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Metabolic abnormalities of the hyperglycemic obese Zucker rat
Metabolic
Abnormalities
of the Hyperglycemic
Obese Zucker Rat
Michael L. McCaleb and Janet Sredy In a cross-sectional study, we evaluated the metabolic profiles of lean (Fa/?) and obese (fa/fa) Zucker male rats at 4 to 8 months of age. Although all of the obese rats (N = 108) demonstrated glucose intolerance, most of the obese rats exhibited only mild elevations of fasted and fed plasma glucose. Only 14 of the obese rats were severely hyperglycemic, which resulted in substantial elevations of glycohemoglobin (GHb) levels. The nerve and lens levels of glucose, sorbitol, and fructose were elevated, and the myo-inositol was depleted in all hyperglycemic obese rats, but not in the euglycemic obese rats. With increasing duration of hyperglycemia, the neural myo-inositol level approached normal, while the lenses became cataractous. All obese rats had increased urinary albumin excretion (UAE), which was dependent on age (r = .45, P < .02) and independent of hyperglycemia, glucosuria, and polyuria. In conclusion, although the euglycemic obese rats exhibited some diabetic abnormalities, the hyperglycemic obese Zucker rat more closely resembled the altered metabolic profile associated with type II diabetes mellitus. Copyright 0 1992 by W.B. Saunders Company
D
IABETES-INDUCED hyperglycemia results in the development of long-term complications involving nerve, eye, and kidney. The clinical expression of diabetic complications, including neuropathy, cataractogenesis, and nephropathy, depends on the duration and extent of hyperglycemia.‘.’ Experimental evidence generated during the last two decades indicates that as a result of hyperglycemia, the excess free glucose in tissues leads to an increased flux of glucose through the polyol pathway and a depletion of cellular myo-inositol. Increased polyol pathway activity has been proposed to lead to a sequela of metabolic changes that are responsible for the pathogenesis of diabetic complications.‘.’ The natural history of diabetic complications in type II diabetes is less well known than that in type I, probably because of difficulty in identifying the precise onset of diabetes.“ The obese Zucker (fa/fa) rat has been proposed to be an animal model of type II diabetes, since this rat exhibits insulin resistance and glucose intolerance.s,6 Generally, however, it is reported that the Zucker rat does not develop severe hyperglycemia,’ which would be necessary for the progression of diabetic complications. Unexpectedly, we found that some of the obese Zucker rats in our colony became overtly hyperglycemic. To elucidate the metabolic and pathophysiologic consequences of hyperglycemia in these animals and to determine whether the Zucker rat might be a suitable model for the study of diabetic complications, we undertook a cross-sectional evaluation of the Zucker rats in our colony.
(Richmond, IN) and water for an additional 2 to 6 months. All rats were infected with pinworms for an undetermined period of time. Initially, the glycohemoglobin (GHb) levels of five lean and 108 obese rats were determined. The GHb level of the lean Zucker rats ranged from 5% to 7%. Fourteen obese rats with high GHh levels (11% to 16%) and 29 obese rats with low GHb levels (5% to 8%) were further evaluated. Blood was collected from the tail-tip of the unanesthetized rats for the determination of plasma glucose, following either 2 hours (fed) or 18 hours (fasted) of food deprivation. Blood samples were also obtained 90 minutes after the administration of glucose (1 g/kg, subcutaneously) in the lb-hour fasted rats. Animals were placed into metabolism cages (Nalgene, Rochester, NY) (ad libitum access to water, but not to food) for 24-hour collection of urine. Total volume of urine was recorded, and aliquots of urine were centrifuged (100 x g, 10 minutes) to remove particulate matter and then frozen at -70°C. The following week, rats were killed by CO, inhalation, and the lens and portions of the sciatic nerve were removed and immediately frozen.
