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Thomas Tai-Seale, Dr.P.H. M.M.S.,M.P.H.,M.A.School of Rural Public HealthTexas A&M Health Science Center

Images in this presentation may be subject to copyright.

601 version

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is the inability of your muscle, fat, and liver cells to use insulin properly.

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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 often used.

• 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, 140-147.

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Here’s the relationship of blood sugar levels and mortality

5DECODE Study Group Lancet 1999, 354: 617-621.

Risk of mortality

Years

N= 25,364 > 30 years old

• Impaired fasting glucose is blood glucose levels between 100-125 mg/dl.

• ADA criteria for diabetes is >125 mg/dl.- this includes many who don’t know they have diabetes.

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(and to a much lesser degree in fat)

So, let’s start at the beginning – where we’d like to stop this disease progression

- and study

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Insulin

Insulin receptor

The pancreas

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.

Muscle cell

The lock is the insulin receptor.

Sugar enters and the muscle then uses it for fuel or stores it as glycogen.

Here’s a muscle cell in red. Note, it has a “lock.”

Insulin is carried in the blood to the lock on the cell surface.

detects sugar(or some amino acids) after a meal and makes insulin in response.

This begins the process to bring a glucose transporting door to the cell surface and open it.

Glu- cose

Tran-sport

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as sugar keeps coming in?

Pancreas

Insulin

Insulin receptors

Muscle cell

But, what would happen to the blood sugar level

And what would happen to the insulin level?

In Phase 1 (a.k.a. early) IR, the extra insulin would open some doors,

if some of the locks were damaged,

That’s right, blood sugar would rise as long as sugar has trouble getting into the cell.

and you wouldn’t’ test hyperglycemic or hyperinsulinemic even though some IR receptors are not working.

It too would rise in response to persistent sugar.

so blood sugar and insulin would fall,

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.

Note: it’s not only malfunctioning receptors on muscle that causes blood sugar to rise, the liver also produces excess glucose in IR.

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Pancreas

Insulin

Insulin receptors

Muscle cell

In Phase 2 IR, there is increased insulin resistance,

and your pancreas responds by making more insulin.

i.e. more locks are damaged,

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.

so blood sugar builds up

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In Phase 3 IR, IR stays the same, but…

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Pancreas

Muscle cell

…the pancreas gets tired of having to make so much insulin.

I’m sick of making all these keys for

this crazy lockInsulin

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.

12Muscle cell

and quits making insulin.

You’d also begin to have to deal with the bad effects of all that excess sugar.

You’d no longer be hyper-insulinemic,

and you’d have to start buying your insulin at the drug store.

I quit!

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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.

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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 Human Design?

• Consider what it would do in times of food scarcity.

• Insulin resistance would cause more glucose to be available to the brain –while muscles could run on fats.

• Thus, it was probably originally adaptive (Landsberg 2006,

Clinical and Experimental Pharmacology and Physiology 33: 863-867.)

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Of course, that was before television!

• As you’ve seen, insulin is secreted in response to glucose.

• It is also released in response to certain free amino acids –though this is not as well studied.

• 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

insulin secretion. • Glutamine can only stimulate insulin release

in the presence of glucose. • The ability of glutamate to stimulate insulin

release is controversial.

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• 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).

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What about fatty acids and insulin release?

• 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.

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The question we will consider next is:

How do the keys and locks get broken?

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Here, I’ll show you…

Well, sometimes, it’s genetic.

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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 dramatically.

This initial increase is due to secretion of preformed insulin, which in a few minutes is significantly depleted.

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 its mRNA.

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These first 5 graphs show the insulin levels (from a OGTT) across a 20 year time series. 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 without genetic risk (yellow) and with risk (orange/red).The horizontal white line is the cut-point for diabetes. Note: even at the start of the 20 year study (furthest left), those who are at risk have elevated insulin levels, but they won’t be diagnosed with diabetes for a long time! Pathophysiology of Insulin Resistance James R. Gavin III, MD, PhD. http://www.medscape.com/viewarticle/442813_9

Now

look

at

this

Insulin levels

Glucose levels

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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 1990; 113:909–915.

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.

Lehtovirta M, Kaprio J, Forsblom C, et al. Insulin sensitivity and insulin secretion in monozygotic and dizygotic twins. Diabetologia 2000; 43:285–

293.

