glucose production ( gp ) is regulated by gluconeogenesis and glycogenolysis . in type 2 diabetes , elevation of gp
an elevation of plasma gluconeogenic substrate precursors such as free fatty acids ( ffa ) or lactate is also seen in type 2 diabetes [ 25 ] .
these findings led to the working hypothesis that elevation of gluconeogenic substrate precursors increases gluconeogenesis and gp .
the findings as a whole indicate that during the pancreatic clamp , when gluco - regulatory hormones and glucose levels are maintained at basal , intravenous ( i.v . )
infusion of ffa or lactate increases hepatic gluconeogensis but , contrary to the hypothesis , not gp because of a compensatory inhibition of glycogenolysis [ 69 ] .
the underlying  nutrient - sensing mechanisms that inhibit glycogenolysis and counteract the direct effects of circulating ffa or lactate on hepatic gluconeogenesis remain to be explored . in this study , we began to assess the underlying nutrient - sensing mechanisms that counteract the direct effect of circulating lactate on hepatic gluconeogenesis in vivo .
the lipid - sensing mechanisms that counteract the direct effect of circulating ffa on hepatic gluconeogenesis have recently been studied .
it is demonstrated that the inhibition of hepatic glycogenolysis induced by circulating ffa to restrain gp is mediated by hypothalamic lipid - sensing mechanisms in rodents .
the hypothalamic lipid - sensing mechanisms to restrain glycogenolysis and gp are disrupted in diet - induced insulin resistance and obesity , leading to hyperglycaemia in response to systemic lipid infusions .
this is in line with previous reports indicating that in patients with metabolic stress conditions such as type 2 diabetes , reciprocal changes in glycogenolysis do not compensate for changes in gluconeogenesis when plasma ffa concentrations are experimentally manipulated .
unlike the lipid - sensing mechanisms in the body , even in metabolic stress patients with hyperglycaemia , an elevation of circulating lactate still does not increase gp because of an inhibition of glycogenolysis .
consistent with this , in contrast to hypothalamic lipid - sensing mechanisms , activation of central lactate metabolism via direct delivery of lactate into the hypothalamus lowers gp in normal and in early onset of diabetic and obese rodents [ 14 , 15 ] .
on the basis of these observations , we postulate that ( i ) the hypothalamic - sensing mechanism of circulating lactate that regulate gp is intact in normal , diabetic and obese individuals , and ( ii ) these hypothalamic  nutrient - sensing mechanisms are designed to counteract the direct stimulatory effect of circulating lactate on hepatic gluconeogenesis to maintain glucose homeostasis .
given that the ability of the hypothalamus to sense circulating lactate to regulate gp and maintain glucose homeostasis is yet to be evaluated , this will be the first study to address our working hypothesis in normal rodents . to examine whether the hypothalamus senses circulating lactate to regulate gp and maintain glucose homeostasis in vivo , we infused i.v .
l - lactate to elevate plasma lactate levels in the presence or absence of direct inhibition of brain lactate - sensing mechanisms . to directly inhibit central / hypothalamic lactate - sensing mechanisms
, we independently infused ( i ) lactate dehydrogenase inhibitor oxamate ( oxa ) into the third cerebral ventricle ( intracerebroventricular  i.c.v . ) , ( ii ) i.c.v .
atp - sensitive potassium ( katp ) channel blocker glibenclamide ( gli ) or ( iii ) oxa directly into the mediobasal hypothalamus ( mbh ) ( fig . 1a and b ) that has been previously described to prevent the inhibitory effects of central lactate administration on gp .
tracer - dilution methodology and the pancreatic clamp technique were used to assess the effect of i.v . and i.c.v./mbh administrations on whole body glucose kinetics independent of changes in circulating gluco - regulatory hormones .
based on this cross - discipline experimental approach , we provided the first evidence , to our knowledge , that direct inhibition of central / hypothalamic lactate - sensing mechanisms by three independent methods increased gp and disrupted glucose homeostasis in response to systemic lactate elevations in vivo .
the hypothalamic metabolism of lactate to pyruvate and the subsequent activation of the katp channels are required to maintain glucose homeostasis in response to systemic lactate elevations .
somatostatin ( srif ) . during the final 30 min . of the clamps , ( c )
intravenous lactate infusion elevated plasma lactate levels compared to control ( saline , sal ) by 2- to 2.5-fold . * p < 0.001 versus sal .
