The genetically obese Zucker rat develops much of the pathophysiology observed in morbidly obese patients including: progressive insulin resistance, glucose intolerance, hyperlipidemia, and hypertension.10,11,40
Gastric bypass in the obese Zucker rat was first described in 1984, but was not used to investigate mechanisms of weight loss until 2002.24,41
Although the Zucker rat is commonly used to study insulin resistance and T2DM, this is the first study examining the effects of RYGB on glucose homeostasis in this model. In patients, the RYGB procedure bypasses >95% of the distal stomach and the proximal jejunum resulting in bypass of the “foregut” and enhanced delivery of undigested nutrients to the ileum. Weight loss after RYGB is commonly ascribed to mechanical restriction of food intake, some degree of malabsorption, and “dumping syndrome” caused by ingestion of concentrated sweets.42
Several anatomic factors limit the restrictive nature of the RYGB procedure in the Zucker rat including the thin walled gastric rumen which is unsuitable for stapling and the location of gastroesophageal junction along the lesser curvature of the stomach. Because of these anatomic factors a 20% gastric pouch with a divided RYGB was used in the current study as problems with staple line disruption were reported when a nondivided RYGB was performed.24
Using this technique the RYGB group sustained significant weight loss compared with the PF and AL groups during the 28 day study period, albeit less than the 30% reduction in total body weight commonly reported after RYGB in humans. Because POD 14 VO2
was similar between the groups, differences in weight loss between the PF and RYGB groups were probably not caused by differences in energy expenditure. The effects of surgery on nutrient absorption were not examined in the current study, but represent a potential cause for differences in weight loss between the PF and RYGB groups.
The relative impact of RYGB compared with pair-feeding on glucose homeostasis was examined by comparing POD 21 OGTTs. The OGTT is commonly used to test for prediabetes or T2DM and determines how quickly glucose is cleared from the blood after a standard glucose load. Using the 1999 World Health Organization and 2004 Expert Committee criteria: fasting plasma glucose levels >100 mg/dL denote impaired fasting glycemia and glucose >200 mg/dL at 120 minutes confirm the diagnosis of T2DM.43
Based on these criteria, all 3 groups demonstrate impaired fasting glycemia and the 120 minutes plasma glucose data indicate T2DM in both the PF and AL groups, whereas the RYGB group has improved to glucose intolerant. Although post-RYGB glucose tolerance is significantly improved relative to obese Zucker controls, it remains somewhat impaired relative to unoperated lean heterozygous Zucker controls (data not shown). Changes in glucose tolerance are noted at 6 to 7 weeks of age in the obese Zucker rat model and worsen over time.10
Consequently, the finding that post-RYGB animals demonstrate some evidence of glucose intolerance on POD 21 is not surprising. The severity and duration of T2DM, as well as the magnitude of postoperative weight loss seem to be predictive of T2DM resolution after RYGB in morbidly obese patients.44
Therefore, the timing of surgical intervention (10 –12 weeks) may have impacted the responsiveness of glucose homeostasis in the current study. Nonetheless, the observation that glucose tolerance remained similar in PF and AL controls suggests decreased nutrient intake alone does not explain the early improvement in glucose homeostasis after RYGB.
Circulating glucose represents a critical nutrient for many tissues. The plasma glucose concentration represents the equilibrium of multiple metabolic processes including: dietary intake and absorption, glucose production (via gluconeogenesis and glycogenolysis), and glucose utilization. During the fasted state the glucose concentration is determined primarily by hepatic glucose output and glucose utilization by peripheral tissues. The postprandial increase in plasma glucose triggers insulin release from pancreatic β
-cells by stimulating fusion of insulin-containing vesicles with the plasma membrane. Circulating insulin acts to reduce hepatic glucose production and increase peripheral GLUT4-mediated glucose uptake by striated muscle and adipose tissue. Muscle represents the principal site of insulin-stimulated glucose transport in vivo accounting for more than 75% of peripheral glucose uptake.14,45
However, post-prandial hyperglycemia also increases glucose uptake by essentially all tissues via noninsulin glucose uptake which is independent of GLUT4 and mediated by mass action effect of the substrate. In the obese Zucker rat, peripheral insulin resistance is because of defective insulin signaling, reductions in the insulin-sensitive GLUT4 expression, and impaired insulin-stimulated GLUT4 membrane translocation.13,14
The release of nonesterified fatty acids and adipokines from adipose tissue is hypothesized to result in decreased responsiveness of peripheral tissues (muscle, liver, adipose) to insulin, a condition referred to as insulin resistance.45
The physiologic response to obesity-related insulin resistance initially involves a compensatory increase in pancreatic β
-cell mass and insulin secretion in the obese Zucker rat.46
Consequently, an assessment of circulating insulin is important in the interpretation of the change in plasma glucose levels during the OGTT. The reduction in fasting insulin observed in the RYGB animals relative to PF and AL controls suggests an improvement in insulin sensitivity as a mechanism for post-RYGB glucose homeostasis. However, these models do not indicate whether this improvement in insulin action occurs at the level of the liver or peripheral tissues. To this end, we used the euglycemic, hyperinsulinemic clamp to directly assess peripheral (mainly skeletal muscle) glucose uptake under maximally insulin stimulating conditions. Our data clearly indicate that RYGB improves the ability of insulin to increase peripheral glucose uptake and this improvement can not be attributed to the reduction in food intake.
