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Frontiers in Physiology logoLink to Frontiers in Physiology
. 2014 Dec 2;5:469. doi: 10.3389/fphys.2014.00469

Short term fat feeding rapidly increases plasma insulin but does not result in dyslipidaemia

Benjamin Barzel 1,2, Jacquelyn M Weir 1, Peter J Meikle 1, Sandra L Burke 1, James A Armitage 1,2,3,, Geoffrey A Head 1,4,*,
PMCID: PMC4251291  PMID: 25520669

Abstract

Although the association between obesity and hypertension is well-known, the underlying mechanism remains elusive. Previously, we have shown that 3 week fat feeding in rabbits produces greater visceral adiposity, hypertension, tachycardia and elevated renal sympathetic nerve activity (RSNA) compared to rabbits on a normal diet. Because hyperinsulinaemia, hyperleptinemia, and dyslipidaemia are independent cardiovascular risk factors associated with hypertension we compared plasma insulin, leptin, and lipid profiles in male New Zealand White rabbits fed a normal fat diet (NFD 4.3% fat, n = 11) or high fat diet (HFD 13.4% fat, n = 13) at days 1, 2, 3 and weeks 1, 2, 3 of the diet. Plasma concentrations of diacylglyceride (DG), triacylglyceride (TG), ceramide and cholesteryl esters (CE) were obtained after analysis by liquid chromatography mass spectrometry. Plasma insulin and glucose increased within the first 3 days of the diet in HFD rabbits (P < 0.05) and remained elevated at week 1 (P < 0.05). Blood pressure and heart rate (HR) followed a similar pattern. By contrast, in both groups, plasma leptin levels remained unchanged during the first few days (P > 0.05), increasing by week 3 in fat fed animals alone (P < 0.05). Concentrations of total DG, TG, CE, and Ceramide at week 3 did not differ between groups (P > 0.05). Our data show plasma insulin increases rapidly following consumption of a HFD and suggests that it may play a role in the rapid rise of blood pressure. Dyslipidaemia does not appear to contribute to the hypertension in this animal model.

Keywords: insulin, leptin, plasma lipids, obesity, hypertension

Introduction

Obesity is associated with increased mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). Accumulating evidence suggests these changes are due to greater circulating concentrations of the adipokine leptin (Burke et al., 2013; Lim et al., 2013) which strongly correlate with RSNA and MAP in animal models of obesity (Prior et al., 2010; Burke et al., 2013). Consumption of a high fat diet (HFD) augments MAP and heart rate (HR) within the first few days of the diet, prior to any change in bodyweight (Burke et al., 2013). However, levels of circulating leptin are proportional to adiposity (Considine et al., 1996) and only begin to increase by the end of the first week of a HFD (Armitage et al., 2012). Thus, rapid changes in cardiovascular parameters suggest that a separate, leptin independent mechanism initiates the pressor response to a HFD. Plasma insulin concentrations increase within hours of meal consumption (Cummings et al., 2001) and are greater in both obese animals and humans (Bagdade et al., 1967; Lim et al., 2013) as well as patients with essential hypertension (Sobotka et al., 2011). Importantly, insulin is known to signal at the arcuate nucleus of the hypothalamus, the same nucleus at which a multitude of peripheral signals, including leptin, act to regulate energy and haemodynamic homeostasis (Benoit et al., 2004). Central administration of insulin attenuates food intake (Air et al., 2002) and augments sympathetic output (Muntzel et al., 1994). We have previously shown that insulin signaling is one of the factors responsible for the development of obesity related hypertension which is later maintained by slowly rising circulating leptin concentrations (Lim et al., 2013).

