Hepatic metabolism of glucose in the adaptation to the transition period in the dairy cow

Transcription

1 Hepatic metabolism of glucose in the adaptation to the transition period in the dairy cow Harald M. Hammon Nutritional Physiology Oskar Kellner Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany

2 Overview Introduction: Importance of glucose supply for milk production 2

3 Glucose metabolism in dairy cows Glucose demand: Maintenance: ca g/(cow x day) 130% of maintenance at the end of pregnancy Milk production: ca g/kg milk A dairy cow with more than 50 kg milk/day has to produce more than kg glucose per day Danfær, 1994 Flachowsky,

4 Glucose metabolism in dairy cows Glucose supply: Intestinal absorption of glucose in ruminants is low 4

5 Passage of starch throughout the gastrointestinal tract in dairy cows Barley 60% Barley 90% Maize 60% Maize 90% Starch passage (kg/day) The increment of 1 g starch/(day x BW) digested in the small intestine leads to Intake g glucose/(day x BW) net portal appearance Duodenum Ileum Faeces Loncke et al., 2009 Reynolds et al.,

6 Passage of starch throughout the gastrointestinal tract in dairy cows Whole body glucose appearance rate (Ra) is about mmol/h ( kg/d) in cows with kg milk/d Splanchnic tissue extracts 15% of Ra glucose Net portal glucose flux: ca. 75 mmol/h (ca. 323 g/d) = ca. 10% of Ra glucose Loncke et. al 2009 Aschenbach et al., 2010; Galindo et al.,

7 Glucose metabolism in dairy cows Glucose supply: Intestinal absorption of glucose in ruminants is low Endogenous glucose production (egp; glycogenolysis and gluconeogenesis) supplies most of the glucose Hepatic net flux of glucose range from 456 to 840 mmol/h (= kg/d) in cows with kg milk/d Mammary gland extracts 73% of Ra glucose Reynolds et al., 2003; Aschenbach et al., 2010; Galindo et al.,

8 Glucose metabolism: Relationship between glucose production and milk production in cows GP, mol/(min x BW 0,75 ) GOx, % of egp Glucose oxidation 45 * Milk yield, kg y = 0.84x + 34 R 2 = 0.61 * P < 0.05 Low Milk Production High Milk Production Hammon et al.,

9 Endogenous glucose production (egp) and Lactation week wk 3 glucose oxidation (GOx) during early lactation in cows wk 9 Milk yield, kg Milk yield * wk 3 wk 9 Glucose production, mmol/(kg x h) 1.4 egp * wk 3 wk 9 * P < 0.05 Dry matter intake, kg 25 Dry matter intake wk 3 wk 9 Glucose oxidation, mmol/(kg x h) GOx * * wk 3 wk 9 Hötger et al.,

10 Irreversible loss rate of glucose without loss in milk lactose in cows around calving ILR of glucose excluding loss in milk lactose, mmol/h Bruckental et al., 1980 Baird et al., 1983 Bennink et al., 1972 Larsen and Kristensen, Time relative to parturition, day Larsen and Kristensen,

11 The increase of endogenous glucose production around calving is not sufficient to cover glucose demands for milk production Dairy cows change substrate utilisation in peripheral tissues to spare glucose Bauman, 2000; Stangassinger and Sallmann, 2004; Larsen and Kristensen,

12 Take home message An increase in hepatic glucose production and a reduction in peripheral glucose utilisation (besides the mammary gland) are the most important changes in glucose metabolism around calving to cover the glucose demand for milk production Glucose absorption in the small intestine is of minor importance 12

13 Overview Introduction: Importance of glucose supply for milk production Hepatic glucose production during the transition from late pregnancy to early lactation 13

14 Hepatic glucose flux around calving Hepatic Glucose Flux [mmol/h] kg/d 3.6 kg/d In cows with ca. 45 kg milk/d Time Days relative around to calving, day Reynolds et al.,

15 Concentration of glycogen in the liver around the time of calving Glycogen in the liver, % of dry matter ca g Glucose Time relative to calving, day Duske et al.,

