Lecture 2: Diabetes Pathogenesis + Insulin Resistance
Diabetes
• Diabetes: metabolic disorder characterised by resistance to actions of insulin, insufficient insulin, or both. The major clinical manifestation is hyperglycaemia
o Hyperglycaemia produces polyuria, polydipsia (increased thirst) and polyphagia
• Type 1: beta cell destruction causing absolute insulin deficiency – immune mediated and idiopathic
• Type 2: ranges from predominantly insulin resistance with relative insulin deficiency, to predominantly insulin secretory defect with insulin resistance (usually both and progressing slowly)
• Other types:
o Gestational diabetes – 5%, cured after parturition, but can have detrimental effects on mother and baby
o Genetic defects of beta cells or insulin action o Exocrine pancreas diseases
o Endocrinopathies – acromegaly
o Drug/chemical-induced such as from glucocorticoids o Infections – cytomegalovirus
o Uncommon forms of immune-mediated – anti IR antibodies
o Other genetic syndromes sometimes associated, e.g. Down’s Syndrome
Type 1 Diabetes: 5%
• 90% have positive markers of immune destruction of β cell: antibodies to islet cells (ICAs), to glutamic acid decarboxylase (GAD), and to insulin (IAAs)
• Usually in child, but also a small number of latent autoimmune diabetes in adults (LADA) o Triggering factor cause immune response and start the T1D pathophysiology which
can be detected. So can we prevent development rather than trying to transplant beta cells by detecting early markers?
Type 2 Diabetes: >90%
• Characterised by insulin resistance (hence often obese) and a relatively deficiency of insulin secretion
o Deficiency not absolute – relative because of resistance so technically not producing enough insulin to be effective
• Usually in central intra obesity with hypertension and dyslipidaemia (high TG, low HDL, high VLDL and cholesterol levels; postprandial hyperlipemia), known as insulin resistance syndrome or metabolic syndrome
o Diabetes closely associated with development of CVD (most diabetics die of CVD)
• Control of blood glucose – focussed on T2D
Pathophysiology
• Insulin resistance means that body cells don’t respond appropriately when insulin is present
• Insulin is a storage hormone – stores energy, but if too much energy (food) consumed, too much stored and you get obesity and T2D
• Other important contributing factors:
o Increased hepatic glucose production (e.g. from glycogen) especially at inappropriate times
o Decreased insulin-mediated glucose transport in muscles (primarily) and adipose tissues (receptor + post-receptor defects)
o Impaired β cell function – loss of early phase of insulin release in response to hyperglycaemic stimulation
▪ Secreting pattern is different between T2D and healthy
• T2D is much more genetic, but doesn’t involve antibodies and doesn’t recommend insulin as first line treatment (has beta cells)
• T1D isn’t as strongly genetically connected, does involve antibodies and does recommend insulin as a first line treatment (no beta cells)
Differences in Type 1 and 2 Diabetes
• T1D
o Mostly in childhood o Insulin deficiency o Auto immune disease o Β cell death
o Minimum organ involvement (no resistance)
o Treat with insulin + islet transplant
• T2D
o Mostly late adults
o Insulin resistance/deficiency o Metabolic disorders
o Β cell dysfunction
o Significant involvement of liver, muscle, fat tissues o Treat with lifestyle, drug,
insulin
Similarities in Type 1 and 2 Diabetes
• T1D
o Hyperglycaemia (mainly insulin low)
o 3 polys (uria, dipsia, phagia) o Increasing rapidly (early
obesity, chronic inflammation)
o Control hyperglycaemia (insulin replacement)
• T2D
o Hyperglycaemia (insulin resistance and low)
o 3 polys (uria, dipsia, phagia) o Increasing rapidly (obesity,
less active, chronic inflammation)
o Control hyperglycaemia (healthy lifestyle and drug, finally insulin
Insulin and Fat metabolism
• Insulin is the principal regulator of fat metabolism
o Processes FFA storage + synthesis into TGs + prevent breakdown o Also regulated glucose uptake into cell into either glycogen or TG o STORAGE HORMONE
Gestational Diabetes Mellitus (4-6%)
• GDM: glucose intolerance, first recognized during pregnancy. Also look at HbA1C which indicates long term glucose levels of pregnant women
• Clinic detection is important as treatment will reduce prenatal morbidity and mortality.
