Effect of diets with different fat contents on the development of diabetes in female Zucker diabetic fatty rat with leptin mutation

The aim of the study was to develop a diet which causes stable hyperglycaemia and development of diabetes in female Zucker diabetic fatty (ZDF) rats. We also examined whether worsened wound healing is caused only by hyperglycaemia or whether it is caused by more factors. Four types of special diets with a different content of fat were fed to eight groups of 3–7 (fa/fa or fa/+) rats. The following diets were used: H1 (24.6% fat), H2 (33.2%), C13004 (25.6%), and St1 (3.4%). We detected significant diet-dependent changes of weight and concentration of glucose in animals with leptin mutation (fa/fa). All examined indicators were significantly (P < 0.001) higher in (fa/fa) animals compared to the fa/+ ones no matter what diet they ate. All diets with high-fat content caused increased glycaemia, but only the diet with 24.6% fat caused a significant (P < 0.01) increase of glycaemia. Our results have proved that this diet is the most suitable to invoke and keep hyperglycaemia. The diet with 25.6% fat is suitable to invoke stable slightly increased glycaemia (10 mmol/l) and hyperinsulinaemia. On the other hand, the diet with 33.2% fat is unsuitable. We did not observe a significant influence of diet on wound healing. We developed a new diet more suitable for induction of stable hyperglycaemia in female ZDF rats than commercially available mixtures. Our study is the first to present recommendations for adjusting a high-fat diet to produce stable hyperglycaemia and hyperinsulinaemia in the rat model. Leptin mutation, glycaemia, insulinaemia, MMP-3, wound healing Type 2 (DM2T) diabetes mellitus (DM) is the most common metabolic disease that arises due to a relative lack of insulin. The cells are not able to intake glucose which results in hyperglycaemia. Long-term hyperglycaemia leads to serious complications including retarded wound healing. Reliable animal models are necessary to observe the disease development and to test suitable medication (Bartosikova et al. 2003; Lukacinova et al. 2008). Zucker diabetic fatty (ZDF) rats can be used as a suitable model (Slavkovsky et al. 2011). Rats with fa/fa genotype develop genetically dependent obesity and subsequently diabetes due to a point missense mutation in extracellular domain of the leptin receptor. It is characterized by non-insulin dependent DM accompanied by hyperglycaemia, neuropathies, nephropathies, insulin resistance, mild hypertension, hypertriglyceridaemia, hypercholesterolaemia, polyphagia, polyuria, polydipsia, protein glycation, and microvascular damage. The development of diabetes in these animals resembles the situation seen in humans: animals gradually evolve from hyperinsulin-euglycaemic state to hyperglycaemic state with relative insulin deficiency (Finegood et al. 2001; Leonard et al. 2005; and Slavkovsky et al. 2011). However, to develop DM due to over nutrition, these rats must be fed on high-energy, high-fat diet. Originally, the rats’ provider Charles River Laboratories International, Inc. ACTA VET. BRNO 2013, 82: 289–296; doi:10.2754/avb201382030289 Address for correspondence: Mgr. Renata Köhlerová, Ph.D. Department of Medical Biochemistry Faculty of Medicine in Hradec Kralove Charles University in Prague Šimkova 870, 500 38 Hradec Králové, Czech Republic Phone: +420 495 816 454 Fax: + 420 495 832 003 E-mail: kohler@lfhk.cuni.cz http://actavet.vfu.cz/ recommended the C13004 diet. In the year 2007 the composition of the C13004 diet had been changed. Our results concerning hyperglycaemia induction by this newly composed diet did not correspond either to the values given by the provider or to the values published with original C13004 composition (Corsetti et al. 2000). The company itself later admitted that the diet was not efficient. While the commercially available diet with 4.5% content of fat (Purina 5001) is enough to develop diabetes in male rats with leptin mutation, the situation in female rats is more complicated. The aim of our study was to develop new diet for rats and compare its effect on development of diabetes mellitus with the standard laboratory diet and the current high fat laboratory C13004 diet. Materials and Methods Animals The experimental protocol was approved by the Animal Welfare Committee of Charles University in Prague, Faculty of Medicine in Hradec Králové (No. 12420/2011-30). In the experiment we used 40 female Zucker diabetic fatty rats that originated from the Charles River Laboratories International, Inc. (USA) and were purchased from Anlab (Prague, Czech Republic). The offspring were bred in the vivarium at the Faculty of Medicine in Hradec