Measuring Insulin Secretion and ResistanceEndocrine RoundsJuly 29, 2015Amanda Brahm, PGY4

Objectives•Review the basics of insulin secretion and

action and the pathophysiology of  insulin resistance

•Review the different laboratory methods of measuring insulin resistance and secretion

•Review the strengths and weaknesses of each method

•Discuss choosing the most appropriate test

Outline

•Review the basics of insulin secretion and action

•Development of insulin resistance

•Overview of biochemical testing/models▫Dynamic vs Single specimen vs Surrogates

•Test selection

Basics of Insulin

Insulin Synthesis• Encoded by INS gene

• Transcribed in pancreatic β-cells

• Processed from preproinsulin -> proinsulin -> insulin

• Packaged in secretory granules along with C-peptide

• C-peptide and insulin released in equimolar concentrations via exocytosis in response to cellular signal (ie glucose)

• Glucose binds to GLUT-2, inducing downstream effects leading to membrane depolarization, calcium influx and release of insulin via secretory granule exocytosis

http://www.medicinehack.com/2011/08/insulin-secretion-local-regulation.html?sa=X&ved=0CBkQ9QEwAmoVChMIsc3fyNr-xgIVWBGSCh1CDQnp

http://research.vet.upenn.edu/Portals/1/lispro%20fig%201.JPG

Insulin released in two phases:▫ First Phase :

Rapid release of preformed insulin from docked and primed secretory granules in response to glucose load (from Rapid Release Pool)

Peaks at 3-5 mins▫ Second Phase:

Mobilization phase of reserve insulin , slower, requires docking and priming of reserve pool of secretory granules

Sustained release, peaks in 60 mins after glucose load

• Degree of glucose stimulus may affect amount of insulin released from secretory granule before membrane retrieval and recycling

Insulin Resistance

Insulin Resistance

• Definition:▫ Decreased responsiveness to the

metabolic actions of insulin

• Continuous variable▫ Difficult to define clear cutoffs

between insulin sensitive and resistant

• Hepatic resistance:▫ Increased glucose production due to

stimulation of gluconeogenesis and impaired inhibition of glycogenolysis

• Peripheral resistance: ▫ In skeletal muscle: decreased

transmembrane glucose transport and glycogen synthesis

▫ In adipose tissue: increased lipolysis (decreased antilipolytic insulin effect

• Initial compensation:▫ Increase B-cell output to maintain normoglycemia until no

longer sufficient then hyperglycemia and T2DM occur▫ Inappropriately high levels of insulin for level of glycemia▫Loss of first phase response

http://dm5migu4zj3pb.cloudfront.net/manuscripts/45000/45680/medium/JCI45680.f5.jpg

Molecular Basis of Insulin Resistance• Down-regulation of insulin-responsive

glucose transporter in adipose tissue

• Impaired translocation to cell surface in skeletal muscle

• Defects in glucose oxidization and glycogen synthesis

• Elevated non-esterified FA seen in obese pts can inhibit peripheral glucose disposal, enhance hepatic glucose output and impair B-cell function

• Possible directly toxic effects of hyperglycemia on beta cells

• Fetal factors http://viim.org/images/insulin-cell-%5BConverted%5D.jpg

Rare, extreme monogenic causes•Type A insulin resistance

▫Insulin receptor defects•Type B insulin resistance

▫Anti-insulin receptor autoantibodies•Type C insulin resistance

▫Post-binding deficits in insulin signaling•Features:

▫Extremely high fasting insulin concentrations (>300pmol/L) and >2000 post-prandially

Conditions Associated with Insulin Resistance

•Metabolic syndrome •HTN•Dyslipidemia (low HDL, high TG, high

sdLDL)•Increased waist circumference•Hypercoaguability•Increased inflammatory cytokines•Obesity

Biochemical Testing

• Dynamic function testing: ▫ Euglycemic clamp▫ Hyperglycemic clamp*▫ Insulin tolerance test▫ Insulin suppression test▫ FSIVGTT + minimal model analysis*▫ OGTT*▫ ISI▫ CIGMA

• Single specimen testing:▫ HOMA*▫ QUICKI▫ Serum insulin +/- glucose▫ C peptide

• Surrogate testing:▫ SHBG▫ IGFBP-1

*Can be used to measure insulin secretion

Testing Options

Dynamic Testing

Testing conditions• Avoid smoking (lowers insulin sensitivity) and alcohol

(impairs gluconeogenesis) for 24 hours prior to test

• Avoid activity that may elevate catecholamines▫ Caffeine morning of test▫ Stress▫ Discomfort▫ Excessive noise during testing

