Age-related Changes in Glycerolipid Formation in Lean and Obese Zucker Rats SUBHASH C. JAMDAR 1, Departments of Internal Medicine and Biochemistry, Medical College of Virginia, Richmond, Virginia 23298 ABSTRACT
Age-related changes in hepatic and adipose glycerolipid formation have been described in Zucker rats. Gtycerolipid formation was measured in vitro in the presence of [ 14C]glycerol-3-phosphate, palmitate, ATP, CoA, and Mg2+ by using liver and adipose tissue homogenates derived from various age groups of animals. Hepatic glycerolipid formation increased after birth to reach a peak value at 1 day of age. This period was followed by a decline in the rates of glycerolipid formation. Hepatic glycerolipid formation increased again at the time of weaning and continued to rise up to 32 days in lean rats and 42-44 days in obese rats. Obesity in rats was recognizable at the age of 32 days and was associated with increased rates of glycerolipid formation in both liver and adipose tissue. As far as the changes in hepatic glycerolipid formation and triglyceride accumulation are concerned, obese rats showed more resemblance to 1-day-old rats than to lean animals of similar age groups. Glycerolipid formation decreased in liver and increased in adipose tissue with age in both lean and obese rats. These studies suggest that hepatic and adipose tissue glycerolipid formation is significantly influenced by age and obesity in Zucker rats. INTRODUCTION
Genetically transmitted obesity has been described in several strains o f rodents (1). In Zucker obese rats, the abnormality is inherited as a u t o s o m a l recessive. The p h e n o t y p i c expression of this trait is n o t apparent at birth but can be recognized at 2-3 weeks of age. By this time, the animals are hyperphagic and show increased b o d y fat and elevated levels of plasma insulin (1). Obesity in this animal m o d e l is accompanied by e n h a n c e d rates o f triglyceride formation in both liver and adipose tissue (2,3). R e c e n t studies from this laboratory d e m o n strate that the period b e t w e e n birth and 3 weeks o f age is active in hepatic triglyceride f o r m a t i o n in Sprague Dawley rats (4). In the present studies, age-related changes in hepatic and adipose tissue triglyceride f o r m a t i o n have been investigated in Zucker rats to d e t e r m i n e whether increased potential o f triglyceride f o r m a t i o n is responsible for the genetic expression of obesity in this animal model.
were purchased f r o m Harriet G. Bird Memorial L a b o r a t o r y , Stow, MA. Birth dates and time of weaning (21 days of age) were recorded carefully. A f t e r weaning, all rats received Purina Chow diet, Ralston Purina Co., St. Louis, MO. The animal colony was maintained in a temperature-controlled r o o m with a 12 hr on, 12 hr off light cycle. F o r d e v e l o p m e n t a l studies, animals from b o t h sexes were selected. Animals were killed by decapitation and b l o o d was collected in heparinized tubes. In some studies, b l o o d was pooled f r o m 2 or 3 animals (newborn animals) to obtain sufficient plasma for tfiglycefide determination. All animals were killed b e t w e e n 9 and 11 a.m. Initial studies were c o n d u c t e d w i t h liver and adipose tissue from albino rats to determine o p t i m u m conditions used in various assays. Sprague Dawley rats o f the same age as the Zucker rats were f r o m our animal colony. The i n c u b a t i o n conditions developed in tissues from albino rats were satisfactory to measure the estefification rates in liver and adipose tissues from Zucker rats.
MATERIALS AND METHODS
Preparation of Homogenates
[ 1,3 -I 4C] glycerol-3-phosphate (sp. radioactivity 30 m C i / m m o l ) was purchased from ICN Chemicals and Radioisotope Division, Irvine, CA. Most of the o t h e r chemicals were of A.R. grade quality and were purchased f r o m the sources r e p o r t e d previously (3,4). Male and female obese (fa/fa) rats and their lean controls (FA/-) were either from our animal c o l o n y or sn
1present address -
Medical Research Institute,
Florida Institute of Technology, Melbourne, Florida
32901. 463
Livers were r e m o v e d , blotted free of b l o o d , and weighed. They were h o m o g e n i z e d (at 4 C) in a Teflon glass h o m o g e n i z e r with 4 vol of buffer (0.25 M sucrose, 1 mM d i t h i o t h r e i t o t , 1 mM E D T A , and 1 mM Tris, pH 7.5) and the resultant h o m o g e n a t e was used in various assays. Gonadal adipose tissues were removed and h o m o g e n i z e d with 3 vol of buffer. The h o m o g e n i z a t i o n was p e r f o r m e d at speed 5 for 30 sec in the cold (4 C) with a T e k m a r Tissumizer ( T e k m a r Instruments, Cincinnati, OH).
