Insulin Action and Its Disturbances in Disease - part 4 ppsx

However, glucose-6-phosphatase [G6Pase] and glu-cokinase [GK] are believed to play prominent roles in the regulation of glucoseproduction by controlling the rate of glucose efflux and upt

different approach Lewis et al.90 also found evidence of resistance to the directsuppressive effect of insulin on hepatic glucose production in T2D In addi-tion, we found that suppression of both plasma FFA and glucagon levels weremarkedly impaired in T2D (Figure 6.3).41 This may reflect impaired insulin-mediated suppression of lipolysis in adipocytes and impaired suppression ofglucagon secretion from the α-cells Since elevated FFA levels per se havebeen shown to stimulate both glycogenolysis as well as gluconeogenesis,91, 92impaired insulin-mediated suppression of FFA may obviously influence hepaticinsulin sensitivity Similarly, because hepatic glucagon sensitivity is normal inT2D,93, 94impaired insulin-mediated suppression of glucagon secretion may alsoinfluence hepatic insulin sensitivity.95 Using the tracer technique in combina-tion with the2H2O technique, Gastaldelli et al have quantitated gluconeogenesis

in obesity and in T2D In obese subjects, the gluconeogenic rate was directlyrelated to the degree of obesity,96 and in clamp studies of type 2 diabetic sub-jects gluconeogenic fluxes were elevated in the basal state and suppression inresponse to insulin was markedly impaired during the clamp.97

Thus, from in vivo studies, there is evidence of hepatic insulin resistance both

in the direct and in the indirect actions (through FFA and glucagon), and both

in the glycogenolytic and in the gluconeogenic pathways

Biochemical defects in hepatic insulin action

Control of hepatic glucose output may occur through regulation of genesis or glycogenolysis However, glucose-6-phosphatase [G6Pase] and glu-cokinase [GK] are believed to play prominent roles in the regulation of glucoseproduction by controlling the rate of glucose efflux and uptake in hepatocytes.The competing activity between the two enzymes has been described as theglucose cycle and represents an important potential site of regulation.98 Glucosecycling has been found to be increased in mild T2D.98 Insulin sensitivity of theglucose cycle is reduced in obese non-diabetic and more so in obese type 2 dia-betic patients,99 suggesting that G6Pase activity is increased in both groups.99This increased activity may be secondary to a decreased insulin-induced sup-pression of the enzyme activity at the level of the liver cell Alternatively, itmay possibly be secondary to the increased peripheral lipolysis and enhancedplasma FFA concentrations, since chronically elevated plasma FFAs have beenshown to enhance liver G6Pase gene expression.100 Moreover, in liver biopsiesfrom type 2 diabetic patients, G6Pase activity has been found to be increased101and GK activity to be reduced.101, 102

gluconeo-Increased hepatic VLDL production

Another important aspect of hepatic insulin resistance is an atherogenic lipidaemia profile characterized by hypertriglyceridaemia, low plasma HDL-cholesterol and raised small dense LDL-cholesterol profile The physiologic

dys-CONCLUSION AND PERSPECTIVES 171

basis for this metabolic dyslipidaemia appears to be hepatic overproduction ofapoB-containing VLDL particles, which may result from a composite set of fac-tors including increased flux of FFAs from adipose tissue to the liver and directlyfrom lipoprotein remnant uptake, increased de novo fatty acid synthesis, pref-erential esterification versus oxidation of fatty acids, reduced post-translationaldegradation of apo-B and overexpression of microsomal triglyceride transferprotein (MTP).103, 104 These conditions, together with resistance to the normalsuppressive effect of insulin on VLDL secretion, act in concert to channel fattyacids into secretory and storage rather than degradative pathways.105, 106

Primary/genetic defects in insulin action in liver

Whether hepatic insulin resistance is a primary trait or a secondary phenomenon

is as yet undetermined However, if hepatic insulin resistance is a secondary nomenon it may be reversible Given the serious consequences of hepatic insulinresistance, both for glucose metabolism and, in particular, for development ofdyslipidaemia, the answer to this question and possible rational treatments might

phe-be quite important

6.4 Conclusion and perspectives

Insulin resistance in glucose disposal and production seems to play an importantrole for the development of the metabolic syndrome and T2D Both diseases dis-pose to cardiovascular disease and cardiovascular mortality Therefore, insulinresistance may be considered as a serious risk factor in the modern society,and because insulin resistance is in itself symptomless it has been named ‘thesecret killer’

In this short description of insulin resistance, and glucose disposal and atic glucose production, we have focused on various aspects of methodologies

hep-to measure insulin resistance, in order hep-to alert researchers and clinicians hep-to theimportance of accurate diagnosis of insulin resistance We have also focused

on the potential cellular mechanisms that could explain the development ofinsulin resistance In skeletal muscle, insulin-mediated glucose disposal is clearlydependent on glycogen synthesis This pathway is impaired, due to hyperphos-phorylation of the key enzyme, glycogen synthase Therefore, regulation ofglycogen synthase activity may be central to our understanding of insulin resis-tance in the metabolic syndrome and T2D We believe that obesity is linked toinsulin resistance, metabolic syndrome and T2D, through the accumulation oflipids, particularly long chain acylCoAs in the skeletal muscle, and that theseintracellular fatty acids and triglycerides may directly inhibit the dephosphory-lation of glycogen synthase and thereby impair glucose disposal

Thus, future studies will need to examine the relationship between ofibril lipid accumulation, skeletal muscle glycogen synthase activity and GLUT4

intramy-translocation Although hepatic insulin resistance may play only a minor role in

the development of the metabolic syndrome per se, the role of the liver in the

dyslipidaemia of the syndrome is important Also, the altered peripheral lation of FFAs and their effect on hepatic glyconeogenesis and glycogenolysis

regu-is a critical factor in the dysregulation of glucose metabolregu-ism in the metabolicsyndrome These latter observations also highlight the importance of a directeffect of peripheral insulin resistance on hepatic glucose production and hepaticinsulin resistance