METHODS
Fourteen of the obese Zucker rats were overtly hyperglycemic, as evidenced by substantial elevations of fasting plasma glucose and blood GHb levels (Table 1). In addition, the hyperglycemic obese rats had levels of plasma glucose, either in the fed state or following a glucose challenge, which were significantly greater than the levels of the euglycemic obese and normal lean rats. Although the age range of the obese euglycemic and hyperglycemic rats was similar, there was a significant difference between the body weights of the two groups (Table 2). The hyperglycemic obese rats demonstrated marked glucosuria and mild polyuria (Table 3). Rates of urinary albumin excretion (UAE) were significantly increased in
Biochemical Methods Glucose, sorbitol, fructose, and myo-inositol in tissue samples were converted to their aldononitrile acetate or acetate derivatives’ and measured by capillary gas chromatography.R The limit of detection was 0.04 nmol/mg tissue. Plasma glucose levels were determined using the hexokinase method on an Abbott VP Analyzer (Irving, TX), and blood levels of GHb were measured by the Glyc-Atlin GHb Kit (Isolab, OH). An enzyme-linked immunosorbent assay (ELISA) method described previously’ was used to determine urinary albumin levels. Data were analyzed using a one-way ANOVA followed by a Tukey’s t test. RESULTS
Animals Male lean (Fa/?) and obese (fa/fa) Zucker rats of 6 to 8 weeks of age were obtained from Indiana University (Indianapolis, IN) during the period of July to November 1986. The rats were maintained with ad libitum access to Purina Rodent Chow 5001
From Wyeth-AyerstResearch, Princeton, NJ. Address reprint requests to Michael L. McCaleb, PhD, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543. Copyright 0 1992 by WB. Saunders Company 00260495/92/4105-0014$03.00/0
522
Metabolism, Vol41,
No
5 (May).
1992: pp
522-525
HYPERGLYCEMIC
OBESE
ZUCKER
RAT
523
Table 4. Sciatic Nerve Carbohydrate
Table 1. Blood Glucose Levels PlasmaGlucose(mg/dL) Fasted
Group
78 t- 3
Lean Euglycemic obese
104*
1
Hyperglycemic obese
201 f 1 I*
Content (nmolimg)
BloodGHb 1%)
Fed
G-i-r
Content
Group
GllK0S.e
Sorbitol
Fructose 0.80 2 0.11
II825
100 + 4
5.6 -t 0.5
Lean
1.99 + 0.04
0.17 + 0.02
170 f 6
115 2 3
6.3 f. 0.2
Euglycemic obese
1.97 +- 0.07
0.21 f 0.01
0.94 i- 0.08
329 + 25*
319 + 25*
Hyperglycemic obese
8.28 + 0.69*
1.81 + 0.21*
5.49 + 0.46*
14.0 t 0.4*
lP < .Ol compared with lean or euglycemic obese.
Abbreviation: GTT, glucose tolerance test. *P < .Ol compared with lean or euglycemic obese.
Table 2. Characteristics Normal
of Animal Groups Hyperglycemic Obese
Euglycemic Obese
Lean
556 + IO*
Body weight (g)
437 2 30
610 + 14t
Age range (mo)
4-6
4-8
5-8
5
29
14*
N
the obese rats; however, the levels of glycemia of the obese rats were not correlated with the UAE rates. As shown in Fig 1, the UAE rates of the obese rats were significantly correlated with the ages of the animals. In the sciatic nerve of hyperglycemic obese rats, which were 5 to 8 months of age, there was a sixfold to eightfold increase in glucose, sorbitol, and fructose (Table 4). With a longer duration of hyperglycemia (6 to 8 months), the neural glucose, sorbitol, and fructose remained elevated. The mean level of neural myu-inositol in the hyperglycemic group (aged 5 to 8 months) was 40% below the levels in euglycemic obese and lean rats (Fig 2, group levels). However, an examination of the levels in the individual rats in the hyperglycemic group showed that neural myoinositol returned to near normal levels with the longer duration of hyperglycemia, eg, 1.25 ? 0.23 nmol/mg versus 1.89 t 0.48 nmol/mg for Smonth and g-month hyperglycemic rats, respectively (Fig 2, individual levels). It should be noted that the normalization of nerve myo-inositol levels occurred in the presence of a small elevation of GHb levels, eg, 12.9% ? 1.7% versus 15.2% * 1.2% for Smonth and g-month hyperglycemic rats, respectively. In the lenses of hyperglycemic obese rats (aged 5 to 7 months, noncataractous) there was a threefold to fivefold increase in glucose and fructose, a 22-fold increase in sorbitol, and a 100% depletion of myo-inositol (Table 5). At 8 months, all hyperglycemic obese rats had developed mature bilateral cataracts that led to lower levels of sorbitol and fructose, presumably due to sugar leakage from ruptured lens cells.
*P < .05 compared with lean or euglycemic obese. tf’ < .Ol compared with lean. *Four animals in this group were cataractous.