Twins often both have IR.

Some ethnic groups have insulin resistance, e.g. Pima Indians

So, if you’ve got it, it may not be all your fault.

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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

Type A Insulin Resistance

Leprechaumism

Rabson-Mendenhall Syndrome

Monogenic causes of IR and diabetes however are rare!Reviewed in Williams Textbook of Endocrinology 10th ed pp 1430-1432. & 11th ed.

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 be activated.

• If calpain 10 is inhibited, the result is insulin resistance and impaired insulin secretion in response to glucose.

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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:A80Seamus, K, et al. Calpain-sensitive pathways in insulin secretion and action: a pathophysiological basis for type 2 diabetes? Diabetes 2000. 49:A62.

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We too may cause IR …

If we look like this.

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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.)

The bigger in the belly you are, the less you can use insulin.

Did you know that it’s not obesity per se that’s related to IR.

It’s abdominal size.

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It’s worse to be an apple than a pear

Apple-shaped people have more intra-abdominal fat than pear-shaped folk.

Look…

Sorry!

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Two people of the same weight, can have very different amounts and types of fat.

Visceral fat is the fat around internal organs. On average, it’s only about 10% of body fat.

Most fat, about 80%, is subcutaneous: just under the skin.

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Why is central obesity worse

than subcutaneous

fat?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)

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.

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Where do the free fatty acids we find in plasma

come from?

As I said, much of the free fatty acids in blood plasma originate from the triacylglyceride

(TAG) stored in fat cells which are regularly broken down by lipolysis.

Another source of plasma free fatty acids are the phospholipid membranes of cells, whose fat is released into blood by the enzyme phospholipase A2.

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.

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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.

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There are four ways more FFA can get into circulation.

From Holm 2003, Biochemical Society Transactions Volume 31, part 6.

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.) .

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.

Is there evidence that FFA causes IR?

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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 steady.

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.)

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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…

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Free fatty acids are of different types and shapes

Sometimes we symbolize them like this.

Sometimes like this.

And other ways too…

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We need something simpler for this presentation, let’s use just one shape and call it…

Fat

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Nucleus

Muscle cell

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.

Fat

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 Met 2004 89(2) 463-478).

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M

Nucleus

Muscle cellThe 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...

Fat

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

M

M

M

43It’s the boiler-room of the

cell

Now, the mitochondria is amazing

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After a meal, here’s what happens:

Look, here’s one inside a cell

Su

gar

Then fat and acetyl –coA enter the 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.

Fats and sugars move into the cell.

Fat

Sugars are digested to acetyl coA in the cytoplasm.

Citric Acid Cycle

BetaOxida-

tion

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In the mitochondria, fats are digested in the beta oxidation 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.

Fats

The citric acid cycle is also known as the tricarboxylic acid (TCA) or Krebs cycle

If you pay me a dollar, I’ll show you the cycle. Pay here.

Citric Acid Cycle

BetaOxida-

tion

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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.)

Sugars

Citric Acid Cycle

BetaOxida-

tion

Fats

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But what happens if you have too much fat and

sugar enter the citric acid cycle?

The citric acid cycle starts working fast.

But some steps happen faster than others, so some products build up at the slower steps.

Citric Acid Cycle

FatSu

gar

One build-up product of particular note is “product 1” which is called “citrate.”

citrate

Appendix 3: The CAC chemical names

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Once made, citrate can flow back into the cytoplasm.

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.

Citric Acid Cycle

citrate

Fat

and through a series of steps, shut off the mitochondrial membrane enzyme, CPT-1 (Remember that?)

So that long-chained fats can no longer get into the mitochodria!Now that would make sense. It would slow down the fuel supply

so things can work at normal pace.

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The trouble is that now fat starts to build up in the muscle cytoplasm.

Citric Acid Cycle

Fat

speeding up the process of forming intracellular TAG.

Fat

Fat

Fat

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 glycerol (DAG).

Glycerol

Glycerol

And it is often inserted into the cell membrane.

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It may help to recall that the cell membrane actually looks more like

this

Fat

Fat

Glycerol

So it’s easy for diacyl glycerol (DAG) to slide in.

