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1Montreal Diabetes Research Center, CRCHUM, 2Department of Medicine, University of Montreal
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A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.
Keywords: Biomedical Engineering, Issue 78, Medicine, Anatomy, Physiology, Basic Protocols, Surgery, Metabolic Diseases, Infusions, Intravenous, Infusion Pumps, Glucolipotoxicity, Rat, Infusion, Glucose, Intralipid, Catheter, canulation, canula, diabetes, animal model
Fergusson, G., Ethier, M., Zarrouki, B., Fontés, G., Poitout, V. A Model of Chronic Nutrient Infusion in the Rat. J. Vis. Exp. (78), e50267, doi:10.3791/50267 (2013).
Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.
Chronically elevated levels of glucose and lipids in the circulation have been proposed to contribute to the pathogenesis of type 2 diabetes by altering the function of several organs implicated in the maintenance of glucose homeostasis including, but not limited to, the pancreatic beta-cell (reviewed in 1). The glucotoxicity hypothesis posits that chronic hyperglycemia aggravates the beta-cell defect which gave rise to hyperglycemia in the first place, thus creating a vicious cycle and contributing to the deterioration of glucose control in type 2 diabetes patients. Likewise, the glucolipotoxicity hypothesis proposes that concomitant elevations of glucose and lipid levels, as often observed in type 2 diabetes, are detrimental to the beta cell.
Deciphering the cellular and molecular mechanisms of the deleterious effects of chronically elevated nutrients on pancreatic beta-cell function is key to the understanding of the pathogenesis of type 2 diabetes 1. To that end, a large number of studies have examined the mechanisms of chronic nutrient excess ex vivo in isolated islets of Langerhans or in vitro in clonal, insulin-secreting cell lines However, translation of the findings obtained in these model systems to the whole organism is complex, in particular because the concentrations of fatty acids used in cultured cells or islets rarely match the circulating levels in the vicinity of the beta cells in vivo 2. On the other hand, the mechanisms of beta-cell failure in response to nutrient excess have been examined in rodent models of diabetes, as exemplified by the Zucker Diabetic Fatty rat 3,4, the gerbil Psammomys obesus 5 and the high-fat diet-fed mouse 6. These models, however, are characterized by intrinsic metabolic abnormalities and are not easily amenable to manipulations of blood glucose and/or lipid levels in a more controlled and less chronic setting. To be able to alter circulating nutrient levels in a timeframe of days in otherwise normal animals, we have developed a chronic infusion model in normal rats which enables us to examine the effects of lipids and glucose, alone or in combination, on physiological parameters and function 7,8.
Overview: The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin 9) for 72 hr.
1. Canulation of the Right Jugular Vein and the Left Carotid Artery
2. Post-operative Care (Post-surgical Treatment and Connection of the Catheters)
5. Post-infusion Euthanasia
Out of a series of 42 rats which underwent surgery, 5 rats were lost during the post-operative period and 1 rat was lost during the infusion, representing an overall success rate of 86%. The average body weight of the 37 rats that were eventually infused was 608±5 g before surgery and 588±6 g at the initiation of the infusion (mean±SE; n=37; P<0.0001 by paired t-test). The following representative results were obtained in 2 infusion groups: Saline (SAL), and Glucose + Intralipid (GLU+IL). Figures 2A and 2B show blood glucose and fatty acid levels, respectively, during the 72-hr infusion period. By design, the glucose levels are maintained around 220-250 mg/dl throughout the infusion, based on regular measurements and adjustments of the glucose infusion rate (GIR) as shown in Figure 3. Rodents have a strong capacity to compensate for the glucose infusion by increasing endogenous insulin secretion. Therefore, the GIR must be increased during the course of the infusion for blood glucose levels to be maintained at a relative steady-state. Nevertheless, blood glucose levels tend to decrease towards the end of the infusion, as seen in Figure 2A. Since the animals are infused with caloric nutrients, they spontaneously decrease their food intake (Figure 4).
|Infusion Condition||Syringe 1 (front position)||Syringe 2 (back position)|
|3||Dextrose 70%||Intralipid 20% + Heparin 20U/ml|
|4||Saline||Intralipid 20% % + Heparin 20U/ml|
Table 1. Infusion regimens.
