 24-72 hours, insulin receptors will be
downregulated and the post receptor bonding pathways
will not work well. Insulin will lose the ability to increase
nonoxidative (i.e. glycolytic) glucose disposal and will
also lose the ability to make glycogen –thus providing a
means to diabetes. See Williams (2008).
Some other hypotheses are in the Appendix 7 – Press here 64
The American Diabetes Association
“…there is little evidence that total carbohydrate intake is
associated with the development of type 2 diabetes.”
But the studies they cite don’t conclude this.
• Two are by Salmeron and colleagues in 1997.
• One in Diabetes Care concludes for men:
• “These findings support the hypothesis that diets with a
high glycemic load and low cereal fiber content increase
risk of NIDDM in men. Further, they suggest that grains
should be consumed in a minimally processed form to
reduce the incidence of NIDDM.”
• The other in JAMA makes the same conclusion for
• One was conducted in Sweden and found no
association between dietary intake and
developing diabetes – but the Swedish diet is
different than ours.
• The last one was by Coditz et al, 1992. This one
found “…no association between intakes of
energy, protein, sucrose, carbohydrate, or fiber
and risk of diabetes.” But, they controlled for
BMI. What if the effects are through BMI?
OK, you’ve looked at how excess fatty acids affect muscle.
WHAT ABOUT OTHER TISSUES?
Insulin resistance also develops in
In much the same way as muscle cells.
Recall that Insulin lets things in –
As with muscle cells,
insulin opens the cell
doors after a meal to
Sugar let sugar in
The sugar that is let in
is turned into fat.
& more In addition, insulin also lets fat
in, which is also stored as fat.
and it won’t let fat out (it
But, if insulin resistance develops
Sugars can Sugars
no longer get
into fat cells Sugars
nor can fats
sferase, CPT- Fats
1, is down-
both build up Fats
in the blood
The fats are in the form of free fatty acids
In addition, in insulin resistance, fat cells have increased
lipolysis, resulting leaking higher levels of free fatty acids.
Now note the hyperglycemic effects downstream
• You just saw how insulin resistance
in fat cells cause an increase in FFA.
• Increased FFA oxidation in muscle,
leading to more insulin resistance and
hyperglycemia –as it did with muscle.
Reviewed in Sheenan, 2008
It’s a spiral of worsening effects!
There are other peculiar fat cell effects in diabetes.
• In type 2 diabetes, preadipocytes, mainly in visceral fat, do not mature
properly to adipocytes.
• These preadipocytes are not very insulin-sensitive and do not secrete an anti-
inflammatory cytokine called adiponectin –which is only secreted by fat cells
and levels of which are inversely correlated with body fat percentage in adults.
• Rather, they are hypertrophic (swollen) and secrete proinflammatory cytokines,
especially tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6).
• Because these immature fat cells, won’t grow up: diabetics suffer global
Insulin has a variety of adipose effects: It causes pre-adipocytes to mature into adipocytes. In
addition, it stimulates glucose transport and triglyceride synthesis (lipogenesis). It also inhibits
lipolysis and increases fatty acid uptake by stimulating LPL activity in fat. Kahn BB and Flier JS,
2000. Obesity and insulin resistance. The Journal of Clinical Investigation 106(4). 473-481. See
Appendix 8 for notes on how insulin inhibits lipolysis. 72
One of liver’s main jobs is to keep
blood glucose levels steady.
Liver Usually this is a simple process
When blood sugar is low, the
liver makes and releases glucose.
Liver can release glucose either by releasing stored glucose (i.e.
glycogen) or making glucose in a process called gluconeogenesis.
To break down glycogen, low blood sugar triggers alpha cells in
the pancreas to release a hormone called glucagon. Glucagon binds
to receptors on the liver causing cAMP to be released. (To see the
reaction, press here > Appendix 9) After several steps, this activates
glycogen phosphorylase to initiate the breakdown of stored
glycogen. To make glucose (to see the reaction press here:
Appendix 2) three substrates (pyruvate, oxaloacetate or glycerol )
Pancreas and three enzymes: PEPCK, G-6_Pase, and fructose-1,6-
bisphosphatase are needed Genetic expression of the first two
enzymes can be induced by glucagon – if fasting (ie. low blood
sugar), glucocorticoids (cortisone and cortisol) -if under stress, or
catecholamines (epinephrine, norepinephrine, and dopamine) - by
exercise. Growth hormone secreted by the pituitary and cortisol
also inhibit the uptake of glucose by muscle and fat. Epinephrine
activates glycogen breakdown in the same way as glucagon –as
shown in the Appendix 1. Barthel A and Schmoll D, 2003. Novel
concepts in insulin regulation of hepatic gluconeogenesis. Am J
Physiol Endocrinol Metab 285: E685–E692
One of liver’s main jobs is to keep
blood glucose levels steady.
Liver When blood sugar is high after
a meal, it responds to both the
sugar and the insulin by
(Ferrannini et al 1988 Metabolism 37:79-85.
