Rapid oscillations in omental lipolysis are independent of changing insulin levels in vivo (original) (raw)
Protocol I: basal plasma profiles. In all basal experiments (Figure 1, Table 1), FFA showed a rapid oscillation in plasma. There were an average of 8 ± 2 FFA pulses per 60 minutes (range, 7–11), with an average pulse length of 5 ± 1 minutes (range, 2–15) and average pulse amplitude of 0.06 ± 0.02 mM (range, 0.03–0.12). Insulin also showed a rapid oscillation in plasma in all basal experiments; there were an average of 7 ± 1 insulin pulses per 60 minutes (range, 5–7), with an average pulse length of 8 ± 1 minutes (range, 2–21) and average pulse amplitude of 33.3 ± 31.6 pM (range, 8.0–91.0). This rapid insulin oscillation is similar to that described previously (3, 14, 16). Plasma glycerol also oscillated at a similar frequency to FFA; there were an average of 8 ± 1 glycerol pulses per 60 minutes (range, 6–10), with an average pulse length of 5 ± 1 minutes (range, 2–17) and average pulse amplitude of 0.02 ± 0.01 mM (range, 0.014–0.04). The glucose profile appeared to have very small amplitude rapid oscillations; however, ULTRA analysis was unable to identify any significant glucose pulses.
Rapid plasma FFA oscillations. Six representative dogs are shown during 1-minute sampling for 1 hour. Data are shown as mean ± SD.
Cross-correlation analysis of the systemic FFA and glycerol oscillations showed that the two oscillatory profiles are correlated with a lag time of approximately zero (95% CI). This result was expected since FFA and glycerol are released together during lipolysis. Because adipose tissue is extremely sensitive to small changes in insulin concentration, it is possible that the insulin oscillation is driving the FFA oscillation. This hypothesis was examined by cross correlating the systemic FFA and insulin oscillations. This analysis showed no correlation between the two profiles (CI < 95%), suggesting that it is not the insulin oscillation that is driving the FFA oscillation.
Protocol II: omental lipolysis. For all six dogs (Table 1), portal FFA levels were significantly higher than arterial levels (0.78 ± 0.09 vs. 0.71 ± 0.19 mM, respectively; P < 0.0001), indicating net omental lipolysis. Similarly, portal glycerol levels were significantly higher than arterial levels (0.19 ± 0.04 vs. 0.14 ± 0.04 mM, respectively; P = 0.05). Pulse analysis confirmed oscillations in arterial FFA (8 ± 2 pulses/h; range, 7–11), portal FFA (10 ± 1 pulses/h; range, 8–11), arterial glycerol (8 ± 1 pulses/h; range, 7–10), portal glycerol (8 ± 1 pulses/h; range, 7–10 pulses/h), and arterial insulin (7 ± 1 pulses/h).
Omental lipolysis was calculated from the direct measurement of FFA and glycerol release from the omentum. For all six dogs (Figure 2, Table 1), there were an average of 10 ± 1 omental FFA pulses per 60 minutes (range, 8–11) and 9 ± 1 omental glycerol pulses (range, 7–10) per 60 minutes, with an average pulse length of 6 ± 1 minutes (range, 2–14). The omental FFA oscillation had an amplitude of 0.02 ± 0.01 mmol/min (range, 0.01–0.03), and the omental glycerol oscillation had an amplitude of 0.01 ± 0.003 mmol/min (range, 0.01–0.02). These results suggest that there is pulsatile lipolysis from the omentum. The average FFA/glycerol ratio calculated from the omental profiles was 2.5 ± 1.2 (range, 0.5–3.8), close to the expected ratio of 3 (three fatty acids and one glycerol are released during lipolysis). One dog had a FFA/glycerol ratio of 0.5, which was probably due to lower than expected basal FFA concentrations. This could have been due to increased FFA uptake due to stress, fasting, or increased insulin sensitivity on that day. Without that one dog, the average FFA/glycerol ratio was 2.9 + 0.6 (range, 2.1–3.8). To see whether the arterial insulin oscillation could be driving pulsatile omental lipolysis, the arterial insulin oscillation and the omental FFA profile were cross correlated. Cross correlation showed no relationship between the two profiles (CI < 95%), suggesting that insulin is not the driving force of pulsatile release of FFA from the omentum.
Omental production of FFA (circles) and glycerol (plus symbols). Six representative dogs are shown during 1-minute sampling for 1 hour. Data are shown as mean ± SD. Omental production was calculated from direct measurements taken simultaneously from the portal vein and carotid artery (see the text).
Protocol III: removal of the insulin oscillation. To examine further the hypothesis that insulin is driving the FFA oscillation, the insulin oscillation was removed by an insulin clamp. If insulin is the driving force of the FFA oscillation, then the FFA oscillation should be blunted, irregular, or removed during a constant insulin infusion. Figure 3 shows representative results of the insulin clamp experiments. In all nine dogs that received a constant insulin infusion, the FFA profile showed no difference in pulsatility when compared with the basal profile (Table 2). In the basal state, there were an average of 9 ± 2 FFA pulses per 60 minutes (range, 7–12), with an average pulse length of 6 ± 2 minutes (range, 2–14) and average pulse amplitude of 0.07 ± 0.02 mM (range, 0.05–0.12). When the insulin oscillation was removed by constant insulin infusion there were an average of 9 ± 3 FFA pulses per 60 minutes (range, 6–14), with an average pulse length of 6 ± 2 minutes (range, 2–16) and average pulse amplitude of 0.04 ± 0.02 mM (range, 0.03–0.07).
Effect of insulin oscillation removal on the FFA oscillation. The top half shows one dog during sampling every 2 minutes for 3 hours, and the bottom half shows one dog with sampling every 1 minute for 1 hour. The left column shows the basal FFA and insulin profiles, and the right column shows the FFA and insulin profiles during insulin oscillation removal by insulin clamp.