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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 (PKCq), 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 PKCq is not known; it may not be through citrate. The effect of activating PKCq 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

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Here’s what’s going on at the insulin receptor

• Normally, insulin binds to the extracellular alpha-subunits

• 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 insulin binding.

• This causes a 200-fold increase in catalytic activity.

• 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.

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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.

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Here’s another view.

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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.

From: Hulver MW and Dohm GL, 2004. The molecular mechanism linking muscle fat accumulation toinsulin resistance. Proceedings of the Nutrition Society (2004), 63, 375–380.

A simplified version of the whole process is shown here

Here’s the fats (NEFA) coming into or building up in the cell.

Both DAG and fatty acyl co-A can activate Protein Kinase C.

So glucose builds upin the blood.

PKC alters the insulin receptor, inactivating it.

It also inhibits IRS-1 and as a consequence, GLUT 4, “the cell door” for glucose, is not made and transported. To the cell surface.

They are first turned into fatty acyl-Co-A, then DAG.

The unsimplified process is a bit overwhelming..see

Note that increased NEFA leads to increased fatty acyl-CoA and also leads to increased ceramide (a fat derived from cell membrane – see Appendix 6 here for more information.) which has a negative effect on Akt and thus a negatiove affect on GLUT 4 translocation. Here’ I’ll show you…

Though it’s not shown, FFA also increase oxidative stress and the resulting reactive oxygen species can activate PKC (see Boden and Laakso, 2004)

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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.

SO, IR SEEMS TO OCCUR WHEN THE INTRAMUSCULAR FAT ISN’T OXIDIZED FAST ENOUGH.

But it’s probably not intramuscular FFA that is the problem - per se. For example:

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Endurance trained athletes have high intramuscular TAG, but don’t have IR. (Goodpaster et al 2001 J Clin Endocrin Metab 86:5755-5761.)

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 92(4): 1229-1231.

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www.odranoel.eu/Images/Mitochondria%20red%20i...

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, and have more Type 2 muscle fibers than Type 1 muscle fibers in relation to normal subjects (Diabetes 2005 54:8-14.)

• Type 2 muscle fibers have fewer mitochondria and favor glycolysis as opposed to TCA cycle oxidation.

• A number of proteins regulate the number of mitochodria. • Peroxisome proliferator-activated receptor (PPAR)

gamma– coactivator 1a and 1b - say that three times fast! - is one that is currently being researched. The name is often shortened to PGC-1a and PGC-1b in the literature. 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

(and their promoters) is a cause or effect of IR. (Note: insulin stimulation up-regulates mitochondria.) In addition, muscle types may be inherited or acquired -exercise changes muscle type patterns.

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CHARACTERISTICTYPE 1 Muscle

TYPE 2Muscle

Contraction Slow Fast

Color Red White

Oxidation High Low

Glycolysis Low High

Mitochondria Abundant Sparse

ATPase, pH 9.4 Light Dark

ATPase, pH 4.3 Dark Light

NADH-TR Dark Light

SDH Dark Light

COX Dark Light

Glycogen Scant Abundant

Type 1 fibers are light, Type 2 are dark in this stained slide.

It could be dysfunctional mitochondria

• But are these causes of IR or the effects of FFA/IR? (Roden 2005 Int J Obes (London) 29 (Suppl 2) S111-S115.)

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Subjects with IR or diabetes have a reduced number of genes responsive to PGC-1a that code for oxidative metabolism. (Mootha et al 2003 Nat Genet 34:267-273. Patti et al, 2003, Proc Nat’l Acad Sci USA 100:8466-8471.).

They also have reduced amounts of a inner mitochondrial membrane protein (uncoupling protein-3, UCP-3) that moves fatty acids from the mitochondria to the cytosol to protect the mitochondria from accumulation of NEFA. (Schrauwen et al The FASEB Journal. 2001;15:2497-2502.) and Schrauwen and Hesselink, 2004. Diabetes 53:1412-1417.)

YOU’D BE TIRED TOO IF YOUR MITOCHONDRIA

WEREN’T WORKING RIGHT!

Mitochondrial maladies may be behind the often reported tiredness diabetics suffer.

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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 Control)

• 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).

64Some other hypotheses are in the Appendix 7 – Press here

Note:The American Diabetes Association

recently said

“…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 women.