( d ) plasma glucose , ( e ) plasma insulin , ( f ) plasma adiponectin and glucagon levels were comparable in all groups during the clamps .
adult 8-week - old male sprague dawley rats ( 250280 g ) were obtained from charles river laboratories ( montreal , qc , canada ) and maintained on a standard light - dark cycle with access to rat chow and water ad libitum .
rats underwent stereotaxic surgery to insert single catheters in the third cerebral ventricle ( i.c.v . ) or bilateral catheters into the mbh 2 weeks before the experiments in vivo as previously described [ 14 , 16 , 17 ] .
one week later , catheters were placed in the internal jugular vein and the carotid artery for infusion and sampling during the clamp procedures as previously described [ 14 , 16 , 18 ] .
recovery from surgery was monitored by measuring daily food intake and weight gain for 45 days after surgery .
the study animal protocol was approved by the institutional animal care and use committee of the university health network .
all the rats were restricted to 20 g of food the night before the experiments to ensure the same nutritional status . to groups of conscious unrestrained rats , we infused i.v .
sodium l - lactate ( 100 mol / kg min . , ph 7.0 ) to elevate plasma lactate concentrations by 2- to 2.5-fold as seen in exercise for 4 hrs ( fig .
infusions consisted of the ( i ) lactate dehydrogenase inhibitor oxa ( dissolved in artificial cerebrospinal fluid [ acsf ] to 50 mm [ 5 l / hr ] and was first given as an i.c.v .
bolus [ 3 l ] ) , ( ii ) katp channel blocker gli ( dissolved in 5% dmso to 100 m ; 5 l / hr ) or ( iii ) vehicle ( 5% dmso or acsf ; 5 l / hr ) ( fig .
mbh infusions consisted of ( i ) oxa ( 50 mm ; 0.33 l / hr and was first given as a mbh bolus [ 0.33 l ] ) or ( ii ) vehicle ( acsf ; 0.33 l / hr ) ( fig .
oxa or gli and mbh oxa administered at these concentrations alone do not affect glucose kinetics but were sufficient to abolish the gp - lowering effect of direct brain lactate administrations .
saline ( sal ) or l - lactate infusion and a primed - continuous infusion of [ 3-h]-glucose ( perkin elmer , woodbridge , on , canada ; 40 ci bolus ; 0.4 ci / min ) were initiated at 120 min . and maintained throughout the study to assess the rate of gp and glucose uptake based on the tracer - dilution methodology .
( steady - state basal period ) , the rate of gp ( mg / kg min . ) and plasma glucose levels ( mm ) were 11.9  0.5 and 8.3  0.1 ( i.c.v .
lactate ) , 12.6  0.2 and 8.2  0.2 ( mbh vehicle + i.v .
lactate ) and 12.2  1.2 and 8.9  0.6 ( mbh oxa + i.v .
lactate ) . a pancreatic clamp was performed in the final 2 hrs of the study starting at 240 min ; a continuous infusion of insulin ( 1.5 mu / kg min . ) and somatostatin ( 3 g / kg min . )
was administered , and a variable infusion of a 25% glucose solution was started and periodically adjusted to maintain the plasma glucose concentration at 8 mm .
saline ) were performed , and the gp during the clamps in both groups ( gp : i.c.v .
plasma samples for the determination of lactate , insulin , adiponectin and glucagon concentrations were obtained at 10-min .
somatostatin . in the presence of systemic lactate elevation , direct inhibition of lactate metabolism within the mediobasal hypothalamus ( mbh ) via mbh administration of oxamate ( compared to control ) ( b )
decreased exogenous glucose infusion rate and ( c ) increased gp during the final 30 min .
