Changes in various plasma lipid components, glucose, and insulin in Spermophilus lateralis during...

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Comp. Biochem. Physiol. Vol. IIIB, No. 4, pp, 651~63, 1995 Elsevier Science Ltd

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Changes in various plasma lipid components, glucose, and insulin in Spermophilus lateralis during hibernation

I. Yeh,* C. F. Tam,* E. Catuira,t T. T. Le,t V. Papa,t L. Pena,t M. Vasquez,t C. Vu,t S. Wangt and G. A. Lopezt Departments of *Health and Nutritional Sciences and tBiology and Microbiology, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032, U.S.A.

This study evaluated fat mobilization as related to gluconeogenesis in two age groups of hibernating golden-mantled ground squirrels (Spermophilus lateralis). Our experimental group consisted of a total of 16 male and 15 female squirrels. Plasma samples were collected from selected animals being killed weekly from January to March, and the concentration of triglycerides, glycerol (GY), free fatty acids (FFA), total cholesterol, fipase activity, glucose, and insulin, were determined by biochemical assays and radioimmunoassay. Our results showed a mean FFA/GY ratio of five, which was higher than the predicted value of three, suggesting a significant depletion of GY and an enhanced rate of gluconeogenesis via GY to maintain glucose homeostasis in the hibernating animals. The factor of age did not significantly affect plasma lipid components. However, in the male group, plasma glucose levels were significantly higher for adults than for juveniles. Overall, females had significantly higher plasma glucose levels than males (150 _+ 11 vs. 110 + 8 mg%, P < 0.05). In the adult group, a gender influence was also seen on plasma insulin levels, with females' being higher than males' (66 + 13 vs. 25 _+ 3 plU/ml , P < 0.01). We suggest that during hibernation, female squirrels may have a higher rate of lipolysis and gluconeogenesis along with a lower glucose utilization than their male counterparts. Additionally, adult females may exhibit a higher peripheral insulin resistance during hibernation than adult males, a possibility which merits further study.

Key words: Age; Free fatty acids; Glycerol; Gender; Golden-mantled ground squirrel; Hibernators; Lipase; Triglycerides.

Comp. Biochem. Physiol. 111B, 651-663, 1995.

In t roduc t ion

Fat is the chief form of energy storage in animals due to its high yield of energy per unit weight. Fat utilization and gluconeogenesis are probably the two major processes used to derive energy and glucose by fasting animals

Correspondence to: C. F. Tam, Department of Health and Nutritional Sciences, CSULA, 5151 State University Drive, Los Angeles, CA 90032, U.S.A. Tel. 213-343- 4641; Fax 213-343-2670.

Received 24 August 1994; revised 12 December 1994; accepted 20 December 1994.

Abbreviations: F, female; FFA, free fatty acids; G, glucose; GY, glycerol; I, insulin; LP, lipase activity; M, male; TC, total cholesterol; TG, triglycerides.

including man (Lyman et al., 1982). In human starvation, gluconeogenesis is critically important for sustaining tissues and organs, such as the brain and red blood cells, while some kidney cells displayed an absolute glucose requirement because the carbohydrate reserves are de- pletable in a day (Hochachka and Somero, 1984). Hibernators, which are in a state of starvation (Green et al., 1984) and undergoing an extended period of inactivity (Wickler et al., 1991), experience a change in lean body mass, utilization of fat reserves, and maintenance of tissue glycogen (Hochachka and Somero, 1984).

CBPB II I / ~ F 6 5 1

652 I. Yeh et al.

The golden-mantled ground squirrel, Spermophilus ( = Citellus ) lateralis, exhibits endogenous rhythms of body weight (Pengelley and Asmundson, 1969; Blake, 1972). Weight- gain occurs through summer activity and weight-loss occurs in winter. The changes in body weight are primarily due to changes in total body fat in both the active and hibernating phases (Jameson and Mead, 1964; Dark et al., 1989).

