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*
Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294;
Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1;
**
Department of Medicine, Medical Statistics Division, University of Alabama at Birmingham, Birmingham, AL 35294; and the
Department of Dermatology, Mount Sinai Medical Center, New York, NY 10029
2To whom correspondence should be addressed. E-mail: hardy{at}path.uab.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • fatty acids • energy restriction • aging • adipocytes • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). Energy restriction (ER)3
is recognized as a potent inhibitor of the aging process in rats (4
). It reduces blood glucose concentrations (5
,6
), improves skeletal muscle insulin sensitivity (7
), and reverses hepatic insulin resistance (8
).
Free fatty acids (FFA) are generally elevated in conditions of insulin resistance such as type 2 diabetes and obesity (9
–12
). FFA are present in the circulation as a result of dietary intake and endogenous release of stored fat, primarily from adipocytes. Increased fat intake causes insulin resistance (13
–15
). Although both unsaturated fats and saturated fatty acids have been linked to insulin resistance, there is evidence that saturated fat intake more effectively induces insulin resistance (13
,14
,16
). Furthermore, insulin resistance is greater in adipocytes obtained from rats fed diets high in saturated fatty acids (i.e., palmitate) compared with those from rats consuming diets consisting of mostly mono- or polyunsaturated fatty acids (PUFA) (17
). We have confirmed the potent effect of long-chain saturated fatty acids (LCSFA) on the development of insulin resistance in adipocytes in vitro (16
,18
). These and other data (15
,19
) indicate that FFA cause insulin resistance both in vitro and in vivo. Furthermore, overnight reduction in FFA improves insulin sensitivity in obese patients, type 2 diabetics and nondiabetics (20
). Thus, increased FFA and especially, LCSFA, likely contribute to insulin resistance.
Hepatic insulin resistance in aging rats was demonstrated to be reversed by ER and the mechanism involved decreased visceral fat (8
). This reversal also reduces plasma FFA and glycerol (8
). Similarly, surgical removal of visceral fat reverses hepatic insulin resistance (21
).
Taken together, these data point to adipocytes and increased plasma fatty acids as being important in the development of insulin resistance. It is possible that in addition to the increased insulin and glucose concentrations with age, there is also an increase in LCSFA associated with plasma lipids. To our knowledge, there has been no investigation of whether ER can reduce the LCSFA associated with plasma lipids in aging adult rats. The goal of this study was to determine whether there is an decrease in LCSFA associated with plasma lipids in adult aging rats that are ER compared with those consuming food ad libitum (AL).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male Fisher 344 x BNF1 rats were purchased from Bionetics (Jefferson, AR) National Test Center for Toxicological Research, through a program sponsored by the National Institute on Aging. At Bionetics, AL rats were fed NIH-31 pellets and ER rats were fed 60% of mean energy intake of AL rats starting at 16 wk of age until they reached the required age (22
). At that point, 10 rats for each age group were shipped to us and maintained individually in our animal facility under standard conditions (12-h light/12-h dark cycle, humidity at 50 ± 15%, temperature 22 ± 2°C and 12 air changes/h) for 2 wk to recover from the shipment stress. During that period, rats continued to consume either an AL diet or an ER diet (food intake equaled 60% of AL value) provided by Bionetics. Rats were monitored biweekly for weight and infectious diseases. Rats in each group were weighed and killed by CO2 inhalation followed by decapitation after overnight food deprivation at 5, 18 and 31 mo. All procedures involving rats were conducted in strict compliance with relevant state and federal laws, the Animal Welfare Act, Public Health Services Policy, and guidelines established by the Institutional Animal Care and Use Committee. Epididymal adipocytes were isolated using the collagenase digestion method of Rodbell (23
). After cells were removed for glucose transport studies the remaining cells were rapidly frozen and stored at -80°C. Frozen cells were homogenized then fractionated by differential centrifugation into crude plasma membranes and low density microsomes as previously described (24
).
Plasma analytes.