The association between dyslipidaemia and obesity is important given several lipid species are associated with a number of cardiovascular risk factors (Siri-Tarino et al., 2010). In addition, a single high-fat meal has been shown to reduce endothelial-dependent vasodilation up to 4 h post consumption in healthy normotensive individuals (Vogel et al., 1997). It has been suggested that endothelial-mediated vasodilatory mechanisms are impaired by triacylglycerides (TG) and free fatty acids (Doi et al., 1998; Lundman et al., 2001). Thus, it is possible that diet-induced changes in lipid profiles may play an early role in the development of obesity related hypertension. Lipid profiles have received scant attention in the fat-fed rabbit model of obesity related hypertension and only after several weeks of fat feeding (Eppel et al., 2013). The contribution of dyslipidaemia to the progression of disease is well-documented. Increased circulating ceramide concentrations are known to increase in obesity and are inversely correlated with insulin resistance (Haus et al., 2009). In addition, circulating levels of TG and cholesteryl esters (CE) are also elevated in obesity and have been shown to affect fasting glucose and insulin sensitivity (Sassolas et al., 1981; Cameron et al., 2008). In the present study the effect of HFD consumption on plasma insulin, leptin, and plasma lipid profiles was assessed in order to elucidate the contribution of each to the rapid rise in MAP observed within the first week of the diet.

Materials and methods

Animals and diets

Experiments were approved by the Alfred Medical Research Education Precinct Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for Scientific Use of Animals. Experiments were conducted in 24 conscious male New Zealand White rabbits (2.76–2.90 kg). Rabbits were housed in individual cages with a telemetry blood pressure receiver (model RLA 1020, Data Sciences International, St. Paul, MN, U.S.A) fitted to the door, under controlled light (6:00–18:00) and temperature (22°C ± 2°C) conditions. Rabbits were initially fed a restricted (150 g daily) normal-fat diet (NFD; 4.3 % total fat, 2.63 kcal/g, Specialty Feeds, Glen Forest, Australia) but after baseline recordings were randomized into two dietary groups and given free access to either a NFD or a high-fat diet (HFD; 13.4 % total fat, 3.34 kcal/g, Specialty Feeds) for 3 weeks. Daily food intake was determined by weighing the contents of the food hopper daily as well as weighing the food added.

Experimental procedures

A subset of rabbits underwent a preliminary operation under isoflurane anesthesia (3–4% in 1L/min oxygen; Abbot, Botany, NSW, Australia) following induction with propofol (10 mg/kg, Fresenius Kabi, Pymble, NSW, Australia). A radiotelemetry transmitter (model TA11PA-D70, Data Sciences) and catheter was implanted in the aorta via a small branch of the left iliac artery. Carprofen (3 mg/kg, Pfizer, Noth Ryde, NSW, Australia) was given before and 24 h after surgery for analgesia. After 1 week recovery, baseline MAP and HR were measured in the laboratory both by telemetry and by a catheter in the central ear artery. The telemetry signal was calibrated to the ear artery signal and this adjustment was applied to MAP measured in the home cage to minimize the possibility of drift of the signal with time. Baseline home cage MAP and HR were recorded for 1–2 days before rabbits were allocated to a group to receive either NFD or HFD. Home cage measurements were made continuously over 2 weeks.

Plasma collection and analysis

In order to avoid the effects of recent food consumption, animals were fasted for 4 h before blood samples were collected. Blood was collected before and on days 1, 2, 3, 7, 14, and 21 following the start of the HFD. Small samples of blood were used to measure blood glucose concentrations (Optium Xceed, Abbott, Doncaster, Victoria, Australia). Arterial blood (4 ml) was drawn into vacuum sealed cylinders containing K3EDTA (Vacuette Premium, Greiner bio-one, Wemmel, Belgium) and spun at 4°C for 10 min at 3000 RPM. Plasma aliquots (100 μl) were snap frozen in liquid nitrogen and stored at −80°C until use. Plasma lipid species were extracted into chloroform/methanol and quantified using high performance liquid chromatography-tandem mass spectrometry (Weir et al., 2013). Lipid species identified were diacylglycerides (DG), TG, ceramides (Cer), and CE. Total lipids within each class were calculated from the sum of the individual species. Plasma insulin and leptin concentrations were assessed using an ultra-sensitive insulin ELISA kit (Crystal Chem, Chicago, USA) with rabbit insulin standard and a radio immunoassay multispecies kit (LINCO Research, St Charles, MO, USA), respectively.

