Measurement of fat synthesis in sheep : A thesis submitted in partial fulfilment of the requirements for the degree of Master of Agricultural Science in the University of Canterbury [Lincoln College]

Abstract

The in vivo rate of fat synthesis in ovine adipose tissue was estimated by measuring the rate of incorporation of radioactive plasma glucose into adipose tissue glyceride glycerol during a continuous intravenous infusion of [U- ¹⁴C]-glucose. Adipose tissue samples were removed from a back fat site using local anaesthesia or immediately following slaughter. The rate of incorporation of ¹⁴C radioactivity into glyceride glycerol was linear for at least 24 hours and was used to calculate the rate of incorporation of plasma glucose into adipose tissue glyceride glycerol. In two experiments (Sheep 1 and 2) , the rates were 0.41 and 1.32 nmol glucose min⁻¹g back fat⁻¹. Assuming that plasma glucose was the only source of glycerol 3-phosphate, that there was no partial hydrolysis of triglycerides with subsequent re-esterification of fatty acids and that the rate of incorporation of plasma glucose into back fat adipose tissue was the same as into total body adipose tissue then the rates of glucose incorporation into adipose tissue glyceride glycerol were equivalent to 14 and 28 grams of adipose tissue synthesized per day. The turnovers of glucose in the respective experiments were 24 and 39 µmol glucose min⁻¹ kg-⁰⁷⁵ . Thus, adipose tissue glyceride glycerol synthesis accounted for one and two percent of glucose turnover. Glucose utilization for fatty acid synthesis in Sheep 2 was less than one percent of the glucose turnover. In 24 week old lambs which were either weaned, suckled, weaned and infused with exogenous insulin or suckled and infused with exogenous insulin. the same method of infusing [U- ¹⁴C]-glucose continuously and sampling adipose tissue was used to determine the rates of glucose incorporation into glyceride glycerol. The rates ranged from 0.7 to 4 .5 nmol glucose min⁻¹ g adipose tissue⁻¹-. The mean rate of incorporation of glucose into back fat glyceride glycerol was 1.87 nmol min⁻¹ g tissue⁻¹ (17.7 µmol glucose min⁻¹ lamb⁻¹) which is equivalent to 54 grams of adipose tissue synthesized per day. There were no significant differences between the treatment groups in the rates of glucose incorporation into total body fat glyceride glycerol. The rate of incorporation of glucose into fatty acids, however , was significantly greater in the lambs infused with insulin than those receiving no exogenous insulin infusion (89 and 20 µmol min⁻¹ lamb⁻¹ respectively) . Euglycaemic insulin clamps were used to examine the effect of insulin on the in vivo rate of fat synthesis in four lambs. In these experiments [U- ¹⁴C ) glucose and insulin were infused continuously and the rate of an intravenous infusion of dextrose varied to maintain the plasma glucose concentration at 3 .6 mM. The plasma insulin concentration was 14 ± 2 μU cm ⁻³ during the first 8 hours (period 1) and 160 ± 9 μU cm ⁻³ μU cm ⁻³ during the second 8 hours (period 2). The rates of incorporation of glucose into glyceride glycerol were 1.84 and 4.00 nmol min⁻¹ g back fat⁻¹ in periods 1 and 2 respectively. These rates are equivalent to 53 and 117 grams of adipose tissue synthesized per day . The glucose turnover was 40 μmol min⁻¹ kg-0. 75 during period 1 and 88 μmol min⁻¹ kg-⁰⁷⁵ during period 2. A constant three percent of glucose turnover was used for glyceride glycerol synthesis. Glucose utilization for fatty acid synthesis increased from one to two percent of glucose turnover on progressing from period 1 to period 2. A new method for measuring fat synthesis is described in this thesis. The method provides direct evidence that insulin stimulates fat synthesis in vivo, and demonstrates that a very small proportion of glucose turnover is used for fat synthesis in sheep.