Usually it is also a sign of high risk group for T2D in even next generation (lack of tolerance)
• High weight baby if not treated
• Diagnosis level is 5.1, which is low, but that’s because mother and baby are using the glucose, so a lot is being used, and if >5.1, mother has glucose tolerance problem → if not treated can lead to macrosomia and baby can get T2D
Type 2 Diabetes + Underlying Causes
• Hyperinsulinemia is the major effect (1st) for causing insulin resistance, and can also cause β cell defect
• After β cell defect, you get decreased insulin secretion (2nd)
• Other factors include obesity and β cell dysfunction which can lead to impaired glucose tolerance, insulin resistance and early diabetes
• With impaired insulin action, get increased glucose produce in liver, decreased glucose utilisation in fat and muscle
• Impaired insulin action + β cell dysfunction/death both cause and get caused by one another
Site of Insulin Action
• Combined effects of insulin + hyperglycaemia to promote glucose disposal are dependent on following mechanisms:
o Suppression of endogenous (primarily hepatic) glucose production o Stimulation of glucose uptake by muscle, liver, adipose tissue
Resting Energy Expenditure in Different Tissues
• Heart uses most energy per kg as pumping constantly
• Sk muscle uses the most overall though because it makes up >50% total body mass
• You don’t want hypoglycaemia in the morning - brain constantly needs glucose
Insulin-stimulated glucose metabolism in different tissues
• Glucose uptake and conversion to glycogen mainly happens in red muscle o In muscle glycogen used for energy
o In liver it’s used for circulating glucose levels
• Glucose into lipid mainly in liver, but also a little bit in muscle where its used for metabolism
• Post meal lipogenesis high in liver and adipose tissue where excess glucose stored as TGs.
o Brown fat for energy, whereas white is not used (exercise browns white fat, allowing it to be used for energy)
Tissue Differences in Energy Metabolism
• Muscle: large capacity for substrate utilisation and energy expenditure because >50% BW
• Brain, heart, liver: constant substrate utilisation and energy expenditure
• Brown adipose tissue: large capacity for energy expenditure, but low % BW
• White adipose tissue: large capacity for substrate storage and endocrine function – storage organ, not used
o Exercise increases fat browning and thus increases energy usage
Hepatic Glucose Production
• In the overnight fasted state, the liver produces glucose at the rate of 1.8-2mg/kg/min
• Brain utilises glucose at constant rate of 1-1.2mg/kg/min, accounts for 50-60% of glucose disposal during post-absorptive state and is insulin independent.
• Insulin suppresses hepatic glucose output
• If liver doesn’t perceive insulin signal (thus continues producing glucose), there’s two inputs of glucose into body, one from liver and one from GIT → hyperglycaemia will ensue
• Hypoglycaemia can cause brain damage, so brain causes liver to keep producing glucose, but then it needs to stop or hyperglycaemia
Effect on Liver Cells
• First, insulin promotes glycogenesis from glucose, and inhibits glycogenolysis to glucose
• Second, insulin promotes glycolysis, CHO oxidation and glucose metabolism, while also promoting pyruvate and inhibiting gluconeogenesis
• Third, insulin promotes synthesis/storage of fats as well as synthesis of some apoproteins packaged with VLDL. Insulin also indirectly inhibits fat oxidation, and helps shunt FAs to esterification as TGs and storage as VLDL or lipid droplets – can cause fatty liver disease
• Fourth, insulin promotes protein synthesis, and inhibits protein breakdown (mechanism unknown)
• Insulin decreases hepatic glucose and ketone body production by actions on muscle + adipose tissue that limit precursor availability
• Increase in hepatic glucose output
o Decreased insulin causes decrease inhibitory effect on glucagon secretion, and thus increase glucagon secretion from α cells, which acts on liver to increase gluconeogenesis and glycogenolysis, ultimately elevating plasma glucose levels
Hepatic Glucose Production
• Increase in 0.5mg/kg/min for overnight sleeping hours, the liver of 80kg diabetic with modest fasting hyperglycaemia adds an additional 25g of glucose
• Increase in basal HGP closely correlated with severity of fasting hyperglycaemia
• Thus, in T2D with overt fasting hyperglycaemia (>140mg/dL, 7.8mmol/L), an excessive rate of hepatic glucose output is the major abnormality responsible for the elevated fasting plasma glucose concentration
Insulin action on adipose tissues
• Adipose tissue accounts for 10% of glucose uptake – but still regulated by insulin (major sites liver + skm)
o Accelerating transmembrane glucose transport (via GLUT) o Activating enzymes that direct flow of glucose carbons into FAs
o Promoting lipoprotein lipase synthesis (enzyme produced by adipose tissue and transported to vessels to clear lipids (FFA) in circulation)
• In humans, adipose tissue is NOT predominant site of FA synthesis
• Insulin stimulates TG production from excess glucose and stores TGs in fat cells
