• Open Access

Concentrations of Plasma Metabolites, Hormones, and mRNA Abundance of Adipose Leptin and Hormone-Sensitive Lipase in Ketotic and Nonketotic Dairy Cows

Authors

  • C. Xia,

    1. Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Heilongjiang province, China
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  • Z. Wang,

    Corresponding author
    • Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, JiLin University, Changchun, China
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  • C. Xu,

    1. Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Heilongjiang province, China
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  • H.Y. Zhang

    1. Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Heilongjiang province, China
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Corresponding author: Z. Wang, Department of Clinical Veterinary Medicine, College of Animal Science and Veterinary Medicine, JiLin University, Changchun, 130062, China; e-mail:wangzhe500518@sohu.com.

Abstract

Background

Ketosis is an important metabolic disorder of dairy cows during the transition period. There have been many reports on the etiology of ketosis in periparturient cows, but little is known about its molecular etiology.

Objectives

The objective of this study was to clarify the status of fat mobilization and mRNA abundance of leptin and hormone-sensitive lipase in cows with spontaneous clinical ketosis.

Animals

Ten ketotic Holstein cows and 10 nonketotic Holstein cows were used as the experimental animals.

Methods

Six blood biochemical parameters were evaluated by means of individual analysis method for 2 groups of cows. The mRNA abundance of leptin and hormone-sensitive lipase in tail fat tissue from 2 groups of cows was measured by real-time (RT)-PCR, with a fluorescent Taqman probe and a standard curve.

Results

The plasma concentrations of glucose (P = 0.01), and leptin (P = 0.03), insulin (P = 0.05), and the ratio of insulin to glucagon (P = 0.04) were lower in ketotic compared with nonketotic cows, whereas there were marked increases in the plasma concentrations of nonesterified fatty acid and β-hydroxybutyric acid (P = 0.005). The mRNA abundance of leptin (P = 0.04) and hormone-sensitive lipase (P = 0.02) in the fat tissue of ketotic cows was lower relative to that of nonketotic cows.

Conclusions and Clinical Importance

The ketotic cows showed characteristics of type I ketosis and some adaptive changes to negative energy balance in the plasma leptin concentration and mRNA abundance of fat leptin and hormone-sensitive lipase.

Abbreviations
Lp

leptin

HSL

hormone-sensitive lipase

BHBA

β-hydroxybutyric acid

Glu

glucose

NEFA

nonesterified fatty acids

Ins

insulin

Gn

glucagon

NPY

neuropeptide Y

NEB

negative energy balance

DMI

dry matter intake

Ketosis is a metabolic disorder that occurs in adult cattle. It typically occurs in dairy cows during early lactation when energy demands exceed energy intake and result in a negative energy balance (NEB).[1] When the blood glucose (Glu) concentration is too low, the cow mobilizes body fat. Part of the mobilized body fat will be converted to ketones in the liver (eg, acetone, β-hydroxybutyric acid), which results in increased ketone concentrations in the blood. Once the metabolic and endocrine systems are unable to regulate NEB, ketone body production increases rapidly, and ketosis becomes unavoidable.[2] It is most consistently characterized by anorexia and lethargy. In severe cases of ketosis, additional signs of nervous dysfunction, including abnormal licking, pica, incoordination, and abnormal gait, occur.[1, 2]

The pathogenesis of ketosis in cattle is incompletely understood, but it requires the combination of intense adipose mobilization and a high Glu demand. Thus, the clinicopathologic characterization of ketosis includes high serum concentrations of nonesterified fatty acids (NEFA) and ketone bodies and low concentrations of Glu. The serum ketone bodies are acetone, acetoacetate, and β-hydroxybutyrate.[3] Hormone-sensitive lipase (HSL) is a key enzyme for fat mobilization. Hormones and Glu precursors regulate fat mobilization by controlling HSL activity and mRNA expression.[4] In addition, leptin (Lp), secreted from adipose tissue, has an important role in the regulation of dry matter intake (DMI) and energy balance. Accordingly, Lp has become an attractive topic in neuroendocrinology of cattle in recent years.[5]

Although there is considerable literature relating to serum metabolites and hormones during ketosis in dairy cows, little is known about the mRNA abundance of adipose HSL and Lp in clinical ketosis of cattle. The purpose of this study therefore was to investigate changes in the plasma concentrations of metabolites and hormones, and the mRNA abundance of adipose Lp and HSL in ketotic cows compared to nonketotic

Materials and Methods

All animals used in the study were treated according to International Guiding Principles for Biomedical Research Involving Animals. Ten clinically ketotic cows and 10 nonketotic cows were selected from a commercial dairy farm at Daqing, Heilongjiang province, China for the experiment.

