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Rat liver response elicited by long-term cold exposure
195
J Physiology (1992) 86, 195-200 © Elsevier, Paris
Rat liver response elicited by long-term cold exposure G Liverini, S Iossa, A Barletta Department of General and Environmental Physiology, University qf Naples, Via Me=.~o-Cannone 8, 180134 Naples, Italy (Received 8 February 1992; accepted 21 September)
Summary - We measured mitochondrial protein mass as well as State 4 and 3 respiratory rates using different substrates in isolated liver mitochondria from 30-day cold-exposed rats. In addition, we measured the respiration under different conditions of stimulation in isolated hepatocytes from long-term cold-exposed rats. The results show that long-term cold exposure elicits a significant increase in hepatic mass and mitochondrial protein mass. No variation was found in oxygen consumption of isolated mitochondria and hepatocytes. On the whole, the results indicate that long-term exposure elicits an increase in hepatic mitochondrial protein mass but not in hepatic oxygen consumption. long-term cold exposure / mitochondria / hepatocytes / oxygen consumption / liver
Introduction It is well known that cold acclimatization in the rat induces several modifications which are useful for survival in a cold environment. The increase in heat production is one of the modifications which takes place during cold exposure in order to maintain body temperature. This increase may be due in part to muscle shivering and in part to non-shivering thermogenesis (NST) that is fully developed after 3-4 weeks of cold exposure (Himms-Hagen, 1986). It is widely accepted that the primary site of NST in the rat is brown adipose tissue (Foster and Frydman, 1979). However, other modifications occur during cold acclimatization, such as a large increase in glucose mobilization (Smith and Davidson, 1982) and utilization (Bukowiecki, 1989), together with an increased formation of ketone bodies (Maekubo et al, 1977). There is also evidence of an increased gluconeogenesis (Shiota et al, 1985). In addition, we have shown that several changes take place in the rat liver during short-term cold exposure, such as a significant increase in the mitochondrial protein mass as well as in the metabolic energy turnover (Liverini et al, 1990; Iossa et al, 1991). On the other hand, it has been shown that neither hepatic blood flow (Foster and Frydman, 1979) nor oxygen consumption (Jansky et al, 1974) vary after long-term cold exposure. These
results suggest that the pattern of hepatic variations observed after short-term cold exposure could be modified after long-term cold exposure. It was, therefore, considered of interest to study the response of the liver after 30 days of cold exposure. To this purpose we have examined some respiratory parameters using various substrates in isolated mitochondria, as well as the respiration under different conditions of stimulation in isolated hepatocytes. In addition, we have measured mitochondrial protein mass. From the results, it appears clear that the liver, according to the length of cold exposure, participates in cold adjustments in different ways.
Materials and methods Animals
Male Wistar rats of about 200 g were divided into two groups. One group of rats was placed in a cold room (4 _+ I°C) for 30 days (30-day cold-exposed rats). The rats in the other group were kept at a temperature of 24°C (control rats). All rats were kept individually in wire-mesh cages under an artificial circadian 12:12 light-dark cycle, and had commercial rat chow, available ad libitum. Food intake was measured by giving a weighed amount of food each day (at 10:00). The intake measurements were corrected for spillage, the spilled
196 food being collected on filter paper placed under the cages.
Mitochondrial isolation and incubation Rats were killed by decapitation between 08:30 and 09:30, without any previous food deprivation, to avoid further stress to cold-exposed rats (Luz and Griggio, 1987). Livers were immediately removed, weighed, finely minced and washed with a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris, 1 mM EDTA, 1% fatty acid free bovine serum albumin (pH 7.4). Tissue fragments were gently homogenized with the same medium (1:10, w/v), in a Potter-Elvehjem homogenizer set at 500 rpm (four strokes/rain). The homogenate was filtered through sterile gauze and freed ot: debris and nuclei by centrifugation at 1000 g for 10 rain; the resulting supernatant was again centrifuged at 3000 g for 10 rain. The mitochondrial pellet was washed twice and resuspended in a medium containing 80 mM KCI, 50 mM Hepes (pH 7.0) 5 mM KH2 P04. The degree of purity of the mitochondrial preparation was determined by assaying several marker enzymes, according to the following published procedures: uricase (Plesner and Kalckar, 1963), acid phosphatase (Trouet, 1974), glucose-6-phosphatase (Swanson, 1955), and 5'-AMP phosphatase (Aronson and Touster, 1974). The mitochondrial protein content was determined by the method of Hartree (1972), using bovine serum albumin as the protein standard, while succinic-dehydrogenase activity was measured at 30°C by the method of Lee and Lardy (1965). Mitochondrial oxygen consumption was measured polarographically, with a Clark type electrode (Yellow Springs Instruments, Yellow Springs, OH, USA), maintained in a chamber at 30°C, using a medium containing 80 mM KCI, 50 mM Hepes (pH 7.0) 5 mM KHzPO4, 1% fatty acid free bovine serum albumin. Measurements were made within 2 h, following the isolation of the mitochondria. The mitochondria were allowed to oxidize their endogenous substrates for a few minutes. Substrate was then added to determine State 4 oxygen consumption rate. 6 min later, ADP (at a final concentration of 0.3 raM) was added and State 3 rate was measured. The respiratory control ratio (RCR) was calcualted as the ratio of State 3 and 4, while the ADP/O ratio was calculated as the ratio of the amount of ADP phosphorylated to ATP and the amount of oxygen consumed in this process (Estabrook, 1967). Concentrations of the various substrates used are noted in the tables.
