Tierärztliche Hochschule Hannover
Insulin-Sensitivität und Insulin-Response nach einer einmaligen Dexamethasonbehandlung bei
Milchkühen in der Frühlaktation
INAUGURAL – DISSERTATION zur Erlangung des Grades eines Doktors
der Veterinärmedizin
- Doctor medicinae veterinariae - ( Dr. med. vet. )
vorgelegt von
Marián Kusenda
Bardejov
Slowakei
Wissenschaftliche Betreuung: Univ.-Prof. Dr. Jürgen Rehage Klinik für Rinder
1. Gutachter: Univ.-Prof. Dr. Jürgen Rehage
Klinik für Rinder
2. Gutachterin: Univ.-Prof. Dr. Korinna Huber
Physiologisches Institut
Mojej rodine
Inhaltsverzeichnis Seite
1. EINLEITENDE LITERATURÜBERSICHT 1
1.1. Metabolische Risiken im peripartalen Zeitraum 1
1.2. Prävention und Therapie des „Fatty liver syndroms“ 2 1.3. Dexamethason in der Therapie der Stoffwechselstörungen 3 1.4. Insulinresistenz: Definition und Quantifizierung 4
1.5. Fragestellung und Zielsetzung dieser Arbeit 5
Literatur 7
2. KAPITEL 1 21
Effects of a single dose of dexamethasone-21-isonicotinate and dexamethasone-21-disodiumphosphate on the peripheral insulin action of dairy cows in early lactation
3. KAPITEL 2 46
Evaluation of surrogate indices of insulin sensitivity by hyperinsulinemic- euglycemic glucose clamps in dairy cows
4. ABSCHLIEßENDE DISKUSSION 66
Literatur 70
5. ZUSAMMENFASSUNG 72
6. SUMMARY 75
Abkürzungsverzeichnis
Literaturübersicht, Abschließende Diskussion und Zusammenfassung
ATP-ase Adenosintriphophatase
BHB ß-Hydroxybutyrat
bzw. beziehungsweise
CG Kontrollgruppe (NaCl)
DG-I Dexamethason-Gruppe-I (Dexamethason-21-isonicotinat)
DG-II Dexamethasone-Gruppe-II (Dexamethason-21-dinatriumphosphat)
g Gramm
h Stunden
HEC hyperinsulinämisch-euglycämischer Clamp HOMA Homeostasis model assessment
i. m. intra muskulär
ISI Insulin-Sensitivitätsindex
ITT Insulintoleranz-Test
ivGTT intravenöser Glucosetoleranz-Test
K+ Kalium
KCl Kaliumchlorid
kg Kilogramm
L Liter
log Logarithmus
Min Minute
mL Milliliter
mmol Millimol
mU Milliunit
Na+ Natrium
NaCl Natriumchlorid (Kochsalz) NEFA nicht veresterte Fettsäuren
QUICKI Quantitative insulin sensitivity check index
r Korrelationskoefizient
SD Standardabweichung
sog. sogenannt
TAG Triglyceride
TL Gesamtlipide
x Mittelwert
z. B. zum Beispiel
µg Mikrogramm
Kapitel 1, 2 und Summary
BHB ß-hydroxybutyrate
CG control group (saline)
DG-I dexamethasone group-I (dexamethasone-21-isonicotinate)
DG-II dexamethasone group-II (dexamethasone-21-disodiumphosphate)
FW fresh weight
GLUT-4 glucose transporter
HEC hyperinsulinemic-euglycemic clamp HOMA Homeostasis model assessment HSL hormone sensitive lipase ISI insulin sensitivity index
k insulin elimination rate constant LDA left displacement of the abomasum
QUICKI Quantitative insulin sensitivity check index QUICKI-glycerol QUICKI including glycerol
RQUICKI revised QUICKI
SSGIR steady-state glucose infusion rate SSIC steady-state insulin concentration
TAG triacylglycerid
TL total lipid
1. EINLEITENDE LITERATURÜBERSICHT
1. 1. Metabolische Risiken im peripartalen Zeitraum
Der Zeitraum zwischen dem Ende der Trächtigkeit und der Frühlaktation - „transition period“
(Drackley, 1999) erfordert bei Hochleistungskühen substantielle koordinierte Adaptationsprozesse des Endokriniums. Während der Nährstoffbedarf des Fötus in den letzten Wochen der Trächtigkeit exponentiell steigt, sinkt die Futteraufnahme infolge der hormonellen ante-partalen Umstellungen deutlich (Grummer, 1995; Herdt, 2000; Ingvartsen u. Andersen, 2000). Die nach der Geburt einsetzende Milchproduktion erhöht weiterhin den Energiebedarf; die Futteraufnahme steigt andererseits nach der Kalbung nur sehr allmählich an (Reynolds et al., 2003; Grummer, 2008). Es resultiert eine negative Energiebilanz. Das Energiedefizit wird durch eine Mobilisierung körpereigener Reserven ausgeglichen, und zwar insbesondere durch die verstärkte Mobilisierung der Triglyceride (TAG) im Fettgewebe (Bell, 1995; Herdt, 2000). Damit kommt es zu einem massiven Anstieg der Plasmakonzentration der nicht-veresterten Fettsäuren (NEFA) und einer konsekutiv gesteigerten Aufnahme in der Leber, wo sie teils erneut zu TAG re-verestert werden (Grummer, 1993). Die erhöhte Neubildung der TAG und deren unter Umständen niedrige Ausschleusung aus dem Cytosol (an Lipoproteine gebunden) führt zu einer mehr oder weniger massiven Leberverfettung (Mazur et al., 1989; Grummer, 1993; Gruffat et al., 1996; Drackley, 1999; Grummer, 2008).