0.001 versus lactate with mbh vehicle . central sensing mechanisms of circulating lactate regulate glucose production ( gp ) . in the presence of systemic elevation of lactate , direct inhibition of central lactate metabolism via i.c.v .
administration of lactate dehydrogenase inhibitor oxamate ( oxa ) ( compared to control ) led to a marked ( a ) decrease in exogenous glucose infusion rate and ( b ) increase in gp during the final 30 min . of the clamps .
administration of blocker glibenclamide ( a ) decreased exogenous glucose infusion rate and ( b ) increased gp in response to systemic lactate infusions .
plasma lactate concentrations were determined by a kit in accordance with manufacturer s instructions ( sigma diagnostics .
plasma glucose concentrations were measured by the glucose oxidase method ( glucose analyzer , analox instruments , lunenberg , ma , usa ) .
statistical analysis was done by anova or unpaired student s t - test as appropriate .
adult 8-week - old male sprague dawley rats ( 250280 g ) were obtained from charles river laboratories ( montreal , qc , canada ) and maintained on a standard light - dark cycle with access to rat chow and water ad libitum .
rats underwent stereotaxic surgery to insert single catheters in the third cerebral ventricle ( i.c.v . ) or bilateral catheters into the mbh 2 weeks before the experiments in vivo as previously described [ 14 , 16 , 17 ] .
one week later , catheters were placed in the internal jugular vein and the carotid artery for infusion and sampling during the clamp procedures as previously described [ 14 , 16 , 18 ] .
recovery from surgery was monitored by measuring daily food intake and weight gain for 45 days after surgery .
the study animal protocol was approved by the institutional animal care and use committee of the university health network .
all the rats were restricted to 20 g of food the night before the experiments to ensure the same nutritional status . to groups of conscious unrestrained rats , we infused i.v .
ph 7.0 ) to elevate plasma lactate concentrations by 2- to 2.5-fold as seen in exercise for 4 hrs ( fig
infusions consisted of the ( i ) lactate dehydrogenase inhibitor oxa ( dissolved in artificial cerebrospinal fluid [ acsf ] to 50 mm [ 5 l / hr ] and was first given as an i.c.v .
bolus [ 3 l ] ) , ( ii ) katp channel blocker gli ( dissolved in 5% dmso to 100 m ; 5 l / hr ) or ( iii ) vehicle ( 5% dmso or acsf ; 5 l / hr ) ( fig .
mbh infusions consisted of ( i ) oxa ( 50 mm ; 0.33 l / hr and was first given as a mbh bolus [ 0.33 l ] ) or ( ii ) vehicle ( acsf ; 0.33 l / hr ) ( fig .
oxa or gli and mbh oxa administered at these concentrations alone do not affect glucose kinetics but were sufficient to abolish the gp - lowering effect of direct brain lactate administrations .
saline ( sal ) or l - lactate infusion and a primed - continuous infusion of [ 3-h]-glucose ( perkin elmer , woodbridge , on , canada ; 40 ci bolus ; 0.4 ci / min ) were initiated at 120 min . and maintained throughout the study to assess the rate of gp and glucose uptake based on the tracer - dilution methodology .
( steady - state basal period ) , the rate of gp ( mg / kg min . ) and plasma glucose levels ( mm ) were 11.9  0.5 and 8.3  0.1 ( i.c.v .
lactate ) , 12.6  0.2 and 8.2  0.2 ( mbh vehicle + i.v .
lactate ) and 12.2  1.2 and 8.9  0.6 ( mbh oxa + i.v .
lactate ) . a pancreatic clamp was performed in the final 2 hrs of the study starting at 240 min ; a continuous infusion of insulin ( 1.5 mu / kg min . ) and somatostatin ( 3 g / kg min . )
was administered , and a variable infusion of a 25% glucose solution was started and periodically adjusted to maintain the plasma glucose concentration at 8 mm .
saline ) were performed , and the gp during the clamps in both groups ( gp : i.c.v .
plasma samples for the determination of lactate , insulin , adiponectin and glucagon concentrations were obtained at 10-min .
somatostatin . in the presence of systemic lactate elevation , direct inhibition of lactate metabolism within the mediobasal hypothalamus ( mbh ) via mbh administration of oxamate ( compared to control ) ( b ) decreased exogenous glucose infusion rate and ( c ) increased gp during the final 30 min .