Preparation and incubation of liver cells Rat liver cells were prepared as described by Seglen (1974), except that the rat was anesthetized by the intraperitoneal administration of chloral hydrate (40 mg/100 g bw). The hepatocytes were washed and suspended in a medium containing 120 mM NaC1,
5 mM KC1, 50 mM Hepes, (pH 7.4), I mM KH2PO4. 2 mM CaC12, 1.2 mM MgSO4, 2% fatty acid free bovine serum albumin. The final cell suspension was counted with trypan blue in a Burker chamber in order to assess the viability (routinely > 90%), and samples were taken for the determination of wet weight. Wet weight was determined by centrifuging cell samples in preweighed tubes at 3000 rpm for 5 min and then inverting the tubes; after 15-20 min any fluid on the tube walls was wiped off and the tubes were weighed. The number of parenchymal cells per g wet liver was calculated by estimating the percent contribution of the above cells to total hepatic volume by morphometric analysis, as described previously (Goglia et al, 1985). Hepatocyte oxygen consumption was measured polarographically, with a Clark type electrode maintained ill a chamber at 37°C. Aliquots corresponding to approximately 106 viable cells were incubated in the above suspension buffer with different substrates, at the concentrations reported in the tables.
Materials Collagenase (type IV), ADP, rotenone, succinate, malate, pyruvate, palmitoylcarnitine, glutamate, lactate, hexanoate, oligomycin were purchased from Sigma, St Louis, USA. All other reagents used were of the highest purity commercially available.
Statistics Data are given as means _+ SEM. Analysis of variance was used to determine significant differences among means, while differences between individual means were examined by the two-tailed Student's t-test, when a significant F value was obtained. For derived parameters, the Gaussian law of error propagation was applied.
Results Body weight and some hepatic parameters in control and cold-exposed rats No d i f f e r e n c e in body w e i g h t was o b s e r v e d at the end o f the e x p e r i m e n t a l period b e t w e e n control and 30-day c o l d - e x p o s e d rats (table I), indicating that the latter thrived in the cold, and co u l d be considered c o l d - a c c l i m a t i z e d . Cold e x p o s u r e d o u b l e d rat food intake (from 7 + 0.2 g/day x 100 g body w e i g h t in control rats to 13.5 + 0.4 g/day x 100 g body w e i g h t in c o l d - e x p o s e d rats), in a g r e e m e n t with previous similar o b s e r v a t i o n s (Portet, 1981; Shibata et al, 1989). On the other hand, si g n i f i can t variations in liver w e i g h t occurred; in fact, l i v er weight, ex p r essed per animal or per 100 gram body weight, was significantly
197 Table I. Body mass and hepatic parameters in control and 30-day cold-exposed rats. The values are the means _+ SEM of 20 different experiments.
Body mass, g Liver, g wet weight/animal Liver, g wet weight/100 g bw
Control rats
30-day coldexposed rats
321 _+ 9
317 _+ 10
Table II. The effect of long-term cold exposure on hepatocyte respiration. The values are the means _+ SEM of 10 different experiments and are expressed as gmol 02/rain x g wet liver. Isolated liver cells were incubated at 37°C as described in Materials and methods. Final substrate concentrations were: hexanoate 4 raM, lactate 10 mM, oligomycin 5 lag/m[ Control rats
30-day coldadapted rats
I. None
1.6 _+ 0.1
1.6 _+ 0.1
2. Hexanoate
2.3 ±
2.6 _+ 0.1
3. Hexanoate + lactate
2.9 _+ 1 b
3.3 ±
4. Hexanoate + oligomycin
0.9 _+ 1 b
1.2 ±
Additions 11.9 _+ 0.4 3.7 _+ 0.2
Number of hepatocytes per g wet weight 150 _+ 5
x 10 6
14.3 _+ 0.5 a 4.5 _+ 0.2 a 149 _+ 4
x 10 6
~' P < 0.05 versus control. h i g h e r (+ 20% and +22%, r e s p e c t i v e l y ) in 30-day c o l d - e x p o s e d rats than in c o n t r o l rats, as a l r e a d y shown by o t h e r authors ( F o s t e r and F r y d m a n , 1978). No s i g n i f i c a n t variations in the h e p a t o c y t e n u m b e r per g r a m wet w e i g h t were o b s e r v e d in rats living at the two different e n v i r o n m e n t a l t e m p e r a t u r e s (table I). As the p e r c e n t a g e contribution o f p a r e n c h y m a l cells ( a p p r o x i m a t e l y 83%) to the total hepatic v o l u m e was unaffected by c o l d e x p o s u r e (data not shown), we c o n c l u d e that the n u m b e r o f p a r e n c h y m a l liver cells per g r a m wet liver is a p p r o x i m a t e l y 124 x 106 in both groups. Respiration rates o f isolated hepatocytes in control and cold-exposed rats L i v e r cells from 30-day c o l d - e x p o s e d rats, inc u b a t e d without a d d e d substrate (basal state), exh i b i t e d an o x y g e n c o n s u m p t i o n s i m i l a r to those from c o n t r o l rats (table II). W h e n the cells were p r o v i d e d with h e x a n o a t e , an i n c r e a s e in o x y g e n c o n s u m p t i o n for both a n i m a l groups o c c u r r e d in c o m p a r i s o n to r e s p e c t i v e basal states. A further increase in the r e s p i r a t i o n rate o f the cells from both animal groups was a c h i e v e d by a d d i n g lactate to the i n c u b a t i o n m e d i u m c o n t a i n i n g hexanoate, since lactate is a g l u c o n e o g e n i c substrate which i n c r e a s e s the A D P availability. No effect o f 30-day c o l d e x p o s u r e was found in either o f the a b o v e c o n d i t i o n s . In the p r e s e n c e o f o l i g o m y c i n , an i n h i b i t o r o f ATP synthase, the m i n i m a l rate o f o x y g e n cons u m p t i o n n e c e s s a r y to b a l a n c e proton l e a k and m a i n t a i n a high A~H + was d e t e r m i n e d . O b v i o u s l y , a d e c r e a s e in o x y g e n c o n s u m p t i o n o c c u r r e d in the cells from both groups o f animals.
Ib
b
0.1 b
0.1 a'b
a Significant effect of cold exposure (P < 0.05). b Significant effect of additions (P < 0.05) comparing value 2 with I, or value 3 with 2, or value 4 with 2.