Finally, as mentioned, the increased secretion of lipoproteins from the liverrepresents a vital link between hepatic insulin resistance and the arteriosclerosisand cardiovascular diseases of the metabolic syndrome Therefore, the relation-ship between insulin resistance in the liver and lipoprotein turnover remains animportant area of future research

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glu-of classical glucoregulatory hormones, such as insulin and glucagon Clearly,hypoglycaemia can be sensed directly by the brain and counter-regulatory mech-anisms can be mounted in the CNS to drive glucose levels back toward thenormoglycaemic range Activation of neuroendocrine systems and the auto-nomic nervous system are the main effector pathways invoked by the brain.Combined, these central and peripheral regulatory events result in increasedproduction of glucose by the liver and decreased utilization by peripheral tis-sues Counter-regulatory responses are relevant during prolonged starvation andare particularly important for diabetic patients using insulin, where hypogly-caemia often occurs inadvertently We will herein discuss the role of the brain

in counter-regulation to severe hypoglycaemia and mechanisms whereby theCNS may sense small day-to-day changes in glucose levels This chapter willalso focus on a number of other afferent signals to the CNS, including leptin,insulin and free fatty acids, that may influence glucose homeostasis independent

of their effects on feeding behaviour

Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly

 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6

7.2 Counter-regulation of hypoglycaemia – role of the CNS

Although the brain depends primarily upon glucose for energy, it does not thesize glucose and brain glycogen stores are very limited It is therefore notsurprising that mechanisms are in place to ensure a sufficient supply of glucose

syn-to protect brain function during hypoglycaemia The importance of these nisms in regulating glucose levels from meal to meal or during overnight fasting

mecha-in normal mecha-individuals is not clear, but they are critical durmecha-ing extended fasts,acute insulin-induced hypoglycaemia, prolonged or repeated hypoglycaemia due

to insulinomas or intensive diabetic therapy and hypoglycaemic episodes thatoccur in diabetic patients overnight They may also be important during periods

of prolonged undernutrition such as occurs during cachexia or anorexia nervosa.Counter-regulation of hypoglycaemia involves a compendium of hormonesand neurotransmitters that are released with the goal of providing glucose forbrain utilization while decreasing glucose need in peripheral tissues (Figure 7.1).The primary players involved in counter-regulation are insulin, glucagon,epinephrine, norepinephrine, cortisol and growth hormone A hierarchy existsfor invoking release of these factors.1 – 3 Decreased insulin release occurs whenglucose levels drop to ∼4.5 mM from a normal level of ∼6.0 mM Glucoselevels that trigger decreased insulin release lie just at or below values normallyseen during the postadsorptive state (∼4.5–5.0 mM), so further absence offood leads to compensatory reduction in pancreatic insulin release Increases

in counter-regulatory release of glucagon, epinephrine, norepinephrine, cortisoland growth hormone occur when glucose levels reach∼3.6–3.8 mM Symptoms

of hypoglycaemia that are of neural origin (i.e sweating, hunger, tingling,weakness, dizziness) and cognitive dysfunction appear at glucose levels of

∼3.0 and ∼2.6 mM, respectively Counter-regulatory mechanisms are invoked

at glycaemic thresholds that are higher than thresholds for symptoms ofhypoglycaemia Of particular importance to diabetic patients is the fact thatthese thresholds are not absolute, but instead are dynamic and vary depending

on the antecedent glucose levels Thus, thresholds are lowered in diabeticindividuals receiving intense insulin therapy as they undergo recurring bouts ofhypoglycaemia, and this is thought to be an underlying cause of hypoglycaemiaunawareness.4 – 8

As described above, the earliest response to falling glucose is decreasedpancreatic secretion of insulin, and this is also the major means of regulat-ing circulating glucose levels between meals Further reductions in blood glu-cose stimulate glucagon release from the α-cells of the pancreas, stimulat-ing hepatic glucose production, but unlike insulin glucagon does not influ-ence glucose utilization.9 Decreasing levels of glucose also elicit release ofepinephrine from the adrenal medulla, which stimulates glucose production andlimits glucose utilization through aβ2-adrenergic-receptor-mediated mechanism.Epinephrine also stimulates mobilization of fatty acids and inhibits pancreaticinsulin secretion.10

COUNTER-REGULATION OF HYPOGLYCAEMIA – ROLE OF THE CNS 181

Less critical to the initial counter-regulatory response are norepinephrine(NE), growth hormone and cortisol Circulating NE levels increase markedlyduring hypoglycaemia and mainly reflect release from the sympathetic nervoussystem As discussed later, sympathetic innervation of the liver and pancreasplays a role in controlling glucagon and insulin release, and influences hep-atic glucose production Release of growth hormone from the anterior pituitaryand of cortisol from the adrenal cortex plays a role during prolonged hypogly-caemia, leading to elevation of alternative fuels such as free fatty acids andketones.11 Cortisol and growth hormone, along with catecholamines, may play

a role in the Somogyi phenomenon, wherein hypoglycaemia leads to reboundhyperglycaemia and posthypoglycaemic insulin resistance due to the inputs ofcounter-regulatory hormones outweighing that of insulin.12 – 14 Growth hormone

is also thought to be involved in the ‘dawn phenomenon’, wherein early morninghyperglycaemia occurs in the absence of antecedent hypoglycaemia.15

In response to acute hypoglycaemia, fasting and prolonged starvation, theCNS regulates several efferent signals Key sensory and effector sites are located

in the hypothalamus, the brainstem and in the spinal cord, which communicatewith each other via direct or indirect neuronal circuitries Efferent signals are ofneuronal (dotted lines) and humoral (full lines) nature Hypoglycaemia reducesthe activity of the parasympathetic nervous system (PNS) and stimulates thesympathetic nervous system (SNS), which innervates the adrenals, the pancreasand the liver, and ultimately leads to increased glucose production (GP) by theliver Additional hypothalamic-pituitary hormonal systems play a role duringfasting and prolonged starvation, stimulating release into the circulation of freefatty acids (FFA) and ketones, which serve as alternative fuels Stimulatory orinhibitory effects on hepatic glucose production are indicated by (+) and (−),respectively; DMV= dorsal motor complex of the vagus nerve; PIT = pituitary.The idea that the brain is important in generating the counter-regulatoryresponse to hypoglycaemia was proposed as early as 1849 by Claude Bernard,16who found that puncturing the fourth cerebroventricle caused glucosuria indogs Subsequent investigators observed that damage to the ventral hypothala-mus led to hyperglycaemia or glucosuria.17 In addition, electrical stimulation ofthe ventromedial hypothalamus (VMH) increases blood glucose levels within 3minutes18and intracerebroventricular delivery of 2-deoxyglucose (2-DG), a glu-cose antagonist, stimulates serum glucose levels and increases glucagon, cortisol,epinephrine and norepinephrine levels,19, 20 a response attenuated by hypotha-