• Glucose enters via GLUT4 transporter, which is stimulated from inside the cell to the plasma membrane in response to elevated insulin levels – insulin dependent
• Liver uses GLUT2 which is intrinsically present (glucose sensor)
• Brain uses GLUT1, which is glucose dependent – more GLUT1 when more glucose to get more in the brain)
• Kidney and gut use SGLT2 to absorb glucose into blood → target for diabetic therapies but GIT side effects
Effect of insulin on adipocytes
• First, insulin promotes glucose uptake by recruiting GLUT4 to plasma membrane
• Second, insulin promotes glycolysis, leading to formation of α-glycerol phosphate. Insulin also promotes conversion of pyruvate to FAs
• Third, insulin promotes esterification of α-glycerol phosphate with FAs to form TGs (fat droplets) o Conversely, insulin inhibits hormone-sensitive TG lipase, which would otherwise break
TGs down into glycerol and FAs – insulin promotes fat synthesis and inhibits fat breakdown
• Fourth, insulin promotes LPL synthesis in adipocyte. LPL moves to endothelial cell and breaks down TGs, yielding FAs to adipocyte for esterification and storage in fat droplets as TGs
o LPL is the key hormone for clearing circulating fat
Effects of insulin on muscle
• Muscle is major site of glucose disposal in humans
• Under high glucose and hyperinsulinemia conditions, 80% of total glucose uptake occurs in sk muscle (normal is only about 50%)
• Muscle comprises 50% of body mass, and uptake accounts for majority of glucose in circulation
• Insulin promotes glucose entering glycolysis or glycogenesis, providing energy immediately, or storing it for later energy usage
o Liver on the other hand stores glycogen as a reserve for circulating glucose levels
• Insulin also stimulates AAs into cell to increase protein synthesis → thus, in diabetes, you don’t get muscle as protein synthesis is inhibited
• Also FFA synthesis
Effect of insulin in muscle
• First, insulin promotes glucose uptake by recruiting GLUT4 to plasma membrane
• Second, insulin promotes glycogenesis from glucose
• Third, insulin promotes glycolysis + CHO oxidation. These actions are similar to those in liver, with little or no gluconeogenesis occurring in muscle
• Fourth, insulin promotes protein synthesis and inhibits protein breakdown
Intracellular Signalling employed by insulin and relation to T2D
• Insulin receptor is a tyrosine kinase receptor, that must dimerise and phosphorylate TK side chains and an insulin receptor substrate protein (IRS) to have downstream effects such as metabolic effects, cell proliferative effects and protein synthesis.
• After insulin binds, and dimerization and activation occur, IRS protein activated, and there’s two downstream pathways: PI3K pathway contributes to glucose metabolism and insulin resistance; the MAPK pathway is more involved in cell proliferation and inflammation (also inhibits PI3K pathway), and it’s still intact in insulin resistance
Insulin Signal Transduction Defects in T2D
• Insulin receptor number and affinity
• Insulin receptor tyrosine kinase activity
• Insulin signalling (IRS-1 and PI-3 kinase) defects
Insulin Receptor Number and Affinity
• 20-30% reduction in insulin binding to monocytes and adipocytes from T2D patients, but that’s not too bad as only need about 5% bound
• However, no difference of insulin receptor expression levels found between obesity, diabetes and normal populations in muscle and liver, the major tissues responsible for regulation of glucose homeostasis – surface receptor density? Perhaps the receptors are all internal and not on the surface → insulin receptor, when activated, is internalised and either recycled in the endosome or degraded in the lysosome, so could be a density issue
• A structural gene abnormality in insulin receptor is excluded from a cause of T2D
Possible Mechanism underlying insulin resistance (insulin receptor on cell membrane)
• Surface receptor density? Perhaps the receptors are all internal and not on the surface → insulin receptor, when activated, is internalised and either recycled in the endosome or degraded in the lysosome, so could be a density issue
Insulin receptor TK activity
• Insulin receptor phosphorylation of β-subunit, with subsequent activation of insulin receptor TK, represents first step in insulin actions on glucose metabolism
• Mutagenesis experiments have shown that insulin receptors devoid of TK activity are completely ineffective in mediating insulin stimulation of cellular metabolism
• Impaired insulin receptor TK activity in T2D is acquired secondarily to hyperglycaemia or some other metabolic disturbance, such as hyperlipidaemia
o Insulin resistance can be due to decreased TK activity, which can be after
hyperglycaemia -→ thus, decreased TK activity secondary, not causing hyperglycaemia
Insulin Signalling (IRS-1,2 and PI-3 kinase) Defects
• Insulin failed to increase the association of the p85 subunit of PI3-Kinase with IRS-1 and IRS-2 in muscle, indicating the T2D is characterised by a combined defect in IRS-1 and IRS-2 function.