U n d e r these conditions, the cells from c o l d exp o s e d rats e x h i b i t e d a r e s p i r a t i o n rate signific a n t l y h i g h e r (+ 33%) than cells from control rats. Hepatic mitochondrial protein mass in control and cold-exposed rats Rat liver m i t o c h o n d r i a l protein mass was determ i n e d by a s s a y i n g the succinic d e h y d r o g e n a s e activity in liver h o m o g e n a t e s and in i s o l a t e d liver m i t o c h o n d r i a (table III). It should be noted that our isolation p r o c e d u r e results in a m i t o c b o n d r i a l p o p u l a t i o n which is e s s e n t i a l l y pure, as shown by the d e t e r m i n a t i o n o f some m a r k e r e n z y m e activities of the plasma membrane (5'-AMP p h o s p h a t a s e ) and o f the m a j o r c e l l u l a r organelles: m i t o c h o n d r i a (succinic d e h y d r o g e n a s e ) , p e r o x i somes (uricase), l y s o s o m e s (acid p b o s p h a t a s e ) and m i c r o s o m e s ( g l u c o s e - 6 - p h o s p h a t a s e ) . The results show that, apart from succinic d e h y d r o genase specific activity, only a very low acid p h o s p h a t a s e activity was detectable, while uricase, glucose-6-phosphatase and 5'-AMP p h o s p h a t a s e activities were undetectable. This e n z y m i c pattern r e m a i n e d u n c h a n g e d after 30 d a y s o f cold exposure. A f t e r 30-day cold e x p o s u r e a s i g n i f i c a n t increase o f about 30% (P < 0.05) in m i t o c h o n d r i a l protein content/g wet liver was o b s e r v e d comp a r e d with control rats. W h e n the data are exp r e s s e d per liver on a 1 0 0 g bw basis, this increase is more m a r k e d (+ 59%, P < 0.05).
198 Table Ill. Rat liver mitochondrial mass in long-term cold-exposed rats. The values are the means + SEM of 20 different experiments. Control rats
30-day coldexposed rats
9.6 _+ 0.5
12.1 _+ 0.5 a
0.28 _+ 0.02
0.27 + 0.02
Mitochondrial proteins, mg/g wet liver
34 + 3
45 _+ 4a
Mitochondrial proteins, (mg/liver/100 g bw)
126 _+ 11
202 _+ 18a
Succinic-dehydrogenase activity in the homogenate (gmol/min x g wet liver) Succinic-dehydrogenase activity in isolated mitochondria (~tmol/min x mg protein)
~ P < 0.05 versus controls.
Hepatic mitochondrial oxidative phosphorylation f r o m c o n t r o l a n d c o l d - e x p o s e d rats
The effect of 30-day cold exposure on oxidative phosphorylation of rat liver mitochondria was studied using lipid and non-lipid substrates to involve different modes of transportation into the mitochondria, different dehydrogenases and different sites of entry of reducing equivalents into the mitochondrial respiratory chain. The high values of RCR and ADP/O ratios obtained (table IV) indicated the high quality of the mitochondrial preparation. No significant difference was observed in the RCR and ADP/O values of mitochondria from rats living at the two different environmental temperatures. Liver mitochondria from cold-exposed rats exhibited rates of respiration similar to those from control animals with all the substrates utilized (table IV).
Discussion In this study we measured the oxygen consumption of rat liver cells isolated from control and 30-day cold-exposed rats under different conditions of stimulation of mitochondrial respiration. When we examined the respiration values of isolated rat liver cells from control rats, we observed that the oxygen consumption measured without added exogenous substrate (basal respiration) depended not only on the ATP/ADP ratio,
as is generally believed (Schwenke et al, 1981: Soboll and Stucki, 1985), but also on substrate supply to the electron transport chain (Nobes et al, 1990). In fact, the addition of an external substrate, such as hexanoate, stimulated an increase in endogenous respiration of approximately 44%. This stimulation was much greater than would be anticipated on the basis of the potential ATP demand arising from the metabolic interactions induced by substrate addition (Berry et al, 1983) and is most likely due to improved substrate supply to the electron transport chain, as shown by Nobes et al (1990). On the other hand, the oxygen consumption rate of isolated rat liver cells, with hexanoate added, reflected the intracellular ATP/ADP ratio, as the addition of lactate, which stimulates gluconeogenesis and hence ATP hydrolysis, caused a further increase in respiration. Analysis of the respiration rates obtained with isolated hepatocytes from 30-day cold-exposed rats shows that oxygen consumption is similar to that obtained with hepatocytes from control rats whatever the condition of stimulation (table II). It should be noted that this result is different from that previously obtained after a shorter period of cold exposure (Iossa et al. 1991). In fact, after 15 days of cold exposure we found increased oxygen consumption in isolated liver cells, as well as an increase in both substrate supply and ADP production rate (Iossa et al, 1991). We also measured oxygen consumption in isolated hepatocytes from both control and 30-day cold-exposed rats in the presence of oligomycin, an inhibitor of ATP synthase, and we obtained the minimal rate of oxygen consumption necessary to balance proton leak and maintain an elevated Agn + (La Noue et al, 1984). The decrease in oxygen consumption following oligomycin addition was not attributable to a fall in cellular ATP levels nor to a resulting interference in fatty acid activation, as the concentration of oligomycin employed has been shown not to cause a marked depletion of cellular ATP, probably because of a compensatory increase in the rate of glycolysis (Kraus-Friedmann et al, 1990). Oxygen consumption in the presence of oligomycin depends not only on proton leak but also on mitochondrial mass. As the values of proton leakage obtained measuring respiration in isolated mitochondria with succinate + rotenone + oligomycin were identical in control and cold-exposed rats (see table IV), the 33% increase found in oligomycinlimited respiration of hepatocytes from 30-day
199 Table IV. The effect of long-term cold exposure on hepatic non-lipid substrates. The values are the means + SEM of 10 x mg protein. Mitochondria were isolated and incubated at concentrations were: succinate 10 raM; rotenone 3.75 p.M; 2 Bg/ml; glutamate 10 raM; pyruvate l0 raM.