lamic deafferentation.21 A combination of spinal cord and vagal transectionblocked the counter-regulatory increase of glucose following insulin admin-istration in dogs.22 Moreover, insulin infusion into the carotid artery induces

a hypoglycemic state,23 and preventing neuroglucopenia by infusing glucosethrough the carotid and/or vertebral arteries24, 25 significantly attenuates the glu-coregulatory response to systemic hypoglycaemia Frizzell et al.26 showed thatselective carotid or vertebral artery glucose infusion was not nearly as effective

Liver GP

Brainstem Spinal cord

Insulin

Pancreas

Glucagon Epinephrine

Adrenal

DMV

PNS

FFA Ketones

Figure 7.1 Central efferent responses to hypoglycaemia

as infusion through both arteries in preventing the glucoregulatory response toinsulin-induced hypoglycemia Since vertebral and carotid artery infusion targetdifferent areas of the brain, this finding implies that several distinct regions areinvolved in counter-regulation to hypoglycaemia

7.3 Brain regions involved in counter-regulation

Food intake and energy balance are primarily controlled by the hypothalamusand by the brainstem.27 – 31 As described below, evidence also supports rolesfor these two brain regions in controlling central responses to hypoglycaemia(Figure 7.2)

The importance of the hypothalamus is supported by studies showing thatinjections of the glucose antagonist 3-O-methyl glucose into the ventrolateralhypothalamus results in epinephrine secretion and hyperglycaemia, an effect that

is blocked by functional denervation of the adrenal gland.32 In addition, trical stimulation of the VMH elicits a rapid increase in plasma glucose, which

elec-is attenuated by adrenalectomy and by injection of glucagon antelec-iserum.18 Borg

et al.33 lesioned the VMH, LHA or cortex and then manipulated serum glucoseconcentrations to achieve euglycaemia (6.0 mM) or hypoglycaemia (3.0 mM)

by insulin clamp As expected, hypoglycaemia increased epinephrine, pinephrine and glucagon VMH lesions reduced the magnitude of this response

nore-by about 60 per cent whereas lesions of the LHA or frontal lobe were

inef-fective In less invasive studies, Borg et al.34 reported an increase in plasmaglucose in freely moving rats within 30 minutes of inducing glucopenia in the

BRAIN REGIONS INVOLVED IN COUNTER-REGULATION 183

CER

3V

Figure 7.2 Key regions of the CNS involved in peripheral gluceregulation

VMH by local delivery of 2-DG via microdialysis Delivery of glucose to thesame site had the opposite effect,35 and delivery of 2-DG to the frontal lobes ofthe brain were ineffective.33

The figure above shows a schematic drawing of a sagital section of the rodentbrain Coronal sections of the hypothalamus and caudal brainstem are indicated

by vertical lines and marked as 1
and 2
, respectively CTX= cortex; CER =cerebellum; PIT= pituitary 1
Schematic drawing of key nuclei in a coronalsection of the hypothalamus PVN= paraventricular hypothalamic nucleus;LHA= lateral hypothalamic area; DMH = dorsomedial hypothalamic nucleus;VMH= ventromedial hypothalamic nucleus; ARC = arcuate nucleus; ME =median eminence; OT= optical tract; 3V = third ventricle 2
Schematicdrawing of key nuclei in a coronal section of the caudal brainstem CER=cerebellum; AP= area postrema; NTS = nucleus of the solitary tract; DMV =dorsal motor complex of the vagus nerve; CC= central canal

Ritter et al.36 localized glucoregulatory sites in the hindbrain of awake ratsusing the 5-thio-D-glucose (5TG) glucose analogue Multiple injection sites wereanalysed for hyperglycaemic or hyperphagic responses between 30 min and 4hours post-injection, and many injection sites, including the nucleus of the soli-tary tract (NTS), were associated with increased blood glucose However, in the

same study and in contrast to the results by Borg et al., Ritter et al did not find

any responsive sites in the VMH The explanation for this discrepancy is unclear,

but one possibility is that the 2-DG compound used by Borg et al reached the hindbrain sites identified by Ritter et al., although Borg et al reported that

hypothalamic regions close to the injection site did not contain 2-DG followinginjection In further support of the hindbrain sites, but not of the forebrain region,

Ritter et al.37 showed that blood glucose levels were unaffected by 5TG tions into the third ventricle when flow of cerebrospinal fluid from the third

injec-to fourth ventricle was blocked, yet 5TG injections ininjec-to the fourth ventriclewere still effective Also pointing to the presence of hindbrain glucoresponsiveregions as primary mediators of the counter-regulatory response are findings byDiRocco and Grill27, 38 demonstrating hyperglycaemic responses to systemic

administration of 2-DG in decerebrate rats While further studies are needed toresolve the discrepancy between these studies, the data clearly support the notionthat specific regions within the central nervous system can sense hypoglycemia

In addition, injections of glucose into the carotid artery supplying the brain, inamounts that do not affect systemic glycemia, rapidly increase plasma insulinconcentrations,39 an effect probably mediated by the parasympathetic nervoussystem Combined with the above data, these data demonstrate a role of thebrain in sensing both low and high glucose levels, and the ability of the CNS

to generate an appropriate response affecting peripheral glucose metabolism

All brain neurons become silent when they experience a rapid fall in glucoselevels below 1 mM,42 a response that may be protective in the short term.43 Incontrast to neuronal silencing at very low glucose levels, rare but highly special-ized neurons exist in the CNS that are sensitive to changes in blood glucose thatare only slightly above or below the normal range Generally, two approacheshave been taken to study this in detail One involves single-cell recordings inbrain-slice preparations during exposure to varying concentrations of glucose,the other using implanted electrodes in animals and measuring neuronal activity

in response to changes in blood glucose levels in situ By recording individual neuronal discharge frequencies in anaesthetized cats, Oomura et al.44 reportedthat hypothalamic neurons either became increasingly active (glucose stimu-lated) or increasingly inactive (glucose inhibited) in response to intracarotid

injection of glucose In later studies, Oomura et al.45, 46 showed that about 30per cent of all tested cells in the LHA reduced their firing rates and about 20 per