Figure 1. Photograph of the infusion system showing the rat with the catheters in place and the tether, swivel, and counterbalance arm mounted on the cage grill.
Figure 2. Plasma levels of glucose (A) and free fatty acids (B) during infusion of saline (SAL) or co-infusion of glucose and Intralipid (GLU+IL) in 6-month old Wistar rats. Data are mean ± SEM of 3-4 animals in each group.
Figure 3. Adjustment of the glucose infusion rate during the co-infusion of glucose and Intralipid (GLU+IL) in 6-month old Wistar rats. Data are mean ± SEM of 4 animals.
Figure 4. Average daily food intake of 6-month old Wistar rats infused with saline (SAL) or co-infused with glucose and Intralipid (GLU+IL). Data are mean ± SEM of 3-4 animals in each group. **p<0.01.
Although a number of previous studies have employed chronic infusions of glucose (e.g. 10-15) or lipids (e.g. 16,17) in rodents, to our knowledge the combined infusion of both fuels has only been reported in mice 18. The chronic infusion model presented here offers several advantages to study the effects on nutrient excess on a variety of biological functions in rats. First, it does not involve genetically obese rodents, and since common obesity in humans is polygenic 19, the findings are therefore more likely to be relevant to the effects of nutrient oversupply in the general population. Second, this model offers the possibility to test the effects of different nutrients (particularly sugars or fats) at different circulating levels, by changing their nature and the infusion flow rate. Third, the IV route of administration enables the co-infusion of compounds or drugs together with the nutrients 20. Finally, the relatively short time frame of the experiment (72 hr vs. weeks for high-fat diet induced models) is cost- and time-effective for preclinical studies.
This model also has some disadvantages and limitations. First, as any surgical model is requires highly trained and skilled personnel in aseptic surgery. Second, as mentioned in the Representative Results section the animals tend to compensate for the glucose infusion by increasing endogenous insulin secretion, which requires frequent monitoring of blood glucose levels and adjustments of the GIR to maintain the target blood glucose levels throughout the infusion. Third, the procedure is not easily applicable to mice, which would be required in order to use it in genetically modified animals. In our experience, catheterization of both jugular vein and carotid artery in mice in technically challenging and the easiest approach in these smaller animals is to place a jugular catheter for the infusion and to sample from a peripheral vessel. This, however considerably limits the volume and frequency of sampling during the infusion.
Given the focus of our laboratory, we have used this model to study the mechanisms of glucolipotoxicity in the pancreatic beta-cell 7,8. However, it can be applied to study the effects of nutrient excess in any tissue and organ such as, but not limited to, the heart 21, skeletal muscle, 22, and liver 23.
Vincent Poitout is co-founder of and received research contracts from Bêtagenex Inc., a contract research organization which offers the infusion model presented in this article as a commercial service.
This work was supported by the National Institutes of Health (R01DK58096 to Vincent Poitout). Vincent Poitout holds the Canada Research Chair in Diabetes and Pancreatic Beta-cell Function. Bader Zarrouki received post-doctoral fellowships from Merck and Eli Lilly. Ghislaine Fontés was supported by a post-doctoral fellowship from the Canadian Diabetes Association.
|Intralipid 20%||Fresenius Kabi||JB6023|
|Metricide (Glutaraldehyde 2.6%)||Metrex||11-1401|
|Heparin Sodium 10,000 USP u/ml||PPC|
|CO-EX T22||Instech Solomon||BCOEX-T22|
|Connector 22G||Instech Solomon||SC22/15|
|Swivel 22G||Instech Solomon||375/22PS|
|Y-Connector 22G||Instech Solomon|
|Counterbalance and arm||Instech Solomon||CM375BP|
|23 G blunted needles||Instech Solomon||LS23|
|23 G canulation pins||Instech Solomon||SP23/12|
|Tethers (12 inch)||Lomir||RT12D|
|Infusion jackets||Lomir||RJ01, RJ02, RJ03, RJ04|
|Tether attachment piece||Lomir||RS T1|
|60 ml syringe||BD||309653|
|1 ml syringe||BD||309602|
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