mitochondrial oxidative phosphorylation using lipid and different experiments and are expressed in nmol 02/min 30°C as described in Materials and methods. Substrate palmitoylcarnitine 40 BM; malate 2.5 raM; oligomycin
Succinate + rotenone
Palmitoylcarnitine + malate
Pyruvate + malate
Glutamate + malate
135 + 15 23.8 _+ 1.3 23.1 _+ 1.2 5.7 1.8 _+ 0.1
36.6 + 2.0 6.6 _+ 0.2
15.2 + 1.4 3.6 _+ 0.3
46.8 _+ 3.3 5.0 _+ 0.2
5.5 2.5 _+ 0.1
4.2 2.7 _+ 0.1
9.4 2.8 _+ 0.1
145 _+ 8 25.0 _+ 1.3 24.2 + 1.0 5.8 1.9 _+ 0.1
36.7 _+ 1.7 7.0 _+ 0.5
16.1 _+ 1.1 3.9 _+ 0.2
43.7 _+ 7.6 5.1 _+ 0.2
5.2 2.6 _+ 0.1
4.1 2.8 _+ 0.1
8.6 2.9 _+ 0.1
Control rats
State 3 State 4 State 4 + oligomycin RCR ADP/O 30-day cold-exposed rats
State 3 State 4 State 4 + oligomycin RCR ADP/O
c o l d - e x p o s e d rats (table II) c o u l d be fully exp l a i n e d by an i n c r e a s e in the m i t o c h o n d r i a l m a s s per gram wet liver found in 3 0 - d a y c o l d - e x p o s e d rats (see table III). In fact, after 30 days of cold e x p o s u r e , the m i t o c h o n d r i a l protein content per g r a m wet liver i n c r e a s e d by 33%; m o r e o v e r , due to the c o n c o m i t a n t i n c r e a s e in liver mass in cold e x p o s e d rats (table I), the total hepatic mitoc h o n d r i a l protein content on a 100 g b w basis inc r e a s e d by about 59%. Our results o b t a i n e d from rat liver i s o l a t e d mit o c h o n d r i a also show that m i t o c h o n d r i a l o x i d a t i v e c a p a c i t i e s do not vary after 30 days of cold exposure (table IV). This result is again different from a p r e v i o u s study on rats e x p o s e d to c o l d for shorter p e r i o d s ( L i v e r i n i et al, 1990) showing inc r e a s e d m i t o c h o n d r i a l o x i d a t i v e c a p a c i t i e s after 10 and 15 d a y s o f cold exposure. C o m p a r i n g p h y s i o l o g i c a l c h a n g e s w h i c h occur in the rat liver during s h o r t - t e r m c o l d e x p o s u r e (Liverini et al, 1990; Iossa et al, 1991) with those found in the p r e s e n t study, it a p p e a r s c l e a r that the liver exhibits different a d j u s t m e n t s a c c o r d i n g to the length o f cold exposure. In fact, after shortterm cold e x p o s u r e the i n c r e a s e in the mitoc h o n d r i a l mass and in the m i t o c h o n d r i a l specific r e s p i r a t o r y rates suggests an i n c r e a s e in ATP production, as d e m o n s t r a t e d by the i n c r e a s e d o x y g e n c o n s u m p t i o n found in i s o l a t e d h e p a t o c y t e s . The i n c r e a s e d ATP d i s p o s a l is in g o o d a g r e e m e n t with the i n c r e a s e in h e p a t i c g l u c o n e o g e n e s i s w h i c h is a s s o c i a t e d with c o l d e x p o s u r e and which shows
its m a x i m a l r e s p o n s e in the first days of c o l d exposure ( S h i o t a et al, 1985). A c c o r d i n g l y , recent data o b t a i n e d by Vallerand et al (1990) showed that the m a x i m a l utilization o f g l u c o s e by the skeletal muscles, which are the m a j o r sites o f glucose d i s p o s a l in the rat, is a s s o c i a t e d m o r e with the acute response to c o l d than with a c c l i m a t i o n , suggesting that glucose represents a substrate that is more i m p o r t a n t for contractile activity o f shivering than for n o n - s h i v e r i n g t h e r m o g e n e s i s . On the other hand, after 30 d a y s o f c o l d e x p o s u r e it appears that the increase in the m i t o c h o n d r i a l mass (see table III) does not give rise to i n c r e a s e d ATP p r o d u c t i o n in liver cells, as r e f l e c t e d by the a b s e n c e o f i n c r e a s e d o x y g e n c o n s u m p t i o n in isolated h e p a t o c y t e s (see table II). Thus, it seems that after l o n g - t e r m c o l d e x p o s u r e no variation o f m e t a b o l i c energy t u r n o v e r occurs in the liver; in fact, the i n c r e a s e d m i t o c h o n d r i a l m a s s is the only m o d i f i c a t i o n which persists in 30-day c o l d - e x p o s e d rat liver. The s i g n i f i c a n c e o f this result is at p r e s e n t unclear; however, taking into account that hepatic ketone b o d y p r o d u c t i o n as well as utilization by p e r i p h e r a l tissues s i g n i f i c a n t l y increases after l o n g - t e r m c o l d e x p o s u r e ( M a e k u b o et al, 1977), it is p o s s i b l e that in 30-day c o l d - e x p o s e d rats the i n c r e a s e d m i t o c h o n d r i a l mass could be a function o f p r o v i d i n g ketone bodies to support the fully d e v e l o p e d t h e r m o g e n i c response of the other organs ( F o s t e r and F r y d m a n , 1979; Shiota and M a s u m i , 1988). E x p e r i m e n t s are in progress to c o n f i r m the a b o v e h y p o t h e s i s .
200
Acknowledgments This work was supported by Ministero d e l l ' U n i v e r s i t ~ e della Ricerca Scientifica e Tecnologica, Italy.