Data analysis

MAP and HR, derived from the pressure pulse, were digitized online at 500 Hz using an analog-to-digital data acquisition card (National Instruments 6024E, Austin, Texas, USA) and averaged over 2 s. MAP and HR were collected continuously over each 24 h period and averaged over one hourly intervals. Data were analyzed by split-plot repeated measures ANOVA allowing for between and within animal comparisons using excel version 2010 (Microsoft). MAP and HR were analyzed by repeated measures analysis of variance that allowed for within-animal contrasts. Data collected at a single time point were analyzed using a One-Way ANOVA. Bonferroni corrections were used to control for Type 1 error. A two sided probability of P < 0.05 was considered significant. For all statistics shown we refer to the main effect as a subscript, e.g., Pbaseline pertains to comparisons between groups made prior to the consumption of either diet, Pgroup, refers to comparisons between HFD and NFD-fed rabbits during dietary intervention, Pdiet refers to contrasts between baseline and dietary intervention within both NFD and HFD groups, Ptime, refers to comparisons within each group made between baseline and week 3 time points, Pdietxtime pertains to the interaction between diet and time.

Results

Effect of 3 week fat feeding on plasma insulin, glucose and leptin, food intake and haemodynamics

Baseline plasma insulin concentrations were not different between the dietary groups and averaged 0.46 ± 0.03 ng/ml (Pbaseline > 0.05; Figure 1, Table 1). A 50% increase from baseline in plasma insulin was observed in both NFD and HFD rabbits over the first 2 days of the diet (Pdiet < 0.05 for both groups; Figure 1). A further increase in plasma insulin concentrations on day 3 resulted in 65% greater insulin concentrations in HFD compared with NFD animals at both day 3 and week 1 time points (Pgroup < 0.05; Figure 1). By week 2, insulin concentrations in HFD rabbits had decreased to those observed in NFD rabbits (Pgroup > 0.05; Figure 1). Plasma glucose concentrations at baseline were not different between the dietary groups and averaged 5.5 ± 0.12 mmol/L (Pbaseline > 0.05; Figure 1, Table 1). Plasma glucose concentrations followed a similar pattern to insulin, rising on days 1 and 2 of the diet in both NFD and HFD rabbits (Pdiet < 0.05 for both groups; Figure 1). However, HFD rabbits exhibited a 59% greater increase in plasma glucose concentrations than controls (Pgroup < 0.05). By week 2, glucose concentrations returned to levels observed in NFD rabbits (Pgroup > 0.05; Figure 1). By contrast, plasma leptin concentrations, which were averaged 0.91 ± 0.13 ng/ml at baseline (Pbaseline > 0.05; Figure 1, Table 1), remained unchanged over the first 3 days of the diet in both dietary group (Pdiet > 0.05; Figure 1). However, plasma leptin concentrations in HFD-fed rabbits increased on week 1 of the diet compared with baseline (Pdiet > 0.05; Figure 1) and were 60 % greater than controls by the end of week 3 (Pgroup < 0.05; Figure 1). Food intake was similar in both groups with rabbits consuming 47–51% more food on the first day of both diets (Pdiet < 0.05). Intake in both groups gradually diminished to baseline levels after the first week (Figure 1). HR also increased rapidly on the first day after the start of the HFD to a level 12% greater than baseline (Pdiet < 0.001; Figure 1). HR remained elevated for the first week but had returned to control levels by week 2 (Pdiet > 0.05). By contrast, MAP increased from baseline levels by the 3rd day of the HFD (Pdiet < 0.05; Figure 1) and remained 7–8% elevated at 1–2 weeks (Pdiet < 0.01; Figure 1). Both MAP and HR in HFD fed rabbits were markedly higher over the 2 weeks of measurements than those fed a NFD (Pgroup < 0.001; Figure 1).

Figure 1.

Figure 1

Changes from baseline in levels of plasma insulin (A), leptin (B) and glucose (C) concentrations, food intake (D), mean arterial pressure (E) and heart rate (F) in rabbits fed either a normal fat diet (open circles) or a high-fat diet (closed circles) for 3 weeks. Data are mean difference ± SED, *P < 0.05, **P < 0.01, ***P < 0.001 for differences between dietary groups. Day, D; Week, W.

Table 1.

Baseline concentrations of insulin, glucose, and leptin.

Pre-NFD Pre-HFD Pgroup
Insulin (ng/ml) 0.440 ± 0.036 0.472 ± 0.048 0.61
Glucose (mmol/l) 5.54 ± 0.20 5.42 ± 0.16 0.65
Leptin (ng/ml) 0.751 ± 0.058 0.964 ± 0.146 0.20

Values are mean ± SEM. Pgroup is comparison of normal fat diet (NFD) with high fat diet (HFD).