16 Glucose production in liver around calving: Changes in substrate utilisation for gluconeogenesis Net-flux of substrates for gluconeogenesis Hepatic glucose flux Day 19 ap Day 11 pp Day 33 pp Day 83 pp 4% 3% 6% 4% 24% 1.3 kg/d 24% 2.7 kg/d 69% 66% 2% 2% 2% 1% 10% 20% 3.4 kg/d 3.6 kg/d 76% 87% Propionate Lactate Alanin Glycerol Hepatic glucose release and propionate utilisation for glucose increase with lactation ap = antepartum pp = postpartum Reynolds et al.,

17 Max. contribution of potential precursors to hepatic glucose release Additional amino acid supply is not directed toward increased hepatic glucose synthesis! Propionate Galindo et al., 2015 Lactate NEAA EAA + Glycerol 17 Larsen and Kristensen, 2013

18 Hepatic removal of lactate as percent of total influx Hepatic removal of lactate, % of total influx Time relative to calving, day Larsen and Kristensen,

19 Where does the lactate for gluconeogenesis postpartum come from? Skeletal muscle: Increased glycolysis and decreased glycogenesis and oxidation in the Krebs cycle of muscle tissue after calving and activation of the Cori cycle (Reynolds et al., 2003; Kuhla et al., 2011; Larsen and Kristensen, 2013) Gastrointestinal tract: Increased rumen lactate production from propionate or valerate, after starch feeding, but the conversion is low (Stangassinger, 1997; Aschenbach et al., 2010; Nozière et al., 2010) 19

20 Take home message An increased hepatic lactate utilisation for gluconeogenesis compensates for the relative reduction of propionate utilisation around calving Amino acids and glycerol are obviously of minor importance for propionate compensation 20

21 Overview Introduction: Importance of glucose supply for milk production Hepatic glucose production during the transition from late pregnancy to early lactation Enzymes involved in hepatic gluconeogenesis Changes in gene expression during the transition period and substrate regulation 21

22 Key-enzymes that are regulating gluconeogenesis 22

23 PDH Actetyl-CoA Pyruvate carboxylase (PC): Acet Catalyses the conversion of pyruvate to oxaloacetate: Pyruvate + CO 2 + ATP + H 2 O Oxaloacetate + ADP + P i Activated allosterically by acetyl-coa; Acetyl-CoA inhibits pyruvate dehydrogenase (PDH) at the same time Regulated by transcription, mrna degradation or both; PC is under endocrine control (glucagon, insulin) in monogastric species 23

24 Phosphoenolpyruvate carboxykinase (PEPCK): Catalyses the conversion of oxaloacetate to phosphoenolpyruvate: Oxaloacetate + GTP Phosphoenolpyruvate + CO 2 + GDP Two forms: Cytosolic (PEPCKc; PCK1) and mitochondrial (PEPCKm; PCK2) form; PEPCKc and PEPCKm are evenly distributed in cows Activity is NOT regulated allosterically or via phosphorylation, but by transcription of the PEPCK gene and mrna stability; Textbooks describe substrate and endocrine regulation for PEPCKc, but PEPCKm is constitutively expressed 24

25 Glucose-6-phosphatase (G6Pase; G6PC): Catalyses the conversion of glucose-6-phosphate to glucose: Glucose 6-phosphate + H 2 O Glucose + P i G6Pase catalyses the final step of the gluconeogenesis and is a prerequisite in tissues with endogenous glucose synthesis G6PC is activated by glucose 6-phosphate and is under endocrine control (glucagon) 25

26 Expression of genes involved in endogenous glucose production in the liver of dairy cows with low and high milk production mrna relative to HKG G6Pase * mrna relative to HKG PC * Low Milk High Milk * P < 0.05 P < 0.1 mrna relative to HKG PEPCKc mrna relative to HKG PEPCKm Hammon et al.,

27 Effect of short term feed restriction on mrna expression of gluconeogenic enzymes in liver of dairy cows Feed restricted Control d 1 to d 5 Ad libitum intake d 6 to d % Ad libitum d 11 to d 20 Refeeding * P < 0.05; differences form d 5 Velez and Donkin,