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J Physiology (1992) 86, 195-200 © Elsevier, Paris
Rat liver response elicited by long-term cold exposure G Liverini, S Iossa, A Barletta Department of General and Environmental Physiology, University qf Naples, Via Me=.~o-Cannone 8, 180134 Naples, Italy (Received 8 February 1992; accepted 21 September)
Summary - We measured mitochondrial protein mass as well as State 4 and 3 respiratory rates using different substrates in isolated liver mitochondria from 30-day cold-exposed rats. In addition, we measured the respiration under different conditions of stimulation in isolated hepatocytes from long-term cold-exposed rats. The results show that long-term cold exposure elicits a significant increase in hepatic mass and mitochondrial protein mass. No variation was found in oxygen consumption of isolated mitochondria and hepatocytes. On the whole, the results indicate that long-term exposure elicits an increase in hepatic mitochondrial protein mass but not in hepatic oxygen consumption. long-term cold exposure / mitochondria / hepatocytes / oxygen consumption / liver
Introduction It is well known that cold acclimatization in the rat induces several modifications which are useful for survival in a cold environment. The increase in heat production is one of the modifications which takes place during cold exposure in order to maintain body temperature. This increase may be due in part to muscle shivering and in part to non-shivering thermogenesis (NST) that is fully developed after 3-4 weeks of cold exposure (Himms-Hagen, 1986). It is widely accepted that the primary site of NST in the rat is brown adipose tissue (Foster and Frydman, 1979). However, other modifications occur during cold acclimatization, such as a large increase in glucose mobilization (Smith and Davidson, 1982) and utilization (Bukowiecki, 1989), together with an increased formation of ketone bodies (Maekubo et al, 1977). There is also evidence of an increased gluconeogenesis (Shiota et al, 1985). In addition, we have shown that several changes take place in the rat liver during short-term cold exposure, such as a significant increase in the mitochondrial protein mass as well as in the metabolic energy turnover (Liverini et al, 1990; Iossa et al, 1991). On the other hand, it has been shown that neither hepatic blood flow (Foster and Frydman, 1979) nor oxygen consumption (Jansky et al, 1974) vary after long-term cold exposure. These
results suggest that the pattern of hepatic variations observed after short-term cold exposure could be modified after long-term cold exposure. It was, therefore, considered of interest to study the response of the liver after 30 days of cold exposure. To this purpose we have examined some respiratory parameters using various substrates in isolated mitochondria, as well as the respiration under different conditions of stimulation in isolated hepatocytes. In addition, we have measured mitochondrial protein mass. From the results, it appears clear that the liver, according to the length of cold exposure, participates in cold adjustments in different ways.
Materials and methods Animals
Male Wistar rats of about 200 g were divided into two groups. One group of rats was placed in a cold room (4 _+ I°C) for 30 days (30-day cold-exposed rats). The rats in the other group were kept at a temperature of 24°C (control rats). All rats were kept individually in wire-mesh cages under an artificial circadian 12:12 light-dark cycle, and had commercial rat chow, available ad libitum. Food intake was measured by giving a weighed amount of food each day (at 10:00). The intake measurements were corrected for spillage, the spilled
196 food being collected on filter paper placed under the cages.
Mitochondrial isolation and incubation Rats were killed by decapitation between 08:30 and 09:30, without any previous food deprivation, to avoid further stress to cold-exposed rats (Luz and Griggio, 1987). Livers were immediately removed, weighed, finely minced and washed with a medium containing 220 mM mannitol, 70 mM sucrose, 20 mM Tris, 1 mM EDTA, 1% fatty acid free bovine serum albumin (pH 7.4). Tissue fragments were gently homogenized with the same medium (1:10, w/v), in a Potter-Elvehjem homogenizer set at 500 rpm (four strokes/rain). The homogenate was filtered through sterile gauze and freed ot: debris and nuclei by centrifugation at 1000 g for 10 rain; the resulting supernatant was again centrifuged at 3000 g for 10 rain. The mitochondrial pellet was washed twice and resuspended in a medium containing 80 mM KCI, 50 mM Hepes (pH 7.0) 5 mM KH2 P04. The degree of purity of the mitochondrial preparation was determined by assaying several marker enzymes, according to the following published procedures: uricase (Plesner and Kalckar, 1963), acid phosphatase (Trouet, 1974), glucose-6-phosphatase (Swanson, 1955), and 5'-AMP phosphatase (Aronson and Touster, 1974). The mitochondrial protein content was determined by the method of Hartree (1972), using bovine serum albumin as the protein standard, while succinic-dehydrogenase activity was measured at 30°C by the method of Lee and Lardy (1965). Mitochondrial oxygen consumption was measured polarographically, with a Clark type electrode (Yellow Springs Instruments, Yellow Springs, OH, USA), maintained in a chamber at 30°C, using a medium containing 80 mM KCI, 50 mM Hepes (pH 7.0) 5 mM KHzPO4, 1% fatty acid free bovine serum albumin. Measurements were made within 2 h, following the isolation of the mitochondria. The mitochondria were allowed to oxidize their endogenous substrates for a few minutes. Substrate was then added to determine State 4 oxygen consumption rate. 6 min later, ADP (at a final concentration of 0.3 raM) was added and State 3 rate was measured. The respiratory control ratio (RCR) was calcualted as the ratio of State 3 and 4, while the ADP/O ratio was calculated as the ratio of the amount of ADP phosphorylated to ATP and the amount of oxygen consumed in this process (Estabrook, 1967). Concentrations of the various substrates used are noted in the tables.