Plasma samples from each rat were frozen at -80°C. Glucose and triglyceride (TG) concentrations were measured by enzymatic assays for hexokinase and glycerol, respectively, on the Hitachi 747 analyzer (Boehringer Mannheim/Roche Diagnostics, Indianapolis, IN) at the University Hospital, University of Alabama at Birmingham (Birmingham, AL). Insulin levels were determined by radioimmunoassay (RIA; Washington University, Diabetes Research and Training Center, RIA Core Facility, St. Louis, MO). Total plasma FFA were measured using an enzymatic colorimetric method (CV <3%; Wako Chemicals, Richmond, VA). Individual fatty acids were analyzed essentially as described by Atkinson and Meckling-Gill (25
), following total lipid extraction by the method of Bligh and Dyer (26
), in the presence of butylated hydroxytoluene (2.3 mmol/L). Lipids and FFA were separated by thin layer chromatography using a solvent of n-heptane/isopropyl ether/acetic acid (60:40:3 v/v) on Silica Gel G Redi Plates (Fisher Scientific, Nepean, Ontario, Canada). Fatty acids were methylated along with 2 µL of a 17:0 lipid standard (NuChek Prep, Elysian, MN) as an internal control. Samples were analyzed on a gas-liquid chromatograph (model 5890; Hewlett Packard, Palo Alto, CA) equipped with a 30-m x 0.54-mm J & W column (J & W Scientifics, Folsom, CA), flame ionization detectors and split injection. Helium was the carrier gas. The sample was injected into the column at 160°C. After 8 min, the column temperature was increased at 2°C/min for 30 min and then held constant at 210°C for an additional 10 min. Fatty acid methyl ester (FAME) peaks were identified using authenticated standards and mixtures at 5 g/L (NuChek Prep). Palmitate, myristate and stearate associated with TG, nonesterified fatty acids (NEFA) and phospholipids (PL) were quantitated. We have distinguished between FFA and NEFA because the analytical methods differ.
2-Deoxyglucose uptake.
Glucose uptake was performed in triplicate as previously described (16
). Briefly, adipocytes (1–2 x 108 cells/L) were resuspended in Krebs-Ringer phosphate (KRP) buffer, with 0.45 mmol/L BSA, and 1.5 mmol/L pyruvate and incubated with or without 1 nmol/L (final concentration) insulin at 37°C for 15 min. 1-[3H]-2-deoxy-D-glucose (6000 Ci/L; 2-deoxyglucose final concentration of 34 µmol/L) was then added to the mixture and transport was measured after 3 min. Cells were separated by centrifugation through dinonyl phthalate oil, and 1-[3H]-2-deoxyglucose uptake was quantified by scintillation counting. Nonspecific 2-deoxyglucose uptake was measured in the presence of 50 µmol/L cytochalasin B.
Immunoblots.
Briefly, subcellular fractions were prepared as described above. Protein concentrations were determined using the BioRad DC protein concentration determination kit (BioRad, Hercules, CA). Protein (15–30 µg) was boiled in an equal volume of 2X Laemmli buffer (with ß-mercaptoethanol) and separated by 10% SDS-PAGE. Proteins were then transferred to Immobilon P membrane (Millipore, Bedford, MA) and probed with anti-insulin-sensitive glucose transporter (GLUT4) antibody (polyclonal; East Acres Biologicals, Southbridge, MA) and visualized with anti-rabbit antibody conjugated to alkaline phosphatase (BioRad, Richmond, CA). Total GLUT4 (crude plasma membrane plus low density microsomes) was compared among ages and treatments by densitometric analysis within blots. The percentage changes or differences from the values in 5-mo-old ER rats were used for comparisons.
Statistical analysis.
Data are presented as mean ± SEM. Differences in body and organ weights, circulating fatty acids, TG, insulin, glucose levels, and adipocyte glucose transport parameters between the AL and ER rats over time were analyzed with a repeated measures fixed effect mixed model (27
). The Shapiro-Wilk statistic for all variables except glucose was <0.05, indicating that the test for normality failed. Thus, the dependent variables were transformed into the natural log for normality. The effects of treatment group (AL vs. ER), time (age) and treatment x age interactions were assessed. When the interaction was not significant, indicating that the treatment groups did not have different slopes among the levels of the dependent variable, the independent effects of treatment and age were assessed. This was done for plasma insulin concentrations and myristate associated with PL. Posthoc contrasts of individual treatment means at each age were performed to examine the differences between treatment groups at each age based on least squares means from the predicted equations. In analyses of TG and FFA, glucose and insulin were included as covariates in the repeated measures model. In all tests, a P-value < 0.05 was deemed significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Rats that consumed food ad libitum (AL) weighed more than the ER rats at all ages (Table 1
). The rate of increase in weight gain was faster in the AL than in the ER rats, although both groups gained weight over time. Groups differed at all three ages tested (P < 0.014). The rate of increase in organ weight for heart, kidney, liver, lung, spleen and thymus was significantly greater for the AL compared with the ER rats. The adrenal and pancreas weights were greater in the AL than in the ER rats but did not change over time. Testes and cerebrum weights were greater in AL than in ER rats and increased similarly in both groups with time.