• In animal models, 80% decrease in insulin-induced IRS-1 phosphorylation and a greater than 90%
reduction in insulin-stimulated PI3-kinase activity
• Thus, IRS plays a role in insulin resistance
IRS Ser/Thr phosphorylation + Insulin Resistance
• Upon insulin binding, you get activation of IRS, which then activates the PI3K pathway and activates PKB and PKC
• mTOR is disinhibited and activates S61K, which causes insulin action on glucose metabolism
• IRS kinases phosphorylate other sites on the IRS (Ser or Thr, rather than Tyr), which can inactivate the IRS, and prevent insulin action
MAPK pathways in insulin receptor
• Profound insulin resistance of PI3-Kinase signalling pathway contrasts markedly with insulin’s ability to stimulate MAP kinase pathway activity in insulin-resistance T2D and obese non- diabetics
• Maintenance of MAPK pathway may be important in development of insulin resistance o MAPK pathway still intact despite insulin resistance → in early diabetes, you get
increased insulin, and thus increased MAPK activity, to increase proliferation and inflammation in diabetes, resulting in a change to the organ. It also inhibits the IRS-PI3K pathway, conferring more insulin resistance
• MAPK can phosphorylate IRS-1 on Ser residues – which has been implicated in desensitising insulin receptor signalling
• Continued MAPK activity, when IRS-1 function already impaired, could worsen insulin resistance
Pathological Outcomes
• Insulin resistance in metabolic pathway (PI3-K), with its compensatory increase in β-cell function and hyperinsulinemia, leads to excessive stimulation of MAPK pathway in vascular tissue
• This results in proliferation of vascular smooth muscle cells, increased collagen formation, increased production of growth factors + inflammatory cytokines, potentially explaining accelerated rate of atherosclerosis in T2D
T2D – not a mild disease
• There are numerous irreversible secondary pathologies caused by T2D, such as diabetic retinopathy, nephropathy, neuropathy, and CVD and stroke
Summary
• Insulin resistance in peripheral tissues, especially liver and muscle, is an important factor contributing to development of T2D
• Cellular signalling is a reduced insulin receptor TK activity, reduced IRS and PI3K signalling, and increased MAPK pathways (inhibits PI3K pathway) → mechanism for insulin resistance
• Exercise and weight control are very effective in correcting insulin resistance
• We want to recover insulin sensitivity, so often in early T2D, we inject insulin to increase insulin levels, so you don’t get increased insulin secretion, and don’t have pancreas causing
hyperinsulinemia, thus preventing diabetes
Pancreatic Islets
• In human: β cells 60%, α cells 30%, somatostatin cells 5%; polypeptide cells and ghrelin cells
• In rodents: β cells 75%; α cells 15%; other cells: 10%
• Insulin inhibits glucagon secretion, but glucagon activates insulin secretion. Somatostatin inhibits both
• In humans there’s not a clear distribution of islets, but all can affect one another
• Following a meal, you see spikes in blood glucose and blood insulin, but no real change in glucagon
• This could be due to glucagon’s short half life (t1/2=5 min)
• There are two phases to insulin secretion: an early transient spike, followed by a sustained plateau (body naturally dealing with hyperglycaemia, not regulated by the glucose ingestion like first phase)
• Phase 1: glucose stimulated/dependent insulin release
o Glucose enters beta cell via intrinsically present GLUT2 transporter
o The glucose is oxidised to ATP, increasing ATP:ADP ratio, causing the ATP dependent K+
channel to close, causing a build up of positive K+ inside along the membrane
o Membrane eventually depolarises, causing opening of voltage gated Ca channels, influx of Ca which causes vesicles of insulin to be exocytosed into circulation
o If K-ATP channel open, no release, but if you stimulate cAMP release, then you do get insulin release. Therefore, insulin has ATP sensitive and cAMP pathways that don’t require ATP (two pathways to get released)
• Insulin usually released as preproinsulin, cleaved to proinsulin, and then insulin with C peptide o If you want to measure insulin levels, measure C peptide → insulin binding receptors
then get internalised so they won’t get picked up in a blood test unlike C peptide
Insulin levels after oral glucose vs IV glucose
• Insulin levels are much higher after oral glucose than IV
o Oral glucose is absorbed in the gut and delivered straight into the portal system and influences other hormones
o IV glucose circulates before reaching the pancreas, so has a smaller response
• Diabetic patients don’t have any real spike in insulin secretion despite rising glucose levels
→ insulin resistance
Insulin after IV glucose – two phases
• First phase immediately after glucose load is ATP dependent phase → glucose dependent
• Second phase – insulin in normal condition trying to restore levels
• In controls, maximum insulin response is reached when only 5% of insulin receptors are occupied
• In T2D, same maximal response can be reached, but requires a much higher insulin concentration – hence why you get hyperinsulinemia
o This is due to downregulation of receptors and impairment of post-receptor signalling in T2D
Beta Cell Dysfunction
• Early stage
o Β cell response to glucose becomes slow and missing first phase
o Β cell number increased to compensate insulin needs due to insulin resistance
• Late stage
o B cell response to glucose becomes much slower and both phases of response are affected;
o B cell number decreased significantly (apoptosis and necrosis) and insulin deficiency occurs → need insulin injection to correct glucose levels
o Structure change in islet with β cells proportion reduced and α cell proportion increased
Glucagon
• Glucagon released stimulated by stress hormones and it results in elevated glucose levels, by acting on liver to increase gluconeogenesis and glycogenolysis
• It activates insulin, and is inhibited by insulin, so as glucagon activates insulin secretion, its own concentration lowers due to inhibition from insulin
• SYNTHESIS: has the same pathway as GLP-1 (intestinal L cells rather than pancreatic α) so GLP-1 can have an add on effect to stimulate insulin release
Glucagon in plasma
• Half life is 5 min in circulation – goes straight from pancreas to liver
• If α cells are slow to respond to low glucose, you get hypoglycaemia
• In peripheral blood, it’s much lower than in portal venous blood; not only a dilution, but also 25% destroyed during passage through liver
• Kidney is other important site of degradation
Mechanism of Glucagon Action
• Glucagon degrades glycogen to glucose in hepatocytes to increase circulating glucose levels. It also stimulates gluconeogenesis
• Glucagon uses a GPCR, with AC, cAMP and PKA to release glucose into circulation
Effect of Glucagon in liver
• First, glucagon inhibits glycogenesis and promotes glycogenolysis
• Second, glucagon promotes net gluconeogenesis – the hormone inhibits glycolysis and carbohydrate oxidation
• Third, glucagon promotes fat oxidation. The hormone inhibits the activity of acetyl coA carboxylase. Glucagon indirectly stimulates fat oxidation because the decreased levels of malonyl CoA relieve the inhibition of malonyl CoA on catalase or CAT
Regulation of Glucagon Secretion
• Still unknown how increased glucose causes decrease in glucagon secretion, but it’s clear glucagon has an anti-hypoglycaemic effect, i.e. as blood glucose levels drop, glucagon levels rise
Summary
• At the present, manifestations of diabetes cannot be explained solely by insulin deficiency, and abnormal α cell function is an important determinant of the magnitude of hyperglycaemia and hyperketonemia found in diabetes
• Fasting hyperglycaemia and insulin requirements are lower in pancreatectomised patients.