GLUCOSENSING NEURONS 185

cent were activated in response to local intrahypothalamic delivery of glucose

in rats In contrast, approximately 35 per cent of examined VMH cells wereactivated and only a few were inhibited Using similar methods, 45 per cent

of tested neurons in the NTS increase firing frequency in response to locallyinjected glucose.47

An elegant and more recent investigation has studied this in further detail.Silver and Erecinska48 measured blood glucose, brain extracellular glucose andneuronal firing rates in anaesthetized rats while gradually increasing or decreas-ing circulating blood glucose levels within the physiological range In the LHA,increasing glucose inhibited 33 per cent of the tested neurons while about sevenper cent were activated and 60 per cent were unresponsive The investigatorsclassified the cellular responses into four groups The predominant type graduallydecreased firing as glucose rose (maximal firing rate at 3 mM blood glucose),becoming completely inhibited at 10–12 mM In the VMH, most cells weresilent at blood glucose of 3–4 mM and progressively increased their activity

as glucose rose to∼15 mM, and could not be inhibited by higher glucose els No cells in the VMH were inhibited by glucose, consistent with earlierreports.45 In summary, this work by Silver and Erecinska suggests that highlyspecialized cells in the hypothalamus alter firing rates in response to very small,physiological changes in blood glucose levels

lev-The study by Silver and Erecinska could not entirely exclude the ity that circulating factors other than glucose were mediating the effect on thehypothalamic neurons Furthermore, it could not be determined whether theaffected cells were directly influenced by extracellular glucose, or whether theywere indirectly modulated via synaptic inputs from true glucosensing cells Otherinvestigators49, 43 have addressed this question by using thin brain slices and

possibil-patch clamp recordings, while controlling glucose concentrations present in themedium Neurons were found that were directly inhibited or directly stimulated

by glucose as well as other neurons that were activated or inhibited via tic modulation, presumably by the true glucosensing neurons Several additionalbrain regions harbouring glucosensing cells have been reported using similarmethods, including the arcuate nucleus (ARC),50 the paraventricular nucleus ofthe hypothalamus (PVN),51 and the hindbrain.52 These in vitro studies demon-

presynap-strate that specific brain regions contain specialized neurons that respond tophysiologically relevant changes in extracellular glucose levels However, itremains to be determined whether these specific cells play a role in regulat-ing peripheral glucose metabolism, either in the counterregulatory response tohypoglycemia or within meal-to-meal variation of blood glucose levels.The exact cellular mechanism by which glucosensing neurons detect changes

in extracellular glucose is not fully understood Evidence suggesting that thalamic glucose-stimulated neurons utilize an ATP-sensitive K+ channel was

hypo-first reported by Ashford et al.53, 54 They showed that blocking the K+-ATPchannel activates neurons in isolated hypothalamic slices Furthermore, injection

of another K+-ATP blocker, glibenclamide, into the VMH impairs the terregulatory increase in blood glucose after insulin-induced hypoglycaemia,and decreases blood glucose in normoglycemic rats.55 In pancreatic β-cells,membrane-bound K+-ATP channels are comprised of a pore-forming subunit(Kir6.2) through which potassium ions travel out of the cell, and of a regulatoryunit (SUR1) that binds synthetic sulfonylureas (tolbutamide, glibenclamide),which close the channel and lead to increased insulin secretion.56 The SUR reg-ulates Kir6.2 in response to the intracellular ATP/ADP ratio Thus, stimulatingβ-cells with glucose increases the ATP/ADP ratio, inhibiting Kir6.2 activity, andcausing accumulation of intracellular K+ Influx of calcium ions via Ca2+ chan-nels finally triggers insulin secretion.57This model has led to the hypothesis thathypothalamic glucose-stimulated neurons have significant similarities to pancre-atic β-cells The neuronal model envisions that glucose induces depolarization

coun-of the neuron by closing K+-ATP channels, leading to increased firing rates andincreased cellular Ca2+ at axon terminals, ultimately causing release of neu-rotransmitters and neuropeptides Less is known about how glucose-inhibitedneurons sense glucose, since these cells become hyperpolarized with increasingglucose levels

Lee et al have shown by single-cell PCR that glucosensing neurons express

ATP-sensitive potassium channels.58 Additional evidence for a role of the K+ATP channel in glucosensing by the brain arises from recent results of Miki

-et al.59 Mice lacking the Kir6.2 gene were devoid of glucose-stimulated

neu-rons in brain slices containing the VMH Furthermore, in response to systemichypoglycaemia or neuroglucopenia, the ability to increase circulating glucagonand glucose levels was greatly impaired Based on this, the authors concludedthat K+-ATP channels in VMH-glucose-stimulated neurons are required forglucose responsiveness and that K+-ATP channels in this brain region areessential for maintenance of glucose homeostasis While the first conclusion

is clearly supported by the data, the latter must be considered speculative, since

it is doubtful that the VMH is solely responsible for the counterregulatoryresponse Also, the K+-ATP channel (Kir6.2) is widely expressed throughoutthe brain and is not restricted to the VMH.42, 60–62 Thus, presence of this

channel is not sufficient to act as the only critical component of ing neurons

glucosens-Of higher potential for use in defining glucosensing neurons is the atic form of hexokinase, i.e glucokinase (GK) This enzyme is rate limitingfor glycolysis in the β-cell because its Km, in contrast to the Km of otherhexokinases, lies within the physiological range for blood glucose.63 The CNSsites of expression include the VMH, DMH, PVN, ARC, LHA and the caudalbrain stem.42, 64–67 This expression pattern thus resembles that of glucosensingneurons and opens the possibility that GK is expressed in these cells In dis-sociated neurons from the VMH, about 70 per cent of both glucose-inhibitedand stimulated cells are affected by inhibition of GK,66 while non-glucosensing

pancre-CONTROL OF PERIPHERAL ORGANS INVOLVED IN GLUCOREGULATION 187

neurons are largely unaffected This suggests that GK is not expressed in glucosensing cells, although this requires further investigation since GK expres-sion is relatively wide as described above Although GK is expressed in bothglucose-inhibited and glucose-stimulated neurons and may be a component ofthe glucosensing mechanism, the question remains that if GK is expressed inboth cells, what then distinguishes the two types of neuron?