Table 3. Urinary Parameters
Volume (mL/d)
Group
Albumin (mg/lOOg body M/d)
Glucose (mg/dL)
Lean
7.4 t 0.9
22 k 2
0.2 k 0.1
Euglycemic obese
9.5 t 0.6
33 + 7
14.4 zk 2.4’
18.8 ? 1.98
11,287 2 4,073$
21.2 2 2.5t
Hyperglycemic obese
*P < .05 compared with lean. tP < .Ol compared with lean. W’ < .05 compared with lean or euglycemic obese. §P < .Ol compared with lean or euglycemic obese.
80
o
Euglycemic Obese
o
Hyperglycemic
DISCUSSION
Obese
The streptozocin (STZ)-induced diabetic rat has become a primary animal model of type I diabetes for the evaluation of the development of diabetic complications.‘“’ It has been demonstrated in multiple experiments that elevated blood
I r = 0.45
0
p < 0.02
01 3
.
m .
m .
Q
4
5
6
-
1
7
.
1 8
.
I 9
4
5
6
7
Age ,mo”th*)
8
9
e-p < ““1
I;ompwa
Euglycemlc
to
Lean
or
Obese
Age (months) Fig 1.
UAE of individual obese Zucker rats, aged 4 to 8 months.
Fig 2. Individual and group levels of myoinositol in the nerve of lean and obese Zucker rats.
524
McCALEB AND SREDY
Table 5. Lens Carbohydrate
Content Content (nmollmg)
Group
GlUCOSe
Sorbitol
Fructose
myo-lnositol
Lean
0.82 + 0.19
0.97 k 0.17
2.12 + 0.20
1.43 f 0.23
Euglycemic obese
1.05 + 0.12
1.58 2 0.44
2.48 + 0.45
1.37 f 0.23
Hyperglycemic obese
3.48 2 0.50*
35.40 3 4.25f
11.55 -t 0.84*
ND
Cataractous hyperglycemic obeset
6.61 2 1.95X
8.90 k 2.50’
6.55 f 1.04’
ND
Abbreviation: ND, not detected. *P < .Ol compared with lean or euglycemic obese. tN = 4, age was 8 months.
glucose levels result directly in high tissue levels of glucose. In the lens, nerve, retina, and kidney, glucose is subsequently converted to sorbitol and fructose.‘-3.9-‘2There is strong evidence to support the hypothesis that activation of the polyol pathway has pathophysiologic consequences in these tissues. For example, by inhibiting the enzyme, aldose reductase, with a variety of specific inhibitors, diabetesinduced nerve dysfunction, retinal basement membrane thickening, cataracts, and renal abnormalities were substantially diminished.‘* The progression of diabetic complications in type II diabetes is less well known than that in type I, even though complications are evident clinically in both types of patients.13 The obese Zucker rat has been proposed as an animal model of type II diabetes; however, there have been only a few reports of the development of diabetes-like pathologies in these animals.‘4-‘a Therefore, using the lean and obese Zucker rats available in our colony, we performed a cross-sectional evaluation of various metabolic and functional parameters that are usually associated with the development of diabetic complications. The occasional occurrence of overt fasting hyperglycemia in some obese Zucker rats derived from the Indianapolis animal colony has been reported previously.” Here, we demonstrate that the animals that have markedly elevated fasting plasma glucose levels also exhibit significantly increased GHb levels. Consistent with clinical studies in obese, euglycemic patients,” the GHb levels of obese, euglycemic, but glucose-intolerant rats were not different from those of lean normal rats, and there were no changes in tissue polyol levels. However, the hyperglycemic Zucker rats had substantial elevations of glucose in the lens and sciatic nerves. As observed previously in the STZ-induced hyperglycemic rat,“1’,‘9,z0the hyperglycemic Zucker rat also had dramatic, and similar, increases in the tissue levels of sorbitol and fructose. In four of the older hyperglycemic rats in which mature cataracts were evident, and hence a loss of lenticular integrity, polyol elevation was still evident. Of special interest was the significant decrease in tissue levels of myo-inositol in the hyperglycemic rats. In all hyperglycemic rats, elevated levels of lens sorbitol and fructose were accompanied by a total depletion of myoinositol. In the nerves of hyperglycemic rats, there was an age-dependent difference in the myo-inositol levels. The myoinositol levels were progressively higher in the older hyperglycemic rats, suggesting that myo-inositol returns to normal with increasing duration of diabetes. These results