o In such individuals and in insulin-dependent diabetics whose glucagon secretion is suppressed with somatostatin, hyperglycaemia and hyperketonemia following acute withdrawal of insulin are markedly diminished
• Failure to suppress glucagon secretion appropriately after meal ingestion increases hyperglycaemia in people with impaired glucose tolerance and diabetes
• In insulin-dependent diabetics, acute suppression of glucagon secretion decreases plasma glucose to concentrations only slightly above normal, and chronic suppression markedly improves diabetic control
• Finally, failure of hypoglycaemia to stimulate glucagon secretion in people with T1D and T2D and marked β cell dysfunction, increases the risk for severe hypoglycaemia in these people
• Thus, abnormalities in α cell function play an important role in pathophysiology of metabolic abnormalities in diabetes mellitus and also its management
o In sleep, glucose is only coming from the liver (not ingesting any), which is in response to glucagon detecting low blood glucose levels
• SO FAR PATHOLOGICAL CHANGES IN ISLETS o Dysfunction of beta and alpha cells o Delayed insulin response to glucose
o Every step of glucose transportation and metabolism, and key molecules involved in insulin secretion has been linked to beta cell dysfunction
o Dysfunction of alpha cell is not clear
Lecture 4: Hyperinsulinemia
Fact of Diabetes
• Every five minutes, one person is diagnosed in Australia – 280 more people every day
• 1.2 million Australians with diabetes
• Around half a million more Australians are estimated to have undiagnosed diabetes
Cause and Consequence of T2D
• Secondary pathologies like cardiomyopathy, retinopathy and fatty liver disease are the main problem of T2D
• Hyperinsulinemia occurs early in diabetes. You get insulin resistance from constant hyperglycaemia, which causes increased insulin secretion (hyperinsulinemia). This also causes β cell dysfunction due to the high insulin concentrations
Biphasic insulin secretion pattern in healthy and T2D
• Currently, glucose levels are used for evaluation of diabetes as insulin is not easy to measure
• 1st phase is glucose dependent (K channel, ATP etc.), absent in early T2D
• 2nd phase is thus much greater in early T2D than in healthy
• Late T2D has no real change in insulin release after a meal
Graphene Field Effect Transistor Aptameric nanosensor
• Kinetics of nano-graphene simple insulin meter is similar to glucose meter
• Uses a physical character to measure hormone levels
Diabetes and Obesity
• Insulin resistance and hyperinsulinemia (compensation for hyperglycaemia in obesity) progressing to later insulin deficiency
• Current treatment:
o Stimulation of insulin secretion o Facilitate insulin sensitivity o Decrease glucose uptake
• Problem:
o Already hyperinsulinemia
o Facilitate fat accumulation (insulin is a storage hormone)
Growth Hormone
• GH: major metabolic regulator – actions opposite to insulin (uses energy, rather than store)
• GH promotes the deposition of lean muscle mass at the expense of adipose tissue – i.e. more muscle and less fat
GH secretion in obesity
• GH release is pulsatile
• The daily production rate of GH in normal weighted people is much higher than in obese
• Obesity associated with reduced total GH secretion and reduced peak GH secretion
• Increased BMI (increased fat) correlated with decreased GH
Insulin and GH on lipid
• GH balances insulin action, critical biological role in adults o GH uses FFAs to provide energy (insulin stores)
• Hyperglycaemia causes hyperinsulinemia and a decrease in GH release, to maximise insulin action – maintaining glucose + FFAs
o With insulin resistance, we want to decrease GH to increase insulin action, but we don’t know how that happens
Reduced GH in overweight and obesity
• Under obesity with insulin resistance, reduced GH may enhance insulin sensitivity
• GH stimulating metabolism by using glucose and fat and synthesis of proteins (muscle) – anti- obesity
• Reduced GH causes further obesity
GH + Regulation
• Likely happening at the hypothalamus or anterior pituitary
• GH acts on fat, muscle, bone and liver metabolic processes
Pulsatile GH secretion in obesity – Diabetes
• As in humans, dietary induced weight gain is associated with a reduction in pulsatile GH secretion – when measuring, make sure to take multiple measurements due to pulsatility. Half life important to consider too
• Increased adiposity and insulin correlates with the progressive suppression of pulsatile GH secretion during weight gain
Actions of GH + Insulin
• Reduced GH may enhance insulin action and obesity
• Insulin increases fat storage and decreases GH levels
Regulation of energy balance
• Neurons in hypothalamus with MC4R: POMC and NPY/AgRP o POMC: inhibits food intake, stimulates MC4R
o NPY/AgRP: stimulate food intake, inhibits MC4R
• Hormones from pancreas, stomach, adipose tissue influence these feeding centres
GH secretion in obesity: MC4R + GH secretion
• Maintenance of GH pulses compared with other types of obesity with same BMI
• Is pubertal growth (regardless of pubertal onset) in MC4R+/- individuals a consequence of excess GH secretion
• Is MC4R regulating pulsatile GH secretion?