The cows were considered to have clinical ketosis if they showed marked signs of clinical ketosis and plasma β-hydroxybutyric acid (BHBA) concentration >1.40 mmol/L. If the cows had no signs of clinical ketosis and normal plasma BHBA concentrations (<1.00 mmol/L), they were considered to be healthy.[1-3] Further description of the ketotic and nonketotic cows is shown in Table 1.

Table 1. Description of the study in the ketotic and nonketotic groups.
GroupsParityMilk Yield (kg/day)Body Condition ScoreDry Matter Intake (kg/day)Postpartum Days
Ketotic3 ± 110.50 ± 4.362.50 ± 0.257.22 ± 3.4423 ± 4
Nonketotic4 ± 121.34 ± 0.483.00 ± 0.2515.67 ± 0.5222 ± 3
P-value.32 .006 .04.008.15

Blood samples from ketotic and nonketotic cows were collected from the jugular vein around 5:00 pm before feeding. Plasma was separated immediately into 1.5 mL tube by centrifugation at 1,500 × g for 10 min after blood collection. Plasma samples were stored at −80°C until analysis. After blood samples were taken, adipose tissue samples (50–100 mg) were collected from alternating sides of the tail base by surgical biopsy, frozen immediately in liquid nitrogen, transported to the laboratory and stored at −80°C until analyzed for Lp and HSL mRNA abundance.

Plasma Glu concentration was measured by means of the oxidase method and NEFA by the acyl-CoA synthetase/oxidase method with a commercially available kit. Plasma BHBA concentration was measured with high-performance liquid chromatography (HPLC-10 AVP).[6] Plasma leptin (Lp), insulin (Ins), and glucagon (Gn) were assayed with a commercially available radioimmunoassay kit.

As for methods of RNA extraction, cDNA generation, DNA contamination control, PCR conditions of Lp and HSL including forward and reverse primers, annealing temperature, number of cycles and gene length, and the mRNA abundance of Lp and HSL assessed by means of a real-time RT-PCR, a fluorescent TaqMan probe, and a standard curve in an ABI prism 7000 Sequence Detection System have been described previously.[7] Standard curve and fluorescent quantitation PCR were performed according to the manufacturer's instructions. The mRNA abundance of the 2 genes was analyzed by LINE GENNEK software.

The data were analyzed using a Student's t-test with Excel software. The results were expressed as mean ± SD.

Results

Table 2 displays the results from ketotic cows and those from nonketotic cows: the plasma concentration of Glu (1.67 ± 0.42 mmol/L νersus 2.90 ± 0.30 mmol/L) was less in ketotic versus nonketotic cows (P = .01), whereas the concentrations of plasma NEFA (1085 ± 81 μmol/L νersus 543 ± 77 μmol/L) (P = .008) and BHBA (3.46 ± 0.60 mmol/L νersus 0.60 ± 0.12 mmol/L) (P = .005) were increased in ketotic cows. Furthermore, the concentrations of plasma Lp (3.50 ± 0.51 ng/mL νersus 4.86 ± 0.56 ng/mL) (P = .03) and insulin (Ins) (9.30 ± 1.16 IU/mL νersus 11.6 ± 1.50 IU/mL) (P = .05) were lower in ketotic versus nonketotic cows, as was ratio of insulin to glucagon (P = .04). In addition, the mRNA abundance of Lp (241 ± 63 νersus 326 ± 70) (P = .04) and HSL (354,000 ± 81,000 νersus 478,000 ± 86,000) (P = .02) was significantly lower in ketotic cows compared with nonketotic cows.

Table 2. Concentration of plasma metabolites, hormones, and mRNA abundance of HSL and Lp in the study cows (mean ± SD).
GroupsGlu (mmol/L)NEFA (μmol/L)BHBA (mmol/L)Lp (ng/mL)Ins (IU/mL)Gn (pg/mL)Ratio of Ins to GnmRNA Abundance
LpHSL
  1. Lp, leptin; HSL, hormone-sensitive lipase; BHBA, β-hydroxybutyric acid; Glu, glucose; NEFA, non-esterified fatty acids; Ins, insulin; Gn, glucagons.