non-7.5 Central control of peripheral organs involved

in glucoregulation

The liver

The liver is richly innervated by both sympathetic and parasympatheticnerves.68, 69 The sympathetic fibres derive from the splanchnic nerves andtheir postganglionic fibres originate from the celiac ganglia Parasympatheticinnervation arises from both the left and right vagus nerves The majority

of the nerve supply enters along the common hepatic artery and portalvein Stimulation of the vagus nerve increases the activity of liver glycogensynthase, the rate-limiting enzyme in glycogen synthesis from glucose-6-phosphate.70 This effect is not influenced by pancreatectomy, suggesting thatthis occurs directly in the liver and is not mediated by changing insulinlevels Systemic infusion of glucose increases vagal efferent activity, arelationship that is linear over the physiological range of circulating glucoseconcentrations.71 In contrast, stimulation of the splanchnic nerves depletesglycogen reserves and increases serum glucose levels.72, 73 Furthermore,

splanchnic nerve stimulation in rabbits activates two glycogenolytic enzymes,phosphorylase and glucose-6-phosphatase, within 30 seconds, suggesting a directeffect on liver glucoregulation.73, 74Moreover, decreases in serum glucose levels

in response to a carotid artery insulin injection have been ascribed to directneural effects on liver glucose production and glucose uptake.23 Altogether,this data points to a role for the CNS in regulating liver glucose metabolism,although the exact quantitative impact of this regulation under physiologicalcircumstances is unclear

The pancreas

As the primary source of insulin and glucagon, the pancreas is of obviousimportance in regulating peripheral glucose levels Regulation of insulin andglucagon release from the pancreas by the central nervous system arises fromthree inputs, two of which are neural while one is hormonal: (1) parasympatheticinnervation, (2) sympathetic innervation and (3) sympathoadrenal input Inner-vation of the pancreas by the parasympathetic nervous system is accomplished

by the vagus nerve and consists mainly of cholinergic input,75 although thereappears to be some peptidergic innervation as well, namely vasoactive intestinal

peptide and gastric releasing peptide.76 Postganglionic sympathetic input entersthe pancreas in conjunction with the arterial blood vessels to enter as part ofthe mixed autonomic nerve There may also be preganglionic sympathetic effer-ents that enter the pancreas directly and innervate intrapancreatic sympatheticganglia.78, 79These sympathetic nerve fibres contain mostly norepinephrine, butmay also include neuropeptides such as neuropeptide Y and galanin.77

Insulin-induced decreases in blood glucose decrease the firing rate of thepancreatic branch of the vagus nerve.80 In contrast, carotid artery infusion ofisotonic glucose stimulates coeliac–pancreatic vagus firing rate and intracarotidinfusion is more effective than intravenous administration.80 Stimulation ofparasympathetic inputs increases insulin release in the dog and the baboon81, 19

and increases glucagon release from α-cells in dogs and calves.82, 83

Further-more, stimulation of the mixed pancreatic nerve increases insulin levels in thepancreatic duodenal vein and vagal stimulation increases insulin release in per-fused preparations of pancreas, responses that are blocked by administration

of the anticholinergic drug atropine.10 Stimulation of sympathetic input or ofthe splanchnic nerve decreases insulin release, likely via the α-adrenoreceptor,and increases glucagon release.84 – 88 Norepinephrine release from the pancreaticsympathetic nervous system increases with increased severity of glucopenia89and ganglionic blockade inhibits this response.90 Pancreatic sympathetic nerveactivity is stimulated by 2-DG administration to the lateral cerebroventricles.91Finally, denervation of the pancreas blocks the response to systemically admin-istered 2-DG and intrapancreatic arterial infusion of 2-DG fails to reproduce thepancreatic norepinephrine response, clearly supporting a central role in theseprocesses.91

The adrenal glands

As mentioned above, the adrenal glands provide input for glucoregulation both viaepinephrine release and via secretion of glucocorticoids Regarding the former,the adrenals receive sympathetic input through the greater and lesser splanchnicnerves and lumbar ganglia of the abdominal sympathetic chains.69 The vagusdoes not appear to contribute directly.69 Cannon92 first showed that hypogly-caemia elicited epinephrine release, a response later shown to increase progres-sively with the magnitude of glucopenia.2, 89, 93, 94 Additionally, epinephrinerelease in response to hypoglycemia or to the 3-O-methylglucose is blocked

by isolating the adrenal glands from neural input.94, 95, 90, 96The hypothalamusappears to be involved in the sympathoadrenal response to hypoglycaemia sincehypothalamic deafferentation reduces the adrenomedullary response to 2-DG.21Indeed, VMH lesions increase adrenal nerve activity and catecholamine release,while LHA stimulation and lesions increase and decrease adrenal nerve activity,respectively.97In contrast, VMH stimulation did not affect adrenal nerve activity.Intracerebroventricular administration of 2-DG increased adrenal nerve activity,

ADDITIONAL AFFERENT SIGNALS TO THE CNS 189

a response blocked by LHA lesions but unaffected by VMH lesions However,stimulation of the VMH prior to 2-DG treatment reduced the 2-DG-inducedincrease in adrenal nerve activity.97 Based on this data, the authors concludedthat the LHA is sensitive to 2-DG and comprises a major part of the sym-pathoadrenal response, but that the VMH response may depend on antecedentadrenal nerve activity and be mediated by other neuronal structures that func-tion as relay points of integrating sites between the VMH and sympatheticefferents Also, adrenalectomy influences the insulin and glucose response toVMH stimulation.18 Thus, the brain is an important component of the pathwaysinfluencing sympathoadrenal epinephrine release during hypoglycemia

