Flow Cytometric Analysis of the Effects of High Protein Diet On Isolated Rat Liver Mitochondria

Abstract. We have investigated changes that occur in mitochondria obtained from the livers of rats that had been maintained on a high protein diet (80% casein instead of 20%) for 6 months. Liver homogenates were separated by centrifugation into a mitochondrial fraction, a nuclear fraction and the supernatant fluid of the nuclear fraction (nuclear wash). Rhodamine-123 was used to selectively stain mitochondria depending upon their membrane. potential. the stained organelles were processed through a flow cytometer where the fluorescent stains were excited by the 488 nm wavelength of a laser and the resultant fluorescence signals analysed. After 6 months on a high protein diet, mitochondria displayed an increase in the fluorescence associated with rhodamine-123 uptake in both mitochondrial and nuclear wash fractions, while mitochondrial fluorescence in the nuclear fraction showed a heterogeneous distribution. This was interpreted as an increase in membrane potential in most of the liver mitochondria under these nutritional conditions, with a certain degree of heterogeneity. These functional changes may be correlated with morphological alterations previously reported and show the usefulness of flow cytometry for biochemical analysis of isolated mitochondria.     

Long-Term Effects of Subacute Ruminal Acidosis (SARA) on Milk Quality and Hepatic Gene Expression in Lactating Goats Fed a High-Concentrate Diet

Purpose

The mechanism underlying the decline in milk quality during periods of feeding high-concentrate diets to dairy ruminants is not well documented. The aim of this study was to investigate the metabolic changes in the liver that contribute to the input of substrate precursors to the mammary gland after feeding a high-concentrate diet to lactating goats for a long period.

Experimental Design

Eight mid-lactating goats with rumen cannulas were randomly assigned to two groups. For 9 weeks, the treatment group was fed a high-concentrate diet (60% concentrate of dry matter, HC) and the control group was fed a low-concentrate diet (40% concentrate of dry matter, LC). Ruminal fluid, plasma, and liver tissues were sampled, microarray techniques and real-time polymerase chain reaction were used to evaluate metabolic parameters and gene expression in liver.

Results

Feeding a 60%-concentrate diet for 9 weeks resulted in a significant decrease in rumen pH. Changes in fat and protein content also occurred, which negatively affected milk quality. Plasma levels of leptin (p = 0.058), non-esterified fatty acid (p = 0.071), and glucose (p = 0.014) increased markedly in HC group. Plasma cortisol concentration was significantly elevated in the treatment group (p<0.05). Expression of the glucocorticoid receptor protein gene was significantly down-regulated (p<0.05) in the liver. The expression of genes for interleukin 1β, serum amyloid A, C-reactive protein, and haptoglobin mRNA was significantly increased (p<0.05) in the HC group. GeneRelNet analysis showed that gene expression involved in inflammatory responses and the metabolism of lipids, protein, and carbohydrate were significantly altered by feeding a high-concentrate diet for 9 weeks.

Conclusions

Activation of the acute phase response and the inflammatory response may contribute to nutrient partitioning and re-distribution of energy in the liver, and ultimately lead to a decline in milk quality.     

Effects of Fluoridated Milk on Root Dentin Remineralization

Background

The prevalence of root caries is increasing with greater life expectancy and number of retained teeth. Therefore, new preventive strategies should be developed to reduce the prevalence of root caries. The aim of this study was to investigate the effects of fluoridated milk on the remineralization of root dentin and to compare these effects to those of sodium fluoride (NaF) application without milk.

Methods

Thirty extracted human molars were divided into 6 groups, and the root cementum was removed from each tooth. The dentin surface was demineralized and then incubated with one of the following six solutions: Sodium chloride NaCl, artificial saliva, milk, milk+2.5 ppm fluoride, milk+10 ppm fluoride and artificial saliva+10 ppm fluoride. Serial sections were cut through the lesions and investigated with polarized light microscopy and quantitative morphometry, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The data were statistically evaluated using a one-way ANOVA for multiple comparisons.

Results

The depth of the lesion decreased with increasing fluoride concentration and was the smallest after incubation with artificial saliva+10 ppm fluoride. SEM analysis revealed a clearly demarcated superficial remineralized zone after incubation with milk+2.5 ppm fluoride, milk+10 ppm fluoride and artificial saliva+10 ppm fluoride. Ca content in this zone increased with increasing fluoride content and was highest after artificial saliva+10 ppm fluoride incubation. In the artificial saliva+10 ppm fluoride group, an additional crystalline layer was present on top of the lesion that contained elevated levels of F and Ca.

Conclusion

Incubation of root dentin with fluoridated milk showed a clear effect on root dentin remineralization, and incubation with NaF dissolved in artificial saliva demonstrated a stronger effect.