In hibernating S. lateralis, the respiratory quotient (RQ) value, which has been reported lower than 0.8 (Kayser, 1961; Snapp and Heller, 1981), strongly suggests lipid catabolism. Endo- genous and exogenous lipids are transported in the circulatory system by lipoproteins. Plasma VLDL+LDL-cholesterol (very low density and low density lipoprotein-cholesterol) levels are increased, but no changes in total plasma cholesterol levels occur during hibernation in S. lateralis (Russom et al., 1992). Fatty acids derived from triglycerides after hydrolysis by lipoprotein lipase during lipolysis in adipose tissues are transported in the blood as an albumin-free fatty acid complex (Mayes, 1983; Voet and Voet, 1990). In the 13-lined ground squirrel (Spermophilus tridecemlineatus), the concentration of albumin in the blood increases 4-fold in early fall and is maintained throughout winter (Galster and Morrison, 1966).

Oxidative energy is mainly derived from non- carbohydrates in the golden-mantled ground squirrel during hibernation (Tashima et al., 1970). Gluconeogenesis (the resynthesis of glucose from most amino acids, lactate, and glycerol, mainly in the liver and, to a small extent, in the kidney) is the main pathway for regeneration of depleted carbohydrate reserves in animals (Hochachka and Somero, 1984; Voet and Voet, 1990). Glycerol, a by-product of lipolysis, is a precursor of glucose via gluconeogenesis (Lyman et al., 1982; Voet and Voet, 1990). The transport of glycerol across the liver cell membrane is a very fast carrier- mediated process that does not depend on hormonal influence (Kraus-Friedmann, 1984). Several studies have demonstrated that the glycerol released from lipolysis of triglycerides is the principal precursor for glucose resynthesis via gluconeogenesis in hibernating squirrels (Burlington and Klain, 1967; Galster and Mor- rison, 1975; Gehnrich and Aprille, 1988; Green et al., 1984).

Since no changes in the levels of blood glucose have been reported to occur in S. lateralis during hibernation (Tashima et al., 1970; Twente and Twente, 1967), it has been suggested that blood glucose homeostasis is maintained throughout the hibernating period. The maintenance of blood glucose is attributed

to the predominant lipolysis and gluconeogenesis in hibernators (Burlington and Klain, 1967; Galster and Morrison, 1966, 1975; Gehn- rich and Aprille, 1988; Green et al., 1984). Glucose can be formed in the liver by way of glycogenolysis or gluconeogenesis when the animals are in a fasting state during the hibernating period, of which dietary carbohydrate (glucose) can be discounted as a source. During hibernation, liver and cardiac muscle glycogen in S. tridecemlineatus (Zimny and Tyrone, 1957) and muscle glycogen in the hedgehog (A1-Badry and Taha, 1983) are increased, possibly to serve as a source of energy for arousal. Glycogen granules have been found to be more abun- dant in hibernating than in warm S. lateralis (McKeever, 1964). Also, a decreased glycogen phosphorylase activity in the liver and kidney of torpid S. lateralis has been reported by Brooks and Storey (1992). Similarly, the reported great reduction in phosphorylase activity, which would lead to a decrease in the rate of glycogenolysis in the Arctic ground squirrel, is consistent with the maintenance of a relatively high concentration of tissue glycogen in hibernators (South and House, 1967). This would, in turn, make gluconeogenesis the major source in supplying blood glucose during hibernation (Burlington and Klain, 1967; Burlington, 1972).

The concentration of pancreatic insulin has been found to be significantly reduced in hibernating Citellus lateralis (Bauman et al., 1987). Data obtained from several studies in other hibernating mammals have demonstrated that insulin resistance does occur during hibernation (Castex et al., 1987; Melnyk and Martin, 1985; Moreau-Hamsany et al., 1988).

Changes in lean body mass, utilization of fat reserves, and the maintenance of tissue glycogen in hibernators appear rather similar to that in man during starvation (Hochachka and Somero, 1984). The golden-mantled ground squirrel, S. lateralis can be considered an excellent animal model for the study of the inter- relationships between the mechanisms which regulate obesity, adipose tissue mass, and lipid metabolism as well as maintain blood glucose homeostasis in a state of long-term starvation.

In view of the lack of study in exploring the gender and/or age effects on fat mobilization as related to gluconeogenesis in hibernating golden-mantled ground squirrel (S. lateralis), this study aims to investigate.