Release of cortisol (humans) or corticosterone (rodents) is increased duringhypoglycaemia.11 This reflects hypothalamic output of corticotropin-releasinghormone (CRH), which in turn stimulates adrenocorticotrophic hormone (ACTH)release from the anterior pituitary, ultimately leading to increased glucocorti-coid secretion from the adrenals As mentioned earlier, increased cortisol releaseprobably plays a minor role in glucoregulation, mainly during the later stages

of prolonged hypoglycaemia However, there is some indication that CRH itselfinfluences sympathoadrenal activity, since CRH administration prior to hypo-glycemia blunts the counter-regulatory epinephrine response, a result not observedafter prior treatment with ACTH or corticosterone.98

7.6 Additional afferent signals to the CNS regulating

peripheral glucose metabolism

Pancreatic and hepatic glucosensing

Russek99 first postulated that specific receptors in the liver monitor glucoselevels and send information via the vagus nerve to brain regions importantfor controlling food intake These glucosensing entities appear to be localizedspecifically to the portal vein100and histological studies have revealed extensiveafferent innervation of the portal vein adventitia.101 – 103 Portal vein glucose infu-sion decreases the firing rate of the hepatic branch of the afferent vagus nerve

in perfused liver preparations104 and discharge rates of hepatic vagal afferentsare inversely proportional to portal vein glucose concentrations.105Furthermore,systemic infusion of 2-DG increases the hepatic vagal afferent discharge rate.106Interestingly, fluctuations in portal vein glucose levels influence the firing rate

of neurons in the LHA and NTS.107 Thus, hepatoportal vagal afferents carryinformation regarding portal vein glucose levels to hypothalamic areas knownfor generating a counter-regulatory response (Figure 7.3) This figure depictsfactors and pathways that can act on the CNS to influence peripheral glu-cose metabolism, independent of long-term effects on energy intake Glucose issensed by specialized glucosensing neurons located primarily in the hypotha-lamus and in the caudal brainstem Neurons that are regulated by leptin are

BLOOD GLUCOSE

Liver

Pancreas Fat

Leptin

NTS Hypothalamus

Vagal afferents

Brainstem

Figure 7.3 Central afferent signals involved in regulation of peripheral glucose metabolism

located in the same regions of the brain and may overlap directly with thosethat sense glucose Moreover, both insulin and FFA may act in similar regions

of the hypothalamus to affect peripheral glucose metabolism Glucosensory rons of the vagus nerve are also present in the pancreas and in the portal vain

neu-of the liver, transmitting information to the CNS about local glucose levels.NTS= nucleus of the solitary tract; FFA = free fatty acids

Perseghin et al.108 assessed the importance of neural input from and to theliver in glucoregulation by examining liver transplant patients They observedthat glucose levels in liver transplant patients were maintained in the lower phys-iological range within a few weeks of transplant These authors also observedthat fasting glucose levels and glucose production were lower, that glucose pro-duction during insulin-induced hypoglycaemia was significantly less and that

the counterregulatory response was blunted in transplant patients Bolli et al.109

pharmacologically blocked counter-regulatory hormone influences on glucoseproduction and observed that counter-regulatory hormones account for practi-cally all of the glucose produced at blood glucose levels of 50 mg/dl, but thathepatic glucose production increased twofold over controls at blood glucose lev-els of 30 mg/dl As mentioned previously, peripheral hypoglycaemia induced byinsulin leads to large increases in epinephrine and norepinephrine release Thisincrease is blunted by about 50–60 per cent in rats wherein the portal vein isdenervated.110 It has been estimated that the liver can produce anywhere from

12 to 50 per cent of circulating glucose during hypoglycaemia independent ofcounter-regulatory hormone influence.111 – 115 Confounding many of these stud-ies is the fact that, during severe hypoglycaemia, the liver can produce glucose

ADDITIONAL AFFERENT SIGNALS TO THE CNS 191

in the absence of neural or counterregulatory hormone input.109, 115–118 Thus,the relative importances of neural influences in the liver on glucoregulation aredifficult to assess, but probably account for less than 25 per cent of hepaticglucose production during moderate hypoglycaemia

The relative importance of the CNS in generating a response to hypoglycaemia

in the pancreas has not been addressed in detail Clearly, decreases in blood cose can be directly detected within the pancreas and a response generated bytheα- and β-cells However, it is possible that pancreatic vagal afferents sendinformation regarding local glucose levels to the brain since intravenous glucose

glu-or 2-DG increases and intravenous insulin decreases the pancreatic vagal afferentfiring rate.80In pancreas transplant patients, glucose levels are normal, suggestingthat humoral regulation of pancreatic function is sufficient for dealing with nor-mal day-to-day changes in glucose levels However, deficits in glucoregulation

during hypoglycemia have been noted in these patients Diem et al.119 reportedthat, although glucose recovery improved in diabetics with pancreatic transplants,recovery of hepatic glucose production during hypoglycaemia increased by only

34 per cent over baseline in transplant patients compared with 58 per cent in

control individuals Battezzati et al.120 observed that, in response to mild glycaemia, hepatic glucose production initially decreased and then returned tobaseline in controls by 1 h, but was still depressed at 2 h in transplant patients

hypo-despite normal glucagon and epinephrine responses Kendall et al.121 showedthat in type 1 diabetic transplant patients subjected to stepped hypoglycaemia theglucagon response and symptom awareness were normalized, but the epinephrineand norepinephrine responses were muted or absent Thus, it appears that neuraloutflow or input from the pancreas influences hepatic glucose production, thoughthe relative importance it has in counter-regulation remains to be defined