support previous views that neural myo-inositol depletion in diabetic rats is only transient.“-” Although the mechanism for this return to normal is unknown, it is possible that the normalization of nerve myo-inositol was caused by an elevation in plasma myo-inositol in the renal-impaired rats. An elevation in the plasma myo-inositol will increase the concentration of myo-inositol in the sciatic nerve.” Additionally, renal-impaired, 14-month STZ-diabetic rats have been shown to have an elevated plasma myo-inositol concentration.23 However, an elevation in plasma myo-inositol may not be the only mechanism, since in 4-month and 6-month STZ-diabetic rats a normal sciatic nerve myo-inositol concentration was associated with a normal plasma myoinositol concentration.‘9’21 The early stages of clinical diabetic nephropathy are characterized by an elevation of UAE. The albuminuria in both type I and type II diabetic patients progresses to overt proteinuria, and eventually results in end-stage renal failure.“’ The STZ-diabetic rat also exhibits a progressive increase in UAE, which appears to be dependent on hyperglycemia, since treatment of the diabetic animals with an aldose reductase inhibitor prevents the albuminuria.’ In contrast to the progression of albuminuria in STZ-diabetic rats, the obese Zucker rats exhibited elevations of UAE that were independent of hyperglycemia, glucosuria, and polyuria. It has been reported previously that the obese Zucker rat excretes large amounts of proteinI and our results demonstrate that the proteinuria is dependent on age and not on glycemia. Recently, it has been suggested that glomerulosclerosis may frequently be due to atherosclerotic lesionsz5 As an extension of this hypothesis, the nephropathy of the obese Zucker rat was proposed to be a direct consequence of the animal’s extreme hyperlipidemia.2b However, we cannot address this specific hypothesis, since blood lipid levels were not determined in the present study. In conclusion, although the euglycemic obese Zucker rat exhibits some abnormalities, the hyperglycemic obese Zucker rat more closely resembles the altered metabolic profile associated with type II diabetes mellitus.
ACKNOWLEDGMENT
We would like to thank Michael Houlihan, Brenda Mihan, and Diane Sawicki for their excellent technical assistance, and Ann Marie Lucchesi for her skillful secretarial support.
HYPERGLYCEMIC
OBESE
ZUCKER
525
RAT
REFERENCES 1. Winegrad AI: Does a common complications of diabetes? Diabetes
mechanism 36:396-406,
induce the diverse 1987
12. Harrison HE, Stribling D, Armstrong FM, et al: Aldose reductase in the etiology of diabetic complications: 1. Introduction. J Diabetic Complications 3:6-11, 1989
Dependent Diabetes Mellitus. Amsterdam. The Netherlands. Elsevier Science 1989, pp 71-87 14. Dosso A, Rungger-Brindle E. Rohner-Jeanrenaud F, et al: Ocular complications in the old and glucose-intolerant genetically obese (fa/fa) rat. Diabetologia 33:137-144, 1990 15. Lash JM, Sherman WM, Hamlin RL: Capillary basement membrane thickness and capillary density in sedentary and trained obese Zucker rats. Diabetes 38:854-860,1989 16. Kasiske BL. Cleary MP, O’Donnell MP, et al: Effects of genetic obesity on renal structure and function in the Zucker rat. J Lab Clin Med 106:598-604,1985 17. Clark JB, Palmer CJ, Shaw WN: The diabetic Zucker fatty rat (41611). Proc Sot Exp Biol Med 173:68-75, 1983 18. Lapolla A, Poli T. Meneghini F, et al: Glycated serum proteins and glucose tolerance. Acta Diabetol Lat 25:325-332. 1988 19. Cameron NE, Leonard MB, Ross IS. et al: The effects of sorbinil on peripheral nerve conduction velocity, polyol concentrations and morphology in the streptozotocin-diabetic rat. Diabetologia 29:168-174, 1986 20. Sredy J, Sawicki D, Hicks D, et al: Lack of peripheral nerve myoinositol depletion in long-term diabetic rats. Fed Proc 46:2011, 1987 21. Stewart MA, Sherman WR. Kurien MM. et al: Polyol accumulation in nervous tissue of rats with experimental diabetes and galactosemia. J Neurochem 14:1057-1066, 1967 22. Greene DA, De Jesus PV, Winegrad AI: Effects of insulin and dietary myoinositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes. J Clin Invest 55:1326-1336, 1975 23. Greene DA, De Jesus PV. Winegrad Al: Motor nerve conduction velocity in acute and chronic streptozotocin diabetes. Diabetes 23:357, 1974 24. Viberti GC, Walker JD: Diabetic nephropathy: Etiology and prevention. Diabetes Metab Rev4:147-162, 1988 25. Kasiske B. O’Donnell MP, Schmitz PG. et al: Renal injury of diet-induced hypercholesterolemia in rats. Kidney Int 37:880-X91, 1990
1.1. Eschwege E. Lacroux A, Papoz L, et al: Epidemiology of complications of NIDDM, in Alberti KGMM, Mazze R (eds): Frontiers of Diabetes Research: Current Trends in Non-Insulin-
26. Kasiske B, O’Donnell MP, Cleary MP, et al: Treatment of hyperlipidemia reduces glomerular injury in ohese Zucker rats, Kidney Int 33:667-672,198s