1. If there are changes in the plasma concentration of triglycerides, glycerol, free fatty acids (FFA), total cholesterol, lipase activity, glucose, and insulin during hibernation, this may imply the extent to which lipolysis

Plasma lipids, glucose and insulin in squirrels 653

contributes to gluconeogenesis in the maintenance of blood glucose in the golden- mantled ground squirrel, S. lateralis.

2. If gender and age differences influence lipid mobilization during torpor in this species,

3. If any correlation exists between changes in the plasma concentration of triglycerides, glycerol, FFA, lipase activity, total cholesterol, glucose, and insulin in hibernating S. lateralis.

Materials and Methods 1. Experimental animals

The wild ground squirrels enter hibernation at snowfall and rouse from hibernation (emergence) at snowmelt (Bronson, 1980, McKeever, 1964). No squirrels were observed from late November to early March at 5000 ft elevation or to April at 6000 ft elevation in years of normal snowfall (McKeever, 1964). It has been reported that the emergence time of golden-mantled ground squirrels trapped from four different elevations demonstrated that adult wild squirrels emerge in late March to late May, whereas juvenile squirrels emerge in late June to early August (Bronson, 1980). During the period of January to March, the ground squirrel is in the deep torpid stage and near the emergent stage. The animals rouse more frequently at the beginning and at the end of hibernation (Pengel- ley and Fish, 1961). We designed our experimental environment as closely as possible to the environment of the wild for our experimental animals. Under our experimental design, by placing the animals from November to March in a cold room, they would have completed their normal hibernation cycle.

In August, 16 male and 15 female golden- mantled ground squirrels, S. lateralis, trapped from the mountains near Redding, California, were transported to and housed individually in wire cages in an animal room with a light:dark (LD) cycle of 12:12 at 22°C and ambient relative humidity. Distilled water and Wayne Rodent Blox (24% protein, 6% fat, 4.5% fiber, and approximately 2.2mEq Na) were provided ad libitum. Age identification was made on the first day the animals arrived according to body mass, body length, and appearance (Davis, 1984; McKeever, 1964). Animals were considered to be juveniles until they reached their first reproductive season (April-May) (McKeever, 1964). Of the 31 squirrels, there were seven juveniles and nine adults in the male group, and four juveniles and nine adults in the female group, whereas two female squirrels could not be identified with certainty as belonging to either the juvenile or adult groups. We did not include these two female

squirrels in our data analyses pertaining to influences of the factor of age. In the fall (November), the squirrels with similar body weights placed in their individual wire cages were moved to a cold room at 6°C under an LD cycle of 0:24. Distilled water and Wayne Ro- dent Blox were continually provided ad libitum during hibernation. Cotton was available for nesting. We finished the sacrifice of all animals by late March.

2. Plasma component analyses

Between January and March, the squirrels were selected by gender, age, and similarities in weight, and they were killed by decapitation weekly while in deep torpor. Blood was collected immediately following decapitation and placed in vials containing sodium heparin. The blood was centrifuged at 5000rpm for 15 min and the plasma portion was removed by the Pasteur pipette. The plasma samples were immediately frozen at -20°C until assayed, and were recentrifuged at 5000 rpm for 5 min on the day of assay.

Plasma triglyceride and glycerol concentrations were determined enzymatically as described by Sigma Diagnostics Procedure No. 337-B (St. Louis, MO), which was a modified method of McGowan et al. (1983). The concentration of FFA was measured using the non-esterified (or free) fatty acids NEFA C test kit (Code No. 990-75401), an enzymatic method developed by Wako (Osaka, Japan). Total cholesterol and glucose concentrations in plasma samples were determined enzymatically as described by Sigma Diagnostics Procedure No. 352-20, modified from the method of Allain et al. (1974), and No. 16-20, modified from Bondar and Mead (1974). Plasma lipase activity was measured as described by Sigma Diag- nostics Procedure No. 805, based on the colori- metric method of Imamura et al. (1989). The determination of insulin concentration was per- formed by radioimmunoassay as described by ICN Diagnostics Catalog No. 07-160102 (Costa Mesa, CA). Each sample was assayed in dupli- cate at 37°C or at room temperature according to the methods of measurement.