Leptin

Leptin, the fat-derived hormone discovered in 1994,122 circulates at levels portional to body fat mass and delivers information to the brain about energystores.29, 30, 123–125 Mutations in leptin or its receptor cause morbid obesityand severe insulin resistance.122, 126 In addition to decreasing food intake and

pro-body weight, leptin influences neuroendocrine function, reproduction, adaptiveresponses to fasting, bone development, blood pressure, energy expenditure,sensory nerve input and autonomic outflow Pertinent to this review is recentdata suggesting that leptin also influences peripheral glucose homeostasis viaactions in the CNS, independent of changes in feeding and body weight Kamo-

hara et al.127 showed that intracerebroventricular (ICV) delivery of small doses

of leptin to fasted mice acutely increased glucose turnover and whole bodyglucose uptake Leptin-induced glucose uptake into muscle was nearly ablatedfollowing denervation of the muscle tissue, suggesting that the effect occurredvia autonomic efferent signals Furthermore, ICV injection of leptin rapidly

regulates hepatic glucose fluxes128 and leptin improves insulin sensitivity inlipodystrophic rodents and patients, independent of feeding.129 – 132

Functional leptin receptors are found in the ARC, the VMH and the DMH,and to a lesser degree in the PVN and the LHA.133 – 136 Outside the hypothala-mus, expression can be detected in the caudal brain stem.137, 138, 134Consistentwith this, leptin affects the firing rates of neurons in isolated brain slices from theARC, the VMH and the NTS.139 – 141 Thus, these locations overlap with centresthat are involved in regulating energy homeostasis and the autonomic nervoussystem,142 and with sites containing glucosensing neurons Indeed, microinjec-tion of leptin into the VMH, but not the LHA, of freely moving rats increasedglucose uptake in peripheral tissues, including brown adipose tissue (BAT), heartand skeletal muscle.143 Subsequent studies showed that the effect on BAT ismediated by the sympathetic nervous system.144 It remains to be determinedwhether physiological changes in leptin levels induce the same effects andwhether other sites in the CNS have similar capacities

Neuropeptide Y (NPY) and proopiomelanocortin (POMC) cells in the ARC

of the hypothalamus have received particular attention due to their key role inregulating energy homeostasis.145NPY potently stimulates food intake146 – 148andNPY neurons co-express the melanocortin receptor antagonist, agouti-related pep-tide (AgRP).149The POMC-derived neuropeptide,α-melanocyte stimulating hor-mone (α-MSH), induces robust anorexigenic responses in rodents.150 – 152 BothNPY/AgRP and POMC neurons are directly regulated by leptin via the leptinreceptor, but in opposing fashions.153, 154Leptin stimulates POMC neurons whileNPY/AgRP neurons are inhibited.139When leptin levels are low (fasting, leptin-

deficient mice), pomc gene expression decreases, indicating that the melanocortin

system mediates at least some of the effects of leptin.155, 156This conclusion is

sup-ported by powerful pharmacological and genetic evidence.157 – 160NPY and AgRPexpression is strongly activated in the absence of leptin.161When leptin levels arehigh (fed state, during leptin administration), POMC expression increases whileNPY and AgRP expression decreases.162, 156

Both the GK enzyme and the K+-ATP (Kir6.2/SUR1) channel are expressed

in POMC139, 66, 163 and in NPY neurons.164, 62 However, the importance of

Kir6.2 channels in leptin action is unclear since leptin still inhibits food intake

in Kir6.2−/− mice,59 although it is possible that other aspects of leptinspleiotrophic actions could be affected in these mice Firing rates of POMCneurons are stimulated by glucose163 and NPY cells are inhibited.164 Thus,both leptin and glucose probably inhibit orexigenic NPY peptide release andstimulate anorexigenic α-MSH release Evidence also suggests that centraladministration of melanocortin receptor agonists rapidly affects peripheralglucose metabolism,165 providing a link between the activity of POMC neuronsand the regulation of glucose and energy homeostasis, a view that is supported

by additional anatomical, genetic, pharmacological and electrophysiologicalstudies.166, 153, 167, 145, 123, 163

ADDITIONAL AFFERENT SIGNALS TO THE CNS 193

Insulin

Insulin plays a critical role in regulating glucose homeostasis via direct actions

on insulin receptors expressed in muscle, liver and adipocytes Insulin receptormRNA is also expressed in the brain, including in the cerebral cortex, the cere-bellum, the dentate gyrus, layers of the pyriform cortex and of the hippocampus,the choroid plexus and the ARC of the hypothalamus.168 – 170 In anaesthetizedrats, insulin injected into the carotid artery immediately decreases systemic bloodsugar23 and delivery of insulin into the VMH or the LHA of rats rapidly affectsneuronal discharge frequency.45 ICV injection of insulin reduces food intake andbody weight in baboons and rodents171, 172 and administration of anti-insulin

antibodies into the rat hypothalamus increases food intake.173 In more recentstudies, complete loss of neuronal insulin receptors by conditional knockout

in mice or partial loss by hypothalamic injection of insulin receptor anti-senseoligonucleotides results in hyperphagia and increased bodyweight.174 – 176Insulingiven ICV into awake rats rapidly inhibits glucose production by the liver,175, 176

supporting a centrally mediated effect of insulin on glucose metabolism Whilethe above studies were mostly chronic and/or pharmacological in nature, a laterstudy shows that minute amounts of insulin delivered into the brain arteries offasted dogs rapidly alter peripheral glucose homeostasis,177 strongly supporting

a physiological role for central insulin signalling

Neurons that are inhibited by insulin are present in the ARC and VMH.Like leptin, insulin activates ATP-sensitive K+ channels in hypothalamic brainslices178 and a role of K+-ATP channels in decreasing hepatic glucose produc-tion in response to insulin has recently been reported.176 Interestingly, insulin-sensitive neurons also have glucosensing capabilities.45, 178 Moreover, insulindoes not affect the activity of neurons from rats lacking functional leptin recep-tors, suggesting that aspects of insulin action in the CNS require leptin signalling,and opening the possibility that receptors for insulin and leptin are co-expressed