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Plasma glucose concentration was greater in AL rats than in ER rats at 5, 18 and 31 mo (P < 0.05). Insulin did not demonstrate a significant treatment effect; however, it was greater in the AL rats than in the ER rats (P < 0.002) when the nonsignificant age x treatment effect was dropped (Table 2
).
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). The significant treatment x age interaction was due to the greater rate of increase in TG in AL rats compared with ER rats over time. At 31 mo, the ER rats’ TG concentrations were approximately one-third that of the AL rats.
The rate of change of FFA over time (treatment x age interaction) was different in FFA from AL compared with ER rats (P = 0.0103; Table 2
). At 5 mo, the ER rats had a higher FFA than the AL rats (P = 0.0125), but by 31 mo, the AL mice had a higher FFA than the ER mice. Neither insulin nor glucose was a significant factor in predicting the change in FFA over time by treatment group (P = 0.5493 and 0.2824, respectively).
Stearate and myristate associated with TG were both greater in the AL than in the ER rats (P < 0.0001). The treatment difference for palmitate associated with TG indicated that palmitate was less in AL rats compared with ER rats. Nevertheless, when all three fatty acids were combined, the plasma TG LCSFA in the AL rats was greater than in the ER rats (P < 0.0001).
Individually, each of the LCSFA was greater in NEFA in the AL rats compared with the ER rats (P < 0.0001). When all three LCSFA were combined, they were greater in the NEFA of AL rats than in the ER rats at every age (Table 2)
. Thus, whether analyzed individually or combined, LCSFA associated with NEFA were greater in the AL than in the ER rats (P < 0.0001; Table 2
).
Myristate associated with PL tended to be less in AL than in ER rats (P = 0.147) and when the nonsignificant treatment x age interaction was dropped, this trend was significant (P = 0.0028; Table 2
). Stearate associated with PL was slightly greater in the AL than in the ER rats (P < 0.0001), whereas palmitate was less in AL than in ER rats (P < 0.0001). When the three fatty acids were combined, LCSFA associated with PL in the AL rats was less than in the ER rats (P < 0.0001; Table 2
).
The combined LCSFA in NEFA, TG and PL for all ages and treatments shows that overall 56 ± 3% reside in NEFA, whereas 24 ± 2% are in PL and 20 ± 2% are in TG (mean ± SEM of combined LCSFA data shown in Table 2
). The SEM indicated that these percentages were remarkably consistent in lipid fractions. In addition, the combined LCSFA associated with lipids was greater in AL than in ER rats (P < 0.0001).
Adipocyte glucose transport.
The fold increase in insulin-stimulated glucose transport in adipocytes was less in the AL than in the ER rats (43%±3; Fig. 1A
, P < 0.0026). However, this was largely because of a greater basal glucose transport in the AL rats (P < 0.0066; 87% ± 3; the mean increase in basal glucose transport in AL rats for three ages compared with ER rats; Fig. 1
B). Adipocyte GLUT4 was not less in the AL group at 5 mo (Fig. 1
C) but became progressively lower than in adipocytes from the AL rats at 18 mo (63% ± 7; P = 0.0018) and 31 mo (80% ± 5; P = 0.0013). There was no change in adipocyte GLUT4 concentration with age in the ER rats (Fig. 1
C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), which likely are due, at least in part, to decreased lipoprotein lipase activity in aging rats (29
). Importantly, this decrease in lipoprotein lipase activity may prolong tissue exposure to fatty acids by keeping TG concentrations elevated for longer periods. Because TG deliver fatty acids to tissues, they are an important reservoir of circulating fatty acids.