0.001 versus lactate with mbh vehicle . central sensing mechanisms of circulating lactate regulate glucose production ( gp ) . in the presence of systemic elevation of lactate , direct inhibition of central lactate metabolism via i.c.v .
administration of lactate dehydrogenase inhibitor oxamate ( oxa ) ( compared to control ) led to a marked ( a ) decrease in exogenous glucose infusion rate and ( b ) increase in gp during the final 30 min .
administration of blocker glibenclamide ( a ) decreased exogenous glucose infusion rate and ( b ) increased gp in response to systemic lactate infusions .
plasma lactate concentrations were determined by a kit in accordance with manufacturer s instructions ( sigma diagnostics .
plasma glucose concentrations were measured by the glucose oxidase method ( glucose analyzer , analox instruments , lunenberg , ma , usa ) .
statistical analysis was done by anova or unpaired student s t - test as appropriate .
here we first examined whether selectively blocking the central effects of lactate through the i.c.v .
administration of lactate dehydrogenase inhibitor oxa or katp channel blocker gli is sufficient to alter the metabolic response of i.v .
. systemic infusion of lactate at 100 mol / kg min . for 4 hrs elevated plasma lactate by 2- to 2.5-fold compared to i.v .
1c ) . in the final 2 hrs of the lactate elevation , we performed pancreatic - insulin clamps with tracer dilution methodology to assess the effects of i.v .
lactate on glucose kinetics independent of changes in plasma gluco - regulatory hormones ( fig .
lactate infusion did not increase gp during the pancreatic clamps when plasma glucose ( fig .
1e and f ) were maintained near basal levels . to assess whether central sensing mechanisms restrain plasma lactate from elevating gp , we administered i.c.v .
f ) , central oxa administration decreased exogenous glucose infusion rate required to maintain euglycaemia ( fig .
this decrease in glucose infusion rate was due entirely to an elevation of gp ( fig .
together , these data indicate that direct inhibition of central lactate metabolism disrupted glucose homeostasis and increased gp in the presence of systemic lactate elevations . to complement the function findings obtained with i.c.v .
katp channels blocker gli to alternatively negate central lactate effects and examine whether central sensing mechanisms restrain plasma lactate to increase gp . similar to the effects of i.c.v .
oxa , central gli administration decreased the exogenous glucose infusion rate required to maintain euglycaemia ( fig .
2a ) in the presence of similar degree of plasma lactate elevation as observed in the other groups ( fig .
the drop in glucose infusion rate was caused by an increase in gp ( fig .
these findings indicate that direct inhibition of central katp channels disrupted glucose homeostasis in response to systemic lactate elevation .
to investigate the central neuroanatomical localization of the cns - sensing mechanisms of circulating lactate on gp , we negated the lactate - sensing mechanisms specifically within the mbh .
lactate infusions ( 100 mol / kg min . ) with the administration of oxa ( 50 mm ) bilaterally into the mbh ( fig .
, mbh oxa administration decreased glucose infusion rate required to maintain euglycaemia in the same degree of plasma lactate elevation ( fig .
again , the decrease in glucose infusion rate was due to increased gp ( fig .
3d ) . thus , direct inhibition of lactate metabolism within the mbh disrupted glucose homeostasis and increased gp in the presence of systemic lactate infusions .
it remains to be assessed whether the hypothalamic restraining effect on gp in response to circulating lactate is mediated by an inhibition of hepatic glycogenolysis and/or gluconeogenesis .
the hypothalamus has been recently demonstrated to detect a rise in both hormones and nutrients to regulate peripheral metabolic processes [ 9 , 15 , 1929 ] . specifically , direct activation of either ( i ) insulin / leptin signalling pathways [ 19 , 2125 , 28 , 29 ] or ( ii ) lipid / lactate metabolism [ 9 , 15 , 30 ] in the hypothalamus regulates gp .
furthermore , the demonstration of the ability of the hypothalamus to sense circulating lipids to lower hepatic glycogenolysis and restrain gp ( 9 ) has added to the existing knowledge of nutrient - sensing mechanisms in the body .