3. Analyses of data

Data were analyzed using the commercial statistical program SPSS for Windows v 6.0. All results were expressed as mean ± SE. Statistical comparisons of the gender or age difference in plasma measurements were evaluated by both Student's unpaired t-test and Mann-Whitney U/Wilcoxon rank sum W tests. The results were found to be similar between the parametric and the non-parametric tests. For simplicity, only results obtained from Mann-Whitney

654 I. Yeh et al.

Table 1. Gender differences in the plasma concentration of triglycerides (TG), glycerol (GY), free fatty acids (FFA), F F A / G Y ratio, total cholesterol (TC), lipase activity (LP), glucose (G), and insulin (I) in the hibernating golden-mantled ground

squirrels, Spermophilus lateralis

Component Male Female Male + female

TG (mg%) 225.06 + 38.52 153.49 + 29.29 189.28 + 24.76 (13) (13) (26)

GY ( u M ) 639.68 + 331.12 645.93 + 385.83 642.80 + 249.08 (13) (13) (26)

FFA (#Eq/l) 1548.00 ± 218.26 1381.46 + 116.39 1464.73 + 122.32 (13) (I 3) (26)

F F A / G Y 4.69 ___ 0.56 5.26 _+ 0.63 4.98 + 0.42 (13) (13) (26)

TC (rag%) 353.91 ___ 26.26 322.20 + 22.93 338.05 + 17.37 (13) (13) (26)

LP (U/l) 102.92 + 13.47 78.30 ___ 9.48 90.61 _+ 8.44 (13) (13) (26)

G (mg%)* 110.11 +_ 8.33 149.50 + 11.44 129.13 + 7.82 (15) (14) (29)

I (/~IU/ml) 37.53 ± 10.60 54.63 + 10.06 45.74 +__ 7.38 (13) (12) (25)

Values are means + SE. The number of animals is given in parentheses. *P < 0.01 difference between male and female.

U/Wilcoxon rank sum W tests were presented for the comparisons of the gender and age difference in this study. Mann-Whitney U/ Wilcoxon rank sum W tests were also used to test the effects of gender differences in each month and between months, and between different age groups. Comparisons of changes in the plasma compounds between months during hibernation were made by one-factor ANOVA. The Pearson correlation coefficients were used to express any bivariate relationship among the variables. Differences were considered significant at a level of P ~< 0.05.

Results

Two of the initial 31 experimental animals were not included in the final data analyses because they either never hibernated (one adult female), or awoke before sacrifice (one adult male).

1. Plasma lipid components and lipase activity

Plasma levels of triglycerides, glycerol (GY), free fatty acids (FFA), FFA/GY ratio, total cholesterol, and lipase activity in our total hibernating golden-mantled ground squirrels (S. lateralis) were 189 +_ 25 mg%, 643 ± 249 #M, 1465 ± 122#Eq/1, 5.0 ± 0.4, 338_+ 17mg%, and 91 ___ 8 U/l, respectively (Table l). Overall, plasma FFA/GY ratio tended to be higher, and plasma triglyceride, FFA, total cholesterol levels and lipase activity tended to be lower in the female squirrels than in their male counterparts, although no significant differences were observed (Table 1). When the gender factor was not considered, the juvenile squirrels had

higher plasma triglyceride levels and FFA/GY ratio, and lower plasma glycerol and FFA levels, and lipase activity than the adults; however, no significant differences were observed (Table 2). When the age factor was not taken into consideration, some changes were observed, although there were no significant differences between male and female animals in the changes of plasma lipase activity and glycerol levels (as shown in Figs 1 and 2, respectively) and other lipid components such as, triglycerides and total cholesterol at various time points during hibernation (unpublished results). In the female group, plasma glycerol levels were significantly higher in January than in March (437 ± 138 vs. 202 +_ 19 #M, P < 0.05; Fig. 2), whereas the FFA/GY ratio was lower in January than in March (4.0 _+ 0.5 vs. 6.4 ± 0.7, P < 0.05; Fig. 3).

