Albumin is the most abundant plasma protein in mammals and plays an important role as a carrier for a variety of molecules. Albumin possesses a high binding affinity for certain divalent cations, bilirubin, free fatty acids (FFA) and other molecules, including xenobiotics. Due to its high abundance and low molecular weight in relation to other major plasma proteins, albumin is responsible for about 80% of the total plasma oncotic pressure [1]. Therefore, albumin is a key regulator of fluid distribution between the plasma and interstitial compartments in physiological conditions; although the absence of plasma albumin can be well compensated by increased liver secretion of other proteins which help to maintain nearly normal plasma oncotic pressure [2]. Other less understood functions may also be ascribed to albumin, since its plasma depletion and redox modification have been demonstrated in several pathological conditions [3–5].

Both humans and rats (Nagase rats, NAR) suffering from congenital analbuminemia present with minor clinical symptoms despite the ascribed roles of albumin [6–8]. Abnormalities in fat storage and in lipid metabolism and/or transport are hallmarks of analbuminemic NAR and humans [9–15]. Plasma triglyceride (TG) levels are 3- to 5-fold higher in female NAR than in control Sprague-Dawley rats (SDR). A more severe dyslipidemia in female NAR seems to be partially driven by estrogen [13]. Compared to male, female NAR also develop more efficient adaptations to the lack of plasma albumin and do not present a deficit in total plasma proteins [2]. In analbuminemic plasma, very low density lipoprotein triglycerides (VLDL-TG) accounts for the largest fraction of total triglycerides [15]. Conversely, plasma FFA levels are severely decreased in NAR compared to control [14]. In addition, analbuminemic rats and human subjects present with hypercholesterolemia [6, 9, 11, 12]. This lipemic phenotype predisposes the affected individuals to a higher risk of developing cardiovascular disease, while low levels of FFA may contribute to other metabolic-related alterations, such as the fatigue experienced by analbuminemic individuals [7, 11, 12, 16].

Studies addressing the basis of hypertriglyceridemia in NAR have shown conflicting results. Catanozzi et al. [17] reported a faster triglyceride secretion rate from the liver and a slightly faster VLDL-protein plasma clearance rate. In contrast, others have demonstrated that NAR present with a slower plasma clearance rate of chylomicron-TG [16] or a similar clearance rate of VLDL- and chylomicron-TG [18]. In addition, lower post-heparin plasma lipoprotein lipase (LPL) activity has been described in NAR [13, 19]. Regarding the hepatic production of triglycerides, Joles et al. [9] showed that the liver lipogenesis rate is not different between NAR and SDR. Therefore, no clear conclusions can be drawn concerning the main metabolic processes contributing to NAR hypertriglyceridemia. Differences in criteria for animal group pairing (by body mass or by age), sex, basal states (fasted vs. fed) and methodology may account for these data discrepancies.

Despite the known roles of albumin as a FFA acceptor and carrier [20], there are no data on the mechanism underlining the low levels of plasma FFA found in NAR. It is still unknown whether the FFA deficit is a consequence of lipolysis inhibition, either directly by lower LPL activity [13, 19] or by a deficiency in plasma albumin, or by both. Abnormalities, such as lower body mass and adiposity [10] and the exercise and starvation intolerance observed in NAR [21, 22], seem consistent with a condition of lower FFA availability and flux into tissues [16, 23].

In this work, we found that liver lipogenesis and hepatic triglyceride output are decreased in fasted NAR compared to control. We also show that the low levels of plasma FFA are due to an inhibition of intravascular lipolysis caused by an acceptor (albumin) deficiency in NAR. All together, our data indicate that hepatic triglyceride production does not contribute to NAR fasting hypertriglyceridemia.

The liver lipogenesis rate (596 ± 40 vs. 929 ± 124 μmol ³H₂O/g/h) and the hepatic TG secretion rate into the plasma (4.25 ± 1.00 vs. 7.04 ± 1.68 mg/dL/min) were significantly (P ≤ 0.05) reduced in the 20-h fasted NAR compared to the SDR controls (Figure 1). In overnight sucrose-fed female (n = 4 each group) and male (n = 6 each group) rats, the hepatic TG secretion rates into the plasma were not significantly different between NAR and SDR: 5.27 ± 0.73 vs 6.08 ± 0.56 mg/dL/min for females, and 2.95 ± 0.77 vs 3.67 ± 0.82 mg/dL/min for males. Therefore, irrespective of feeding state and sex, liver triglyceride output does not explain the higher TG plasma levels in NAR.

Data from early in vitro studies with purified LPL [20, 27] showed that albumin allows lipolysis to proceed by acting as a FFA acceptor. Here, we show that an intravascular injection of albumin into NAR elicited an almost linear increase in the plasma FFA levels over time (Figure 2A). NAR plasma FFA levels increased 4-fold from a baseline of 0.36 ± 0.05 to nearly the control levels of 1.34 ± 0.16 mEq/L 90 min after the injection (P ≤ 0.05) (Figure 2A). SDR and NAR groups were also injected with saline to control for possible changes in plasma volume, which actually did not cause significant fluctuations in plasma FFA levels (Figure 2A). A heparin injection, which is known to increase the plasma activity of LPL [28], promptly increased (P ≤ 0.05) the plasma FFA levels in NAR but had no effect on SDR (Figure 2B). The highest increase in plasma FFA levels in NAR was observed 30 min after the heparin injection (from a baseline of 0.52 ± 0.12 to 1.40 ± 0.41 mEq/L, P ≤ 0.05) and decreased thereafter (Figure 2B). Despite the lack of albumin acting as a FFA acceptor in NAR, the heparin injection was effective in acutely increasing plasma FFA levels, probably because lipoproteins can carry an additional load of FFA [29]. The lack of an observable effect of heparin on SDR's plasma FFA levels is probably due to low concentrations of substrate (TG-rich lipoproteins) after the long fasting period.