28 Effect of fasting and glucosuria on gluconeogenesis and glucose irreversible loss rate in goats Fed Fasted The increase of PC mrna abundance during mmol/l Fasted and glucosuria fasting in dairy cows is probably not a result of Glucose in blood plasma mmol/(h x kg 0.75 ) Gluconeogenesis Glucose ILR 1) C3-Recycling ) ILR = Irreversible Loss Rate increased hepatic gluconeogenesis! Stangassinger and Bendisch,

29 mrna Abundance of pyruvate carboxylase (PC) and cytosolic phosphoenolpyruvate carboxykinase (PEPCKc) during the time of calving in the liver of dairy cows PC PEPCKc Greenfield et al.,

30 mrna Abundance of PC, PCK1, PCK2 and G6PC (G6Pase) during the time of calving in the liver of dairy cows G6PC mrna PC mrna PCK1 mrna PCK2 mrna Time relative to calving, day Weber et al.,

31 Expression of PCK1 and PCK2 depends on substrate availability NADH is needed for ongoing gluconeogenesis Synthesis of glyceraldehyd-3-phosphate 31

32 Take home message PC, PEPCKc and PEPCKm and G6Pase are key-enzymes that are involved in hepatic regulation of gluconeogenesis Fasting and the transition period around calving differently affect gene expression of these enzymes: There is an immediate increase of PC and PEPCKm, but a delayed increase of PEPCKc after calving These differences may be best explained by the shift in substrate utilisation with a reduced propionate portion and an increased lactate portion: Lactate PC, PEPCKm Propionate PEPCKc 32

33 Overview Introduction: Importance of glucose supply for milk production Hepatic glucose production during the transition from late pregnancy to early lactation Enzymes involved in hepatic gluconeogenesis Changes in gene expression during the transition period and substrate regulation Endocrine control 33

34 Endocrine regulation of gluconeogenesis in ruminants As in monogastric species, glucagon, catecholamines and cortisol stimulate endogenous glucose production (gluconeogenesis and glycogenolysis) and insulin is the main inhibitor (McDowell, 1983; Brockman and Laarveld, 1986; Danfær, 1994; Donkin, 1999) In ruminants, glucose synthesis from propionate seems to be less under endocrine (insulin) control (when compared to lactate or amino acids) (Donkin and Armentano, 1995, Smith et. al., 2008) 34

35 Plasma glucose and related hormones in dairy cows around time of calving Glucose, mmol/l Glucagon/Insulin, µg/l mol/mol Glucagon, ng/l Time relative to calving, day Weber et al.,

36 Effects of insulin treatment on gluconeogenic enzyme mrna expression in liver before and after calving Euglycemic-hyperinsulinemic clamps in dairy cows at week 5 antepartum (ap) and Insulin week 3 postpartum (pp): 40.0 basal steady state Time of clamp: wk 5 antepartum wk 3 postpartum Insulin, µg/l basal steady state * Insulin Insulin, µg/l Time, min * Glucose, mmol/l basal steady state 5 4 Time, min 2 Glucose * * Time, min Liver biopsy at wk 5 ap and wk 3 pp *P < 0.05 Weber et al.,

37 Effects of insulin treatment on gluconeogenic enzyme mrna expression PC mrna PC PCK2 mrna PEPCKm * * Insulin: P < 0.05 Time: P < Insulin: Insulin: PCK1 mrna Antepartum PEPCKc * Postpartum * G6PC mrna Antepartum G6Pase * Postpartum * Insulin: Antepartum Postpartum Insulin: Antepartum Postpartum 37 Weber et al., 2017

38 Endogenous glucose production (egp) postpartum before and after the euglycemic-hyperinsulinemic clamp 1.5 egp egp, mmol/(kg x h) % of pre-clamp egp 0.0 Insulin: - + Postpartum Weber et al.,

39 Glucagon differently affects hepatic mrna abundance of PEPCK and PC in mid-lactation dairy cows Control Glucagon Glucagon PEPCKc Insulin PC She et al.,