AL rats did not have increased circulating FFA compared with ER rats, although there was a treatment effect. In addition, unlike glucose and insulin, FFA had significant age and treatment x age effects. It is likely that the two dietary states influenced FFA concentrations differently. ER would be expected to increase FFA to provide an energy source to muscle, especially after overnight food deprivation. This would explain the relatively high FFA concentrations in ER rats of all ages. AL rats, in contrast, gained body weight more rapidly than ER rats (Table 1)
and were not energy-deprived. Barzilai and Rossetti (30
) have shown that body weight is correlated with FFA concentrations in aging rats. Because the body weight of the AL rats increased more than that of the ER rats, it is possible that increased body weight contributes to the increase in FFA with aging in AL rats.
Another consideration for how fatty acids influence blood glucose and insulin concentrations is the type of fatty acid. As previously stated, LCSFA are more effective than mono- or PUFA at inducing insulin resistance (13
,14
,16
). Thus, it is possible that the type of fatty acid as well as its concentration contributes to plasma insulin and glucose concentrations. Interestingly, the combined LCSFA were greater in the AL than in the ER rats. The reason for this difference is not clear. It is possible that a decrease in fatty acid desaturase activity and expression may account for some of the difference. Desaturase activity has been proposed to decline with age (31
), and this has been confirmed in some studies (32
,33
). Other studies indicate that desaturase changes do not necessarily follow a constant decline with age (34
,35
). Specifically, 
-9 (33
) and -6 (32
) desaturase activities have been demonstrated to decline with age. A decline in the activity of -9 desaturase would be expected to increase stearic acid (36
). Further investigation is required to determine whether desaturase activity is maintained by ER.
Studies in humans indicate that an increased percentage of plasma PL saturated fatty acids was associated with increased fasting plasma insulin (37
). We did not find similar results in this study, although there was a treatment effect of LCSFA associated with PL. It is unclear why we did not observe an increase in LCSFA associated with PL in AL rats at each age. One possible explanation is that the study referred to in humans measured PL-associated saturated fatty acids as the percentage of total fatty acids associated with PL while we measured concentrations. It is possible that in the human study, the concentration of PL-associated saturated fatty acids did not increase with insulin concentrations but rather, the amount of other fatty acids decreased, resulting in a greater percentage of saturated fatty acids. It is also possible that there are species differences in concentrations, activities and fatty acid preference of fatty acid desaturases or enzymes responsible for the transfer of fatty acids to PL moieties.
The LCSFA associated with TG were generally higher in the aged AL rats except in the 31-mo-old group. The decreased LCSFA association with TG in the 31-mo-old rats from both groups compared with the 18-mo-old rats was unexpected. TG synthesis depends at least in part on the availability of fatty acids (38
) and 31-mo, FFA concentrations did not differ from those in the 18-mo-old rats. There could have been reduced lipogenesis in the ER group, possibly due to a change in preference of enzymes involved in TG synthesis such as diacylglyceride acyltransferase or there was a change in plasma clearance rate of LCSFA containing TG at 31 mo. Alternatively, there may have been increased lipogenesis in the AL group because of carbon excess (carbohydrate and amino acids) that was used for de novo fatty acid synthesis. Further studies are required to address this issue.
Generally, the internal organs measured increased with aging; however, there were exceptions. In ER rats, the adrenals and thymus did not increase in weight with age. These data per se are difficult to interpret. It is interesting that the adrenals are the major source for insulin counter-regulatory hormones such as epinephrine and norepinephrine.