In den ersten Laktationswochen sind etwa 20 bis zu über 50% der Milchkühe in moderatem bis schwerem Umfang von einem derartigen „Fatty liver syndrome“ betroffen (Gerloff et al., 1986; Mazur et al., 1988; Jorritsma et al., 2000; Jorritsma et al., 2001; Raoofi et al., 2001).
Unter physiologischen Bedingungen werden die von der Leber aufgenommen NEFA in den Mitochondrien vollständig über den oxidativen Stoffwechsel abgebaut. Übersteigt die Anflutung von NEFA aus der Peripherie die Einschleusungskapazität in den Citratzyklus, so können diese lediglich partiell oxidiert werden; als alternativer Stoffwechselweg bietet sich dann die Ketogenese im Mitochondrium des Hepatozyten an. So entsteht unter anderem ß- Hydroxybutyrat (BHB; Herdt, 2007). Die Prävalenz der Ketose kann in den ersten zwei Monaten nach der Abkalbung mehr als 30% erreichen (Duffield, 2000).
Die negativen Konsequenzen einer exzessiven Lipomobilisation mit Leberverfettung und/oder Ketose bestehen in direkten Gesundheitsschäden und indirekt induzierten, schlechteren Produktions- und Reproduktionsergebnissen und sind intensiv untersucht worden (Grummer, 1993; Rehage et al., 1996; Wensing et al., 1997; Wentink et al., 1997; Drackley, 1999;
Jorritsma et al., 2000; Ingvartsen et al., 2003; Bobe et al., 2004; Geelen u. Wensing, 2006;
Mulligan u. Doherty, 2008; Duffield et al., 2009).
1. 2. Prävention und Therapie des „Fatty liver syndroms“
Die Prävention und Therapie des „Fatty liver syndroms“ (Gruffat et al., 1996) sind aufgrund der entstehenden Kosten durch Tierverluste, Leistungsminderung und Behandlungskosten von großer Bedeutung (Kelton et al., 1998; Duffield, 2000; Bobe et al., 2004). Im Wesentlichen konzentrieren sich die prophylaktischen und therapeutischen Maßnahmen auf drei Ziele: (1) Reduktion der Konzentration der im Blut zirkulierenden NEFA durch eine Hemmung der Lipolyse im Fettgewebe; (2) Unterstützung der vollständigen NEFA-Oxidation in der Leber, und (3) Erhöhung der Ausschleusungsrate der an Lipoproteine gebundenen TAG aus der Leber (Grummer, 2008).
Im Rahmen der Prophylaxe belegen mehrere Studien positive Effekte der Nutzung verschiedener Fütterungssupplemente in der peripartalen Phase. So soll die Verfütterung von Propylenglykol (Nielsen u. Ingvartsen, 2004), Niacin (Waterman et al., 1972; French, 2004), Monensin (Duffield et al., 1998; Zahra et al., 2006), Methionin (Hayirli et al., 2001) wesentlich die Lipolyse hemmen - mit konsekutivem Abfall der NEFA und BHB sowie einer verminderten Konzentration der TAG im Lebergewebe.
Eine Hemmung der Leberverfettung und Ketogenese bzw. die Steigerung der NEFA- Oxidation soll vor allem durch die Verfütterung spezifischer Fettsäuren begünstigt werden.
Entsprechende Effekte z. B. von Leinsamenöl wurden auf der Grundlage von in vitro- Studien postuliert (Mashek u. Grummer, 2003). Diese Effekte wurden experimentell bei Infusionsversuchen an Milchkühen bestätigt (Mashek et al., 2005). Im Fütterungsversuch mit Calciumsalzen der Fettsäuren wurde ebenfalls eine positive Wirkung beobachtet (Patton et al., 2004).
Die Ausschleusung der in der Leber re-veresterten TAG soll durch die peripartale Supplementation von Pansen-geschütztem Cholin erhöht werden können (Elek et al., 2004;
Cooke et al., 2007).
In der Therapie von Fettleber und Ketose ist die Wahl des Mittels und dessen Erfolg abhängig vom Grad der Erkrankung (Bobe et al., 2004). In der Praxis kommen gegenwärtig bei leicht erkrankten Tieren vor allem glucoplastische Substanzen zum Einsatz. Die Wirksamkeit von Propylenglycol (Christensen et al., 1997; Pickett et al., 2003), Natriumpropionat (Schultz, 1952), Calciumpropionat (Goff et al., 1996) und Glycerin (Pieper u. Pieper, 2009) wurde wissenschaftlich bewiesen. Bei moderat bis schwer kranken Tieren hat sich vor allem die Infusion von Glucose (als Bolusinfusion z. B. 500 mL 40%; als Dauerinfusion z. B. 2 L 40%
über 24 h) etabliert (Stöber u. Scholz, 1991). Von der Gruppe metabolisch wirksamer Hormone sind nur die Glucocorticoide zugelassen und haben sich vor allem als Therapeutikum bei den ketotischen Kühen durchgesetzt (Wierda et al., 1987; Shpigel et al., 1996).