Preparation and incubation of liver cells Rat liver cells were prepared as described by Seglen (1974), except that the rat was anesthetized by the intraperitoneal administration of chloral hydrate (40 mg/100 g bw). The hepatocytes were washed and suspended in a medium containing 120 mM NaC1,
5 mM KC1, 50 mM Hepes, (pH 7.4), I mM KH2PO4. 2 mM CaC12, 1.2 mM MgSO4, 2% fatty acid free bovine serum albumin. The final cell suspension was counted with trypan blue in a Burker chamber in order to assess the viability (routinely > 90%), and samples were taken for the determination of wet weight. Wet weight was determined by centrifuging cell samples in preweighed tubes at 3000 rpm for 5 min and then inverting the tubes; after 15-20 min any fluid on the tube walls was wiped off and the tubes were weighed. The number of parenchymal cells per g wet liver was calculated by estimating the percent contribution of the above cells to total hepatic volume by morphometric analysis, as described previously (Goglia et al, 1985). Hepatocyte oxygen consumption was measured polarographically, with a Clark type electrode maintained ill a chamber at 37°C. Aliquots corresponding to approximately 106 viable cells were incubated in the above suspension buffer with different substrates, at the concentrations reported in the tables.
Materials Collagenase (type IV), ADP, rotenone, succinate, malate, pyruvate, palmitoylcarnitine, glutamate, lactate, hexanoate, oligomycin were purchased from Sigma, St Louis, USA. All other reagents used were of the highest purity commercially available.
Statistics Data are given as means _+ SEM. Analysis of variance was used to determine significant differences among means, while differences between individual means were examined by the two-tailed Student's t-test, when a significant F value was obtained. For derived parameters, the Gaussian law of error propagation was applied.
Results Body weight and some hepatic parameters in control and cold-exposed rats No d i f f e r e n c e in body w e i g h t was o b s e r v e d at the end o f the e x p e r i m e n t a l period b e t w e e n control and 30-day c o l d - e x p o s e d rats (table I), indicating that the latter thrived in the cold, and co u l d be considered c o l d - a c c l i m a t i z e d . Cold e x p o s u r e d o u b l e d rat food intake (from 7 + 0.2 g/day x 100 g body w e i g h t in control rats to 13.5 + 0.4 g/day x 100 g body w e i g h t in c o l d - e x p o s e d rats), in a g r e e m e n t with previous similar o b s e r v a t i o n s (Portet, 1981; Shibata et al, 1989). On the other hand, si g n i f i can t variations in liver w e i g h t occurred; in fact, l i v er weight, ex p r essed per animal or per 100 gram body weight, was significantly
197 Table I. Body mass and hepatic parameters in control and 30-day cold-exposed rats. The values are the means _+ SEM of 20 different experiments.
Body mass, g Liver, g wet weight/animal Liver, g wet weight/100 g bw
Control rats
30-day coldexposed rats
321 _+ 9
317 _+ 10
Table II. The effect of long-term cold exposure on hepatocyte respiration. The values are the means _+ SEM of 10 different experiments and are expressed as gmol 02/rain x g wet liver. Isolated liver cells were incubated at 37°C as described in Materials and methods. Final substrate concentrations were: hexanoate 4 raM, lactate 10 mM, oligomycin 5 lag/m[ Control rats
30-day coldadapted rats
I. None
1.6 _+ 0.1
1.6 _+ 0.1
2. Hexanoate
2.3 ±
2.6 _+ 0.1
3. Hexanoate + lactate
2.9 _+ 1 b
3.3 ±
4. Hexanoate + oligomycin
0.9 _+ 1 b
1.2 ±
Additions 11.9 _+ 0.4 3.7 _+ 0.2
Number of hepatocytes per g wet weight 150 _+ 5
x 10 6
14.3 _+ 0.5 a 4.5 _+ 0.2 a 149 _+ 4
x 10 6
~' P < 0.05 versus control. h i g h e r (+ 20% and +22%, r e s p e c t i v e l y ) in 30-day c o l d - e x p o s e d rats than in c o n t r o l rats, as a l r e a d y shown by o t h e r authors ( F o s t e r and F r y d m a n , 1978). No s i g n i f i c a n t variations in the h e p a t o c y t e n u m b e r per g r a m wet w e i g h t were o b s e r v e d in rats living at the two different e n v i r o n m e n t a l t e m p e r a t u r e s (table I). As the p e r c e n t a g e contribution o f p a r e n c h y m a l cells ( a p p r o x i m a t e l y 83%) to the total hepatic v o l u m e was unaffected by c o l d e x p o s u r e (data not shown), we c o n c l u d e that the n u m b e r o f p a r e n c h y m a l liver cells per g r a m wet liver is a p p r o x i m a t e l y 124 x 106 in both groups. Respiration rates o f isolated hepatocytes in control and cold-exposed rats L i v e r cells from 30-day c o l d - e x p o s e d rats, inc u b a t e d without a d d e d substrate (basal state), exh i b i t e d an o x y g e n c o n s u m p t i o n s i m i l a r to those from c o n t r o l rats (table II). W h e n the cells were p r o v i d e d with h e x a n o a t e , an i n c r e a s e in o x y g e n c o n s u m p t i o n for both a n i m a l groups o c c u r r e d in c o m p a r i s o n to r e s p e c t i v e basal states. A further increase in the r e s p i r a t i o n rate o f the cells from both animal groups was a c h i e v e d by a d d i n g lactate to the i n c u b a t i o n m e d i u m c o n t a i n i n g hexanoate, since lactate is a g l u c o n e o g e n i c substrate which i n c r e a s e s the A D P availability. No effect o f 30-day c o l d e x p o s u r e was found in either o f the a b o v e c o n d i t i o n s . In the p r e s e n c e o f o l i g o m y c i n , an i n h i b i t o r o f ATP synthase, the m i n i m a l rate o f o x y g e n cons u m p t i o n n e c e s s a r y to b a l a n c e proton l e a k and m a i n t a i n a high A~H + was d e t e r m i n e d . O b v i o u s l y , a d e c r e a s e in o x y g e n c o n s u m p t i o n o c c u r r e d in the cells from both groups o f animals.
Ib
b
0.1 b
0.1 a'b
a Significant effect of cold exposure (P < 0.05). b Significant effect of additions (P < 0.05) comparing value 2 with I, or value 3 with 2, or value 4 with 2.