Several studies have concluded that insulin resistance in aging is closely associated with increased adiposity (39
–41
). Barzilai and Rossetti (30
) found that increases in fat mass after 4 mo of age were not associated with a further decrease in insulin responsiveness. Although insulin responsiveness and adiposity were not assessed in this study, neither fasting plasma glucose nor insulin concentrations increased with increasing age in either treatment group in spite of both groups gaining weight. These results are consistent with the results of Barzilai and Rossetti (30
). Adipocytes account for a relatively modest amount of glucose disposal; nevertheless, if they are insulin-resistant, then less fat can be processed into TG in adipocytes. This leads to increased circulating FFA, increased plasma TG and insulin resistance in other tissues known to be affected by FFA such as muscle and liver (15
,19
). Thus, fat cells and their state of insulin responsiveness may be important in regulating plasma lipids. Experiments have shown that although both glucose transporter-1 (GLUT1) and GLUT4 decrease with age when expressed per unit surface area, GLUT1 actually increases on the plasma membrane when expressed per cell (42
). Although we did not directly measure GLUT1, increased adipocyte basal glucose transport observed in the AL rats may be due to increased GLUT1. Importantly, ER adipocytes retained their GLUT4 and insulin-sensitive glucose transport with age. In humans with type 2 diabetes, there is a marked reduction in GLUT4 in isolated adipocytes (43
). Also, GLUT4 expression is reduced in adipose tissue of older Zucker rats as well as in other animal models of type 2 diabetes such as the yellow Avy/a and the KKAy mice (44
). It is possible that the decrease in GLUT4 observed in aging AL rats is due in part to increased exposure of adipocytes to LCSFA.
In summary, these studies indicate that concentrations of LCSFA are significantly lower in the plasma lipids of mature male ER rats compared with those consuming food AL. This reduction of LCSFA may contribute to increased adipocyte GLUT4 and reduced plasma glucose concentrations in the ER compared with the AL rats.
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FOOTNOTES |
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3 Abbreviations used: AL, ad libitum; ER, energy restricted; FFA, free fatty acids; GLUT1, glucose transporter-1; GLUT4, insulin-sensitive glucose transporter; LCSFA, long-chain saturated fatty acids; NEFA, nonesterified fatty acids; PL, phospholipids; TG, triglycerides.
Manuscript received 1 April 2002. Initial review completed 7 May 2002. Revision accepted 12 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Couet, C., Delarue, J., Constans, T. & Lamisse, F. (1992) Age-related insulin resistance: a review. Horm. Res. 38:46-50.[Medline]
2. Davidson, M. B. (1979) The effect of aging on carbohydrate metabolism: a review of the English literature and a practical approach to the diagnosis of diabetes mellitus in the elderly. Metabolism 28:688-705.[Medline]
3. Defronzo, R. A. (1979) Glucose intolerance and aging evidence for tissue insensitivity to insulin. Diabetes 28:1095-1101.[Medline]
4. Yu, B. P. (1994) How diet influences the aging process of the rat. Proc. Soc. Exp. Biol. Med. 205:97-105.[Abstract]
5. Santos, R. F. & Azhar, C. (1988) Prevention of insulin resistance by environmental manipulation as young rats mature. Horm. Metab. Res. 21:55-58.
6. Mondon, C. E. & Sims, C. (1986) The effect of exercise training on insulin resistance in sedentary year old rats. J. Gerontol. 41:605-610.[Medline]
7. Davidson, R. T., Arias, E. B. & Cartee, G. D. (2002) Energy restriction increases muscle insulin action but not IRS-1-, IRS-2-, or phosphotyrosine-PI3-kinase. Am. J. Physiol. Endocrinol. Metab. 282:E270-E276.
8. Barzilai, N., Banerjee, S., Hawkins, M., Chen, W. & Rossetti, L. (1998) Caloric restriction reverses hepatic insulin resistance in aging rats by decreasing visceral fat. J. Clin. Invest. 101:1353-1361.[Medline]
9. Lillioja, S., Foley, J., Bogardus, C., Mott, D. & Howard, B. V. (1986) Free fatty acid metabolism and obesity in man: in vivo and in vitro comparisons. Metabolism 35:505-514.[Medline]
10. Jensen, M. D., Haymond, M. W., Rizza, R. A., Cryer, P. E. & Miles, J. M. (1989) Influence of body fat distribution on free fatty acid metabolism in obesity. J. Clin. Invest. 83:1168-1173.
11. Fraze, E., Donner, C. C., Swislocki, A. L., Chiou, Y.-A. & Chen, Y.-D. I. (1985) Ambient plasma free fatty acid concentrations in noninsulin-dependent diabetes mellitus: evidence for insulin resistance. J. Clin. Endocrinol. Metab. 61:807-811.[Abstract]
12. Swislocki, A.L.M., Chen, Y.-D.I., Golay, A., Chang, M.-O. & Reaven, G. M. (1987) Insulin suppression of plasma-free fatty acid concentration in normal individuals and patients with type 2 (non-insulin-dependent) diabetes. Diabetologia 30:622-626.[Medline]
13. Marshall, J., Hamman, R. & Baster, J. (1991) High fat, low carbohydrate diet and the etiology of non-insulin-dependent diabetes mellitus: San Luis Valley Diabetes Study. Am. J. Epidemiol. 134:590-603.