When the gender and age factors were not considered in the total group, plasma glycerol

Table 2. Influence of age on the plasma concentration of triglycerides (TG), glycerol (GY), free fatty acids (FFA), F F A / G Y ratio, total cholesterol (TC), lipase activity (LP), glucose (G), and insulin (I) in the hibernating golden-

mantled ground squirrels, Spermophilus lateralis

Component Juvenile Adult

TG (mg%) GY (/~M) FFA (#Eq/l) FFA/Gy TC (mg%) LP (U/l) G (mg%) I (#IU/ml)

214.23 + 48.93 (9) 179.90 + 32.03 (15) 318.83 _+ 88.56 (9) 898.99 + 422.02 (15)

1434.46 + 226.32 (9) 1538.30 + 163.19 (15) 5.26 +_ 0.60 (9) 4.66 + 0.62 (15)

351.99+35.71 (9) 334.30+ 19,32 (15) 87.13 + 12.25 (9) 90.69 + 11.82 (15)

111.49+12.13 (11) 136.71+ 10.02 (16) 50.27 + 16.12 (9) 45.63 + 8.45 (14)

Values are means + SE. The number of animals is given in parentheses.


.= a.

175

150 I

100

50

Male

Female

n=2

n=7

n=3

0 I ! I

January February March

Durstion of Hibernation

Fig. 1. Influence of gender on plasma lipase activity in the golden-mantled squirrel, Spermophilus lateralis, at various time points during hibernation.

levels and FFA/GY ratio changed in proportion with the length of hibernation (Table 3). From January to March, plasma glycerol and FFA levels of the overall hibernating ground squirrels declined significantly from 1244 -t- 829 to 278 -t- 58/~M (P < 0.05) and from 1967 _+ 353 to 1396___ 156#Eq/1 (P < 0.05), respectively. On the contrary, the FFA/GY ratio in the plasma of pooled gender/age animals increased significantly from 3.51 _+ 0.76 to 5.76-t- 0.54 (P < 0.05) from January to March (Table 3). The levels of plasma triglycerides and total cholesterol as well as the plasma lipase activity were not significantly different during the length of hibernation (Table 3). The effect of gender on plasma ratio of FFA/GY at various time points in hibernating S. lateralis is shown in Fig. 3.

2. Plasma glucose and insulin

Plasma levels of glucose and insulin in our total hibernating golden-mantled ground squirrels were 129 _ 8 mg% and 46 _+ 7 #IU/ml, respectively (Table 1). Gender had a significant

influence on plasma glucose levels in the hibernating S. lateralis. Female squirrels had significantly higher plasma glucose levels than males (150+ 11 vs. 110+_ 8mg%, P < 0.01; Table 1). This difference was mostly contributed to by the juvenile group in which females showed a higher mean plasma glucose level than their male counterparts (141 + 18 vs. 95-I- 13 mg%, P < 0.05; Fig. 4). There was no significant difference between months in plasma glucose levels for all animals throughout their hibernation (Table 3). When the age factor was not considered, female squirrels showed consistently higher plasma glucose levels than males as hibernation progressed, but not significantly for the months of January and February (Fig. 5). With respect to the age factor, a significant difference in plasma glucose levels in the male group was observed (Fig. 4), although there was no significant difference for gender-pooled animals (Table 2). In the male group, adult squirrels showed a higher mean plasma glucose level than their juvenile counterparts (124_ 9

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O-

4200

3500

2800

2100

1400

700

n = 3 n = 4 n = 7

I I I


n = 2

n = 3

~; Male

Female

* P<O.05

656 I. Yeh et al.

Duration of Hibernation

Fig. 2. Influence of gender on plasma glycerol levels in the golden-mantled ground squirrel, Spermophilus lateralis, at various time points during hibernation.

vs. 95 +_ 13 mg%, P = 0.05; Fig. 4). In contrast, there was no significant age difference in plasma glucose levels in the female group.