• 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?

WHAT ABOUT OTHER TISSUES?

OK, you’ve looked at how excess fatty acids affect muscle.

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Insulin resistance also develops in

fat cells

In much the same way as muscle cells.

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Recall that Insulin lets things in – not out

As with muscle cells, insulin opens the cell doors after a meal to let sugar inThe sugar that is let in is turned into fat.

Sugar

FatIn addition, insulin also lets fatin, which is also stored as fat.

Fat

and it won’t let fat out (it inhibits lipolysis).

& more

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But, if insulin resistance develops

Sugars can no longer get into fat cells (no GLUT transporter), nor can fats (as carnitine palmitoyltransferase, CPT-1, is down-regulated) so both build up in the blood stream.

Sugars

Fats

Sugars

Sugars

Fats

The fats are in the form of free fatty acids

Fats

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. This causes: • Increased FFA oxidation in muscle,

leading to more insulin resistance and hyperglycemia –as it did with muscle.

Reviewed in Sheenan, 2008

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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.

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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.

• 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 inflammation.

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OK, now we’ll look at FFA and the liver

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Liver

Pancreas

One of liver’s main jobs is to keep blood glucose levels steady.

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 ) 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

Usually this is a simple process

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One of liver’s main jobs is to keep blood glucose levels steady.

Liver

Liver is freely permeable to blood sugar (unlike muscle). High blood sugar normally triggers beta cells in the pancreas to release insulin. 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.

When blood sugar is high after a meal, it responds to both the sugar and the insulin by stopping gluconeogenesis. (Ferrannini et al

1988 Metabolism 37:79-85.

Pancreas

It also absorbs about a third of the carbohydrate from the meal. Ferrannini op cit.

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Here’s a nice diagram of the process

health.howstuffworks.com/diabetes1.htm

To “see” the normal process, start with high blood sugar (at top) and follow the yellow arrows.

High blood sugar causes pancreatic secretion of insulin which causes liver to store glucose (make glycogen) and help restore blood sugar levels.

Glucagon works the exact opposite of insulin – it releases glucose.

With low blood sugar follow the blue arrows.

Low blood sugar causes pancreatic release of glucagon which causes the liver to breakdown glycogen and raise blood sugar to normal levels.

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.

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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 glucose.

Together, these effects can increase plasma glucose at a time when the liver should be removing glucose. More

terrible news!!

2. FFAs also increase the activity of the enzyme that controls the release of glucose from the liver.

* Pyruvate carboxylase and phosphoenolpyruvate

Exton et al, 1966 J Biol Chem 244 4095-4102;

Bahl et al, 1997 Biochem pharmacol 53, 67-74.

Glucose-6-phosphatase 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.

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

heart disease.• Further, the increased lipid production within

liver leads to a pathologic condition known as “fatty liver.”

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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, hepatotoxins …

Normal, fatty and cirrhotic liver.

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We return now to where we started – with pancreatic beta cells – which

make insulin.

What is the effect of FFA on the pancreas?

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• We already know that elevated Plasma FFAs predict the development of glucose intolerance and diabetes (Charles et al, 1997. Diabetologia 40:1101-1106.)

http://www.nlm.nih.gov/medlineplus/images/cholesterol.jpg

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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

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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 FFA

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 of insulin.

6. These seem to be adaptive 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).

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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 –environment.

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.

BUMMER! 84

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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 49 399-408.)

• 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.

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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).

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SO, GIVEN ALL YOU’VE LEARNED, WHAT MUST WE DO TO IMPROVE THE PUBLIC’S HEALTH?

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Here’s my thoughts1. We need more money for basic research; it’s

hard to prevent and treat a disease that’s not understood.

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 restricts lipolysis.

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Stem Questions1. 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 following

8. Ultimately IR is probably caused by …9. The figure at left describes…

10. T/F IR is NOT always associated with obesity.

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Stem 211. 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?

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Stem 318. PPAR gamma is…

19. As opposed to a disease of FFA, IR may be a malfunction of the following organelle…

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 diabetes.”

22. Besides muscle cells IR develops in …

23. Adiponectin is best described as…

24. Proinflammatory cytokines secreted by fat cells include…

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Stem 4• 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

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The End