Effect of HFD feeding on plasma lipid profiles

After 3 weeks of diet, total plasma DG, TG, Cer, and CE concentrations were not different between the dietary groups (Pgroup > 0.05; Figure 2). Specific DG, TG, and CE species did not change over the 3-week diet in either dietary group (Ptime > 0.05 for both NFD and HFD, Tables 25). By contrast, plasma Cer 16:0, 20:0, and 22:0 concentrations increased in HFD-fed rabbits over the 3 week period (Ptime > 0.05; Table 2) yet this was unlikely due to the consumption of the HFD (Pdiet > 0.05; Table 2) as the overall interaction between diet and time did not reach statistical significance (Pdiet× time > 0.05; Table 2). Individual cholesteryl ester species at week 3 were not different between the dietary groups (Pgroup > 0.05; Table 3). Similarly, DG (Pgroup > 0.05; Table 4) and TG (Pgroup > 0.05; Table 5) lipid species were not different between the dietary groups.

Figure 2.

Figure 2

Total Plasma concentrations of diacylglycerides (DG; A), triacylglycerides (TG; B), cholesteryl esters (CE; C) and ceramides (Cer; D) species in normal fat-fed (NFD; white bars) and high fat diet-fed (HFD; gray bars) after 3 weeks of diet. Data are mean ± SEM.

Table 2.

Ceramide species at baseline and week 3 in both NFD and HFD–fed rabbits.

NFD Week 0 NFD Week 3 HFD Week 0 HFD Week 3 Pdiet Ptime Pdiet×time
n 9 10 10 12
Ceramide species Mean SE Mean SE Mean SE Mean SE
Cer 16:0 189 28 253 28 189 16 287 21 1 0.01 1
Cer 18:0 131 18 136 17 139 13 179 26 1 1 1
Cer 20:0 168 22 206 27 159 12 239 21 1 0.05 1
Cer 22:0 608 93 754 108 550 47 882 84 1 0.05 1
Cer 24:1 440 71 633 94 395 43 510 50 1 0.21 1
Cer 24:0 833 141 971 174 665 56 885 95 1 1 1
Total Cer 2368 361 2952 430 2098 172 2983 271 1 0.18 1

Cer, Ceramides; NFD, normal fat diet; HFD, high fat diet.

Table 5.

Triacylglycerides at baseline and week 3 in both NFD and HFD–fed rabbits.