The hibernating adult female golden-mantled ground squirrels (S. lateralis) showed significantly higher plasma insulin levels than their male counterparts (66 _+ 13 vs. 25 _+ 3/~IU/ml, P < 0.01), but these differences were not seen in the juvenile group (Fig, 6). When the gender or age factor was not considered, however, no significant difference in plasma insulin levels was observed in different sexes or age groups (Tables 1 and 2). Also, no significant difference in the plasma insulin levels was seen when animals were pooled for gender/age at various time points during hibernation (Table 3).

3. Correlation of bivariate among variables in the total group of squirrels

Significant correlations in plasma glycerol and FFA levels (r = 0.586, P < 0.005), plasma

glycerol levels and F F A /G Y ratio (r = -0 .667, P <0.001), plasma FFA and insulin levels ( r=0 .804 , P < 0 . 0 0 1 ) , and plasma lipase activity and total cholesterol levels (r = 0.425, P < 0.05) were observed in the total group of squirrels.

Discussion 1. Plasma lipid components

The high F F A /G Y ratio of five in the hibernating S. lateralis observed in our present study (Table 1) compared with the predicted ratio of three for equal utilization of FFA and glycerol from triglyceride (Galster and Morrison, 1975), suggests that depletion of GY could occur at a greater rate than the utilization of FFA. Glycerol is a preferential substrate of gluconeogenesis in hibernators (Galster and Morrison, 1975; Gehnrich and


._o r r

LL

7.5

7

]; Male * n=7

Female

• P < 0 . 0 5

n=4

n = 3

n = 2

0 I I I



Fig. 3. Influence of gender on plasma ratio of free fatty acids to glycerol in the golden-mantled ground squirrel, Spermophilus lateralis, at various time points during hibernation.

Aprille, 1988; Green et al., 1984), in rabbits (Hagen, 1963), and in human (Bortz et al., 1972; Steele et al., 1971), although it can be considered as a source of energy through oxidation. Using ~4C-glycerol as a substrate in human studies, Bortz et al. (1972) found that plasma glycerol was nearly or completely converted to glucose in both lean and obese subjects during starvation. By observing the depletion of glycerol as well as the elevated FFA/GY ratio in this study (Tables 1 and 3, Figs 2 and 3), we suggest that an enhanced rate of gluconeogenesis could exist via GY to maintain glucose homeostasis rather than that GY is oxidized to a greater extent than FFA in the hibernating S. lateralis. Furthermore, the FFA/GY ratio in the plasma of our pooled gender/age animals

increased significantly from January to March (Table 3), suggesting that a peak increase in the rate of gluconeogenesis may be required for permanent arousal in spring. However, due to the limited experimental design of this study, the data obtained can only imply, but by no means prove, the extent to which lipolysis contributes to gluconeogenesis in the maintenance of blood glucose in the golden-mantled ground squirrel, S. lateralis.

Our results regarding total plasma cholesterol levels are also consistent with those (350 mg%) reported in previous studies from our laboratory (Russom et al., 1992), although the reasons for the observed lack of changes in this par- ameter throughout hibernation are unclear at this time.

658 I. Yeh et al.

Table 3. Changes in the plasma levels of triglycerides (TG), glycerol (GY), free fatty acids (FFA), FFA/GY ratio, total cholesterol (TC), lipase activity (LP), glucose (G), and insulin (I) in the golden-mantled ground squirrel, Spermophilus lateral& at

various time points during hibernation

Component January February March

TG (rag%) 124.22 _+ 43.61 184.60 + 48.09 214.85 + 35.78 (5) (7) (14)

GY (#M)*? 1243.61 + 829.22 942.42 _ 718.24 278.42 _+ 58.22 (5) (7) (14)

FFA (~Eq/l)* 1967.37 _+ 353.43 1244.00 + 162.62 1395.59 + 156.27 (5) (7) (14)

FFA/GY* 3.51 + 0.76 4.46 + 0.80 5.76 + 0.54 (5) (7) (14)

TC (rag%) 303.41 _+ 39.84 321.55 _+ 29.06 358.68 _+ 25.14 (5) (7) (14)

LP (U/l) 93.46 +_ 23.47 75.22 _+ 12.05 97.28 _+ 12.14 (5) (7) (14)

G (mg%) 127.53 _+ 12.58 136.77 + 16.04 125.69 + 11.91 (6) (8) (15)

I (#IU/ml)~ 71.50 + 35.50 31.73 _+ 8.52 49.77 +_ 10.46 (2) (8) (15)

Values are means _+ SE. The number of animals is given in parentheses. *P < 0.05 between January and March. t P < 0.05 between January and February. :~P < 0.05 between February and March.