o If POMC MC4R not responding, get increased food intake → obesity
MC4RKO mice are GH deficient during sustained rapid linear growth
• MC4R KO mice have decreased GH, associated with an increased body weight and body length.
These are due to hyperinsulinemia due to the hyperglycaemia from increased food intake (POMC can no longer inhibit food intake as can’t stimulate MC4R)
GH secretion in obesity: insulin and sensitivity
• Mice with MC4R KO have increased food intake, increased insulin secretion, probably causing the decreased GH (rather than MC4R KO directly)
• Increased insulin causes increased fat accumulation, but the mouse still responds to insulin so glucose and FFA levels are normal, but decreased GH
o Sustaining growth, regardless of obesity associated GH deficiency because glucose levels are normal still, and get increased fat storage
Obese and non-obese glucose and insulin to meals in humans
• Obese humans showed normal glucose, but massive differences in insulin – hyperinsulinemia in obese
o Hyperinsulinemia can cause insulin resistance, proliferation in early diabetes and can cause β cell apoptosis
MC4RKO mice developed hyperinsulinemia + insulin resistance
• Early on, insulin sensitivity is okay despite hyperinsulinemia, so glucose regulation early is okay (still respond to increase glucose). Progressively get insulin resistance with increasing
hyperinsulinemia
• Hyperinsulinemia may cause this reduction of GH to keep a good sensitivity of tissue to insulin (physiological response to a short term but pathophysiological in a long term)
• Hyperinsulinemia will promote lipid storage in adipose, liver + muscle
• It will reduce metabolism (activities) in muscle due to low GH
• Hyperinsulinemia (and obesity) may eventually cause insulin resistance (mechanism?)
IRS Ser/Thr phosphorylation and insulin resistance, as well as internalisation causing resistance
• Hyperinsulinemia causes insulin resistance (via Ser/Thr phosphorylation) and insulin receptor has a recycling problem – normally receptors recycled back to plasma membrane after being internalised upon insulin binding, but in hyperinsulinemia, you get insulin receptor degradation instead.
Hyperphagia in MC4R KO mice
• Hyperinsulinemia is a consequence of hyperphagia?
o Mice don’t respond to inhibitory hormone due to MC4R KO on POMC neurons, causing hyperphagia, which causes hyperglycaemia and thus hyperinsulinemia
Pair-feeding on insulin levels
• Reversal of hyperinsulinemia achieved through pair-feeding (restricting food intake to that seen in WT mice)
• Night-time metabolic activity still lower so slight increase in BW still
• Reduced GH back to normal when no hyperinsulinemia so hyperinsulinemia is causing decreased GH levels
Regulation of insulin secretion in response to glucose
• Diazoxide: Stop hyperinsulinemia by opening up K+ channel (ATP can’t close it)
• So insulin secretion doesn’t increase + GH recovery increases
o With no hyperinsulinemia, get increased GH and less fat as less insulin o Keeps metabolic balance
Effect of SGLT2 inhibitor on circulating and urine glucose levels
• SGLT2 inhibitor (dapagliflozin) causing significant increase in glucose excretion from kidney, decrease in hyperglycaemia and reduction in hyperinsulinemia
• These changes improve insulin sensitivity and recover GH and metabolic balance
• MC4RKO have β cell proliferation. With drug, get decreased glucose, decreased need for insulin so proliferation decreases, improves insulin sensitivity, and get increased GH (With reduced hyperinsulinemia)
Insulin/GH
• Normal insulin (energy saving) and GH (energy spending) balance keep metabolic balance
• Hyperphagia or obesity causes hyperinsulinemia to compensate insulin resistance to keep normal glucose/FFAs but causing obesity and diabetes
• Hyperinsulinemia causes reduction of GH to facilitate insulin action on energy storage → obesity
• Reduced insulin levels may recover GH profile to maintain normal metabolic balance