1. 3. Dexamethason in der Therapie der Stoffwechselstörungen
Vor mehr als 60 Jahren berichtete Shaw (1947) über Behandlungserfolge bei der Ketose der Kühe mittels Verabreichung von Nebennierenextrakten und führte damit erfolgreich die Glucocorticoide in die Stoffwechselbehandlung beim Rind ein. Dexamethason gehört heute zu den am häufigsten benutzten, synthetischen Formulierungen. Die klinische Wirksamkeit wurde wiederholt nachgewiesen und wird vor allem dem Anstieg des Glucosespiegels sowie der Beeinflussung der NEFA- und BHB-Konzentrationen im Blut zugeschrieben (Andersson u. Olsson, 1984; Wierda et al., 1987; Shpigel et al., 1996; Jorritsma et al., 2004; Fürll u.
Jäckel, 2005). Die Mechanismen sind jedoch weitgehend unbekannt. Als Erklärung des hyperglycämischen Effekts wurde dem Dexamethason (und generell den Glucocorticoiden) in früheren Jahren eine Beeinflussung der Glucoseproduktion unterstellt. Diese Theorie wurde bestätigt von mehreren älteren, an Ratten durchgeführten in vivo- (Seitz et al., 1976; Allan u.
Titheradge, 1984; Fleig et al., 1984) und in vitro-Studien (Exton et al., 1976; Krone et al., 1976; Sistare u. Haynes, 1985; Jones et al., 1993), die eine Steigerung der Gluconeogenese-
Untersuchungen an Kälbern mittels Clamp-Technik in Kombination mit der Infusion von Tracer-markierter Glucose (Scheuer et al., 2005) und Studien an erwachsenen Kühen mit katheterisierten hepatischen Gefäßen (Starke et al., 2009) keine Änderungen der endogenen Glucoseproduktion nach einer Applikation von Dexamethason festgestellt. Diese Ergebnisse lassen vermuten, dass die Dexamethason-induzierte Hyperglycämie eher durch eine verminderte periphere Glucoseaufnahme – und damit eine sog. Insulinresistenz - verursacht ist, wie sie bereits auch bei Mensch (Tappy et al., 1994; Nicod et al., 2003; Binnert et al., 2004) und Pferd (Tiley et al., 2007, 2008; Haffner et al., 2009) beschrieben wurde.
Beim Rind existieren nur zwei relevante Arbeiten, die den Einfluss der Glucocorticoide auf die periphere Insulinwirkung beschreiben. So induziert eine einmalige Flumethason-Injektion bei drei Monate alten Kälbern (Sternbauer et al., 1998) bzw. eine wiederholte Dexamethason- Verabreichung bei neugeborenen Kälbern (Scheuer et al., 2005) eine deutliche Verminderung der peripheren Glucoseaufnahme und damit eine periphere Insulinresistenz. Vergleichbare Untersuchungen an Milchkühen fehlen bislang, obwohl der Glucosestoffwechsel ruminierender Rinder sich von dem nicht ruminierender Kälber wesentlich unterscheidet.
1. 4. Insulinresistenz: Definition und Quantifizierung
Eine Insulinresistenz ist definiert als eine Stoffwechselsituation, bei der eine physiologische oder erhöhte Insulinkonzentration lediglich einen subnormalen biologischen Effekt induziert (Kahn, 1978; Rizza et al., 1981a,b; Muniyappa et al. 2008). Eine Insulinresistenz kann sich als (a) verminderte Insulin-Sensitivität manifestieren, bei der eine höhere Insulinkonzentration erforderlich ist, um einen halbmaximalen biologischen Effekt zu erreichen und als (b) eine verminderte Insulin-Response, die durch eine verminderte maximale biologische Antwort auf die Insulinstimulation charakterisiert ist, oder (c) als eine Kombination einer verminderten Insulin-Sensitivität und Insulin-Response (Rizza et al., 1981a).
Die „Goldstandard-Methode“ zur Quantifizierung der peripheren Insulinresistenz in vivo ist der hyperinsulinämisch-euglycämische Clamp (HEC). Dieser ermöglicht eine präzise und direkte Messung der Insulin-stimulierten Glucose-Utilisation im peripheren Gewebe unter definierten steady-state Bedingungen (DeFronzo et al., 1979). Die Durchführung dieser
Insulinkonzentrationen erlaubt die Darstellung einer Dosis-Wirkung Kurve und ermöglicht so die Unterscheidung zwischen einer gestörten Insulin-Sensitivität bzw. Response (Rizza et al., 1981a). Die HEC-Methode wurde häufig und in verschiedenen Variationen beim Rind angewendet (Hostettler-Allen et al., 1994; Blum et al., 1999; Mashek et al., 2001; Sternbauer u. Luthman, 2002; Scheuer et al., 2005).
Alternativ zu dem relativ aufwendigen HEC wurden in der Humanmedizin indirekte Parameter etabliert, die eine einfachere Erfassung der Insulinresistenz ermöglichen sollen.
Auch diese haben in der Buiatrik bereits Anwendung gefunden. Dazu gehört der intravenöse Glucosetoleranz-Test (ivGTT; Opsomer et al., 1999; Holtenius et al., 2003; Pires et al., 2007) und der Insulintoleranz-Test (ITT; Pires et al., 2007; Kerestes et al., 2009). Da sich bei Anwendung dieser Methoden die Konzentrationen der zu messenden Metaboliten (insbesondere Glucose) ständig ändern und somit ein steady-state nicht erreicht wird, spiegeln die Ergebnisse die Wirkung des Insulins an den peripheren Geweben nur bedingt wieder.