U n d e r these conditions, the cells from c o l d exp o s e d rats e x h i b i t e d a r e s p i r a t i o n rate signific a n t l y h i g h e r (+ 33%) than cells from control rats. Hepatic mitochondrial protein mass in control and cold-exposed rats Rat liver m i t o c h o n d r i a l protein mass was determ i n e d by a s s a y i n g the succinic d e h y d r o g e n a s e activity in liver h o m o g e n a t e s and in i s o l a t e d liver m i t o c h o n d r i a (table III). It should be noted that our isolation p r o c e d u r e results in a m i t o c b o n d r i a l p o p u l a t i o n which is e s s e n t i a l l y pure, as shown by the d e t e r m i n a t i o n o f some m a r k e r e n z y m e activities of the plasma membrane (5'-AMP p h o s p h a t a s e ) and o f the m a j o r c e l l u l a r organelles: m i t o c h o n d r i a (succinic d e h y d r o g e n a s e ) , p e r o x i somes (uricase), l y s o s o m e s (acid p b o s p h a t a s e ) and m i c r o s o m e s ( g l u c o s e - 6 - p h o s p h a t a s e ) . The results show that, apart from succinic d e h y d r o genase specific activity, only a very low acid p h o s p h a t a s e activity was detectable, while uricase, glucose-6-phosphatase and 5'-AMP p h o s p h a t a s e activities were undetectable. This e n z y m i c pattern r e m a i n e d u n c h a n g e d after 30 d a y s o f cold exposure. A f t e r 30-day cold e x p o s u r e a s i g n i f i c a n t increase o f about 30% (P < 0.05) in m i t o c h o n d r i a l protein content/g wet liver was o b s e r v e d comp a r e d with control rats. W h e n the data are exp r e s s e d per liver on a 1 0 0 g bw basis, this increase is more m a r k e d (+ 59%, P < 0.05).
198 Table Ill. Rat liver mitochondrial mass in long-term cold-exposed rats. The values are the means + SEM of 20 different experiments. Control rats
30-day coldexposed rats
9.6 _+ 0.5
12.1 _+ 0.5 a
0.28 _+ 0.02
0.27 + 0.02
Mitochondrial proteins, mg/g wet liver
34 + 3
45 _+ 4a
Mitochondrial proteins, (mg/liver/100 g bw)
126 _+ 11
202 _+ 18a
Succinic-dehydrogenase activity in the homogenate (gmol/min x g wet liver) Succinic-dehydrogenase activity in isolated mitochondria (~tmol/min x mg protein)
~ P < 0.05 versus controls.
Hepatic mitochondrial oxidative phosphorylation f r o m c o n t r o l a n d c o l d - e x p o s e d rats
The effect of 30-day cold exposure on oxidative phosphorylation of rat liver mitochondria was studied using lipid and non-lipid substrates to involve different modes of transportation into the mitochondria, different dehydrogenases and different sites of entry of reducing equivalents into the mitochondrial respiratory chain. The high values of RCR and ADP/O ratios obtained (table IV) indicated the high quality of the mitochondrial preparation. No significant difference was observed in the RCR and ADP/O values of mitochondria from rats living at the two different environmental temperatures. Liver mitochondria from cold-exposed rats exhibited rates of respiration similar to those from control animals with all the substrates utilized (table IV).
Discussion In this study we measured the oxygen consumption of rat liver cells isolated from control and 30-day cold-exposed rats under different conditions of stimulation of mitochondrial respiration. When we examined the respiration values of isolated rat liver cells from control rats, we observed that the oxygen consumption measured without added exogenous substrate (basal respiration) depended not only on the ATP/ADP ratio,
as is generally believed (Schwenke et al, 1981: Soboll and Stucki, 1985), but also on substrate supply to the electron transport chain (Nobes et al, 1990). In fact, the addition of an external substrate, such as hexanoate, stimulated an increase in endogenous respiration of approximately 44%. This stimulation was much greater than would be anticipated on the basis of the potential ATP demand arising from the metabolic interactions induced by substrate addition (Berry et al, 1983) and is most likely due to improved substrate supply to the electron transport chain, as shown by Nobes et al (1990). On the other hand, the oxygen consumption rate of isolated rat liver cells, with hexanoate added, reflected the intracellular ATP/ADP ratio, as the addition of lactate, which stimulates gluconeogenesis and hence ATP hydrolysis, caused a further increase in respiration. Analysis of the respiration rates obtained with isolated hepatocytes from 30-day cold-exposed rats shows that oxygen consumption is similar to that obtained with hepatocytes from control rats whatever the condition of stimulation (table II). It should be noted that this result is different from that previously obtained after a shorter period of cold exposure (Iossa et al. 1991). In fact, after 15 days of cold exposure we found increased oxygen consumption in isolated liver cells, as well as an increase in both substrate supply and ADP production rate (Iossa et al, 1991). We also measured oxygen consumption in isolated hepatocytes from both control and 30-day cold-exposed rats in the presence of oligomycin, an inhibitor of ATP synthase, and we obtained the minimal rate of oxygen consumption necessary to balance proton leak and maintain an elevated Agn + (La Noue et al, 1984). The decrease in oxygen consumption following oligomycin addition was not attributable to a fall in cellular ATP levels nor to a resulting interference in fatty acid activation, as the concentration of oligomycin employed has been shown not to cause a marked depletion of cellular ATP, probably because of a compensatory increase in the rate of glycolysis (Kraus-Friedmann et al, 1990). Oxygen consumption in the presence of oligomycin depends not only on proton leak but also on mitochondrial mass. As the values of proton leakage obtained measuring respiration in isolated mitochondria with succinate + rotenone + oligomycin were identical in control and cold-exposed rats (see table IV), the 33% increase found in oligomycinlimited respiration of hepatocytes from 30-day
199 Table IV. The effect of long-term cold exposure on hepatic non-lipid substrates. The values are the means + SEM of 10 x mg protein. Mitochondria were isolated and incubated at concentrations were: succinate 10 raM; rotenone 3.75 p.M; 2 Bg/ml; glutamate 10 raM; pyruvate l0 raM.
