Oral administration of immunoglobulin G-enhanced colostrum alleviates insulin resistance and liver injury and is associated with alterations in natural killer T cells

Abstract

Insulin resistance and metabolic syndrome are chronic inflammatory conditions that lead to hepatic injury and non-alcoholic steatohepatitis (NASH). Bovine colostrum has therapeutic effects in a variety of chronic infections. However its effectiveness in NASH was never studied. Natural killer T (NKT) cells have been shown to be associated with some of the pathological and metabolic abnormalities accompanying NASH in leptin-deficient (ob/ob) mice. In the present study, we used hyperimmune bovine colostrum to treat hepatic injury and insulin resistance and we also assessed the effects on NKT cells. We used ob/ob mice that were fed for 6 weeks with either 0·1 mg bovine colostrum prepared from non-immunized cows, 0·1 mg hyperimmune colostrum raised against a bacterial lipopolysaccharide (LPS) extract or 0·001, 0·1 or 1 mg of immunoglobulin (Ig)G purified from hyperimmune colostrum (IgG-LPS). NKT cells were phenotyped by flow cytometry, and hepatic injury and insulin resistance were assessed by measuring fasting glucose levels, glucose tolerance tests and liver enzymes. Fat accumulation was measured in the liver and plasma. Oral administration of hyperimmune colostrums decreased alanine aminotransferase (ALT) serum levels and serum triglycerides compared to controls. Glucose intolerance was also improved by the hyperimmune colostrum preparations. These results were accompanied by a decrease in serum tumour necrosis factor (TNF)-α levels following oral treatment with 0·1 or 1 mg of IgG-LPS. The beneficial effects of hyperimmune colostrums were associated with an increase in the number of splenic NKT cells. These data suggest that oral administration of hyperimmune colostrum preparations can alleviate chronic inflammation, liver injury and insulin resistance associated with NASH.

Fig. 1

Enzyme-linked immunosorbent assay (ELISA) procedure for determining assay-specific antibody titre in colostrum preparations. An indirect ELISA was used to evaluate the specific antibody activity of immunoglobulin (IgG)–lipopolysaccharide (LPS) and Imm124-E colostrum preparations against the antigens used in both the multi-strain and single-strain vaccines. ELISA plates were coated with antigens, as described in the Methods section. Fourfold dilutions (1/250, 1/1000, 1/4000, 1/16 000, 1/64 000 and 1/256 000) of each sample and control were prepared. The optical density (OD) was read at 450 nm on a plate reader.

Fig. 2

(a) High-dose immunoglobulin (IgG)–lipopolysaccharide (LPS) abrogated hepatic injury in ob/ob mice. Serum ALT levels were measured in mice after 4 weeks of oral treatment with various colostrum preparations or water (vehicle). Data are shown as the mean ± standard deviation (s.d.) of nine to 13 mice in each group. Comparisons were made to water and to colostrum control. Similar results were obtained for two independent experiments. (b) High-dose IgG–LPS and Imm124-E decreased hepatic TG content in ob/ob mice. Eight weeks after oral treatment with various colostrum preparations or vehicle, mice were killed and their livers were harvested immediately. Triglycerides (TGs) were extracted from snap-frozen livers and then assayed spectrophotometrically. The number of milligrams of TGs in each sample was calculated on the basis of liver mass, and the amounts are expressed as percentages (mg TGs/g liver). Data are shown as the mean ± s.d. of six to 10 mice for each group. Comparisons were made to water and to colostrum control.

Fig. 3

(a) High-dose immunoglobulin (IgG)–lipopolysaccharide (LPS) and Imm124-E decreased serum triglycerides (TGs) in ob/ob mice. Serum TGs levels were measured in mice after 4 weeks of oral treatment with various colostrum preparations or vehicle. Data are shown as the mean ± standard deviation (s.d.) of three to six mice for each group. *P < 0·05, **P < 0·01 compared with water and with colostrum control. (b) Colostrum treatment did not affect serum cholesterol levels in ob/ob mice. Total serum cholesterol levels in ob/ob mice were determined after 4 weeks of oral treatment with various colostrum preparations or vehicle. Data are shown as the mean ± s.d. of six to 12 mice for each group. Similar results were obtained for two independent experiments. Comparisons were made to water and to colostrum control.

Fig. 4

(a) Colostrum preparations decreased fasting plasma glucose levels in ob/ob mice. Fasting glucose levels were monitored after 1, 3 and 5 weeks of treatment with various colostrum preparations. Glucose levels were measured in tail vein blood by a standard glucometer. Data are shown as the mean ± standard deviation (s.d.) of six mice for each group. *P < 0·05, **P < 0·01 compared with water and with colostrum control. (b) High-dose immunoglobulin IgG–LPS ameliorated glucose intolerance in ob/ob mice. Oral glucose tolerance test (OGTT) was performed during week 5 after an overnight fast. Glucose was administered orally (1·25 g/kg body weight), and serum glucose levels in tail vein blood were measured every 15 min for 3 h using a standard glucometer. Glucose values are presented in absolute values. Data symbols represent average ± s.d. For each time-point, six mice were analysed. *P < 0·05, **P < 0·01 compared with water and with colostrum control. (c) High-dose IgG–LPS ameliorated glucose intolerance in ob/ob mice. Oral GTTs were performed during week 5 after an overnight fast. Glucose was administered orally (1·25 g/kg body weight), and serum glucose levels in tail vein blood were measured every 15 min for 3 h using a standard glucometer. Results are presented as area under the curve (AUC). Data symbols represent average ± s.d. For each time-point, five to six mice were analysed.

Fig. 5

Medium and high-dose immunoglobulin (IgG)–lipopolysaccharide (LPS) decreased serum tumour necrosis factor (TNF)-α in ob/ob mice. Serum TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA). Data are shown as the mean ± standard deviation (s.d.) of five to six mice for each group. Comparisons were made to water-treated mice. Significance in Student's t-test was considered as P < 0·05.

Fig. 6

(a) High- and low-dose immunoglobulin (IgG)–lipopolysaccharide (LPS) and Imm124-E promoted NKT cells in spleens of ob/ob mice. Eight weeks after treatment with various colostrum preparations or vehicle, mice were killed and their spleens removed. Splenocytes were prepared as described in the Methods section. One million cells were analysed for the expression of CD3 and NK1.1. The numbers of purified CD3⁺NK1.1⁺ cells were calculated. Data are shown as the mean ± standard deviation (s.d.) of six mice for each group. *P < 0·05, compared with water and with colostrum control.