Zwar erfordern ivGTT und ITT einen geringeren Aufwand von Material, Zeit und Arbeit, dennoch ist eine Durchführung nur unter experimentellen Bedingungen an einer begrenzten Zahl von Probanden möglich. Das gleiche Problem stellte sich auch in der Humanmedizin, wo im Rahmen epidemiologischer Studien über Diabetes-Prävalenz und prädisponierende Faktoren unkomplizierter zu erfassende Parameter notwendig waren. Dafür wurden von verschiedenen Autoren die Konzentrationen der NEFA, Glucose, des Insulins und des Glycerols bei nüchternen Probanden herangezogen, die logarithmiert in spezifische Formeln Eingang fanden (z. B. HOMA, Matthews et al., 1985; QUICKI, Katz et al., 2000; revised QUICKI, Perseghin et al., 2001; QUICKI-Glycerol, Rabasa-Lhoret et al., 2003). Es ist gegenwärtig jedoch völlig offen, ob derartige abgeleitete Hilfsparameter auch für die Beurteilung der Stoffwechselkonstellation von hochleistenden Milchkühen verwendbar sind.
1. 5. Fragestellung und Zielsetzung dieser Arbeit
Hauptziel der vorliegenden Arbeit war: (1) die Effekte einer einmaligen Dexamethasonbehandlung auf die periphere Insulin-Wirkung bezüglich des Glucose- und
Methoden zur Erfassung der Insulinresistenz durchzuführen. Die Arbeit wurde in zwei Studien eingeteilt.
In der ersten Studie wurden mittels der fünf konsekutiven hyperinsulinämisch- euglycämischen clamps bei klinisch gesunden, nicht behandelten sowie bei klinisch gesunden mit zwei gängigen Dexamethasonpräparaten behandelten Kühen in der Frühlaktation folgende Fragen untersucht:
1. Unterscheidet sich die Insulin-stimulierbare Glucoseaufnahme im peripheren Gewebe zwischen den mit Dexamethason behandelten und nicht behandelten Milchkühen?
2. Gibt es eventuelle Unterschiede in der Insulin-Sensitivität, Insulin-Response oder in deren Kombination?
3. Sind Unterschiede in der Abnahme der NEFA während der bei HEC entstehender Hyperinsulinämie zwischen den Gruppen nachweisbar?
4. Wie wirkt sich die Dexamethasonbehandlung auf den Lipidgehalt in der Leber aus?
5. Gibt es Unterschiede in dem Ausmaß der Wirkung von Dexamethason-21-isonicotinat und Dexamethason-21-dinatriumphosphat?
In der zweiten Studie, sollen bereits in der Humanmedizin etablierte Indices zur Erfassung der Insulinsensitivität hinsichtlich ihrer Aussagekraft für die Milchkühe evaluiert werden, wobei die Ergebnisse des HEC als Goldstandard dienen.
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2. KAPITEL 1
To be submitted
Tabellen und Abbildungen befinden sich am Ende des Manuskripts
Effects of a single dose of dexamethasone-21-isonicotinate and dexamethasone-21- disodiumphosphate on the peripheral insulin action of dairy cows in early lactation
Effects of a single dose of dexamethasone-21-isonicotinate and dexamethasone-21- disodiumphosphate on the peripheral insulin action of dairy cows in early lactation
M. Kusenda, A. Starke, M. Kaske, A. Haudum, M. Piechotta, and J. Rehage
Clinic for Cattle, University of Veterinary Medicine Hannover, 30173 Hannover, Germany
ABSTRACT
The aim of the present study was to characterise the changes in the peripheral insulin sensitivity and response after a single dose of two different dexamethasone formulations in dairy cows in early lactation and to assess the effect of the treatment on the hepatic lipid content.
Eighteen clinically healthy, multiparous German Holstein cows (2-4 weeks post partum, five days after right flank omentopexy performed due to left displacement of abomasum) were assigned to one of three treatment groups: dexamethasone group-I (DG-I; 40 µg/kg dexamethasone-21-isonicotinate i.m.; n = 6), dexamethasone group-II (DG-II; 40 µg/kg dexamethasone-21-disodiumphosphate i.m.; n = 6), control group (CG, 15 mL saline i.m., n = 6). After a withdrawal period of 14 hours for concentrate and corn silage, with each animal, five consecutive hyperinsulinemic-euglycemic clamps (HEC-I, II, III, IV and V with increasing doses of bovine insulin 0.1, 0.5, 2, 5, 10 mU/kg/min; each 120 min) were performed one day after administration of the drugs. A liver biopsy was performed immediately before treatment as well as two days afterwards. After dexamethasone treatment, mean plasma glucose concentrations were significantly higher in DG-I-cows (5.04 ± 0.66 mmol/L; mean ± SD) and DG-II-cows (7.27 ± 0.95 mmol/L) compared to CG- cows (3.52 ± 0.72 mmol/L). The increase was significantly higher in DG-II-cows compared to DG-I-cows (P < 0.001). Steady-state glucose infusion rates (SSGIR) were significantly lower in DG-I-cows (P < 0.01) and DG-II-cows (P < 0.05) compared to CG-cows during HEC-II and in DG-I-cows compared to CG-cows during HEC-IV (P < 0.05). The maximal insulin response, defined as SSGIR during HEC-V, was significantly lower in DG-I-cows compared to DG-II-cows (P < 0.05), but there were no differences between dexamethasone-treated and
significantly lower in dexamethasone-treated cows compared to CG-cows during HEC-I and HEC-II (P < 0.05). Dexamethasone treatment reduced the insulin-mediated relative decrease of circulating NEFA during HEC, but not before HEC-II (P < 0.05). The total lipid (TL) and triacylglycerides (TAG) content in the liver of DG-I- and DG-II-cows decreased two days after treatment. The relative decrease of TL and TAG in the liver of dexamethasone-treated cows was higher compared to CG-cows (P < 0.001).