Ketotic1.67 ± 0.421085 ± 813.46 ± 0.603.50 ± 0.519.30 ± 1.16180 ± 390.052 ± 0.029241 ± 63354000 ± 81000
Nonketotiic2.90 ± 0.30543 ± 770.60 ± 0.124.86 ± 0.5611.6 ± 1.50185 ± 450.063 ± 0.033326 ± 70478000 ± 86000
P-value.01.008.005.03.05.36.04.04.02

Discussion

In this study, the low concentration of plasma Lp and low mRNA abundance of Lp and HSL in adipose tissue in ketotic cows indicated some adaptive responses to NEB.[2] However, in this study the reduction in plasma concentration of Glu and the high plasma concentrations of NEFA and BHBA in ketotic cows indicate that they were still experiencing NEB and increased fat mobilization, which might be related to failure to regulate energy balance or perhaps other environmental and management factors not measured in this study.

Our study showed that ketosis occurred around 23 days after calving and was accompanied by hypoglycemia, high blood β-hydroxybutyric acid, high NEFA, low body condition score (BCS), low milk yield, and apparent clinical signs of ketosis, compared with nonketotic cows. These characteristics are consistent with previous reports of type I ketosis, which is the classic form of ketosis that occurs in cows that are 3–6 weeks postcalving because cows are at their highest milk energy outflow at this time. It is named type I ketosis because of its similarities to type I diabetes mellitus. Insulin is low in type I diabetes because of chronic hypoglycemia caused by a shortage of Glu precursors. Cows with type I ketosis are able to make Glu from precursors (mostly propionate and amino acids). The limiting factor is the supply of Glu precursors. Blood ketone concentrations become very high and blood Glu concentrations very low under these conditions.[2, 3, 8] Furthermore, low plasma concentrations of insulin (Ins), Glu and low ratio of insulin (Ins) to glucagon (Gn) were observed in the ketotic cows (Table 2). Some studies have demonstrated that a low ratio of Ins to Gn (<0.10) often is associated with NEB during early lactation, which stimulates fat mobilization. If cows develop severe hypoglycemia during early lactation, a substantial amount of NEFA should be mobilized from their adipose tissue, which might exceed the oxidative capacity of liver, thus increasing the formation of ketone bodies.[1, 2] Therefore, in this study the affected cows had all clinical and biochemical attributes of type I ketosis, and also experienced NEB and fat mobilization.

The plasma concentration of Lp is closely related to body fat and the feeding level of the cows during lactation, increases during gestation, and reaches its lowest point at calving. High-yielding cows have low plasma Lp concentrations, and high DMI during lactation. The Lp mRNA abundance in fat tissue is known to decrease in malnourished sheep and cattle, and to increase in animals that are overfed.[5, 9] Lp is a potent inhibitor of appetite that is mainly dependent on regulation of neuropeptide Y (NPY) in the hypothalamus.[10] In Table 2, plasma concentration and adipose mRNA abundance of Lp are shown to be decreased in ketotic cows, which should lead to an increase in appetite that should decrease ketone bodies and resolve ketosis. However, in Table 1, the ketotic cows are shown to have low BCS and DMI, which are not helpful for recovery of the ketotic cows because of lack of dietary energy supply.

Finally, previous research has demonstrated that HSL expression generally is upregulated in early lactation in some cases.[11]. However, in Table 2 the mRNA abundance of HSL in ketotic cows is shown to be significantly lower than in nonketotic cows. In addition, in Table 2 plasma NEFA concentration is shown to be increased in ketotic cows because of a low ration of insulin to glucagon that can stimulate fat mobilization and release NEFA as a result of the increased activity of HSL.[1, 2, 8] Because different mechanisms are involved in the regulation of lipolysis caused by the activity and mRNA expression of HSL, other factors that may also affect the activity of mRNA expression of HSL should be considered such as epinephrine, other catecholamines, and dexamethasone, which were not measured in our experiment.

In summary, our data demonstrate that the ketotic cows in this study had characteristics of type I ketosis and some adaptive changes in plasma Lp concentration and mRNA abundance of fat Lp and hormone-sensitive lipase. However, they still are in a state of NEB and increased fat mobilization, which might be related to other factors involved in the development of ketosis.

Acknowledgments

This work was supported by the Chinese National Nature Science Foundation (30230260) and Heilongjiang Province Science and Technology Department (GZ02B033). The authors thank Dr Daryl V Nydam who modified the manuscript carefully, who works in College of Veterinary Medicine, Cornell University.

The authors thank TaKaRa Company in Japan for manufacturer's instructions of fluorescent quantitation PCR, Xiehe Medicine Technology Limited Company in Tianjin of China for commercial kits of plasma Glu and NEFA, and Yulan Biotechnology Research Institute in Shanghai of China for plasma Lp, Ins, and Gn.

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