2. Plasma glucose, triglycerides, lipase activity, and insulin

In hibernating S. lateralis, fat storage is less for females than for males (Blake, 1972). Since the weight loss rate was higher in both juvenile and adult female squirrels than in juvenile males during hibernation in a previous study done in this laboratory (Catuira et al., 1994), this may indicate the fact that male squirrels possess more residual fat than females when they rouse from hibernation (Michener and Locklear, 1990). In addition, our present results (Table 1) showed that plasma triglycerides levels in female squirrels were decreased, although not significantly over those in males. This difference can be explained, in part, by the greater weight loss rate in female squirrels than juvenile males during hibernation (Catuira et al., 1994).

The observed plasma glucose levels (Table 1) in pooled gender/age hibernating S. lateralis are consistent with those reported by other studies (Twente and Twente, 1967; Tashima et al., 1970). We observed in the total group a significantly higher plasma glucose level in the female than in the male squirrels (Table 1). Also, the juvenile and adult female animals showed significantly higher plasma glucose levels than the juvenile males (Fig. 4). We suggest that the factor of gender may have more influences on the concentrations of plasma glucose in the juvenile hibernators while they make physio- logical adjustments for their continued growth and reproduction (Table 1 and Fig. 4). The increase in plasma FFA is associated with a reduction in the rate of glucose oxidation in

humans (Saloranta et al., 1993), as well as FFA uptake being reported to be much greater in female than in male human hepatocytes (Sorrentino et al., 1988). When compared with the males, we suggest that the hibernating female ground squirrels may have a greater rate of gluconeogenesis via a higher lipolytic rate to make more glycerol available for gluconeogenesis and/or a lower glucose utilization by an elevating FFA to spare glucose from being utilized as fuel during torpor. We also suggest that female sex hormones could play a role in modulating plasma glucose levels in the female hibernators.

Normal function of circulating lipoprotein lipase requires insulin (Brodsky and Devlin, 1994) and the occurrence of insulin resistance during hibernation has been reported (Castex et al., 1987; Melnyk and Martin, 1985; Moreau- Hamsany et al., 1988). However, studies on the effects of gender and/or age on the occurrence of insulin resistance during hibernation were not available. In the adult group, although no significant difference was obtained, there were observed differences between males and females in plasma glucose (male vs. female: 124 + 9 vs. 150 _ 17mg%, P =0.38; Fig. 4), triglyceride levels (male vs. female: 228 _+ 54 vs. 138 _+ 34 mg%, P = 0.23), and plasma lipase activity (male vs. female: 113+21 vs. 71+9U/1, P = 0.15), with adult females having significantly higher plasma insulin levels (66 + 13 #IU/ml) than adult males (25_+3#IU/ml, P < 0.01; Fig. 6). In human studies, females had higher glucose tolerance curves than males, in


175

150

100

50

K

K

~ ~ n=8

n=4

Juvenile Adult

Age

• Male

Female

] Male & Female

• k p<O.05

Fig. 4. Influence of gender and age on plasma glucose levels in the hibernating golden-mantled ground squirrels, Spermophilus lateralis.

particular, with advancing age (Gordon, 1964). Based upon our study, the adult female squirrels had significantly higher plasma insulin levels and tended to have higher (although not significant) plasma glucose levels than their adult male counterparts during hibernation. We suggest that female sex hormones and/or the factor of age could play a role during torpor in regulating plasma glucose and/or insulin levels in the adult female squirrels. We propose that the adult female ground squirrels may have higher peripheral insulin resistance than their male counterparts during torpor. By proposing the possibility of a higher insulin resistance being present in the adult female hibernating squirrels, we further suggest that their white adipose tissue might have a higher resistance to insulin and enhanced sensitivity to the lipolytic hormones, such as glucagon and norepinephrine. In other words, the greater insulin resistance as proposed