2. Dyck PJ, Zimmerman BR, Vilen TH, et al: Nerve glucose, fructose, sorbitol. myo-inositol, and fiber degeneration and regeneration in diabetic neuropathy. N Engl J Med 319:542-548. 1988 3 Greene D: The pathogenesis and prevention of diabetic neuropathy and nephropathy. Metabolism 37:25-29, 1988 4. Dowse GK, Zimmet PZ: The prevalence and incidence of non-insulin-dependent diabetes mellitus, in Alberti KGMM, Mazze R (eds): Frontiers of Diabetes Research: Current Trends in Non-Insulin-Dependent Diabetes Mellitus. Amsterdam, The Netherlands, Elsevier Science, 1989, pp 37-59 5 Bray GA: The Zucker-fatty 153 1977
rat: A review. Fed Proc 36:148-
6 Terrettaz J, Jeanrenaud B: In vivo hepatic and peripheral insulin resistance in genetically obese (faifa) rats. Endocrinology 112 1346-1351,1983 7 Guerrant GO, Moss CW: Determination of monosaccharides as aldononitrile. O-methyloxime, alditol, and cyclitol acetate derivatives by gas chromatography. Anal Chem 56:633-638,1984 8 Kemper C, Dvornik D: Prevention of neural myoinositol depletion in diabetic rats by aldose reductase inhibition with tolrestat (42372). Proc Sot Exp Biol Med 182:505-510, 1986 9. McCaleb ML. Sredy J, Millen J, et al: Prevention of urinary albumin excretion in 6 month streptozocin-diabetic rats with the aldose reductase inhibitor tolrestat. J Diabetic Complications 2 10-18,198X 10. Simard-Duquesne N, Greselin E, Dubuc J, et al: The effects of a new aldose reductase inhibitor (tolrestat) in galactosemic and diabetic rats. Metabolism 34:885-892, 1985 1 I. Stribling D, Mirrlees DJ. Harrison HE, et al: Properties of ICI 128,436, a novel aldose reductase inhibitor, and its effects on diabetic complications in the rat. Metabolism 34:336-344, 1985
Abnormalities
of the Hyperglycemic
Obese Zucker Rat
Michael L. McCaleb and Janet Sredy In a cross-sectional study, we evaluated the metabolic profiles of lean (Fa/?) and obese (fa/fa) Zucker male rats at 4 to 8 months of age. Although all of the obese rats (N = 108) demonstrated glucose intolerance, most of the obese rats exhibited only mild elevations of fasted and fed plasma glucose. Only 14 of the obese rats were severely hyperglycemic, which resulted in substantial elevations of glycohemoglobin (GHb) levels. The nerve and lens levels of glucose, sorbitol, and fructose were elevated, and the myo-inositol was depleted in all hyperglycemic obese rats, but not in the euglycemic obese rats. With increasing duration of hyperglycemia, the neural myo-inositol level approached normal, while the lenses became cataractous. All obese rats had increased urinary albumin excretion (UAE), which was dependent on age (r = .45, P < .02) and independent of hyperglycemia, glucosuria, and polyuria. In conclusion, although the euglycemic obese rats exhibited some diabetic abnormalities, the hyperglycemic obese Zucker rat more closely resembled the altered metabolic profile associated with type II diabetes mellitus. Copyright 0 1992 by W.B. Saunders Company
D
IABETES-INDUCED hyperglycemia results in the development of long-term complications involving nerve, eye, and kidney. The clinical expression of diabetic complications, including neuropathy, cataractogenesis, and nephropathy, depends on the duration and extent of hyperglycemia.‘.’ Experimental evidence generated during the last two decades indicates that as a result of hyperglycemia, the excess free glucose in tissues leads to an increased flux of glucose through the polyol pathway and a depletion of cellular myo-inositol. Increased polyol pathway activity has been proposed to lead to a sequela of metabolic changes that are responsible for the pathogenesis of diabetic complications.‘.’ The natural history of diabetic complications in type II diabetes is less well known than that in type I, probably because of difficulty in identifying the precise onset of diabetes.“ The obese Zucker (fa/fa) rat has been proposed to be an animal model of type II diabetes, since this rat exhibits insulin resistance and glucose intolerance.s,6 Generally, however, it is reported that the Zucker rat does not develop severe hyperglycemia,’ which would be necessary for the progression of diabetic complications. Unexpectedly, we found that some of the obese Zucker rats in our colony became overtly hyperglycemic. To elucidate the metabolic and pathophysiologic consequences of hyperglycemia in these animals and to determine whether the Zucker rat might be a suitable model for the study of diabetic complications, we undertook a cross-sectional evaluation of the Zucker rats in our colony.