• Constant hyperinsulinemia is a pathophysiological factor contributing to metabolic unbalance, insulin resistance and diabetes
• The best way is to enhance glucose-stimulated insulin secretion to reduce long-term hyperinsulinemia
Proposed mechanisms of KCNH6 and K
ATPin regulating insulin secretion
• Block the voltage gated K channel that restores RMP after depolarisation
• No effect at low glucose levels as not being depolarised, and KATP channel is open
• Only get an effect at high glucose levels as cell will depolarise and then need to be brought back to RMP
• Insulin secretion not increased at normal levels, only released in hyperglycaemia and get sustained insulin release because cell remains in depolarised state (can’t get back to RMP)
Blockade of KCNH6 or K
ATPon [Ca
2+]i and insulin secretion
• A selective islet specific KCNH6 blocker would be developed to improve first phase GSIS (only stimulate insulin release when you have high glucose levels) to prevent hyperinsulinemia and T2D. Only works on this specific K channel
Smart nano-particles containing insulin secreted only to high glucose stimulation
• A novel insulin-releasing nano-system with adjustable glucose responsive threshold and multiple ON-OFF insulin release may help to rebuild coupling of glucose to insulin
• pH sensitive (high glucose – decreased pH and therefore insulin secretion), but hypoglycaemia can be a problem
Summary
• Hyperinsulinemia contributes to diabetes progress and better assay of insulin is needed
• Reduce hyperinsulinemia – recovery of GH – re-establish health balance of insulin-GH – maintain normal body weight and composition – delay the progress of diabetes
• Best way is to stimulate insulin secretion only when glucose is high – Kv blocker and nano-insulin system, or to reduce hyperglycaemia to rebuild coupling of glucose to insulin secretion
Lecture 5: Peripheral View of Obesity – adipose tissue derived adipokines + FFAs
Defining obesity
• Typically done by BMI
Obesity associated diseases
• Chronic diseases associated include T2D, hypertension, dyslipidaemia, insulin resistance, CVD and cancer
• Obesity not always associated with metabolic diseases
Adipose Tissue Expansion
• Beneficial adipokines:
o FGF21: stimulating hepatic gluconeogenesis
o Leptin: suppressing food intake, promoting lipolysis
o Adiponectin: insulin sensitizing, suppressing hepatic glucose output, anti-inflammatory and anti-fibrotic
• Metabolically healthy obesity: increase in adipocytes via hyperplasia, which is healthy and adaptive, whereby tissue maintains vascularity/circulation and adipokine levels
• Metabolically unhealthy obesity: increase in adipocytes via hypertrophy, which leads to hypoxia (poor vascularisation) → upregulation of pro-fibrosis and necrosis of adipocytes, get immune infiltration → impair adipose tissue function, causing increased levels of glucose and lipids in circulation, as well as deposition of fat in liver and muscle (metabolic issues)
Different types of adipose tissue
• White adipose tissue (WAT): white adipocytes, large, unilocular lipid droplets; small mitochondria; no UCP1 (no energy/heat generation); energy storage (neutral lipids)
• Brown adipose tissue (BAT): brown adipocytes, small, multilocular lipid droplets; large
mitochondria (well developed); express UCP1 so therefore → thermogenesis (heat production)
• A fat organ may display a dynamic spectrum of colour
o Colour change is possible due to temperature (cool temperature favours brown fat for heat production – changes fat type – reversible)
Putative batokines and target organs
• BAT adipokines (batokines) have effects on bone, WAT, brain, liver, pancreas and heart
• Protect against obesity and insulin resistance
WAT secreted adipokines
• Normal adipose tissue is stable
• Enlarged adipose tissue has decreased anti-inflammatory cytokines, increased FFAs, increased pro-inflammatory cytokines which then contributes to metabolic issues like inflammation, insulin resistance and dyslipidaemia.