NFD Week 0 NFD Week 3 HFD Week 0 HFD Week 3 Pdiet Ptime Pdiet×time
n 9 10 11 13
TG Species Mean SE Mean SE Mean SE Mean SE
TG 14:0 16:0 18:2 3755 695 3729 521 2661 636 3812 495 1 1 1
TG 14:0 16:1 18:1 1644 426 3188 562 1728 463 1548 226 1 1 1
TG 14:0 16:1 18:2 432 94 557 74 585 202 600 76 1 1 1
TG 14:0 18:0 18:1 344 58 301 56 365 108 304 49 1 1 1
TG 14:0 18:2 18:2 514 90 493 114 729 313 767 110 1 1 1
TG 14:1 16:0 18:1 569 148 1139 249 742 196 584 130 1 1 1
TG 14:1 16:1 18:0 1798 450 3235 581 1729 375 1762 260 1 1 1
TG 14:1 18:0 18:2 117 35 303 54 4881 4747 193 30 1 1 1
TG 14:1 18:1 18:1 1378 299 1894 253 4834 3611 1644 186 1 1 1
TG 15:0 18:1 16:0 2032 209 1417 309 1809 419 1072 138 1 0.92 1
TG 15:0 18:1 18:1 1228 149 1075 216 2602 1586 754 93 1 1 1
TG 16:0 16:0 16:0 3150 560 2154 491 2434 591 3199 697 1 1 1
TG 16:0 16:0 18:0 2100 346 1811 675 1377 189 3107 616 1 1 1
TG 16:0 16:0 18:1 25852 3856 19383 3561 15841 3018 22531 3518 1 1 1
TG 16:0 16:0 18:2 12162 1992 7170 1940 7109 1269 15046 2945 1 1 0.23
TG 16:0 16:1 18:1 12109 2080 16866 3160 10526 2406 11433 1657 1 1 1
TG 16:0 18:0 18:1 7491 1216 4312 679 5718 807 5389 1143 1 1 1
TG 16:0 18:1 18:1 50498 5980 41240 5833 34880 7887 38074 4161 1 1 1
TG 16:0 18:1 18:2 35618 4652 23555 5079 24236 4482 36135 4229 1 1 0.56
TG 16:0 18:2 18:2 11604 1763 8206 2297 8113 1453 16218 2561 1 1 0.30
TG 16:1 16:1 16:1 173 41 284 39 291 130 191 25 1 1 1
TG 16:1 16:1 18:0 521 66 430 55 1047 639 528 73 1 1 1
TG 16:1 16:1 18:1 1723 299 1877 269 1293 302 1910 281 1 1 1
TG 16:1 18:1 18:1 2441 552 3941 619 2040 489 2530 313 1 1 1
TG 16:1 18:1 18:2 6301 1096 5597 980 5109 1078 7057 932 1 1 1
TG 17:0 16:0 16:1 4652 557 2903 503 3843 829 2066 300 1 0.15 1
TG 17:0 18:1 14:0 3653 450 2117 562 12151 9170 1141 203 1 1 1
TG 17:0 18:1 16:0 2101 257 1443 337 4914 3326 1402 192 1 1 1
TG 17:0 18:1 16:1 4237 499 3808 577 3463 941 2425 251 1 1 1
TG 17:0 18:1 18:1 2622 603 2397 375 2664 572 1902 440 1 1 1
TG 17:0 18:2 16:0 3291 423 2115 287 2559 532 1921 262 1 1 1
TG 18:0 18:0 18:0 71 26 31 7 1121 1084 55 11 1 1 1
TG 18:0 18:0 18:1 555 92 440 93 15842 15377 734 120 1 1 1
TG 18:0 18:1 18:1 5408 836 4963 817 31439 27087 6779 933 1 1 1
TG 18:0 18:2 18:2 1713 227 1334 352 4942 3562 2033 532 1 1 1
TG 18:1 14:0 16:0 4784 940 4858 781 3477 863 3857 641 1 1 1
TG 18:1 18:1 18:1 8080 1312 9679 1041 6304 1327 8604 873 1 1 1
TG 18:1 18:1 18:2 5822 917 6548 1358 4423 779 9515 1414 1 0.53 1
TG 18:1 18:1 20:4 345 67 1053 803 352 123 2421 763 1 0.82 1
TG 18:1 18:1 22:6 169 32 232 115 2570 2448 472 102 1 1 1
TG 18:1 18:2 18:2 4289 798 5209 1225 3480 616 7900 1428 1 0.68 1
TG 18:2 18:2 18:2 605 120 825 265 490 89 1440 341 1 0.72 1
TG 18:2 18:2 20:4 314 164 509 197 229 74 797 352 1 1 1
Total TG 238265 31727 204621 29850 246940 72879 231851 27372 1 1 1

TG, triacylglycerides, NFD, normal fat diet, HFD, high fat diet.

Table 3.

Cholesteryl esters at baseline and week 3 in both NFD and HFD–fed rabbits.