Králové, Charles University by mating non-obese heterozygous (fa/+) carrier parents or by mating fa/fa obese males with fa/+ females. The rats were kept under standard conditions. They received food and drinking water ad libitum. Leptin mutation genotypization All rats were genotyped using a modified PCR-RFLP method. DNA was isolated from a piece of the tail or the phalanx obtained at the age of 3 weeks using a DNeasy Blood and Tissue Mini Kit (Qiagen, Hilden, Germany). The methods were described in detail by Slavkovsky et al. (2011). Types of diets Rats at the age of 4 weeks were divided into eight groups of 3–7 animals. There were 4 groups of fa/fa and 4 groups of fa/+ rats. According to type of diet (different amount of fat), there were following groups: H1 (24.6% fat), H2 (33.2%), C13004 (25.6%) and St1 (3.4%); each for fa/fa and fa/+ rats. The St1 is standard laboratory diet (ST-1, VELAS, a.s., Lysá nad Labem, Czech Republic) and C13004 is commercial high fat laboratory diet (Research Diets, New Brunswick, NJ, USA). The other diets H1 and H2 (Table 1) were based on mixture of St1, pork fat, corn starch (maizena), and sugar. The ingredients were mixed together and water was added as necessary. The final mixture was shaped into loaves and dried for 48 h in an oven heated to 60 °C. Weights of rats were regularly measured every two weeks since the age of 4 weeks from birth (week 0); i.e. 6 × during the 12-week period. A portable glucose meter, AccuChek-Go (Roche, Basel, Switzerland), was used for glucose measurement using a drop of blood from the tail vein every two weeks, same as the measuring of weight. The rats were not anaesthetized during glucose measurement because anaesthesia could increase glycaemia (Saha et al. 2005). Measurements of insulin concentration Blood was collected from the orbital plexus of rats. Heparinized blood was centrifuged and plasma was stored at -70 °C until it was analysed. Insulin was measured using the Rat Insulin ELISA kit (H-type, Shibayagi, Japan) 290 Table 1. Composition of diets used for feeding Zucker diabetic fatty rat in our experiment. Composition H1 (%) H2 (%) C13004 (%) St1 (%) Proteins 14.4 12.0 14.6 24.0 Carbohydrates 44.8 39.8 46.6 50.5 Fats 24.6 33.2 25.6 3.4 Fibrous material 2.2 1.9 1.4 4.4 Ash 3.9 3.3 4.9 6.8 Moisture 10.1 9.9 7.8 12.5 Diets C1304 and St1 are commercial, diets H1 and H2 are self-prepared, St1 is a low fat diet. according to the manufacturer’s instructions. Concentration of insulin in unknown samples was calculated using the calibration curve of insulin standards (y = 0.0341x + 0.0393, R2 = 0.9995). Wound induction and evaluation Wounds in rats were induced at the age of 16–18 weeks as described by Slavkovský (2011). Briefly: A fullthickness excision circular skin wound of 2 cm in diameter was made on the back. Wounds were allowed to heal naturally. The wounds were photographed (EOS D350, Canon, Tokyo, Japan) immediately after induction (Day 0) and then every second or third day. The wound area was measured using ImageJ software (NIH) calibrated to standard length using a millimetre ruler. The relative wound size was expressed according to the following formula: % wound area = wound area on Day n/wound area on Day 0. The animals were sacrificed on Day 10 and their granulation tissue (GT) was collected at the same time. The sample was stored in RNA stabilizing solution (Qiagen) and frozen to -70 °C. Evaluation of MMP-3 expression in the granulation tissue RNA was isolated from the GT sample (RNeasy Lipid Tissue Mini Kit, RNeasy Fibrous Tissue Mini Kit Qiagen). The amount of RNA was measured using spectrophotometer while the quality was detected using electrophoresis. Total RNA (1 mg) was transcribed to cDNA using the cDNA archive kit (Applied Biosystems, Foster City, California). Synthesized cDNA (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems) was used to study MMP-3 gene expression using rt RT-PCR method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rat gene (TaqMan Gene Expression Assays Applied Biosystems) was used as housekeeping gen (endogenous control). The results were analyzed using the 7500 Fast System SDS Software and then processed in Microsoft Excel 2007. Statistical analysis Statistics were calculated using the STATISTICA 10.0 data analysis software (StatSoft ČR, s. r. o., 2011). The differences between the control and experimental group were evaluated by Mann-Whitney U test. Differences within the groups were evaluated by Kruskal-Wallis test (P < 0.001 or P < 0.05). Results Weight gain The weights of fa/fa and fa/+ animals were approximately the same in week 0 (Fig. 1). The weight of both groups grew during the time, but weight of fa/fa animals grew faster. 291