In conclusion, both dexamethasone formulations exhibited comparable effects on glucose and lipid metabolism. The administration of dexamethasone induced insulin resistance due to reduced peripheral insulin sensitivity. Peripheral insulin response appeared to not be affected.
Although both dexamethasone formulations impaired the antilipolytic effect of insulin, the lipid content (TL and TAG) in the liver decreased after treatment.
Key words: dexamethasone, hyperinsulinemic-euglycemic clamp, insulin resistance, liver, NEFA, cattle
INTRODUCTION
During early lactation excessive lipomobilisation with subsequent development of ketosis and fatty liver is a common metabolic health disorder in high yielding dairy cows (Bobe et al., 2003). Glucocorticoids such as dexamethasone are frequently used in treatment protocols for ketosis. The efficacy has been clearly demonstrated in experimental (Andersson and Olsson, 1984; Wierda et al., 1987; Jorritsma et al., 2004) and clinical field studies (Shpigel et al., 1996).
However, the mechanism causing the well known hyperglycemic effect of glucocorticoids is still unclear. It appears unlikely that hyperglycemia is caused by increased glucose production since neither phosphoenolpyruvate carboxykinase nor pyruvate carboxylase, whose represent the key enzymes of gluconeogenesis, were stimulated by dexamethasone in neonatal calves (Hammon et al., 2003; Hammon et al., 2005). Moreover, dexamethasone does not affect endogenous glucose production in calves (Scheuer et al., 2005) and adult cattle (Starke et al., 2009).
Dexamethasone-induced hyperglycemia in dairy cows could also be due to peripheral insulin resistance. The complex effects of glucocorticoids on insulin resistance with subsequent alterations in insulin-mediated whole-body glucose uptake and the antilipolytic action of insulin have been demonstrated in humans (Tappy et al., 1994; Nicod et al., 2003; Binnert et al., 2004) and horses (Tiley et al., 2007, 2008; Haffner et al., 2009). A flumethasone- and dexamethasone-induced insulin resistance was moreover reported in milk fed calves (Sternbauer et al., 1998; Scheuer et al., 2005). However, since marked differences in digestion and regulation of glucose homeostasis exist between monogastrics and ruminating cattle (Van Soest, 1996), it remains unclear if dexamethasone-induced hyperglycemia is also mainly due to insulin resistance in early lactating dairy cows.
The gold standard method to quantify the insulin resistance in vivo is the hyperinsulinemic- euglycemic clamp (HEC; DeFronzo et al., 1979). A dose response curve between plasma insulin concentration and biological effects can be assessed by consecutive clamps with increasing insulin infusion rates and allows the differentiation between insulin sensitivity and insulin response (Debras et al., 1989; Sano and Fujita, 2006).
The objective of the present study was to evaluate the effects of dexamethasone on whole- body glucose uptake and the antilipolytic effect of insulin as indicators of insulin resistance by means of HEC in dairy cows. Secondly, the effects of two commonly used commercial dexamethasone formulations are compared, since it is reported that administration of dexamethasone as isonicotinate leads to less pronounced hyperglycemia than a formulation as phosphate (Andersson and Olsson, 1984; Wierda et al., 1987).
MATERIALS AND METHODS
The study was performed at the Clinic for Cattle, University of Veterinary Medicine Hannover, and conducted according to the guidelines of the Research Animal Act (research permit number 33-42502-06/1084) of the Lower Saxony Federal State Office for Consumer Protection and Food Safety, Oldenburg, Germany.
Animals
Eighteen clinically healthy, multiparous German Holstein cows were included in the study.
All animals had been admitted to the clinic due to a left displacement of the abomasum (LDA) between the second and fourth week after calving (18.5 ± 5.5 d post partum;
mean ± SD). The cows ranged from 3 to 8 yr of age (5.0 ± 1.4 yr), weighted between 430 and 684 kg (554 ± 71 kg) and had a BCS between 2.25 and 3.5 (2.81 ± 0.30) on a 1-5 scale (Edmonson et al., 1989). Experiments were carried out five days after surgical correction of the LDA (right flank omentopexy; Dirksen, 1967). Selection criteria were (a) undisturbed general condition subsequent to surgery, (b) undisturbed dry matter intake, (c) daily milk yield >15 L (22.1 ± 5.5 L), (d) plasma β-hydroxybutyrate (BHB) <2.0 mmol/L, (e) no significant other diseases, (f) no treatment with glucocorticoids within the last seven days and no application of glucose or glucoplastic substances within the last 24 h. Cows were kept free in single pens and were fed hay, corn silage and concentrates according to milk yield. Water was available ad libitum.
Study design
In a randomised, blinded case control study each cow was investigated for three subsequent days (Table 1). On d 1 (i.e. five days after LDA-surgery) liver biopsies were taken, both jugular veins were catheterized (Cavafix® Certo® Splittocan® 358, B. Braun, Melsungen AG, Melsungen) and blood samples obtained to assess the basal pre-treatment values.