• Keep physical activity to a minimum (increases peripheral glucose disposal)

Hyperinsulinemic Euglycemic Clamp

• Gold standard test for insulin sensitivity

• IV infusions:▫ Insulin at constant 6mU/kg/min▫ 20% dextrose glucose adjusted rate to maintain euglycemia▫ K-Phos

• Glucose measured every 5-10 mins ▫ Adjust glucose infusion to maintain BG between 5-

5.5mmol/L

• Will reach steady state - glucose levels will plateau▫ Hepatic glucose production completely suppressed▫ Max glucose disposal rates in skeletal and adipose tissues

http://dx.doi.org/10.3803/jkes.2009.24.2.75

• At steady state (usually last 30-60 mins of test):▫ glucose infusion rate = peripheral glucose disposal rate (M value) expressed

in mg/kg body weight/min

• Higher values =greater insulin sensitivity:▫ >7.5 mg/kg/min = insulin sensitive▫ <4.0 mg/kg/min = insulin resistant▫ 4.0-7.5 mg/kg/min = pre-diabetic

• Disposal mainly occurs into skeletal muscle with minimal uptake in adipose tissue, therefore measurement may overestimate resistance in obese individuals ▫ Can be corrected for by normalizing disposal rates in lean body mass,

however this correction has been criticized as adipose tissue has been shown to significantly contribute to glucose disposal in morbidly obese patients.

▫ Measurement of lean body mass can be determined with DXA (dual x-ray absorptiometry) or bioimpedance.

• Can calculate insulin sensitivity index to compare to minimal model:▫ SIClamp = GDR/(SSPG×(steady state I – fasting I))

Limitations of HIEG Clamp• Adjusting infusion rate based on venous glucose may overestimate

of insulin sensitivity (as glucose infusion rates increase, there is a greater difference between arterial and venous glucose) ▫ Can use arterial draws or arterialized venous draws to avoid

• Must achieve complete suppression of hepatic glucose production, or will underestimate glucose disposal rate

• Time (approx 2-3 hours), labour and cost intensive

• Lack of standardization across studies in terms of duration of clamps, insulin infusion rate, and normalization for lean mass which limits comparisons

• Cannot assess insulin secretion/β-cell function

• Non-physiologic

Hyperglycemic clamp• Glucose infusion to rapidly achieve hyperglycemia at

11.0mmol/L (“priming”) and maintained for 2 hours

• Monitor insulin levels at both initial onset (early phase) and once steady state reached (late phase)

• At steady state, rate of glucose infusion required to maintain glucose at 11.0mmol/L allows estimation of insulin secretion

• Can assess insulin sensitivity/resistance by calculating Glucose disposal rate=infusion rate/Insulin level

• Arginine can be bolused at the end of the clamp to estimate maximal insulin secretion response

Limitations to hyperglycemic clamp

•Less precise than HIEG clamp

•Time, labour and cost intensive

•Non-physiologic

Insulin Tolerance Test• Insulin administered (0.1U/Kg body weight)

• Measure decline of plasma glucose concentration at -15,-5, 3,6,9,12,15,20 and 30 mins

• At 30 mins glucose injected and rate of glucose disappearance calculated

• Limitations: ▫ Initiation of counter-regulatory response to avoid hypoglycemia

will interfere with analysis

▫ Risks associated with hypoglycemia limit use

▫ Does not differentiate between hepatic vs skeletal muscle insulin resistance

Insulin suppression test• After overnight fast, somatostatin (250µg/h) is infused

to suppress endogenous insulin production.

• Glucose at 6mg/kg body weight/min and insulin 50mU/min are infused at a constant rate for 150 mins

• Glucose and insulin measures are taken at 30 min intervals for the first 2.5 hours then at 10 min intervals between 150-180mins

• The SSPG values from the last 30 mins of infusion time can be used to estimate insulin sensitivity, with the higher the concentration of glucose, the more insulin resistant

Limitations to IST•Risk of hypoglycemia in very insulin sensitive

subjects

•Could induce glycosuria in some subjects, underestimating insulin resistance

•Time, labour and cost intensive, although less than clamp techniques

FSIVGTT• Frequently Sampled IntraVenous Glucose Tolerance Test

• Several different protocols and analysis options

• Generally 4 samples taken for fasting insulin and glucose prior to IV bolus of glucose given over 1min