mitochondrial oxidative phosphorylation using lipid and different experiments and are expressed in nmol 02/min 30°C as described in Materials and methods. Substrate palmitoylcarnitine 40 BM; malate 2.5 raM; oligomycin
Succinate + rotenone
Palmitoylcarnitine + malate
Pyruvate + malate
Glutamate + malate
135 + 15 23.8 _+ 1.3 23.1 _+ 1.2 5.7 1.8 _+ 0.1
36.6 + 2.0 6.6 _+ 0.2
15.2 + 1.4 3.6 _+ 0.3
46.8 _+ 3.3 5.0 _+ 0.2
5.5 2.5 _+ 0.1
4.2 2.7 _+ 0.1
9.4 2.8 _+ 0.1
145 _+ 8 25.0 _+ 1.3 24.2 + 1.0 5.8 1.9 _+ 0.1
36.7 _+ 1.7 7.0 _+ 0.5
16.1 _+ 1.1 3.9 _+ 0.2
43.7 _+ 7.6 5.1 _+ 0.2
5.2 2.6 _+ 0.1
4.1 2.8 _+ 0.1
8.6 2.9 _+ 0.1
Control rats
State 3 State 4 State 4 + oligomycin RCR ADP/O 30-day cold-exposed rats
State 3 State 4 State 4 + oligomycin RCR ADP/O
c o l d - e x p o s e d rats (table II) c o u l d be fully exp l a i n e d by an i n c r e a s e in the m i t o c h o n d r i a l m a s s per gram wet liver found in 3 0 - d a y c o l d - e x p o s e d rats (see table III). In fact, after 30 days of cold e x p o s u r e , the m i t o c h o n d r i a l protein content per g r a m wet liver i n c r e a s e d by 33%; m o r e o v e r , due to the c o n c o m i t a n t i n c r e a s e in liver mass in cold e x p o s e d rats (table I), the total hepatic mitoc h o n d r i a l protein content on a 100 g b w basis inc r e a s e d by about 59%. Our results o b t a i n e d from rat liver i s o l a t e d mit o c h o n d r i a also show that m i t o c h o n d r i a l o x i d a t i v e c a p a c i t i e s do not vary after 30 days of cold exposure (table IV). This result is again different from a p r e v i o u s study on rats e x p o s e d to c o l d for shorter p e r i o d s ( L i v e r i n i et al, 1990) showing inc r e a s e d m i t o c h o n d r i a l o x i d a t i v e c a p a c i t i e s after 10 and 15 d a y s o f cold exposure. C o m p a r i n g p h y s i o l o g i c a l c h a n g e s w h i c h occur in the rat liver during s h o r t - t e r m c o l d e x p o s u r e (Liverini et al, 1990; Iossa et al, 1991) with those found in the p r e s e n t study, it a p p e a r s c l e a r that the liver exhibits different a d j u s t m e n t s a c c o r d i n g to the length o f cold exposure. In fact, after shortterm cold e x p o s u r e the i n c r e a s e in the mitoc h o n d r i a l mass and in the m i t o c h o n d r i a l specific r e s p i r a t o r y rates suggests an i n c r e a s e in ATP production, as d e m o n s t r a t e d by the i n c r e a s e d o x y g e n c o n s u m p t i o n found in i s o l a t e d h e p a t o c y t e s . The i n c r e a s e d ATP d i s p o s a l is in g o o d a g r e e m e n t with the i n c r e a s e in h e p a t i c g l u c o n e o g e n e s i s w h i c h is a s s o c i a t e d with c o l d e x p o s u r e and which shows
its m a x i m a l r e s p o n s e in the first days of c o l d exposure ( S h i o t a et al, 1985). A c c o r d i n g l y , recent data o b t a i n e d by Vallerand et al (1990) showed that the m a x i m a l utilization o f g l u c o s e by the skeletal muscles, which are the m a j o r sites o f glucose d i s p o s a l in the rat, is a s s o c i a t e d m o r e with the acute response to c o l d than with a c c l i m a t i o n , suggesting that glucose represents a substrate that is more i m p o r t a n t for contractile activity o f shivering than for n o n - s h i v e r i n g t h e r m o g e n e s i s . On the other hand, after 30 d a y s o f c o l d e x p o s u r e it appears that the increase in the m i t o c h o n d r i a l mass (see table III) does not give rise to i n c r e a s e d ATP p r o d u c t i o n in liver cells, as r e f l e c t e d by the a b s e n c e o f i n c r e a s e d o x y g e n c o n s u m p t i o n in isolated h e p a t o c y t e s (see table II). Thus, it seems that after l o n g - t e r m c o l d e x p o s u r e no variation o f m e t a b o l i c energy t u r n o v e r occurs in the liver; in fact, the i n c r e a s e d m i t o c h o n d r i a l m a s s is the only m o d i f i c a t i o n which persists in 30-day c o l d - e x p o s e d rat liver. The s i g n i f i c a n c e o f this result is at p r e s e n t unclear; however, taking into account that hepatic ketone b o d y p r o d u c t i o n as well as utilization by p e r i p h e r a l tissues s i g n i f i c a n t l y increases after l o n g - t e r m c o l d e x p o s u r e ( M a e k u b o et al, 1977), it is p o s s i b l e that in 30-day c o l d - e x p o s e d rats the i n c r e a s e d m i t o c h o n d r i a l mass could be a function o f p r o v i d i n g ketone bodies to support the fully d e v e l o p e d t h e r m o g e n i c response of the other organs ( F o s t e r and F r y d m a n , 1979; Shiota and M a s u m i , 1988). E x p e r i m e n t s are in progress to c o n f i r m the a b o v e h y p o t h e s i s .
200
Acknowledgments This work was supported by Ministero d e l l ' U n i v e r s i t ~ e della Ricerca Scientifica e Tecnologica, Italy.
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