464
S.C. JAMDAR TABLE I Changes in Hepatic and Plasma Triglyceride Concentrations as a Function of Age a Trigly ceride
Group
Lean Obese
Lean Obese
Lean Obese
Lean Obese
No. and age of animals
Body weight (g)
18hr (12) 1 day (8) 4-6 days (23) 11 days (11) 15 days (5) 20 days (8) 22 days (8) 32 days (4) 32 days (4) 42-44 days (8) 42-44 days (8) 67-69 days (5) 67-69 days (5) 75-79 days (4) 75-79 days (4)
5.5 -+ O.S 6.0_+ 0.5 10 _+ 1 18 • 1 27 • 3 33 +_ 3 42 +_ 3 75 • 8 83 • 9 114 • 17 111 • 19 192 • 45 268 b • 30 287 • 11 326 • 28
Liver weight (g) 0.23 +-0.02 0.21• 0.02 0.30 • 0.02 0.50 • 0.04 1.2 • 0.1 1.3 • 0.2 1.6 • 0.1 3.0 • 0.2 4.0 • 0.4 S • 1 5 • 1 8 • 1 11 b • 2 12 • 1 15 b • 1
Liver (rag/g) 18 30 7 6.0 1.2 3 3 6.2 b 2.6 6.5 b 1.7 5b 1.5 9b
-+ 3 • 4 -+4 + 0.5 • • 0.3 • 1 • 1 -+ 1.2 +_ 1.2 • 2.4 _+0.8 • 2 _+0.4 • 3
Plasma (rag %) 110 •
20
140 • 18 141 _+ 19 237 114 76 79 90 138 71 197 b 75 323 b
• 85 • 52 _+ 10 • 20 • 40 • 22 + 32 + 38 • 23 _+ 104
aEach value is mean • S.D. from the number of rats referred to in parentheses. bp
Age-related changes in hepatic and adipose glycerolipid formation have been described in Zucker rats. Gtycerolipid formation was measured in vitro in the presence of [ 14C]glycerol-3-phosphate, palmitate, ATP, CoA, and Mg2+ by using liver and adipose tissue homogenates derived from various age groups of animals. Hepatic glycerolipid formation increased after birth to reach a peak value at 1 day of age. This period was followed by a decline in the rates of glycerolipid formation. Hepatic glycerolipid formation increased again at the time of weaning and continued to rise up to 32 days in lean rats and 42-44 days in obese rats. Obesity in rats was recognizable at the age of 32 days and was associated with increased rates of glycerolipid formation in both liver and adipose tissue. As far as the changes in hepatic glycerolipid formation and triglyceride accumulation are concerned, obese rats showed more resemblance to 1-day-old rats than to lean animals of similar age groups. Glycerolipid formation decreased in liver and increased in adipose tissue with age in both lean and obese rats. These studies suggest that hepatic and adipose tissue glycerolipid formation is significantly influenced by age and obesity in Zucker rats. INTRODUCTION
Genetically transmitted obesity has been described in several strains o f rodents (1). In Zucker obese rats, the abnormality is inherited as a u t o s o m a l recessive. The p h e n o t y p i c expression of this trait is n o t apparent at birth but can be recognized at 2-3 weeks of age. By this time, the animals are hyperphagic and show increased b o d y fat and elevated levels of plasma insulin (1). Obesity in this animal m o d e l is accompanied by e n h a n c e d rates o f triglyceride formation in both liver and adipose tissue (2,3). R e c e n t studies from this laboratory d e m o n strate that the period b e t w e e n birth and 3 weeks o f age is active in hepatic triglyceride f o r m a t i o n in Sprague Dawley rats (4). In the present studies, age-related changes in hepatic and adipose tissue triglyceride f o r m a t i o n have been investigated in Zucker rats to d e t e r m i n e whether increased potential o f triglyceride f o r m a t i o n is responsible for the genetic expression of obesity in this animal model.