Leptin, the heavy hormone
• Leptin (Greek for thin) is a non-glycosylated 16kDa protein encoded by obese (ob) gene o Increased adipocytes, increased leptin, decreased food intake and increased energy
expenditure
• Ob/ob mice: autosomal recessive mutation and cannot produce leptin – marked obesity + hyperphagia with mild transient diabetes
• Db/db mice: autosomal recessive defects in leptin receptor gene, but can still produce leptin, though leptin now non-functional due to faulty receptor – marked obesity and hyperphagia, with severe diabetes
Parabiosis Experiments
• Db/Db over produces leptin, which the WT and Ob/ob mice respond to, hence why they don’t eat and starve to death
• But ob/ob can respond, and WT can produce, so both are okay
• Therefore, same satiety factor working (leptin)
Leptin, the satiety hormone
• See a greater mass decrease with higher leptin concentrations, both peripherally and centrally – therefore leptin (adipose derived hormone) is transversing BBB and having an effect centrally to decrease food intake
• Enters brain by saturable system independent of insulin
Leptin, the central regulator
• Leptin produced by adipose tissue, binds to leptin receptor on neuron in the arcuate nucleus of hypothalamus
• STAT dimer moves to nucleus and upregulates transcription of POMC
• POMC is activated, and POMC-derived peptide activates MC4R receptors on neurons in PVN to reduce food intake
Leptin treatment in humans
• Lipodystrophy (loss of adipose tissue) occurs in HIV-infected individual treated with highly active antiretroviral therapy (HAART)
• Leptin therapy reverses pathological changes in fat distribution → increase in limb fat (which was reduced) and decrease in truncal fat (which was increased)
Leptin Resistance – a major hurdle
• Normally negative feedback from SOCS3 gene to regulate leptin resistance
• In obesity, get chronic overnutrition-mediated lipotoxicity which causes ER stress, which impairs negative feedback via inflammation, causing upregulation of leptin effects, eventually leading to resistance
Key Points
• Leptin is an adipokine, secreted in proportion to body fat mass
• Adipose tissue derived leptin is able to cross BBB to target on hypothalamic neurons that regulate food intake
• Leptin is a thin hormone, able to suppress food intake
• Leptin therapy is effective in congenital leptin deficient and HIV-associated lipodystrophy patients
• Leptin resistance is a major hurdle for ordinary obesity treatment
• Cause of leptin resistance remains largely unknown but may be associated with FFA induced lipotoxicity
WAT secreted adipokines
• Increased adiposity also causes increased FFA levels (as well as adipokines, IL1/6 and leptin) which causes metabolic issues
Free fatty acids (FFAs)
• Carboxylic acid linked to an aliphatic chain of variable length o Short C2-C6 – fibre fermentation by intestinal microbiome o Medium C7-C12
o Large >C12
• Dietary sources:
• Palmitic acid – 20-30% of FFA in circulation and high levels leads to metabolic issues
Metabolic Fate of FFA
• Levels increased from dietary intake, lipolysis and de novo hepatic lipogenesis (convert CHO to lipids)
• Then once levels are increased, it can be oxidised to ATP, used to synthesise phospholipids and steroids. Can also be stored as TGs through lipogenesis
Important FFA derived metabolites
• PIP2 is a lipid in cell membrane, which when cleaved by PLC turns into DAG + IP3
• Ceramide – derived from sphingosine alcohol and LCFA o Sometimes from sphingomyelinase pathway o Mainly from de novo from palmitic acid o Synthesis occurs in ER
o Ceramide is an important second messenger
Insulin PI3K-AKT(PKB) signalling pathway
• Insulin binds receptor, cross phosphorylation of TK occurs, IRS activated + phosphorylated – uses in lipid synthesis, glycogen synthesis, protein synthesis and glucose uptake
• IRS (adaptor protein) causes PI3K to bind and phosphorylate PIP2, activating it
PLC-IP3 and PI3K-AKT(PKB) pathway
• In GPCR, PIP2 cleaved by PLC, into DAG and IP3
• In Tyr-K receptor, PI3K converts PIP2 to PIP3 (via IRS). PIP3 acts on PDK1. PDK1 phosphorylates/activates AKT/PKB
• Insulin-activated PKB/AKT causes GLUT4 translation to the membrane and glycogenesis.
Palmitic Acid induces insulin resistance (IR)
• FFA binds receptor (GPCR), activates PLC, hydrolyses PIP2 to DAG and IP3.
• IP3 binds Ca channel on ER, causing Ca release into cytosol, which activates PKC, and also is associated with increased ceramide
• Active PKC phosphorylates IRS-Ser, desensitising insulin action, causing insulin resistance
• If too much FFA, can also enter through FAT/CD36 transporter, which leads to increased ER stress, and increased ROS causing mitochondrial and ER dysfunction
Not all fats are bad
• Butyrate (SCFA) reduces appetite and activated BAT via gut-brain neural circuit o Decreased appetite
• An increased dietary supply of medium chain FAs during early weaning in rodents prevents excessive fat accumulation in adulthood
o Decreased weight gain
• Oleic acid elicits beneficial effects on insulin sensitivity, and the dietary palmitic acid:oleic acid ratio impacts diabetes risk in humans
SCFA on islet
• FFA2 and FFA3 related to β cell
o FFA2: positive insulin secretion o FFA3: negative insulin secretion
• Thus increased F2, causes increased transcription of β cells (β cell compensation)
• F2KO thus cannot cope with high fat or high sugar or pregnancy because it can’t positively stimulate beta cell function or proliferation – can’t compensate
Oleic Acid prevents palmitic acid induced insulin resistance
• Oleic acid causes decreased FFA influx and modulates the genes responsible for inflammatory factors, and modulates insulin sensitivity.
Key Points
• FFAs have different types and functions
• The mechanisms by which FFAs cause insulin resistance, although not completely known, include generation of lipid metabolites (DAG/ceramide) to impair insulin signalling and inducing cellular stress, leading to mitochondria and endoplasmic reticulum dysfunction
• SCFA, MCFA and polyunsaturated LCFA may be a potential target for treating obesity