NFD Week 0 NFD Week 3 HFD Week 0 HFD Week 3 Pdiet Ptime Pdiet×time
n 9 10 11 13
Cholesteryl esters Mean SE Mean SE Mean SE Mean SE
CE 14:0 7697 1061 8329 1187 6480 1096 5407 453 0.73 1 1
CE 15:0 14345 2862 11136 2272 10088 2064 6288 946 0.63 1 1
CE 16:2 481 102 517 101 344 86 621 70 1 1 1
CE 16:1 36966 6848 56509 14258 28942 6014 30401 4080 0.97 1 1
CE 16:0 166404 29325 153289 29209 127649 25054 134902 19118 1 1 1
CE 17:1 9896 1933 6440 1188 7643 1094 5736 805 1 0.95 1
CE 17:0 11718 2693 6294 1127 8364 1989 5420 892 1 0.50 1
CE 18:3 17329 3009 20249 5470 13419 2731 21319 3599 1 1 1
CE 18:2 253823 46115 224743 32202 197578 35925 273220 36054 1 1 1
CE 18:1 154389 27782 154154 30973 96569 19247 121237 14200 1 1 1
CE 18:0 22633 5433 12713 2946 14992 4003 13617 2003 1 1 1
CE 20:5 894 257 1211 327 946 221 1382 258 1 1 1
CE 20:3 1113 229 1374 238 805 153 1182 168 1 1 1
CE 20:4 24310 5865 21934 3782 17486 3941 24641 3141 1 1 1
CE 20:2 204 38 244 51 214 55 239 42 1 1 1
CE 20:1 367 73 444 120 3211 2937 289 43 1 1 1
CE 20:0 477 89 308 62 1812 1449 259 46 1 1 1
CE 22:5 901 212 1116 339 2859 2087 1227 235 1 1 1
CE 22:4 293 79 280 63 256 62 262 37 1 1 1
CE 22:1 91 22 117 30 76 26 80 14 1 1 1
CE 22:0 221 32 177 36 372 210 144 25 1 1 1
CE 24:0 171 40 90 20 312 175 137 27 1 1 1
COH 125399 20715 147173 25523 98990 17953 154609 16540 1 1 1
Total CE 849914 142086 828575 138733 639050 113456 802445 92730 1 1 1

CE, cholesteryl esters; NFD, normal fat diet; HFD, high fat diet.

Table 4.

Diacylglycerides at baseline and week 3 in both NFD and HFD –fed rabbits.

NFD Week 0 NFD Week 3 HFD Week 0 HFD Week 3 Ptime Ptime Pdiet×time
n 9 10 11 13
DG Species Mean SE Mean SE Mean SE Mean SE
DG 14:0 14:0 28 4 34 5 23 4 25 5 1 1 1
DG 14:0 16:0 378 52 448 54 343 47 375 42 1 1 1
DG 14:1 16:0 57 9 109 11 74 10 68 14 1 1 0.61
DG 16:0 16:0 1720 221 1753 201 1439 129 1689 213 1 1 1
DG 14:0 18:1 670 139 888 121 632 113 618 72 1 1 1
DG 14:0 18:2 436 70 402 80 386 59 518 46 1 1 1
DG 16:0 18:0 993 110 871 109 837 81 1024 101 1 1 1
DG 16:0 18:1 7054 1323 7679 932 6111 717 6785 739 1 1 1
DG 16:0 18:2 5986 877 4836 976 4203 629 7382 999 1 1 0.36
DG 16:1 18:1 1214 203 2012 227 1641 513 1223 147 1 1 1
DG 18:0 18:0 212 16 185 42 277 119 255 23 1 1 1
DG 18:0 18:1 1425 187 1444 151 1123 175 1384 110 1 1 1
DG 18:0 18:2 1184 143 1001 189 895 114 1431 145 1 1 0.35
DG 18:1 18:1 5021 767 6195 749 4079 691 4460 384 1 1 1
DG 16:0 20:3 90 16 92 12 201 118 97 14 1 1 1
DG 18:1 18:2 7275 1046 7253 1480 6040 711 8615 771 1 1 1
DG 16:0 20:4 156 19 112 21 123 15 198 36 1 1 0.49
DG 18:1 18:3 1069 159 1112 234 1648 767 1262 110 1 1 1
DG 18:2 18:2 1702 253 1670 481 1274 193 2647 302 1 0.62 0.45
DG 18:0 20:4 197 112 84 8 202 125 105 14 1 1 1
DG 18:1 20:3 184 28 171 26 347 223 164 18 1 1 1
DG 16:0 22:5 130 17 83 15 76 18 104 20 1 1 1
DG 18:1 20:4 374 59 288 49 270 37 425 59 1 1 0.56
DG 16:0 22:6 29 4 18 4 34 12 28 5 1 1 1
Total DG 37583 5289 38739 5294 32277 3603 40884 3828 1 1 1

DG, diacylglycerides, NFD, normal fat diet, HFD, high fat diet.

Discussion

The main findings of the present study were that alongside elevations in blood pressure and HR, plasma glucose and insulin concentrations were increased within the first 3 days of a HFD, remaining elevated for the first week of the diet and returning to control levels thereafter. Notably, circulating leptin concentrations were unaltered by a HFD at day 3 but were markedly increased by week 3 whilst in the same time period, no evidence of dyslipidaemia was found. Together, these data suggest hyperinsulinemia rapidly develops after the commencement of a HFD and is a likely mechanism by which haemodynamics and sympathetic tone may change rapidly in the fat-fed rabbit model of obesity related hypertension.