were purchased f r o m Harriet G. Bird Memorial L a b o r a t o r y , Stow, MA. Birth dates and time of weaning (21 days of age) were recorded carefully. A f t e r weaning, all rats received Purina Chow diet, Ralston Purina Co., St. Louis, MO. The animal colony was maintained in a temperature-controlled r o o m with a 12 hr on, 12 hr off light cycle. F o r d e v e l o p m e n t a l studies, animals from b o t h sexes were selected. Animals were killed by decapitation and b l o o d was collected in heparinized tubes. In some studies, b l o o d was pooled f r o m 2 or 3 animals (newborn animals) to obtain sufficient plasma for tfiglycefide determination. All animals were killed b e t w e e n 9 and 11 a.m. Initial studies were c o n d u c t e d w i t h liver and adipose tissue from albino rats to determine o p t i m u m conditions used in various assays. Sprague Dawley rats o f the same age as the Zucker rats were f r o m our animal colony. The i n c u b a t i o n conditions developed in tissues from albino rats were satisfactory to measure the estefification rates in liver and adipose tissues from Zucker rats.
MATERIALS AND METHODS
Preparation of Homogenates
[ 1,3 -I 4C] glycerol-3-phosphate (sp. radioactivity 30 m C i / m m o l ) was purchased from ICN Chemicals and Radioisotope Division, Irvine, CA. Most of the o t h e r chemicals were of A.R. grade quality and were purchased f r o m the sources r e p o r t e d previously (3,4). Male and female obese (fa/fa) rats and their lean controls (FA/-) were either from our animal c o l o n y or sn
1present address -
Medical Research Institute,
Florida Institute of Technology, Melbourne, Florida
32901. 463
Livers were r e m o v e d , blotted free of b l o o d , and weighed. They were h o m o g e n i z e d (at 4 C) in a Teflon glass h o m o g e n i z e r with 4 vol of buffer (0.25 M sucrose, 1 mM d i t h i o t h r e i t o t , 1 mM E D T A , and 1 mM Tris, pH 7.5) and the resultant h o m o g e n a t e was used in various assays. Gonadal adipose tissues were removed and h o m o g e n i z e d with 3 vol of buffer. The h o m o g e n i z a t i o n was p e r f o r m e d at speed 5 for 30 sec in the cold (4 C) with a T e k m a r Tissumizer ( T e k m a r Instruments, Cincinnati, OH).
464
S.C. JAMDAR TABLE I Changes in Hepatic and Plasma Triglyceride Concentrations as a Function of Age a Trigly ceride
Group
Lean Obese
Lean Obese
Lean Obese
Lean Obese
No. and age of animals
Body weight (g)
18hr (12) 1 day (8) 4-6 days (23) 11 days (11) 15 days (5) 20 days (8) 22 days (8) 32 days (4) 32 days (4) 42-44 days (8) 42-44 days (8) 67-69 days (5) 67-69 days (5) 75-79 days (4) 75-79 days (4)
5.5 -+ O.S 6.0_+ 0.5 10 _+ 1 18 • 1 27 • 3 33 +_ 3 42 +_ 3 75 • 8 83 • 9 114 • 17 111 • 19 192 • 45 268 b • 30 287 • 11 326 • 28
Liver weight (g) 0.23 +-0.02 0.21• 0.02 0.30 • 0.02 0.50 • 0.04 1.2 • 0.1 1.3 • 0.2 1.6 • 0.1 3.0 • 0.2 4.0 • 0.4 S • 1 5 • 1 8 • 1 11 b • 2 12 • 1 15 b • 1
Liver (rag/g) 18 30 7 6.0 1.2 3 3 6.2 b 2.6 6.5 b 1.7 5b 1.5 9b
-+ 3 • 4 -+4 + 0.5 • • 0.3 • 1 • 1 -+ 1.2 +_ 1.2 • 2.4 _+0.8 • 2 _+0.4 • 3
Plasma (rag %) 110 •
20
140 • 18 141 _+ 19 237 114 76 79 90 138 71 197 b 75 323 b
• 85 • 52 _+ 10 • 20 • 40 • 22 + 32 + 38 • 23 _+ 104
aEach value is mean • S.D. from the number of rats referred to in parentheses. bp