(Richmond, IN) and water for an additional 2 to 6 months. All rats were infected with pinworms for an undetermined period of time. Initially, the glycohemoglobin (GHb) levels of five lean and 108 obese rats were determined. The GHb level of the lean Zucker rats ranged from 5% to 7%. Fourteen obese rats with high GHh levels (11% to 16%) and 29 obese rats with low GHb levels (5% to 8%) were further evaluated. Blood was collected from the tail-tip of the unanesthetized rats for the determination of plasma glucose, following either 2 hours (fed) or 18 hours (fasted) of food deprivation. Blood samples were also obtained 90 minutes after the administration of glucose (1 g/kg, subcutaneously) in the lb-hour fasted rats. Animals were placed into metabolism cages (Nalgene, Rochester, NY) (ad libitum access to water, but not to food) for 24-hour collection of urine. Total volume of urine was recorded, and aliquots of urine were centrifuged (100 x g, 10 minutes) to remove particulate matter and then frozen at -70°C. The following week, rats were killed by CO, inhalation, and the lens and portions of the sciatic nerve were removed and immediately frozen.
METHODS
Fourteen of the obese Zucker rats were overtly hyperglycemic, as evidenced by substantial elevations of fasting plasma glucose and blood GHb levels (Table 1). In addition, the hyperglycemic obese rats had levels of plasma glucose, either in the fed state or following a glucose challenge, which were significantly greater than the levels of the euglycemic obese and normal lean rats. Although the age range of the obese euglycemic and hyperglycemic rats was similar, there was a significant difference between the body weights of the two groups (Table 2). The hyperglycemic obese rats demonstrated marked glucosuria and mild polyuria (Table 3). Rates of urinary albumin excretion (UAE) were significantly increased in
Biochemical Methods Glucose, sorbitol, fructose, and myo-inositol in tissue samples were converted to their aldononitrile acetate or acetate derivatives’ and measured by capillary gas chromatography.R The limit of detection was 0.04 nmol/mg tissue. Plasma glucose levels were determined using the hexokinase method on an Abbott VP Analyzer (Irving, TX), and blood levels of GHb were measured by the Glyc-Atlin GHb Kit (Isolab, OH). An enzyme-linked immunosorbent assay (ELISA) method described previously’ was used to determine urinary albumin levels. Data were analyzed using a one-way ANOVA followed by a Tukey’s t test. RESULTS
Animals Male lean (Fa/?) and obese (fa/fa) Zucker rats of 6 to 8 weeks of age were obtained from Indiana University (Indianapolis, IN) during the period of July to November 1986. The rats were maintained with ad libitum access to Purina Rodent Chow 5001
From Wyeth-AyerstResearch, Princeton, NJ. Address reprint requests to Michael L. McCaleb, PhD, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543. Copyright 0 1992 by WB. Saunders Company 00260495/92/4105-0014$03.00/0
522
Metabolism, Vol41,
No
5 (May).
1992: pp
522-525
HYPERGLYCEMIC
OBESE
ZUCKER
RAT
523
Table 4. Sciatic Nerve Carbohydrate
Table 1. Blood Glucose Levels PlasmaGlucose(mg/dL) Fasted
Group
78 t- 3
Lean Euglycemic obese
104*
1
Hyperglycemic obese
201 f 1 I*
Content (nmolimg)
BloodGHb 1%)
Fed
G-i-r
Content
Group
GllK0S.e
Sorbitol
Fructose 0.80 2 0.11
II825
100 + 4
5.6 -t 0.5
Lean
1.99 + 0.04
0.17 + 0.02
170 f 6
115 2 3
6.3 f. 0.2
Euglycemic obese
1.97 +- 0.07
0.21 f 0.01
0.94 i- 0.08
329 + 25*
319 + 25*
Hyperglycemic obese
8.28 + 0.69*
1.81 + 0.21*
5.49 + 0.46*
14.0 t 0.4*
lP < .Ol compared with lean or euglycemic obese.