It also absorbs about a third of the
carbohydrate from the meal. Ferrannini op cit.
Liver is freely permeable to blood sugar (unlike muscle). High blood
sugar normally triggers beta cells in the pancreas to release insulin.
Pancreas Insulin binds to receptors on the liver (and muscle – as we saw)
causing glucose uptake in liver (and muscle).
As glucose is taken into liver cells it binds to and inhibits glycogen
phosphorylase - which normally breaks down stored starch) The result
is that glycogen is not broken down and sugar (glucose) is not
released into blood. Instead, glucose is stored as glycogen in the
liver. As glucose is not added to blood, glucose levels return to
normal. Hepatic glucose activates glucose kinase to convert glucose to
G-6-P. G-6-P is converted to G-1-P by phosphoglucomutase and is
then converted to glycogen.
Here’s a nice diagram of the process
To “see” the
With low blood
sugar follow the
start with high
blood sugar (at
top) and follow
Low blood sugar
release of glucagon
High blood which causes the
sugar causes liver to breakdown
pancreatic glycogen and raise
secretion of blood sugar to
insulin which normal levels.
causes liver to
store glucose Glucagon works the
(make glycogen) exact opposite of
and help restore insulin – it releases
blood sugar glucose.
Trouble is: In Insulin Resistance, glucagon is continually released, causing
glucose to be released, and this adds to the already accumulating hyperglycemia
due to insulin resistance. It’s bad on top of bad.
Note also what happens when there’s excess FFA around?
1. Heapatic FFA uptake is increased by mass action, leading
to increased FFA oxidation, leading to increased acetyl
CoA which stimulates the two rate limiting enzymes* in
gluconeogenesis while providing ATP for forming more
* Pyruvate carboxylase and phosphoenolpyruvate
Exton et al, 1966 J Biol Chem 244 4095-4102;
Bahl et al, 1997 Biochem pharmacol 53, 67-74.
2. FFAs also increase the activity of the enzyme that
controls the release of glucose from the liver.
Massillon et al 1996. Diabetes 46 153-157.
3. FFAs also interfere with the insulin receptor on liver,
resulting in the inability of insulin to stop
gluconeogenesis. Lam et al 2003 Am J Physiol 284 E863-E873.
Together, these effects can increase plasma glucose at a time when the
liver should be removing glucose. More terrible news!!
Here’s a final terrible blow
• In insulin resistance the liver over secretes VLDL
–which is of course triglyceride rich.
• Triglycerides are an independent risk factor for
• Further, the increased lipid production within
liver leads to a pathologic condition known as
In severe fatty liver, fat comprises
as much as 40% of the liver’s
weight (as opposed to 5% in a
normal liver), and the weight of the
liver may increase from 3.31 lb (1.5
kg) to as much as 11 lb (4.9 kg).
Minimal fatty changes are
temporary and asymptomatic;
severe or persistent changes may
cause liver dysfunction. Fatty liver
is usually reversible by simply
eliminating the cause –usually
alcohol, obesity, malnutrition,
diabetes, Cushing’s syndrome,
Normal, fatty and cirrhotic liver.
We return now to where we started – with
pancreatic beta cells – which make insulin.
What is the effect of FFA on the pancreas?
• We already know that elevated Plasma
FFAs predict the development of glucose
intolerance and diabetes (Charles et al, 1997. Diabetologia 40:1101-1106.)
But what do we know about FFAs and the pancreas?
1. We know that short-term (acute) elevations of FFAs (oleic, linoleic, lauric, or
palmitic) when directly injected into the pancreas immediately stimulate
insulin secretion (Crespin et al, 1973, J Clin Invest 52:1979-1984).
2. nsulin immediately
But what do we know about FFAs and the pancreas?
1. We know that after about 24 hours of fasting FFAs are the primary fuel
2. If then given a glucose challenge (at time 0) – as when we eat – the FFA
augments the acute primary response release of preformed insulin…
3. But once insulin is released, it’s antilipolytic and clearing effect removes
4. …and curtails any further co-stimulation of the beta cell. So, you don’t see
the typical secondary response to glucose (McGarry and Dobbins, 1999)
5. During fasting
FFA are required
to maintain at
least a basal level
6. These seem to be
functions of FFA
to fasting (McGarry
and Dobbin, 1999).
We also know that the more saturated and longer the FFA,
the greater the insulin response – while fasting to a
glucose challenge (McGarry & Dobbins 1999).
Our trouble is that we are not in an environment
where fasting is a regular occurrence. We are in
a food rich – especially saturated fat rich –
Thus, the adaptive effects of low levels of FFA
during fasting which act to enhance insulin
secretion and glucose absorption when the fast
is broken now works against us.