A considerable body of evidence suggests insulin acts centrally to increase both blood pressure and sympathetic tone (Landsberg, 1996; Straznicky et al., 2010; Ward et al., 2011; Lim et al., 2013). There is a strong association between obesity, hyperinsulinemia and, at a later stage, insulin resistance (Weyer et al., 2001; Yuan et al., 2001). Of note is the apparent delay between the engagement of sympathetic nerve activity in obesity and the development of insulin resistance (Flaa et al., 2008) suggesting sympathetic overactivity may occur in response to very early changes in plasma insulin. Indeed central injections of insulin into the paraventricular nucleus of the hypothalamus produce large increases in lumbar sympathetic nerve activity (Ward et al., 2011). In the present study we observed a near two-fold increase in plasma glucose and insulin concentrations within 3 days of starting the HFD. Importantly, increases in MAP and HR in HFD-fed rabbits also began in the first few days of consumption as do increases in RSNA (Armitage et al., 2012; Burke et al., 2013) suggesting that circulating insulin may be involved in augmenting MAP early in the diet. In support of this are the findings that central administration of an insulin antagonist attenuated MAP after 1 week of a HFD (Lim et al., 2013). It is important to note that in the present study, plasma leptin concentrations in HFD-fed rabbits remained unchanged over the first 3 days of the diet but had increased by week 3. These results help explain our previous findings that central administration of a leptin antagonist to HFD-fed rabbits failed to elicit a reduction in either haemodynamic or sympathetic parameters at week 1 of the diet but produced large sympathoinhibitory and depressor responses at week 3 (Lim et al., 2013). Combined, these observations imply plasma insulin is involved in the remodeling of sympathetic tone within the first few days of consuming a HFD whilst leptin acts as a sympathoexcitatory signal later on in the diet, presumably once adiposity is increased. As both plasma glucose and insulin concentrations normalized by week 2 of the diet, the present observations point to sympathetic output preceding insulin resistance. Moreover, the apparent lack of effect of central administration of insulin on RSNA has been observed by others (Ward et al., 2011) and may in part be due to the direct effect of insulin on baroreflex gain (Pricher et al., 2008).

The present study also sought to establish the presence of dyslipidemia in our obese rabbit model and any subsequent contribution to the development of hypertension observed in these animals. In humans, dyslipidemia is a prominent feature of metabolic syndrome (Bays, 2009) and often appears in conjunction with hypertension (Nguyen et al., 2008). An example of the consequences of dyslipidemia can be found in greater total plasma ceramide concentrations which are known to occur in obesity whilst specific ceramide species are strongly associated with insulin resistance (Haus et al., 2009). In the present study, plasma concentrations of 4 lipid classes (Cer, CE, DG, and TG) were unchanged after 3 weeks of HFD. Our findings are in agreement with those made by Eppel and colleagues who observed no change in total plasma cholesteryl, and total plasma TG in rabbits fed a HFD for 9 weeks (Eppel et al., 2013) and suggests large changes in lipid profiles may take longer to develop in the rabbit model (Hamilton and Carroll, 1976). However, given the rapid haemodynamic and hormonal responses to dietary fat content, we expected to find changes in the expression of individual lipid species which would have been indicative of altered lipid metabolism. It is likely that our study was not powered to detect minute perturbations in the expression of specific plasma lipid species, contributing to our findings that plasma lipid profiles are unchanged by a diet high in fat. However, given that other parameters found in plasma, including insulin and leptin, can be measured accurately, our design is unlikely to be a confounding factor. Thus, our findings discount dyslipidemia as a likely mechanism by which hypertension occurs during 3 weeks of a HFD.

In conclusion, our findings demonstrate plasma insulin is a likely mechanism by which rapid increases in MAP occur over the first few days of consumption of a HFD. In addition, dyslipidaemia does not appear to develop after 3 weeks of fat feeding suggesting plasma lipid profiles do not play a role in the genesis of hypertension in our animal model but may contribute to the development of comorbidities associated with obesity at a later stage.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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