Type 2 (DM2T) diabetes mellitus (DM) is the most common metabolic disease that arises due to a relative lack of insulin.The cells are not able to intake glucose which results in hyperglycaemia.Long-term hyperglycaemia leads to serious complications including retarded wound healing.Reliable animal models are necessary to observe the disease development and to test suitable medication (Bartosikova et al. 2003;Lukacinova et al. 2008).
Zucker diabetic fatty (ZDF) rats can be used as a suitable model (Slavkovsky et al. 2011).Rats with fa/fa genotype develop genetically dependent obesity and subsequently diabetes due to a point missense mutation in extracellular domain of the leptin receptor.It is characterized by non-insulin dependent DM accompanied by hyperglycaemia, neuropathies, nephropathies, insulin resistance, mild hypertension, hypertriglyceridaemia, hypercholesterolaemia, polyphagia, polyuria, polydipsia, protein glycation, and microvascular damage.The development of diabetes in these animals resembles the situation seen in humans: animals gradually evolve from hyperinsulin-euglycaemic state to hyperglycaemic state with relative insulin deficiency (Finegood et al. 2001;Leonard et al. 2005;and Slavkovsky et al. 2011).
However, to develop DM due to over nutrition, these rats must be fed on high-energy, high-fat diet.Originally, the rats' provider Charles River Laboratories International, Inc. recommended the C13004 diet.In the year 2007 the composition of the C13004 diet had been changed.Our results concerning hyperglycaemia induction by this newly composed diet did not correspond either to the values given by the provider or to the values published with original C13004 composition (Corsetti et al. 2000).The company itself later admitted that the diet was not efficient.While the commercially available diet with 4.5% content of fat (Purina 5001) is enough to develop diabetes in male rats with leptin mutation, the situation in female rats is more complicated.
The aim of our study was to develop new diet for rats and compare its effect on development of diabetes mellitus with the standard laboratory diet and the current high fat laboratory C13004 diet.

Animals
The experimental protocol was approved by the Animal Welfare Committee of Charles University in Prague, Faculty of Medicine in Hradec Králové (No. 12420/2011-30).In the experiment we used 40 female Zucker diabetic fatty rats that originated from the Charles River Laboratories International, Inc. (USA) and were purchased from Anlab (Prague, Czech Republic).The offspring were bred in the vivarium at the Faculty of Medicine in Hradec Králové, Charles University by mating non-obese heterozygous (fa/+) carrier parents or by mating fa/fa obese males with fa/+ females.The rats were kept under standard conditions.They received food and drinking water ad libitum.

Leptin mutation genotypization
All rats were genotyped using a modified PCR-RFLP method.DNA was isolated from a piece of the tail or the phalanx obtained at the age of 3 weeks using a DNeasy Blood and Tissue Mini Kit (Qiagen, Hilden, Germany).The methods were described in detail by Slavkovsky et al. (2011).