Abbreviation: GTT, glucose tolerance test. *P < .Ol compared with lean or euglycemic obese.
Table 2. Characteristics Normal
of Animal Groups Hyperglycemic Obese
Euglycemic Obese
Lean
556 + IO*
Body weight (g)
437 2 30
610 + 14t
Age range (mo)
4-6
4-8
5-8
5
29
14*
N
the obese rats; however, the levels of glycemia of the obese rats were not correlated with the UAE rates. As shown in Fig 1, the UAE rates of the obese rats were significantly correlated with the ages of the animals. In the sciatic nerve of hyperglycemic obese rats, which were 5 to 8 months of age, there was a sixfold to eightfold increase in glucose, sorbitol, and fructose (Table 4). With a longer duration of hyperglycemia (6 to 8 months), the neural glucose, sorbitol, and fructose remained elevated. The mean level of neural myu-inositol in the hyperglycemic group (aged 5 to 8 months) was 40% below the levels in euglycemic obese and lean rats (Fig 2, group levels). However, an examination of the levels in the individual rats in the hyperglycemic group showed that neural myoinositol returned to near normal levels with the longer duration of hyperglycemia, eg, 1.25 ? 0.23 nmol/mg versus 1.89 t 0.48 nmol/mg for Smonth and g-month hyperglycemic rats, respectively (Fig 2, individual levels). It should be noted that the normalization of nerve myo-inositol levels occurred in the presence of a small elevation of GHb levels, eg, 12.9% ? 1.7% versus 15.2% * 1.2% for Smonth and g-month hyperglycemic rats, respectively. In the lenses of hyperglycemic obese rats (aged 5 to 7 months, noncataractous) there was a threefold to fivefold increase in glucose and fructose, a 22-fold increase in sorbitol, and a 100% depletion of myo-inositol (Table 5). At 8 months, all hyperglycemic obese rats had developed mature bilateral cataracts that led to lower levels of sorbitol and fructose, presumably due to sugar leakage from ruptured lens cells.
*P < .05 compared with lean or euglycemic obese. tf’ < .Ol compared with lean. *Four animals in this group were cataractous.
Table 3. Urinary Parameters
Volume (mL/d)
Group
Albumin (mg/lOOg body M/d)
Glucose (mg/dL)
Lean
7.4 t 0.9
22 k 2
0.2 k 0.1
Euglycemic obese
9.5 t 0.6
33 + 7
14.4 zk 2.4’
18.8 ? 1.98
11,287 2 4,073$
21.2 2 2.5t
Hyperglycemic obese
*P < .05 compared with lean. tP < .Ol compared with lean. W’ < .05 compared with lean or euglycemic obese. §P < .Ol compared with lean or euglycemic obese.
80
o
Euglycemic Obese
o
Hyperglycemic
DISCUSSION
Obese
The streptozocin (STZ)-induced diabetic rat has become a primary animal model of type I diabetes for the evaluation of the development of diabetic complications.‘“’ It has been demonstrated in multiple experiments that elevated blood
I r = 0.45
0
p < 0.02
01 3
.
m .
m .
Q
4
5
6
-
1
7
.
1 8
.
I 9
4
5
6
7
Age ,mo”th*)
8
9
e-p < ““1
I;ompwa
Euglycemlc
to
Lean
or
Obese
Age (months) Fig 1.
UAE of individual obese Zucker rats, aged 4 to 8 months.
Fig 2. Individual and group levels of myoinositol in the nerve of lean and obese Zucker rats.
524
McCALEB AND SREDY
Table 5. Lens Carbohydrate
Content Content (nmollmg)
Group
GlUCOSe
Sorbitol
Fructose
myo-lnositol
Lean
0.82 + 0.19
0.97 k 0.17
2.12 + 0.20
1.43 f 0.23
Euglycemic obese
1.05 + 0.12
1.58 2 0.44
2.48 + 0.45
1.37 f 0.23
Hyperglycemic obese
3.48 2 0.50*
35.40 3 4.25f
11.55 -t 0.84*
ND
Cataractous hyperglycemic obeset
6.61 2 1.95X
8.90 k 2.50’
6.55 f 1.04’
ND
Abbreviation: ND, not detected. *P < .Ol compared with lean or euglycemic obese. tN = 4, age was 8 months.