Long-term exposure of the pancreas to high
FFA – as you see with overweight – leads to
enhanced insulin secretion at low glucose
levels (giving higher basal insulin levels), but
suppression of making new insulin, and
impaired ability of beta cells to respond to high
glucose concentrations (McGarry and Dobbin, 1999;
Carpentier et al, 1999, Kashyap et al, 2003…). Thus,
increased FFA > decreased insulin secretion.
1. We’ve also known since the late 1960s
that if long chain FFA (oleic acid) is
infused with infused glucose, the
insulin levels are dramatically raised
above glucose alone – and the rise in
insulin is accompanied by falling
glucose in healthy fasting dogs.
Greenough et al 1967 Lancet ii 1334-1336.
2. The increase in glucose-stimulated
insulin release to short-term exposure
to long chain FFA has now been
demonstrated many times (e.g. Paolisso et
al, 1996. Diabetologia 38:1295-1299).
3. However, if the exposure to increased
FFA (e.g. a twofold elevation) is longer
than a day (in the studies sited, 48
hours) in rats (Sako and Grill, 1990 Endocrinology
127: 1580–1589) or healthy non-obese
young men (e.g. Carpentier et al 1999. Am J
Physiology 276: E1055-E1066) – the FFA induced
augmented insulin response to
glucose is lost.
• If you are obese, but not diabetic, the
ability of long-term administration of FFA
to stimulate insulin release when
challenged with glucose is markedly
decreased. (Carpentier et al 2000 Diabetes 49 399-
• If, you are not diabetic and not obese
(BMI 25, avg age 43) BUT, you have a
first degree diabetic relative who is
diabetic, the higher your plasma level of
FFA, the less insulin you secrete in the
first 10 minutes – the acute response
(Paolisso et al, 1998).
• The good news is that in this study if you
treat with a drug (acipimox –a niacin
derivative) that reduces FFA, the acute
insulin response increases.
There is also evidence that FFA that are richer in saturated than
polyunsaturated fats have a greater decrease in insulin release
Stefan et al 2001 Hormone and Metabolism Research 33 :432-438.
OK here’s a good summary
Prolonged experimental elevation of
plasma NEFA in humans reproduces
the cardinal pathophysiological
features of type 2 diabetes, including
reduced insulinmediated glucose
utilization, impaired glucose-
mediated insulin secretion, and
increased endogenous glucose
production (10, 36, 39).
SO, GIVEN ALL YOU’VE
LEARNED, WHAT MUST WE
DO TO IMPROVE THE
Here’s my thoughts
1. We need more money for basic research; it’s
hard to prevent and treat a disease that’s not
2. More people need to know if they are insulin
resistant – why wait until they are diabetic.
3. Assuming the FFA hypothesis is correct, we
need to find ways to lower FFAs.
4. It would seem if we want to burn fat, we must
limit or control insulin release – as insulin
1. T/F IR is commonly measured during routine physicals.
2. The prevalence of IR is…
3. The organ that detects sugar and makes insulin…
4. In Phase 2 IR is characterized by the following blood states…
5. In Phase 3 IR is characterized by the following blood states…
6. Insulin resistance is a disease of …
7. The pancreas will release insulin in response to which of the
8. Ultimately IR is probably caused by …
9. The figure at left describes…
10. T/F IR is NOT always associated with obesity.
11. Central obesity is worse than subcutaneous fat because…
12. Epinephrine, stimulates the following enzyme on central fat cells…
13. Which of the following explains how FFA in the blood can be increased:
• If there is more fat mass over which lipolysis can occur.
• If subjects are stressed, norepinephrine triggers a sequence that
activates hormone sensitive lipase (HSL) in fat cells
• If there’s less insulin or resistance to insulin – as insulin has an
antilipolytic effect on the same process.
• If there is decreased uptake or oxidation of FFA.
14. Excess FFA entering the mitochondria eventually causes insulin resistance
by producing DAG. How?
15. DAG exerts its effects by activating an enzyme named…
16. PKC phosphorylating the insulin receptor results in …
17. Is Intramuscular FFA by itself a bad thing?
18. PPAR gamma is…
19. As opposed to a disease of FFA, IR may be a malfunction of the following
20. A inner mitochondrial membrane protein of concern in mitochondrial
dysfunction in IR is …
21. T/F According to the ADA “…there is little evidence that total
carbohydrate intake is associated with the development of type 2
22. Besides muscle cells IR develops in …
23. Adiponectin is best described as…
24. Proinflammatory cytokines secreted by fat cells include…
• The organ that makes, stores, and releases glucose into the
blood when blood sugar is low is…
• low blood sugar triggers alpha cells in the pancreas to
release a hormone called ________.
• Glucagon is made … ________.
• Glucagon acts to break down ___ in the liver.
• Glucagon stimulates making __- in the liver.
• Normally, as glucose is taken into liver cells it binds to and
inhibits an enzyme named ____________ - which normally
breaks down …stored starch – thus high glucose inhibits
the beakdown of glucose.
• Excess FFA generally has the effect on liver of….
• The effect of excess FFA on pancreatic beta cells is