14. Tsunehara, C. H., Leonetti, D. L. & Fujimoto, W. Y. (1991) Animal fat and cholesterol intake is high in men with IGT progressing to NIDDM. Diabetes 40:427A.
15. Boden, G. (1996) Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46:3-10.[Abstract]
16. Hunnicutt, J. W., Hardy, R. W., Williford, J. & McDonald, J. M. (1994) Saturated fatty acid-induced insulin resistance in rat adipocytes. Diabetes 43:540-545.[Abstract]
17. Van Amelsvoort, J. M., Van der Beek, A. & Stam, J. J. (1986) Effect of the type of dietary fatty acid on the insulin receptor function in rat epididymal fat cells. Ann. Nutr. Metab. 30:273-280.[Medline]
18. Van Epps-Fung, M., Williford, J., Wells, A. & Hardy, R. W. (1997) Fatty acid-induced insulin resistance in adipocytes. Endocrinology 138:4338-4345.
19. Shulman, G. I. (2000) Cellular mechanisms of insulin resistance. J. Clin. Invest. 106:171-176.[Medline]
20. Santomauro, A., Boden, G., Silva, M., Rocha, D., Santos, R., Ursich, M., Strassmann, P. & Wajchenberg, B. (1999) Overnight lowering of free fatty acids with acipimox improves insulin resistance and glucose tolerance in obese diabetic and nondiabetic subjects. Diabetes 48:1836-1841.[Abstract]
21. Barzilai, N., She, L., Lui, B. Q., Vuguin, P., Cohen, P., Wang, J. & Rossetti, L. (1999) Surgical removal of visceral fat reverses hepatic insulin resistance. Diabetes 48:94-98.[Abstract]
22. Witt, W. M. & Brand, C. D. (1989) A nationally supported study on caloric restriction of rodents. Lab. Anim. 18:37-43.
23. Rodbell, M. (1984) Metabolism of isolated fat cells: effects of hormones on glucose metabolism and lipolysis. J. Biol. Chem. 239:375-380.
24. Hardy, R. W., Ladenson, J. H., Henriksen, E. J., Holloszy, J. O. & McDonald, J. M. (1991) Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT 4). Biochem. Biophys. Res. Commun. 177:343-349.[Medline]
25. Atkinson, T. G. & Meckling-Gill, K. A. (1995) Regulation of nucleoside drug toxicity by transport inhibitors and 
-3 polyunsaturated fatty acids in normal and transformed rat-2 fibroblasts. Cell. Pharmacol. 2:259-264.
26. Bligh, E. G. & Dyer, W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917.
27. Littell, R. C., Milliken, G. A., Stroup, W. W. & Wolfinger, R. D. (1996) SAS System for Mixed Models 1996:633 SAS Institute Inc Cary, NC. .
28. Van Liew, J. B., Davis, P. J., Davis, F. B., Bernardis, L. L., Deziel, M. R., Marinucci, L. N. & Kumar, D. (1993) Effects of aging, diet, and sex on plasma glucose, fructosamine and lipid concentrations in barrier-raised Fisher 344 rats. J. Gerontol. 48:B184-B190.[Medline]
29. Barakat, H. A., Dohm, G. L., Shukla, N., Marks, R. H., Kern, M., Carpenter, J. W. & Mazzeo, R. S. (1989) Influence of age and exercise training on lipid metabolism in Fisher-344 rats. J. Appl. Physiol. 67:1638-1642.
30. Barzilai, N. & Rossetti, L. (1995) Relationship between changes in body composition and insulin responsiveness in models of the aging rat. Am. J. Physiol. 269:E591-E597.