in glucosensing neurons.179 Indeed, insulin receptors have recently been tified in hypothalamic POMC neurons,180 cells that are activated by leptin andglucose Whether POMC neurons increase or decrease firing rates in response toinsulin is unknown, although activation seems more likely since the melanocortinsystem appears to be required for insulin-mediated inhibition of food intake180and fat mass.181 In addition, central administration of melanocortin receptoragonists rapidly reduces serum insulin levels, an effect mediated via the sym-pathetic nervous system.165 However, blockade of melanocortin signalling didnot affect inhibition of liver glucose production by insulin.181 Combined, thesedata suggest that the central melanocortin system regulates peripheral glucosemetabolism via effects on insulin release, but that another system regulates glu-cose production Further studies of POMC neurons will illuminate the role ofthese neurons in insulin action, and of the interplay between insulin, glucoseand leptin signalling in the brain

iden-Fatty acids

Increased quantities of free fatty acids (FFAs) are released from adipocytes underconditions of starvation, diabetes and obesity These molecules can be utilizedinterchangeably with glucose for energy in most tissues, with the notable excep-tion of brain tissue However, FFAs are present in the cerebrospinal fluid182and FFA extracts delivered to the VMH or LHA of anaesthetized rats rapidlyaffect neuronal discharge rates,183 implying a function for FFAs in the CNS.Effects on neuronal activity also occur with purified long chain fatty acidssuch as oleic acid or palmitic acid, the major FFA in blood.45 FFAs activatesome neurons, while inhibiting others Interestingly, the majority of glucosens-ing neurons respond to FFAs, while the majority of non-glucosensing neuronsare unaffected by FFAs, suggesting that sensitivity to FFAs may be relativelyspecific to rare glucosensing neurons, and that these cells integrate multiplemetabolic signals

Like glucose, insulin and leptin, central administration of oleic acid reducesfood intake in rodents184 and alteration of central fatty acid metabolism affectsenergy intake in rodents.185Obici et al.184 showed that central infusion of oleicacid in fasted rats inhibited liver glucose production, suggesting that fatty acidscan act within the CNS to affect peripheral glucose metabolism independent offood intake This inhibition required activation of the K+-ATP channel, pos-sibly via direct binding of long chain fatty acyl CoA esters to the K+-ATPchannel.186, 187 Since the brain does not usually use lipids as a significant fuel,these studies indicate that FFAs can act as afferent signals informing the brainabout metabolic status, although the exact brain regions involved and the cellularmechanisms by which FFAs are sensed remain unclear

Difficult to reconcile, however, is the finding that oleic acid inhibits foodintake and decreases hepatic glucose production, since circulating FFAs increaseduring starvation, a state characterized by increased appetite and hepatic glucoseproduction Moreover, the hyperlipidaemia present in human and rodent obesity

is associated with hyperphagia, not hypophagia Finally, it has been shown thatphysiological increases of systemic FFAs in humans increase glucose productionand induce mild hyperglycaemia.188 – 190 Although the latter effect is presumablymediated by FFAs acting peripherally, these data imply that central actions ofFFAs to decrease glucose production are of minor importance in the regulation

of whole body glucose metabolism

7.7 Conclusions and future perspectives

It is clear that the CNS can detect large changes in glucose availability andrespond appropriately in order to maintain adequate glucose supply for thebrain The most noticeable evidence for this is the rapid counter-regulatory

CONCLUSIONS AND FUTURE PERSPECTIVES 195

response to hypoglycaemia, where neuroendocrine and autonomic efferent nals regulate functions of the pancreas, liver and the adrenals, which combinedwith direct communications between peripheral tissues leads to increased bloodglucose concentrations The brain regions important for these responses includethe brainstem and regions of the hypothalamus, areas that also contain spe-cialized neurons with unique capabilities to detect alterations in extracellularglucose by changing neuronal activity Although likely, there is as yet no directevidence that these ‘glucosensing’ neurons are actually responsible for initiatingthe counterregulatory response Furthermore, specific physiological functions ofthese cells in individual hypothalamic nuclei have yet to be assigned Theseissues need to be examined using new and more conclusive methods, includingsystematic genetic targeting of neurons in each brain region

sig-Inherent to glucosensing neurons are systems enabling them to be enced by small, physiological changes in extracellular glucose levels, suggest-ing that meal-to-meal variation and diurnal rhythms in blood glucose levelscan be sensed by the brain The cellular mechanism by which glucosensingoccurs may require the activity of specialized glucokinase enzymes and K+-ATP channels that are also critical for pancreatic β-cells to regulate insulinrelease in response to glucose However, these proteins are not sufficient tocharacterize glucosensing neurons, since it is evident that their expression isnot restricted to these rare cells in the brain In addition to glucose, circulatinghormones such as leptin and insulin can influence the brain to affect periph-eral glucose metabolism, possibly via regulation of neurons that also haveglucosensing capabilities Further analyses are required to elucidate the glu-cosensing mechanism and the mechanisms that distinguish glucose-stimulatedand glucose-inhibited neurons

influ-Although this review has focused on signals to the brain that affect glucosemetabolism independent of alterations in food intake and body weight, at leastsome glucosensing cells, including POMC and NPY neurons, are also likely

to serve more complex functions such as regulation of food-seeking behaviour,appetite and meal size In addition, vagal sensory input and gut- and stomach-derived hormones such as ghrelin and cholecystokinin may influence food intakeand energy homeostasis via neuronal circuitries that overlap with those of lep-tin and insulin.31, 29, 28, 191, 192 Additional studies are needed to identify themechanisms whereby glucosensing neurons integrate multiple metabolic inputs,and how these cells are connected to efferent systems that regulate glucosehomeostasis

Finally, studies of the pathogenesis of type 2 diabetes have focused on eral tissues (muscle, β-cells, liver and fat) However, central mechanisms canclearly influence glucose metabolism and control fat mass and energy balance,suggesting that defects in the brain may exist that cause or worsen insulinresistance and type 2 diabetes This possibility deserves further investigation

C.B is supported by a grant from the NIH (RO1 DK-60673) and S.M.H byUSDA (2001-35203-10835) We thank Dr A N Hollenberg (BIDMC, Boston)for critically reviewing the manuscript and Dr H Grill (University of Pennsyl-vania) for providing us with helpful advice, and C Romanosky (West VirginiaUniversity) for assistance with organizing the references We apologize if cer-tain authors and papers were not acknowledged in the review due to spacelimitations

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