Subsequently, cows were assigned to one of three treatment groups. They were treated with either 15 mL saline (control group CG; i.m., n = 6) or one of two dexamethasone formulations (DG-I: 40 µg/kg dexamethasone-21-isonicotinate i.m; Boehringer Ingelheim, Germany; n = 6, DG-II: 40 µg/kg dexamethasone-21-disodiumphosphate i.m.; Cp-Pharma, Burgdorf, Germany; n = 6). On d 2 five consecutive HEC were performed. On d 3 a second liver biopsy was taken together with blood samples. All cows received antibiotic treatment from d 1 up to d 3 (Procaine-Penicillin; 30,000 IU/kg/d i.m., q 24 h).
- Table 1 near here -
Hyperinsulinemic-euglycemic clamps
The clamps were performed in a separate experimental building after withdrawal of silage and concentrates for 14 h; water and hay were available to the animals ad libitum at any time.
Three blood samples were collected in 10 min intervals to assess clamp-basal concentrations of insulin, glucose, NEFA and BHB. Thereafter, five consecutive HEC with increasing doses of insulin (bovine insulin 250 mg; Sigma-Aldrich, Germany; HEC-I, II, III, IV and V: 0.1, 0.5, 2, 5 and 10 mU/kg/min, respectively) were performed, each for 2 h. Insulin solutions were infused into the right jugular vein using an infusion pump (Ismatec®, REGLO Digital 2 Kanal, Glattbrug ZH, Switzerland). The blood glucose concentration was determined every 10 min by analysing a sample from the left jugular vein. Blood glucose was clamped at the clamp-baseline level by adjusted infusion of glucose solution (40% wt/vol; Bela-Pharm GmbH, Vechta, Germany) via a second pump into the right jugular vein. To avoid hypokalemia caused by hyperinsulinemia (Boini et al., 2009), potassium chloride was added to the insulin solutions infused during HEC-III (1 mmol KCl/min/cow) and HEC-IV as well as HEC-V (2 mmol KCl/min/cow). At 10, 20, 30, 60, 90, 100, 110 and 120 min after start of each HEC, further blood samples (lithium-heparin; Sarstedt AG&Co., Nümbrecht; Germany)
The obtained plasma was stored at -80 °C in Eppendorf cups (Micro tube 2 mL, PP, Sarstedt AG&Co., Nümbrecht; Germany) until further analysis. After completion of HEC-V, blood samples were taken throughout the following 4 h period (5, 10, 20, 30, 40, 60, 80, 120 180 and 240 min after the end of insulin infusion).
After the last collection of blood cows received glucose infusions over night (10 L saline, 750 mL 40% glucose (wt/vol), 300 mL 1M KCl).
Liver biopsy
Liver biopsies (Bard® MAGNUMTM, Biopsy Instrument, Covington, GA Bard® MAGNUM®, Core Tissue Biopsy Needle, 12 G x 200 mm, BIP GmbH, Türkenfeld, Germany) were aseptically taken under ultrasonographic control (SSA 370 A, Toshiba, Tokio, Japan) in the 10th or 11th intercostal space after local anaesthesia (Procasel 2%, Selectavet, GmbH, Weyarn- Holzolling, Germany). Biopsies were immediately frozen in liquid nitrogen and stored thereafter at -80 °C until analysis.
Analysis
For surveillance of euglycemia during the clamp blood glucose concentration was assessed using a cow site glucometer (Ascensia®, CONTOUR®, Bayer Vital GmbH, Leverkusen, Germany). The relative CV determined by analysing one sample 12 times was 6.4%. For statistical evaluation plasma concentrations of glucose, NEFA and BHB were analysed by an automatic analyser (Cobas Mira Plus System fromRoche Diagnostic, Mannheim, Germany) using commercial enzymatic kits. Insulin concentration was measured by a commercial radioimmunoassay (Insulin RIA DSL-1600, Texas, USA). The intraassay and interassay CV were 5.8% and 14.2%, respectively.
Triacylglycerid (TAG) and total lipid (TL) hepatic content were assessed as described by Starke et al. (2010). In brief, TL was measured gravimetrically, subsequently the extract of lipids was resuspended and after an overnight incubation with lipase, TAG content was assessed enzymatically.
Calculations and statistical analyses
Steady-state glucose infusion rate (SSGIR) was assessed as infusion rate during the last 30
and steady-state NEFA concentrations (SSNC) were defined as mean plasma concentration of three blood samples taken within the last 30 min of each HEC. To demonstrate relative changes in NEFA plasma concentrations during the five HEC, plasma NEFA were additionally expressed as percentage of clamp-baseline NEFA values. The insulin sensitivity index (ISI) was calculated as the ratio of SSGIR (µmol/kg/min) to SSIC (µU/mL) according to Mitrakou et al. (1992). The insulin elimination from plasma was quantified by calculating the rate constant (k) according to y = a × e-k × t + b, where y represents plasma concentration of insulin (µU/mL) at t, a corresponds to the intersection with the y-axis, b represents clamp- basal plasma concentration of insulin (µU/mL) and t (min) is the sampling time after truncation of the insulin infusion. The hepatic lipid content (TL and TAG) is presented for d 1 and d 3 in absolute values and on d 3 as proportion of baseline values of d 1.