31. Takahashi, R. & Horrobin, D. F. (1988) Linoleic acid-induced fatty acid changes in platelet and aorta of the rat: effect of age and cholesterol. Lipids 23:885-890.[Medline]
32. Bordoni, A., Biagi, P. L., Turchetto, E. & Hrelia, S. (1988) Aging influence on -6-desaturase activity and fatty acid composition of rat liver microsomes. Biochem. Intl. 17:1001-1009.[Medline]
33. Kumar, V. B., Vyas, K., Buddhiraju, M., Alshahler, M., Flood, J. F. & Morley, J. E. (1999) Changes in membrane fatty acids and -9 desaturase in senescence accelerated (SAMP8, mouse hippocampus with aging). Life Sci. 65:1657-1662.[Medline]
34. Dinh, T. K., Bourre, J. M. & Durand, G. (1993) Effect of age and 
-linoleic acid deficiency on -6 desaturase activity and liver lipids in rats. Lipids 28:517-523.[Medline]
35. Maniongui, C., Blond, J. P., Ulmann, L., Durand, G., Poisson, J. P. & Bezard, J. (1993) Age-related changes in -6 and -5 desaturase activities in rat liver microsomes. Lipids 28:291-297.[Medline]
36. Tocher, D. R., Leaver, M. J. & Hodgson, P. A. (1998) Recent advances in the biochemistry and molecular biology of fatty acyl desaturases. Prog. Lipid Res. 37:73-117.[Medline]
37. Folsom, A. R., Ma, J., McGovern, P. G. & Eckfeldt, H. (1996) Relation between plasma phospholipid saturated fatty acids and hyperinsulinemia. Metabolism 45:223-228.[Medline]
38. Stals, H. K., Top, W. & Declercq, P. E. (1994) Regulation of triacylglycerol synthesis in permeabilized rat hepatocytes: role of fatty acid concentration and diacylglycerol acyltransferase. FEBS Lett 343:99-102.[Medline]
39. Lawrence, J. C., Jr, Colvin, J., Cartee, G. D. & Holloszy, J. O. (1989) Effects of aging and exercise on insulin action in rat adipocytes are correlated with changes in fat cell volume. J. Gerontol. 44:B88-B92.[Medline]
40. Cefalu, W. T., Wang, Z. Q., Werbel, S., Bell-Farrow, A., Crouse, J. R., III, Hinson, W. H., Terry, J. G. & Anderson, R. (1995) Contribution of visceral fat mass to the insulin resistance of aging. Metabolism 44:954-959.[Medline]
41. Kohrt, W. M., Kirwan, J. P., Staten, M. A., Bourey, R. E., King, D. S. & Holloszy, J. O. (1993) Insulin resistance in aging is related to abdominal obesity. Diabetes 42:273-281.[Abstract]
42. Ezaki, O., Fukuda, N. & Itakura, H. (1990) Role of two types of glucose transporters in enlarged adipocytes from aged obese rats. Diabetes 39:1543-1549.[Abstract]
43. Després, J.-P. & Marette, A. (1999) Obesity and insulin resistance, epidemiologic, metabolic, and molecular aspects. Reaven, G. M. Laws, A. eds. Insulin Resistance: The Metabolic Syndrome X 1999:51-81 Humana Press Totowa, NJ. .
44. Tsakiridis, T., Marette, A. & Klip, A. (1996) Glucose transporters in skeletal muscle of animal models of diabetes. Shafrir, E. eds. Lessons from Animal Models of Diabetes V 1996:141-159 Smith-Gordon & Co. Ltd London, UK. .
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 J. L. Quiles, J. J. Ochoa, M. C. Ramirez-Tortosa, J. R. Huertas, and J. Mataix Age-related mitochondrial DNA deletion in rat liver depends on dietary fat unsaturation. J. Gerontol. A Biol. Sci. Med. Sci., February 1, 2006; 61(2): 107 - 114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hagopian, M.-E. Harper, J. J. Ram, S. J. Humble, R. Weindruch, and J. J. Ramsey Long-term calorie restriction reduces proton leak and hydrogen peroxide production in liver mitochondria Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E674 - E684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Ramsey, K. Hagopian, T. M. Kenny, E. K. Koomson, L. Bevilacqua, R. Weindruch, and M.-E. Harper Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats Am J Physiol Endocrinol Metab, January 1, 2004; 286(1): E31 - E40. [Abstract] [Full Text] [PDF]
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