in the adult female hibernators could imply that their white adipose tissue emphasizes lipolysis by the lipolytic hormones more than those in the adult male counterparts. The increased rate of lipolysis in the white adipose tissues, as evi- denced by the finding that female S. lateralis stored less fat than males during hibernation (Blake, 1972), could contribute to a greater rate of gluconeogenesis in the adult female hibernators. Hence, a higher plasma glucose level (although not significant) was found in these animals than the adult males (Fig. 4). It is noteworthy that we have observed a gender/age difference in relation to insulin resistance as proposed in our hibernators.

3. Correlation ofbivariate among variables in the total groups of squirrels

Our observed correlation between plasma glycerol and FFA levels in the hibernating S.

660 I. Yeh et al.

=o

8~

(9

E @ R-

200

15o

lOO

o

Male

Female

• k p<O.05

n=4

=7

n = 3

I

n = 8

i I I



Fig. 5. Influence of gender on plasma glucose levels in the golden-mantled squirrel, Spermophilus lateralis, at various time points during hibernation.

lateralis agrees with similar findings of a significant correlation between plasma FFA and GY levels in humans (Bjorntorp et al., 1969; Bortz et al., 1972). Our observed results indi- cated that the plasma FFA/GY ratio was nega- tively correlated with plasma glycerol levels, but not correlated with plasma FFA levels. We suggest that a preferential utilization of glycerol rather than of FFA for gluconeogenesis may occur during hibernation. Because oxaloacetate (OAA) is an intermediate substrate both in the citric acid cycle and in the pathway of gluconeogenesis, it is also possible that FFA, to form OAA via the citric acid cycle, can be used as a substrate for resynthesis of glucose (Mayes, 1983).

We have previously reported that S. lateralis changes its body weight during pre-hibernation and hibernation (Catuira et al., 1994). The ground squirrel gains weight from overeating in summer (pre-hibernation) and loses weight from

starvation in winter (hibernation). The changes in body weight are primarily due to changes in total body fat in both pre-hibernation and hibernation (Jameson and Mead, 1964; Dark et al., 1989). We found that there was a significant correlation in plasma triglycerides levels and body weights (unpublished results). Melnyk and Martin (1985) suggested that insulin played an important role in the regulation of body weight in the Richardson's ground squirrel. Bjorntorp et al. (1969) reported that with obesity, or increasing body fat, lipid mobilization and lipolysis were increased in man. We found that plasma insulin levels were positively correlated with both body weights (r =0.5099, P < 0.01), and plasma FFA (a lipolytic product) levels (r = 0.804, P < 0.001), and a significant correlation was found between FFA and GY (r = 0.586, P < 0.005) in the hibernating S. lateralis. Since elevated portal FFA concentrations might increase hepatic secretion of


80

70

60

50

30

20

10

n=6

n=7 B Male

Female

] Male & Female

* p<O.01

Juvenile Adult

Age

Fig. 6. Influence of gender and age on plasma insulin levels in the hibernating golden-mantled ground squirrels, Spermophilus lateralis.

lipoproteins and glucose, which contributed to the increase in peripheral insulin concentrations in severely obese humans (Rebuffe- Scrive et al., 1990), the positive correlations between plasma insulin and FFA levels, and plasma lipase activity and plasma cholesterol levels observed in our present study suggest that such a relationship could exist in the hibernating S. lateralis. The golden-mantled ground squirrel can indeed be considered an excellent animal model for the study of the interrelation- ships between the mechanisms which regulate obesity, adipose tissue mass, and lipid metabolism as well as maintain blood glucose homeostasis in a state of long-term starvation. Although we did not include pre-hibernating animals in our present experimental design, our next project will study the changes in the plasma lipid components of the golden-mantled

ground squirrel during weight gain phase (pre- hibernation) and weight loss phase (hibernation).

Acknowledgements--This work was supported in part by a NIGMS-MBRS grant No. 08101. We would like to express our sincere thanks to Dr Eleanor Wilbon and Genevieve Duncan for their editorial comments of this paper.

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