Preparation of isolated bovine adipocytes: validation of use for studies characterizing insulin sensitivity and binding.

The present study was undertaken to develop techniques to isolate bovine adipocytes, to compare their rates of glucose metabolism and insulin sensitivity with adipocytes in adipose tissue, and to determine if isolated bovine adipocytes specifically bind insulin. Cell size and diameter distributions were the same for adipocytes fixed with OsO4 after isolation with collagenase and adipocytes liberated from OsO4-fixed adipose tissue slices. On a per cell basis, lipogenic rates were greater for isolated adipocytes compared with intact adipose tissue. Similar differences were found for glucose oxidation. In short term incubations, glucose oxidation and lipid synthesis were not stimulated by insulin (0-100 ng/ml) in either isolated adipocytes or tissue. Specific binding of [125I]iodoinsulin at 30 C was low (0.8%) in the first group of six beef cattle sampled, but increased with increasing cell concentration. Insulin degradation after 90 min was less than 5%. The specificity of [125I]iodoinsulin binding was studied in a second group of six animals. There was no specific binding of insulin in this group. In summary, bovine adipocytes can be isolated which are metabolically active and provide a valid system for studying hormone binding and action. In the present study, glucose metabolism in bovine adipocytes was not stimulated by insulin in vitro. This insensitivity to insulin was associated with a negligible capacity for insulin binding. These findings suggest that the lack of insulin sensitivity in bovine adipose tissue may be due to an inability to specifically bind insulin. This may be related to the unique metabolism of ruminant adipose tissue, which is less dependent upon glucose for fatty acid synthesis than is adipose tissue from nonruminant species.

Diurnal variations in plasma insulin and growth hormone associated with two stages of lactation in high producing dairy cows.

Plasma concentrations of GH, insulin, glucose, and FFA were measured in high yielding dairy cows at periods of peak milk production and later during periods of increased feed intake. During each period, five cows were prepared with indwelling jugular catheters, and blood was sampled at 10-min intervals for 24 h, followed by hourly sampling for an additional 24 h. During early lactation (30 days post partum), the plasma GH concentration was elevated (13.2 ng/ml) compared to that in later lactation (90 days post partum; 9.8 ng/ml). This increased GH status was due to a greater magnitude of individual secretory spikes (27.3 vs. 20.2 ng/ml) rather than a difference in the frequency of spikes or in baseline plasma levels of GH. As lactation progressed from 30-90 days post partum, milk yield decreased, feed intake increased, and overall plasma concentration of insulin increased (17.3 vs. 29.3 microU/ml), reflecting both an elevated magnitude of hormone secretory spikes (30.2 vs. 55.2 microU/ml) and an elevated baseline concentration (16.8 vs. 27.3 microU/ml). The short term repeatability of overall mean and total 24-h GH and insulin secretion in lactating cows was demonstrated as well as the uniqueness of individual cow GH secretory patterns. Significant differences were not found in either glucose or FFA concentrations between lactation periods. Increased GH and decreased insulin during early lactation are likely to promote the mobilization of adipose tissue stores needed to supplement dietary energy consumption.

Effect of fasting on hormones and metabolites in plasma of fast-growing, lean and slow-growing obese pigs.

Plasma concentrations of glucose, free fatty acids (FFA), insulin and growth hormone (GH) were determined immediately after food removal and then hourly for 24 hours. Blood was sampled from six lean and six obese pigs at 10 weeks of age via indwelling catheters. Plasma glucose decreased but was similar in both pig strains shortly after feed removal; at the end of the 24-hr fast, plasma glucose was higher (P less than .01) in lean pigs. Plasma FFA concentrations were similar in lean and obese pigs and increased five-fold within 24 hr of fasting. Plasma insulin was higher (P less than .05) in obese pigs than in lean pigs immediately after food removal only (21.4 +/- 3.0 vs 9.8 +/- 2.4 microU/ml). Pattern of GH secretion over 24 hr was episodic; average plasma GH was lower in obese pigs than in lean pigs (2.8 +/- .7 vs 9.4 +/- 1.9 ng/ml). In summary, FFA mobilization was similar in lean and obese pigs, GH concentrations were lower in plasma of obese pigs and relative differences in plasma glucose and insulin between pig strains were influenced by time after feed removal.

Changes in concentrations of insulin, growth hormone and metabolites in plasma with spontaneous feeding in lactating dairy cows.

Changes in plasma levels of growth hormone (GH), insulin and metabolites and their relationships to spontaneous feeding in four lactating dairy cows were studied. Cows were fed a 60:40 (concentrate:silage) mixed ration through 100 days postpartum. Jugular blood samples were withdrawn at 10-minute intervals for 24 hours at day 90 postpartum; feeding behavior was monitored by time-lapse photography during this period. Insulin concentration increased immediately upon feeding and remained elevated for up to 50 minutes after meal termination, despite no significant change in plasma glucose levels. Plasma GH was depressed at 20 minutes postmeal and rose thereafter. Feeding appeared to dampen the fluctuations in free fatty acids (FFA) observed during premeal intervals. Time spent eating during a meal was not correlated with concentrations of these humoral factors at any premeal interval. The correlations of GH/glucose and GH/FFA were significant during eating, noneating and all other intervals during 24 hours. Correlation coefficients of other hormone/metabolite relationships were lower during eating as compared to non-eating periods. The consistent GH/metabolite relationships and rapid, feeding-induced changes in insulin, suggest their importance in the control of feed intake and energy balance.

Feeding behavior of lactating dairy cows as measured by time-lapse photography.

Evaluation of feeding behavior of ad libitum-fed lactating dairy cows by time-lapse photography revealed 68% of the total feeding activity occurred between the daylight hours of 0600 and 1800. Cows consumed an average of 12.1 meals/day, each 20.9 min in duration. Only 58% of the total defined meal time actually was spent eating, or 253.6 min/cow per day. Estimated meal size and rate of eating, as well as total daily time spent eating, were greater for cows as compared to animals with lower energy demand. Certain feeding characteristics, such as meal frequency and duration, were variable among animals, suggesting that these behaviors may be characteristics of individual cows. Results by time-lapse photography compared well with direct measurement by weigh-cell apparatus.