Effect of insulin clamp and/or β-adrenergic blockade on the FFA oscillation
The number of pulses per 60 minutes and the average pulse length were not significantly different between the basal experiments and insulin clamp experiments (P = 0.4 and 0.3, respectively), indicating that the rapid insulin oscillation is not required for the FFA oscillation. However, the average FFA pulse amplitude was significantly lower during the insulin infusion protocol (P = 0.02). Given that the FFA concentration tended to be lower during the insulin clamp experiments, the FFA oscillation amplitude was recalculated as a percent of the average FFA concentration. During the basal state, the FFA oscillation amplitude was 13% of the average FFA concentration and 9% during the insulin clamp. This decrement is still significantly different (decrease of 30%; P = 0.04). The fall in FFA amplitude during the insulin clamp experiments was not due to overreplacement of insulin, as insulin was underreplaced (97.1 ± 79.8 pM ambient basal vs. 56.4 ± 25.7 pM during insulin infusion; P = 0.05). It is also possible that the drop in FFA amplitude is due to an under-replacement of glucagon or to the fact that growth hormone was not replaced.
The effect of removing the insulin oscillation on the FFA profile was also examined using the approximate entropy method (see Methods). During the basal state, the FFA profile had an average ApEn value of 0.28 ± 0.15. During constant insulin infusion, the FFA profile had an average ApEn value of 0.23 ± 0.13. These two ApEn values are not significantly different (P = 0.1), which further supports the assumption that the insulin oscillation is not the driving force of the FFA oscillation. In fact, the lower ApEn value and the decrease in amplitude of the FFA oscillation during removal of the insulin oscillation suggests that the FFA profile during the insulin infusion is less random than the basal FFA profile. This suggests that the insulin oscillation may be adding noise to the FFA oscillation.
Protocol IV: β-adrenergic blockade. To examine the possible role of the CNS, adrenergic modulation of the adipocyte was blocked by peripheral infusion of propranolol. The dogs’ heart rate, blood pressure, and glucose showed no significant change from basal. Figure 4 shows representative results of the β-blockade experiments. In three of the nine dogs that received propranolol infusion, ULTRA was unable to identify any significant FFA pulses, suggesting that adrenergic blockade removed the FFA oscillation in these dogs. In the other six dogs, the FFA oscillation remained identifiable by ULTRA, and the number and length of pulses was unchanged when compared with the basal state (Table 2). During β-blockade, there were an average of 10 ± 1 FFA pulses per 60 minutes (range, 10–11), with an average pulse length of 5 ± 1 minutes (range, 2–11) and average pulse amplitude of 0.06 ± 0.04 mM (range, 0.02–0.15). Although the average pulse amplitude was lower during propranolol infusion, when calculated as percent of basal, the amplitudes were not significantly different (14% during basal state versus 14% during β-blockade; P = 0.4).
Effect of β-adrenergic blockade or β-adrenergic blockade plus insulin oscillation removal on the FFA oscillation. The top row shows the basal FFA profiles. The bottom row shows the effect of propranolol infusion (Prop. Inf.) or propranolol infusion plus insulin clamp (Prop. + Ins. Inf.) on the FFA profile. The left half shows two dogs in which propranolol or propranolol plus insulin clamp seemed to remove the FFA oscillation, as determined by ULTRA (see the text). The right half shows two dogs in which propranolol or propranolol plus insulin clamp significantly disrupted the FFA oscillation, but did not remove it.
The effect of β-adrenergic blockade on the FFA profile was also examined using the approximate entropy method. During the basal state, the FFA profile had an average ApEn value of 0.28 ± 0.15. During β-adrenergic blockade, the FFA profile had an average ApEn value of 0.49 ± 0.09. The ApEn value during propranolol infusion was significantly higher than basal (P < 0.01). This suggests that β-adrenergic blockade disrupted the pulsatility of the FFA oscillation.
Protocol V: insulin clamp plus β-adrenergic blockade. To see whether both the rapid insulin oscillation and the sympathetic nervous system play a role in regulating the FFA oscillation, experiments were performed in which the insulin oscillation was removed with an insulin clamp and adrenergic modulation of the adipocyte was blocked by peripheral infusion of the β-blocker propranolol. Figure 4 shows representative results of the insulin clamp plus β-blockade experiments. In three of the six dogs that received insulin clamp plus propranolol, ULTRA was unable to identify any significant FFA pulses, suggesting that the FFA oscillation was removed in these dogs. In the other three dogs, the FFA oscillation remained identifiable by ULTRA, and the number and length of pulses was unchanged when compared with the basal state (Table 2). There were an average of 11 ± 2 FFA pulses per 60 minutes (range, 9–13), with an average pulse length of 4 ± 1 minutes (range, 2–7) and average pulse amplitude of 0.02 ± 0.01 mM (range, 0.02–0.03). Although the average pulse amplitude was lower during insulin clamp plus propranolol infusion, when calculated as percent of basal, the amplitudes were not significantly different (12% during basal state versus 12% during β-blockade; P = 0.5).
The effect of insulin clamp plus β-adrenergic blockade on the FFA profile was also examined using the approximate entropy method. During the basal state, the FFA profile had an average ApEn value of 0.28 ± 0.15. During β-adrenergic blockade, the FFA profile had an average ApEn value of 0.36 ± 0.17. The ApEn value during insulin clamp plus propranolol infusion was significantly higher than basal (P < 0.01). Because these results are no different than those of propranol infusion alone, this suggests that the effect of β-adrenergic blockade on the FFA oscillation is independent of the rapid insulin oscillation.