• Serum insulin and/or c-peptide and glucose are then measured at 25 timed intervals up to 240 mins

• Modified protocols:▫Use of tolbutamide or IV insulin at 20 mins▫Reduced frequency of blood draws to 12▫Use of stable isotope tracers with glucose bolus

To differentiate peripheral uptake and hepatic production

Advantages of FSIVGTT

•Minimally invasive

•Less technically demanding and more cost-effective than other dynamic tests

•Can measure first and second phase insulin response

Limitations for FSIVGTT• Time (3-4h), labour and cost intensive

• Requires very large number of blood draws

• Generally dependent on software tools to interpret data

• Interpretation difficulties in patients with impaired endogenous insulin response and in T2DM

• Indirect measure of insulin resistance

• Non-physiologic

Minimal Model•Computer modeling software that uses results

for FSIVGTT is estimate insulin resistance parameters

•Uses relationships between increasing circulating insulin and decreasing circulating glucose to estimate both SiMM (insulin sensitivity index) and SgMM (glucose effectiveness index)

•Can provide information on both early and late phase of insulin secretion

Limitations of Minimal Model•Oversimplifies physiology of glucose

homeostasis▫Tends to overestimate glucose effectiveness and

underestimate insulin sensitivity

•Less reliable in patients with impaired insulin secretion or significant insulin resistance

Insulin Secretion from FSIVGTT•Acute insulin response (AIR) is calculated from

area under the curve of plasma insulin concentration in first 10 mins following IV insulin load ▫Estimates first phase insulin release

•However, not independent assessment as will also reflect hepatic insulin extraction

OGTT• Simple, widely used and available, similar to clinical

OGTT with more frequent sampling

• More physiologic than IV administration, allows for incretin effect

• Obtain fasting glucose, insulin levels initially than glucose and insulin levels every 30 mins following 75g glucose load

• Several different surrogate indices, validated against the gold standard HEIG have been suggested to estimate insulin resistance based on these values, some incorporating other values such as weight, BMI, and glucose volume of distribution

Avignon, Matsuda and SIisOGGT showed best correlation with HIEG clamp

Limitations to OGTT•More prone to other influences on plasma

glucose such as rate of gastric emptying, glucose absorption, endogenous insulin secretion, response to incretins, and insulin-independent effects of glucose uptake and production

•Poor reproducibility due to variation in glucose absorption, insulin action and secretion

•Require frequent blood sampling over two hours

CIGMA•Continuous Infusion of Glucose with Model

Assessment

•Glucose is infused at a constant rate of 5mg/kg/min

•Blood samples for glucose and insulin are obtained at 50, 55 and 60 minutes

•Model assessment similar to HOMA

Limitations of CIGMA•Low precision of results

•Requires an IV infusion

•Assumes hepatic and peripheral insulin resistance are the same

Single Specimen Testing

Advantages to Single Specimen Testing•Inexpensive

•Easily applied in almost every setting, including large clinical trials

•Appropriate when a direct measure of insulin sensitivity not required, not feasible to obtain or when insulin sensitivity is of secondary interest

HOMA• HOmeostatic Model Analysis of

Insulin Resistance

• Using fasting glucose and fasting insulin and complicated computed non-linear model that allows assessment of the expected β-cell response and insulin resistance

• Recommend using mean of three samples for insulin taken at 5 min intervals, although single sample often used which is acceptable and yields similar results in large data sets

HOMA1: The Original• Used simplified calculations

based on original non-linear solution to yield:

HOMA-IR = (FPIxFPG)/22.5

HOMA-%B = (20xFPI)/(FPG-3.5)

• Good for assessing relative changes or comparing population over time

• Based on older insulin assays

HOMA2: Updated HOMA model• Requires computer model

• Updated for higher plasma glucose values

• Allows for either total or specific insulin assays

• Takes renal losses into account

• Better for assessing absolute resistance or β-cell function

• Allows estimation of:▫Proinsulin secretion▫ Insulin sensitivity ▫Beta cell function

HOMA - Advantages•Especially useful in large epidemiological

studies

•Good correlation with HEIG

•Easy, inexpensive

Limitations to HOMA•Samples must be obtained in steady state

(fasting)

•Cannot assess β-cell function in those on exogenous insulin – may be able to substitute C-peptide but not validated