glucose levels result directly in high tissue levels of glucose. In the lens, nerve, retina, and kidney, glucose is subsequently converted to sorbitol and fructose.‘-3.9-‘2There is strong evidence to support the hypothesis that activation of the polyol pathway has pathophysiologic consequences in these tissues. For example, by inhibiting the enzyme, aldose reductase, with a variety of specific inhibitors, diabetesinduced nerve dysfunction, retinal basement membrane thickening, cataracts, and renal abnormalities were substantially diminished.‘* The progression of diabetic complications in type II diabetes is less well known than that in type I, even though complications are evident clinically in both types of patients.13 The obese Zucker rat has been proposed as an animal model of type II diabetes; however, there have been only a few reports of the development of diabetes-like pathologies in these animals.‘4-‘a Therefore, using the lean and obese Zucker rats available in our colony, we performed a cross-sectional evaluation of various metabolic and functional parameters that are usually associated with the development of diabetic complications. The occasional occurrence of overt fasting hyperglycemia in some obese Zucker rats derived from the Indianapolis animal colony has been reported previously.” Here, we demonstrate that the animals that have markedly elevated fasting plasma glucose levels also exhibit significantly increased GHb levels. Consistent with clinical studies in obese, euglycemic patients,” the GHb levels of obese, euglycemic, but glucose-intolerant rats were not different from those of lean normal rats, and there were no changes in tissue polyol levels. However, the hyperglycemic Zucker rats had substantial elevations of glucose in the lens and sciatic nerves. As observed previously in the STZ-induced hyperglycemic rat,“1’,‘9,z0the hyperglycemic Zucker rat also had dramatic, and similar, increases in the tissue levels of sorbitol and fructose. In four of the older hyperglycemic rats in which mature cataracts were evident, and hence a loss of lenticular integrity, polyol elevation was still evident. Of special interest was the significant decrease in tissue levels of myo-inositol in the hyperglycemic rats. In all hyperglycemic rats, elevated levels of lens sorbitol and fructose were accompanied by a total depletion of myoinositol. In the nerves of hyperglycemic rats, there was an age-dependent difference in the myo-inositol levels. The myoinositol levels were progressively higher in the older hyperglycemic rats, suggesting that myo-inositol returns to normal with increasing duration of diabetes. These results
support previous views that neural myo-inositol depletion in diabetic rats is only transient.“-” Although the mechanism for this return to normal is unknown, it is possible that the normalization of nerve myo-inositol was caused by an elevation in plasma myo-inositol in the renal-impaired rats. An elevation in the plasma myo-inositol will increase the concentration of myo-inositol in the sciatic nerve.” Additionally, renal-impaired, 14-month STZ-diabetic rats have been shown to have an elevated plasma myo-inositol concentration.23 However, an elevation in plasma myo-inositol may not be the only mechanism, since in 4-month and 6-month STZ-diabetic rats a normal sciatic nerve myo-inositol concentration was associated with a normal plasma myoinositol concentration.‘9’21 The early stages of clinical diabetic nephropathy are characterized by an elevation of UAE. The albuminuria in both type I and type II diabetic patients progresses to overt proteinuria, and eventually results in end-stage renal failure.“’ The STZ-diabetic rat also exhibits a progressive increase in UAE, which appears to be dependent on hyperglycemia, since treatment of the diabetic animals with an aldose reductase inhibitor prevents the albuminuria.’ In contrast to the progression of albuminuria in STZ-diabetic rats, the obese Zucker rats exhibited elevations of UAE that were independent of hyperglycemia, glucosuria, and polyuria. It has been reported previously that the obese Zucker rat excretes large amounts of proteinI and our results demonstrate that the proteinuria is dependent on age and not on glycemia. Recently, it has been suggested that glomerulosclerosis may frequently be due to atherosclerotic lesionsz5 As an extension of this hypothesis, the nephropathy of the obese Zucker rat was proposed to be a direct consequence of the animal’s extreme hyperlipidemia.2b However, we cannot address this specific hypothesis, since blood lipid levels were not determined in the present study. In conclusion, although the euglycemic obese Zucker rat exhibits some abnormalities, the hyperglycemic obese Zucker rat more closely resembles the altered metabolic profile associated with type II diabetes mellitus.
ACKNOWLEDGMENT
We would like to thank Michael Houlihan, Brenda Mihan, and Diane Sawicki for their excellent technical assistance, and Ann Marie Lucchesi for her skillful secretarial support.
HYPERGLYCEMIC
OBESE
ZUCKER
525
RAT
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