The effect of exogenous albumin (final concentration of 30 mg/mL) on in vitro lipolysis was also studied. The addition of albumin to NAR serum promoted a nearly 3-fold stimulation in the amount of released FFA (Figure 3). The extent of lipolysis, taken as FFA accumulation above the initial values in NAR serum, increased (P ≤ 0.05) from 120.3 ± 21.1 (saline control) to 310.2 ± 51.5 uEq/L in the presence of exogenous albumin.

Since female NAR present with more prominent abnormalities in lipid metabolism compared to male NAR [12, 30], we also investigated whether intravenous albumin injection would stimulate plasma lipolysis in fasted male NAR. Similarly to female NAR, plasma lipolysis was stimulated in male NAR as indicated by a 2-fold increase (P ≤ 0.05) in plasma free glycerol levels 90 min after the intravenous injection of albumin (from a pre-injection level of 125.0 ± 17.7 to 260.4 ± 31.3 umol/L at the 90^th min after the injection).

Our results indicate that hypertriglyceridemia in analbuminemic rats does not occur as a result of increased hepatic triglyceride synthesis and output (Figure 1). Consequently, a slower removal rate of the plasma triglycerides seems to delineate the origin of NAR hypertriglyceridemia. Triglyceride removal from the circulation by targeted tissues occurs after their breakdown into glycerol and FFA [31]. Most of the FFA (~ 80%) that are derived from the LPL-mediated lipolysis of plasma triglyceride-rich lipoproteins are mixed within the pool of albumin-bound FFA before their transport into tissues [23, 32]. Our data show that plasma lipolysis is stimulated by the exogenous administration of albumin in NAR, both in vivo (Figure 2A) and in vitro (Figure 3). These data support that the lack of albumin limits LPL activity and thus, slows down the removal rate of plasma triglyceride-rich lipoproteins.

Regarding the contribution of hepatic triglyceride production our findings differ from previous studies that reported this process is enhanced in fasted female NAR [13] and in glucose-fed male NAR [17]. However, in sucrose-fed NAR groups, our results are in accordance with data from Joles et al. [9] that showed similar liver lipogenesis rates between fed NAR and controls. Some discrepancies may be due to important differences in study design. First, we paired the rat groups by age (3-month old) and not by body mass as did Catanozzi et al. [17]. It is noteworthy that NAR are lighter than SDR at the same age. Second, the time of the day at which the experiments were conducted must be considered, since circadian rhythms are well known modifiers of sterol and lipid biosynthesis. While we performed the metabolic studies in the morning from 10:00 to 13:00 h, Catanozi et al. [17] evaluated TG secretion rates from 17:00 to 19:00 h. Third, fasting period and/or type of diet prior to the experiments could also interfere in the results. Thus, based on our findings one can conclude that, either during fasting or fed states, hepatic triglyceride production does not contribute to NAR hypertriglyceridemia. However, since we measured liver lipogenesis only, we may not exclude that extra-hepatic lipogenesis may contribute to NAR hyperlipidemia as previously shown by Joles et al. [9].

A plasma FFA deficit (Table 1) has long been described in NAR [14] and is also observed in analbuminemic humans [7]. However, its physiological basis has never been experimentally addressed. Our attempts to stimulate plasma lipolysis, either by releasing the hypothesized lipolysis inhibition (albumin injection) or by increasing plasma LPL activity (heparin injection), caused a significant increase in plasma FFA levels in NAR, although only albumin induced a virtually complete correction of the NAR plasma FFA deficit (Figure 2A). The fast rise in plasma FFA levels induced by the injection of heparin in NAR (Figure 2B) reveals that the restraint on intravascular lipolysis is not due to defective LPL activity. Therefore, the metabolic machinery involved in plasma triglyceride breakdown and FFA release is not severely compromised in NAR and the plasma FFA deficit reflects an impaired lipolysis due mainly to a lack of plasma albumin as an acceptor. However, since lower LPL activity was reported in NAR parametrial adipose and heart tissues [10, 19], it is possible that the lower tissue-specific LPL may constitute another restraint for FFA flux into the NAR tissues [23].

Previous in vitro enzymatic studies using purified LPL clearly demonstrated that albumin acts as a FFA acceptor and allows lipolysis to proceed [20, 27]. Yet, this fundamental role of albumin in the control of lipolysis was not known to operate in vivo. The present results (Figure 2A) expand these previous in vitro findings of albumin playing a permissive role in intravascular lipolysis. To avoid any misinterpretation between albumin having a FFA sparing effect or stimulating lipolysis, we used an in vitro setup to assess the effects of exogenous albumin on the NAR serum lipolysis rate (Figure 3). This result clearly indicates that albumin is affecting lipolysis, i.e. stimulating the appearance of FFA in NAR plasma.

In conclusion, although NAR present with marked liver hypertrophy, the hepatic triglyceride synthesis and output rates do not contribute to the analbuminemic hypertriglyceridemia. Instead, an impaired triglyceride removal from the circulation may cause this alteration. It is proposed that a lack of plasma albumin to act as a FFA acceptor inhibits the intravascular lipolysis rate, which in turn hampers plasma triglyceride breakdown and its plasma removal.