these findings , at least in rodents , suggest that the hypothalamus senses circulating lipids to counteract the direct stimulatory effects of lipids on hepatic gluconeogenesis . more importantly ,
the hypothalamic lipid - sensing mechanisms are disrupted in the early onset of diet - induced obese rodents , leading to a rise in gp and glucose levels induced by circulating lipids .
these observations , if extended to human beings , could in part begin to address the inability of the body to compensate for a lipid - induced increase in gluconeogenesis in type 2 diabetes . in direct contrast to lipid - sensing mechanisms , the body has the ability to inhibit hepatic glycogenolysis and restrain gp to compensate for the circulating lactate - induced increase in hepatic gluconeogenesis in both normal individuals and metabolic stress individuals with hyperglycaemia [ 8 , 12 ] . in an attempt to elucidate the underlying mechanisms of lactate - sensing that are responsible for this metabolic restraining effect on gp , we assessed whether the hypothalamus senses an elevation of plasma lactate by 2- to 2.5-fold for 4 hrs to restrain gp and maintain glucose homeostasis in normal rodents .
lactate infusion on glucose metabolism during pancreatic clamps when gluco - regulatory hormones ( fig .
consistent with previous reports in human beings , an elevation of circulating gluconeogenic substrate precursor lactate did not increase gp .
we postulated that the hypothalamic lactate - sensing mechanisms are responsible for this glucose homeostatic control .
if this is true , direct inhibition of cns / hypothalamic lactate - sensing mechanisms should disrupt glucose homeostasis and lead to an elevation of gp in the presence of systemic lactate infusion . to test this hypothesis ,
we directly inhibited cns / hypothalamic lactate - sensing mechanisms that regulate gp by three independent approaches . during the pancreatic clamps ,
all three experimental interventions in the brain ( comparing to control ) led to a marked increase in gp in response to systemic lactate infusion .
together , these data indicated that hypothalamic sensing mechanisms restrain systemic lactate to increase gp and is required to maintain glucose homeostasis .
this is the first report that demonstrates the ability of the hypothalamus to sense circulating lactate to restrain gp and maintain glucose homeostasis in normal rodents . in light of the fact that ( i ) direct central lactate administration lowers gp in normal and early onset of diabetic and obese rodents [ 14 , 15 ] , ( ii ) an elevation of circulating lactate
does not increase gp in normal individuals and in patients with hyperglycaemia [ 8 , 12 ] , we postulate that the hypothalamic nutrient sensing mechanisms of circulating lactate are intact in at least the early onset of diabetes and obesity . however , future studies are required to address this hypothesis . in summary
, our data suggest that therapeutic strategies designed to activate cns lactate - sensing mechanisms by physiological route could eventually be proven useful to maintain glucose homeostasis in vivo .
 emerging studies indicate that hypothalamic hormonal signalling pathways and nutrient metabolism regulate glucose homeostasis in rodents . 
 although hypothalamic lactate - sensing mechanisms have been described to lower glucose production ( gp ) , it is currently unknown whether the hypothalamus senses lactate in the blood circulation to regulate gp and maintain glucose homeostasis in vivo . to examine 
 whether hypothalamic sensing of circulating lactate is required to regulate gp , we infused intravenous ( i.v . ) 
 lactate in the absence or presence of inhibition of central / hypothalamic lactate - sensing mechanisms in normal rodents . 
 inhibition of central / hypothalamic lactate - sensing mechanisms was achieved by three independent approaches . 
 tracer - dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of i.v . and central / hypothalamic administrations on glucose metabolism in vivo . 
 in the presence of physiologically relevant increases in the levels of plasma lactate , inhibition of central lactate - sensing mechanisms by lactate dehydrogenase inhibitor oxamate ( oxa ) or atp - sensitive potassium channels blocker glibenclamide increased gp . 
 furthermore , direct administration of oxa into the mediobasal hypothalamus increased gp in the presence of similar elevation of circulating lactate . together , these data indicate that hypothalamic sensing of circulating lactate regulates gp and is required to maintain glucose homeostasis . 
Introduction
Methods
Animal preparation
Clamp procedure in rats
Biochemical analyses
Statistical analysis
Result
Discussion