40 Regulation of PEPCKc (PCK1) promotor: In vitro studies in rat hepatoma H4IIE cells 40 Zhang et al., 2016

41 Take home message The degree of inhibition of hepatic gluconeogenic gene expression by insulin differs among enzymes in the transition period: Cytosolic PEPCK and G6Pase show a distinct inhibition Mitochondial PEPCK and PC show a weak or no inhibition Stimulation of gluconeogenic gene expression in liver by glucagon obviously depends on the insulin status and on the glucagon/insulin ratio in blood plasma Stimulation of PEPCKc gene expression by propionate is probably less inhibited by insulin 41

42 Overview Introduction: Importance of glucose supply for milk production Hepatic glucose production during the transition from late pregnancy to early lactation Enzymes involved in hepatic gluconeogenesis Changes in gene expression during the transition period and substrate regulation Endocrine control Inter-individual variation in cows during the transition period 42

43 Inter-individual variation of body condition before calving Different strategies to adapt energy metabolism during early lactation Inter-individual differences are reflected by: Adipose tissue mobilisation Hepatic fat accumulation Feed intake Metabolic and endocrine responses 43

44 Variation of liver fat concentration (LFC) in Holstein cows with same feeding protocol and herd management LFC Back Fat NEFA Thickness LFC, % of DM tissue Time relative to calving, day NEFA, BFT, mmol/l Time relative to to calving, week day LFC: P < 0.05 LFC x Time: P < 0.05 LFC: P < 0.05 Time: P < 0.05 LFC: P < 0.05 (prepartum) Time: P < 0.05 Weber et al.,

45 Dry matter intake (DMI), energy-corrected milk (ECM) and energy balance (EB) in cows with variable fat mobilisation DMI, kg/d DMI LFC: P < 0.05 Time: P < 0.05 Liver fat concentration/body fat mobilisation: High Low Weber et al., ECM, kg/d Time relative to calving, week ECM -50 Time: P < 0.05 LFC: P < Time: P < 0.05 EB, MJ NE L /d Time relative to calving, week Time relative to calving, week EB 45

46 NEFA, glucose and the glucagon/insulin ratio in blood plasma of cows with variable fat mobilisation Liver fat concentration/body fat mobilisation: High Glucose Glucagon/Insulin Low Glucose, mmol/l Glucagon/Insulin, mol/mol Time relative to calving, day Time relative to calving, day LFC: P < 0.05 Time: P < 0.05 LFC: P < 0.05 (postpartum) Time: P < 0.05 Weber et al.,

47 Hepatic mrna abundance of pyruvate carboxylase (PC) in cows with variable fat mobilisation PC mrna PC mrna * * High LFC Low LFC * P < Time relative to calving, day Weber et al.,

48 Hepatic mrna abundance of cytosolic and mitochondrial PEPCK in cows with variable fat mobilisation High LFC Low LFC PEPCKc mrna PEPCKm mrna PCK1 mrna PCK2 mrna Time relative to calving, day Time relative to calving, day Weber et al.,

49 Greater Lean Dairy cows with reduced feed intake and energy fat balance oxidation Fat during the transition period indicate an elevated gene expression of pyruvate carboxylase (PC), but not phosphoenolpyruvate carboxykinase (PEPCK; cytosolic and mitochondrial) The increase in PC mrna abundance after calving might be related to elevated Krebs cycling and fatty acid oxidation, because endogenous glucose production does not differ between fat and lean cows (Weber et al., 2016) and fat cows oxidise more fat (Börner et al., 2013) Respiration Take home quotient message Börner et al.,

50 Thanks to C. Weber C. Schäff U. Kautzsch S. Görs B. Kuhla C. C. Metges C. Reiko C. Fiedler K. Hötger A. K. Lohrenz G. Nürnberg A. Tuchscherer E. Kanitz W. Otten B. Losand M. Röntgen B.Stabenow, K. Witt and their staff R. M. Bruckmaier, University of Berne H. Sauerwein, University of Bonn FBN Colloquium: Colostrum and milk feeding in calves

51 End Leibniz-Institut for Farm Animal Biology FBN Wilhelm-Stahl-Allee Dummerstorf Harald M. Hammon Phone: Fax: hammon@fbn-dummerstorf.de Internet: Thank you for your attention!