•Low precision of estimates

•Only assesses hepatic insulin sensitivity as done in fasting state

QUICKI• QUantitative Insulin-sensitivity ChecK Index

• Uses fasting insulin and glucose values

• Valid for a wider range of insulin sensitivities than HOMA

• Basically the log inverse of HOMA but derived from different set of mathematical parameters

• Reasonable correlation with HEIG, extensively validated in different populations

• Formula:▫ 1/[log(Insulin)+log(Glucose)]

• Correction for fasting non-esterified fatty acids (NEFA) added to QUICKI improves correlations with clamp data

Limitations of QUICKI

•Does not assess response to glucose load

•Difficulty in analyzing data from patients with uncontrolled diabetes or lack of endogenous insulin

Serum fasting insulin +/- glucose•In right context, elevated levels are suggestive

of insulin resistance▫>120 pmol/L under normoglycemic conditions

would suggest resistance

•May be misleading if done without correlating glucose levels

•Will also be influenced by β-cell reserves and rate of insulin degradation

C-Peptide

• Secreted in equimolar concentrations to insulin endogenously

• Longer half life than insulin, avoids hepatic degradation, and may allow for more accurate indicator of β-cell secretion

• Can use C-peptide deconvolution analysis to more accurately reflect pre-hepatic insulin secretion

• Must be interpreted in context of glucose state

• Will be less accurate when insulin levels are changing rapidly

Surrogate Testing

Surrogate Markers •Sex Hormone Binding Globulin (SHBG)

▫Obtained from fasting blood specimen▫Can be influenced by other factors such as

estrogens and testosterone

•Insulin-Like Growth Factor Binding Protein-1 (IGFBP-1)▫Synthesis induced by insulin▫Obtained from fasting blood specimen▫Strong correlation to HEIG,▫More sensitive marker than SHBG▫Assay not widely available

Selecting the most appropriate test

How to Select Method of Measurement•Determine the nature of the study population and

question under investigation

•Consider resource/cost concerns

•Determine which aspects of insulin sensitivity/secretion/resistance are desired

•Standardize factors that than influence test results:▫Physical exercise▫Dietary intake▫Duration of fasting

For research purposes:• HIEG clamp preferred:

▫ Gold standard direct measure▫ Can be used in all populations▫ Can determine different tissue sensitivities (muscle, liver, adipose)

• Hyperglycemic clamp:▫ If require insulin secretion data

• IST:▫ Can be used in various populations ▫ Cannot determine sensitivity in different tissues

• FSIVGTT with Minimal Model ▫ Can measure both insulin sensitivity and glucose-stimulated secretion ▫ Impractical in subjects with major impaired insulin secretion (type 1 and 2 diabetics)▫ Cannot differentiate tissue specificity ▫ Still resource intensive▫ Must use software model

• OGTT▫ Most physiologic▫ Simple and reliable ▫ Measures glucose tolerance, insulin resistance, insulin secretion▫ Not as precise as HEIG for insulin sensitivity▫ Good for larger populations,▫ Limits to comparison due to multiple different formulas in use and poor reproducability

Use of a simple index• Based on single fasting sample

• Advantages:▫ Simple, cost-effective, practical▫ No requirement for glucose load

• Disadvantages:▫ Wide variability in reliability▫ Not all rigorously validated▫ Measure hepatic insulin resistance more than peripheral

• Considerations:▫ Formulas which include at least both insulin and glucose values are

preferable; additional parameters may help improve accuracy (ie NEFA)

▫ Use most up-to-date version of formulae or software▫ Consider using more than one simple index to minimize limitations

Other Considerations•Glycemic status:

▫In T2DM, HEIC clamp or insulin-modified FSIVGTT preferred

•Mathematical considerations:▫Consider the appropriateness of data for use

with a particular formula or model to avoid misinterpretation

•Insulin assay▫Within a study, should use high performance

assay from same source and lot number throughout study

In clinical practice•Use of advanced methods of measuring insulin

resistance or secretion not recommended

•Can use fasting glucose, HgAlc or OGGT with samples at fasting and two hour time points▫Pre-diabetes:

Fasting glucose: 5.6-6.9 mmol/L HgA1c: 6.0-6.49% 2hr OGTT 7.8-11.0 mmol/L

▫T2DM: Fasting glucose: ≥7.0 HgA1c: ≥6.5% 2hr OGTT/random glucose ≥ 11.1 mmol/L

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• 8. Wallace TM, Levy JC, Matthews DR. Use and abuse of HOMA modeling. Diabetes care. 2004;27(6):1487-95.