Types of diets
Rats at the age of 4 weeks were divided into eight groups of 3-7 animals.There were 4 groups of fa/fa and 4 groups of fa/+ rats.According to type of diet (different amount of fat), there were following groups: H1 (24.6% fat), H2 (33.2%),C13004 (25.6%) and St1 (3.4%); each for fa/fa and fa/+ rats.The St1 is standard laboratory diet (ST-1, VELAS, a.s., Lysá nad Labem, Czech Republic) and C13004 is commercial high fat laboratory diet (Research Diets, New Brunswick, NJ, USA).The other diets H1 and H2 (Table 1) were based on mixture of St1, pork fat, corn starch (maizena), and sugar.The ingredients were mixed together and water was added as necessary.The final mixture was shaped into loaves and dried for 48 h in an oven heated to 60 °C.
Weights of rats were regularly measured every two weeks since the age of 4 weeks from birth (week 0); i.e. 6 × during the 12-week period.
A portable glucose meter, AccuChek-Go (Roche, Basel, Switzerland), was used for glucose measurement using a drop of blood from the tail vein every two weeks, same as the measuring of weight.The rats were not anaesthetized during glucose measurement because anaesthesia could increase glycaemia (Saha et al. 2005).

Measurements of insulin concentration
Blood was collected from the orbital plexus of rats.Heparinized blood was centrifuged and plasma was stored at -70 °C until it was analysed.Insulin was measured using the Rat Insulin ELISA kit (H-type, Shibayagi, Japan) 290 according to the manufacturer's instructions.Concentration of insulin in unknown samples was calculated using the calibration curve of insulin standards (y = 0.0341x + 0.0393, R² = 0.9995).
Wound induction and evaluation Wounds in rats were induced at the age of 16-18 weeks as described by Slavkovský (2011).Briefly: A fullthickness excision circular skin wound of 2 cm in diameter was made on the back.Wounds were allowed to heal naturally.The wounds were photographed (EOS D350, Canon, Tokyo, Japan) immediately after induction (Day 0) and then every second or third day.The wound area was measured using ImageJ software (NIH) calibrated to standard length using a millimetre ruler.The relative wound size was expressed according to the following formula: % wound area = wound area on Day n/wound area on Day 0. The animals were sacrificed on Day 10 and their granulation tissue (GT) was collected at the same time.The sample was stored in RNA stabilizing solution (Qiagen) and frozen to -70 °C.
Evaluation of MMP-3 expression in the granulation tissue RNA was isolated from the GT sample (RNeasy Lipid Tissue Mini Kit, RNeasy Fibrous Tissue Mini Kit Qiagen).The amount of RNA was measured using spectrophotometer while the quality was detected using electrophoresis.Total RNA (1 mg) was transcribed to cDNA using the cDNA archive kit (Applied Biosystems, Foster City, California).Synthesized cDNA (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems) was used to study MMP-3 gene expression using rt RT-PCR method.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rat gene (TaqMan Gene Expression Assays Applied Biosystems) was used as housekeeping gen (endogenous control).The results were analyzed using the 7500 Fast System SDS Software and then processed in Microsoft Excel 2007.

Statistical analysis
Statistics were calculated using the STATISTICA 10.0 data analysis software (StatSoft ČR, s. r. o., 2011).The differences between the control and experimental group were evaluated by Mann-Whitney U test.Differences within the groups were evaluated by Kruskal-Wallis test (P < 0.001 or P < 0.05).

Weight gain
The weights of fa/fa and fa/+ animals were approximately the same in week 0 (Fig. 1).The weight of both groups grew during the time, but weight of fa/fa animals grew faster.The average weight of fa/fa was significantly higher compared to fa/+ animals in week 8 (P < 0.001).In week 16 the resulting weight of fa/fa rats was about twice as high as the weight of fa/+ controls.Significant differences between diet groups were observed too (P < 0.05).Both groups of animals fed high fat diet (H2) showed the lowest weight gain and also high mortality (50% for fa/fa and 71.4% for fa/+).