Statistical analyses were carried out using SAS (release 9.1 for Windows, SAS Institute Inc., Cary, N.C., USA). Normality was tested by Shapiro-Wilk-Test (P < 0.05). Results are expressed as mean ± SD. Clamp data were tested for significant differences between groups by means of t-distributed test statistics (PROC GLM of SAS, LSMEANS-statement, Option PDIFF/TDIFF) in case the global F-test turned out to be significant (P < 0.05). Means of blood test results on d 1, d 2, d 3 and hepatic lipid content on d 1 and d 3 were analysed by two-factorial analysis of variance for repeated measurements (Proc GLM, REPEATED statement; factor: group, time and time*group). At each time point, multiple comparisons of group means were performed by the LSMEANS statement (pdiff/tdiff option) when the F-test was significant (P < 0.05). Within different treatments means at different time points were compared with baseline values using the paired t-test.
RESULTS
Blood parameters before and after treatment. On d 1 before treatment mean plasma concentrations of glucose, insulin, NEFA and BHB did not reveal statistically significant differences between DG-I-cows, DG-II-cows and CG-cows (Table 2).
After treatment (d 2) mean plasma glucose concentrations were significantly higher in
additionally, mean plasma glucose was significantly higher in DG-II-cows compared to DG-I- cows.
The dexamethasone treatment caused a significant rise of mean plasma insulin concentrations on d 2 compared to CG-cows, whose plasma insulin concentrations did not change significantly.
Mean plasma NEFA concentrations in DG-II-cows were lower than in CG-cows on d 2, but there were no significant differences in mean plasma NEFA concentrations after treatment within any group (Table 2).
- Table 2 near here -
HEC results. Compared to control cows (CG) the mean SSGIR was significantly lower during HEC-II in DG-I- and DG-II-cows and during HEC-IV in DG-I-cows. The mean SSGIR differed between cows of DG-I and DG-II only during HEC-V.
During HEC-I and HEC-II mean SSIC were significantly higher in dexamethasone-treated cows (DG-I and DG-II) than in CG-cows (Table 3).
- Figure 1 near here -
The peripheral glucose uptake per insulin unit characterised by ISI were in average significantly lower after dexamethasone treatment during HEC-I and HEC-II in comparison to CG-cows. During extremely supraphysiological insulin infusion rates (HEC-III, HEC-IV and HEC-V) no differences in ISI group means were found. There were no differences in mean ISI between DC-I- and DC-II-cows during any HEC (Figure 2).
- Figure 2 near here -
The mean plasma NEFA concentrations decreased in all cows rapidly during the insulin infusion. The absolute values of plasma NEFA did not differ between the cows of all groups during any HEC, but the relative decline of NEFA was significantly less pronounced in DC-I- and DC-II-cows compared to CG-cows starting from HEC-II (Table 3 and Figure 3).
The mean plasma insulin elimination rate constant (k) was 0.0397 ± 0.0077 in DC-I-cows, 0.0441 ± 0.0104 in DC-II-cows, 0.0357 ± 0.0066 in CG-cows and did not differ among the groups.
- Table 3 and Figure 3 near here -
Hepatic TL and TAG content. No significant group differences were found in hepatic
cows (DG-I and DG-I) mean hepatic TL and TAG decreased significantly on d 3 compared to d 1, while no noteworthy changes were seen in CG-cows. The relative changes in DG-I- and DG-I-cows on d 3 compared to d 1 were on average 30 to 40% for TL and 40 to 50% for TAG (Table 4).
- Table 4 near here -
DISCUSSION
The experimental protocol with five consecutive HEC using subsequently increasing insulin infusion rates represents an appropriate approach to quantify insulin resistance. Altogether, five conditions were investigated, each with a specific SSIC and a respective SSGIR and SSNC, describing the biological effect as a function of plasma insulin concentration (Debras et al., 1989; Sano and Fujita, 2006; Gjessing et al., 2010). Thereby it became possible to differentiate between an impaired insulin sensitivity and response.
The main finding of the present study was the dexamethasone-induced peripheral insulin resistance, as indicated by a decrease of SSGIR required to maintain euglycemia and a decrease of ISI representing the peripheral glucose uptake per insulin unit during HEC. This phenomenon was observed during almost all HEC (except of HEC-V in DG-II-cows), yet significant differences were found only during insulin infusion rates of 0.5 mU/kg/min for SSGIR and 0.1 and 0.5 mU/kg/min for ISI. We presume that the dexamethasone-induced insulin resistance is due to impaired insulin sensitivity without any remarkable influence on insulin response (assessed as SSGIR and ISI during the highest insulin infusion rate by HEC- V).
An impairment of insulin sensitivity after a single injection of flumethasone has also been found in calves (Sternbauer et al., 1998). After performing a single HEC with an insulin infusion rate of 1 mU/kg/min, SSGIR was reduced by about 75%. Comparing the results of the present study, SSGIR was reduced after dexamethasone treatment by about 40% during
(2 mU/kg/min). Inconsistencies are also apparent regarding ISI; while Sternbauer et al. (1998) reported a flumethasone-induced reduction of ISI by about 75% in calves, the results of our study in dairy cows found that dexamethasone led to a reduction of 75%, 60%, and 30%
during HEC-I, HEC-II, and HEC-III, respectively. The differences may be explained by the different drugs used and the different metabolic situation of milk fed calves and ruminating cows in early lactation.