Glycaemia
Glucose concentrations are shown in Fig. 2. St1 diet had no effect on glycaemia in fa/fa animals, fa/+ rats of all diet groups had normoglycaemia.The H1 and C13004 diets caused significantly (P < 0.05) higher glycaemia in fa/fa animals than St1 as early as after 8 weeks on the diet.Higher glycaemia was detected after 12 weeks in the fa/fa animals fed all three high fat diets (H1, H2, C13004) compared to the fa/fa animals fed St1, but only after using H1 and C13004 did this increase become significant (P < 0.05).The highest increase in glycaemia was observed in fa/fa animals fed H1 diet (26.8 mmol/l), and was higher than hyperglycaemia induced by C13004 (10.4 mmol/l).

Insulinaemia
Levels of insulinaemia were measured before the induction of the skin wound.Concentrations of insulin in fa/+ animals were low and, regardless of the type of diet, did not exceed 2.22 mg/l.Nevertheless, we observed significant differences (P < 0.001) among the fa/+ animals depending on the implemented diet (Fig. 3A).
The levels of insulinaemia between fa/fa animals were significantly (P < 0.001) higher with much higher variance (Fig. 3B).Higher concentrations of plasmatic insulin were □ □ detected in the animals fed the C13004 diet, but the difference was not significant due to high variance.
The relation between concentrations of glycaemia and insulin is shown in Fig. 3C.Both values were normal in fa/+ rats.The fa/fa rats fed the standard St1 diet with 3.4% of fat had normoglycaemia and 10 × higher concentrations of insulin than fa/+ rats.The rats fed the commercial C13004 diet (25.6% of fat) had high concentrations of insulin and glycemia around 10 mmol/l.On the other hand, the rats fed the prepared H1 diet (24.6% of fat) had low concentrations of insulin and hyperglycaemia (mean mmol/l).No trends were observed in animals fed the diet with 33.2% of fat; these animals responded individually.

Wound closure
Skin wound contraction was decreased in all fa/fa rats compared to fa/+ ones (P < 0.001) regardless of diet and glucose or insulin concentrations (Fig. 4).The wound in fa/+ animals started to contract immediately after induction, whereas the wound in diabetic animals did not reduce or even grew, and the wound reducing started as late as on day 4.The wound reduced by more than two thirds within 10 days in fa/+ rats but only by less than a half in diabetic ones.We observed the slowest wound contraction in animals fed the H2 diet no matter whether they were fa/+ or fa/fa.We observed the fastest wound closure in animals fed the C13004 diet within the fa/fa animals.

Expression of MMP-3
The fa/fa rats had significantly (P < 0.001) higher values of MMP-3 expression compared to the fa/+ rats (Fig. 5).When we evaluated the influence of the diet, we found significant (P < 0.05) difference between fa/fa and fa/+ rats fed the St1 and H1 diets.The differences between fa/+ rats fed different diets were not significant, nor were the differences within the fa/fa group.diet is suitable to develop hyperinsulinaemia and a stable, slightly increased glycaemia (10 mmol/l).Our results suggest that correction of glucose and insulin concentrations into physiological range is not sufficient enough to influence all undesirable symptoms of DM.The wounds in fa/fa rats healed slowly regardless of weight, glycaemia, and insulinaemia.Impaired healing was accompanied by increased expression of MMP-3 in fa/fa rats in comparison to fa/+ rats.

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Fig. 1.Influence of different diets on the body weight development in rats * value is significantly higher in comparison to healthy animals for respective group in the week 8 (P < 0.001) Diets C1304 (25.6 % fat) and St1 (3.4 %) are commercial, diets H1(24.6 %) and H2 (33.2 %) are self-prepared.

Table 1 .
Composition of diets used for feeding Zucker diabetic fatty rat in our experiment.