Studies in humans revealed a decrease of whole-body glucose uptake by 44.6% (Tappy et al., 1994) and 34% (Willi et al., 2002) after dexamethasone treatment. A long term dexamethasone application reduced insulin-stimulated glucose uptake by about 70% in healthy horses (Tiley et al., 2008) and 60% horses with polysaccharide storage myopathy (Firshman et al., 2005). Buren et al. (2008) presented similar results by in vitro experiments on fat and muscle tissue from in vivo dexamethasone-treated rats. Treatment reduced insulin- stimulated glucose uptake in muscle by 30-70% and by approximately 40% in adipocytes.
The mechanisms responsible for the glucocorticoid-induced insulin resistance are still unclear. Studies carried out on rat skeletal muscles and adipocytes demonstrated that the decrease of insulin-stimulated glucose uptake occurs in absence of any consistent effect on insulin receptor number or ligand affinity (Watanabe et al., 1984; Saad et al., 1993;
Venkatesan et al., 1996) and without decreasing the expression of insulin-depended glucose transporter (GLUT-4; Dimitriadis et al., 1997; Buren et al., 2002; Buren et al., 2008).
However, dexamethasone has been shown to impair GLUT-4 translocation to the cell surface (Dimitriadis et al., 1997; Weinstein et al., 1998; Buren et al., 2002). The involved molecular pathways are probably found in the intracellular signaling cascade and include multiple alterations of insulin receptor substrate-1, protein kinase B, protein kinase C and glycogen synthase (Saad et al., 1993; Kajita et al., 2001; Buren et al., 2002; Ruzzin et al., 2005; Brown et al., 2007; Buren et al., 2008).
The effect of dexamethasone treatment on hyperglycemia in the present study is consistent with previous reports (Andersson and Olsson, 1984; Wierda et al., 1987; Shpigel et al., 1996;
was significantly more pronounced after application of dexamethasone-21- disodiumphosphate (+100%) than after dexamethasone-21-isonicotinate (+50%). The observed increase in plasma insulin concentrations after dexamethasone treatment had previously been reported (Jorritsma et al., 2004; Furll and Jackel, 2005). Not surprisingly, having in mind the more pronounced increase of plasma glucose, the concomitant rise of plasma insulin compared to the pre-treatment values was higher after dexamethasone-21- disodiumphosphate (+283%) than after dexamethasone-21-isonicotinate (+186%). However, clamp results of this study gave no evidence that the different effects of the two dexamethasone formulations on hyperglycemia can be explained by development of different degrees of insulin resistance after treatment.
The decrease of plasma NEFA concentrations during HEC represents the insulin-mediated antilipolytic effect. Although dexamethasone did not alter fasting NEFA levels in healthy humans, it decreased the insulin’s ability to suppress NEFA concentration during the clamp (Willi et al., 2002). These findings are in line with the results of the present study.
Hyperinsulinemia decreased plasma NEFA concentrations to roughly 10% of basal values in control cows, while in the dexamethasone-treated cows the NEFA decreased only to 20% and 30% of clamp-baseline values after dexamethasone-21-isonicotinate and dexamethasone-21- disodiumphosphate, respectively. Almost the same result was described by Tappy et al.
(1994) in healthy humans after dexamethasone treatment (decrease to 10% and 20% of baseline for untreated and treated subjects, respectively).
The mechanisms responsible for observed reduction of insulin-mediated decrease of NEFA remain to a large extent mystery. The in vitro studies using human adipocytes assume a direct influence of glucocorticoids on some substances involved in lipolysis, but the results are still very controversial. While in the study from Lundgren et al. (2008) dexamethasone treatment did not affect the content of protein kinase A, perilipin A, and hormone sensitive lipase (HSL), Fain et al. (2008) measured significant stimulation of the same substances in the presence of dexamethasone, leading to increased rates of lipolysis. Similarly, increased lipolysis was observed in isolated adipocytes from dexamethasone-treated rats as a result of
dexamethasone did not induce HSL translocation to the lipid droplet surface in differentiated adipocytes (Xu et al., 2009).
Although dexamethasone may stimulate lipolysis, the missing increase of post-treatment NEFA values in the present and other studies (Shpigel et al., 1996; Jorritsma et al., 2004;
Furll and Jackel, 2005) is not surprising, assuming that dexamethasone concurrently induces an increase of basal lipid oxidation (Tappy et al., 1994). The missing effect of dexamethasone on mean plasma BHB concentrations in the present study is in agreement with results reported previously (Wierda et al., 1987; Shpigel et al., 1996; Jorritsma et al., 2004; Furll and Jackel, 2005).
Dexamethasone treatment induced a significant decrease of both TL and TAG content in the liver, without significant differences in the extent of the effect between both dexamethasone formulations. This result is in agreement with Starke et al. (2009). In contrast, other studies found no effect of dexamethasone isonicotinate on the hepatic TAG content (Jorritsma et al., 2004; Furll and Jackel, 2005). However, these authors used lower dexamethasone dosages and cows presented lower pre-treatment hepatic lipid values (Jorritsma et al., 2004).
In conclusion, results of our study indicate that dexamethasone treatment induces insulin resistance in dairy cows in early lactation, affecting insulin sensitivity, but not insulin response. Although the increase of basal glucose and insulin concentration after dexamethasone-21-disodiumphosphate was higher than after dexamethasone-21-isonicotinate, the extent of insulin resistance did not vary. Dexamethasone treatment impaired insulin- mediated antilipolytic effect, but did not result in hyperlipidemia or hepatic lipidosis. In contrast, a single dose of both dexamethasone formulations decreased the TL and TAG content in the liver.
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