The distribution of body fat between the subcutaneous and visceral depots is an important determinant of insulin action. The relative abundance of visceral fat in particular correlates with insulin resistance and surgical removal of visceral fat has been shown to improve insulin sensitivity.22,47
Therefore, serial MRI scans were performed in a subgroup of animals to determine whether RYGB or decreased nutrient intake preferentially affects either the subcutaneous or visceral fat depots. After RYGB, a 13% reduction in subcutaneous fat was seen with only a 3% reduction in visceral fat. The reduction in subcutaneous fat after RYGB appeared independent of food intake, whereas the post-RYGB decrease in visceral fat was largely because of decreased food intake. This finding is consistent with data from Xu et al in the Zucker rat model where reductions in retroperitoneal and epididymal fat depots after RYGB were primarily related to decreased nutrient intake.24
Collectively these results provide evidence that reductions in the relative abundance of visceral fat do not explain the observed improvements in insulin action and glucose tolerance after RYGB in the Zucker rat model.
An important limitation of the current study is the lack of data on the effects of RYGB on the synthesis and/or secretion of hormones and metabolites from adipose tissue which could potentially influence insulin sensitivity. The release of inflammatory cytokines (eg, TNF, IL-6, MCP-1) from adipocytes and macrophages in visceral fat has been implicated in the pathogenesis of T2DM.48
Likewise, the synthesis and secretion of metabolically active proteins or adipokines (eg, resistin, leptin, visfatin) by adipose tissue represents a potential mechanism for obesity-related insulin resistance that is not addressed by our results.47,49
Consequently, the effects of RYGB on cytokine/adipokine synthesis by adipose tissue represents an important area for future study as a potential contributory mechanism for post-RYGB glucose homeostasis.
Despite this caveat, the observed changes in postprandial gut peptide production provide evidence that alterations in incretin production may contribute to improvements in glycemic control after RYGB. GIP is secreted by K cells in the duodenum and jejunum in response to ingested fat and glucose. GLP-1, a product of the proglucagon gene is secreted by L cells of the distal ileum and colon in response to intraluminal fats and carbohydrates. GIP and GLP-1 are rapidly degraded by dipeptidyl peptidase-4 starting shortly after secretion. Differential processing of the proglucagon gene results in multiple circulating proglucagon peptides. However, only the GLP-1 7–36 amide and Gly-extended forms are bioactive and stimulate insulin release from pancreatic β
-cells. As a result of DPPIV degradation, <25% of newly secreted GLP-1 reaches the portal circulation as the intact, active form and only 10% to 15% reaches the systemic circulation.15
This observation has raised concerns regarding the importance of portal GLP-1 concentrations in regulating glucose homeostasis and represents a potential limitation of the current study which only measured systemic incretin levels.50
Normally, GIP and GLP-1 induce β
-cell proliferation, inhibit apoptosis, and stimulate glucose-dependent β
-cell insulin secretion via specific receptor-mediated pathways.15,16,51
The simultaneous increase in plasma GLP-1 and insulin concentrations observed 30 minutes after gavage in the RYGB group suggests the insulinotropic effect of GLP-1 on pancreatic β
-cells contributes to glycemic control. However, the relative importance of incretin-mediated insulin release versus incretin-mediated improvement in peripheral insulin sensitivity/action in post-RYGB glycemic control is difficult to ascertain because both mechanisms are likely involved. Although there do not seem to be GLP-1 receptors in either muscle or liver, extrapancreatic effects of GLP-1 on insulin-independent glucose disposal/metabolism in liver and muscle have been described.17,18
More recently, GLP-1 receptors on vagal afferents in the portal vein were shown to improve glucose tolerance without altering the concentration of circulating insulin.50
Consequently the increase in total and intact GLP-1 7–36 amide observed after RYGB potentially acts by multiple mechanisms to improve glucose homeostasis. The increase in total plasma GLP-1 after gavage may be because of hyperplasia of intestinal endocrine cells. Increased expression of proglucagon, proconvertase 1/3, and chromogranin genes has been described in the transposed ileal segment after ileal transposition and supports a potential role for gut endocrine hyperplasia as a contributing factor.52
Although fasting and postgavage plasma GIP concentrations were similar to controls after RYGB, the insulinotropic effects of GIP seem to be decreased in T2DM as a result of reductions in β
-cell GIP receptors and postreceptor defects in β
-cell GIP signaling.19,53–55
Consequently, evaluating the effects of RYGB on GIP bioactivity will require a more detailed analysis of the relative abundance of GIP receptors and postreceptor signaling events in pancreatic β
-cells. The increase in postprandial PYY observed after RYGB is consistent with changes in PYY noted after RYGB in morbidly obese patients.56,57
Although PYY is a known satiety factor posited to mediate postsurgical reductions in appetite and improved satiety, there is no evidence that postgavage changes in PYY contribute directly to post-RYGB improvements in glucose homeostasis. The finding of paradoxical hyperglucagonemia after RYGB in the current study is consistent with results of Laferrere et al who observed a post-OGTT increase in glucagon 1 month after RYGB in morbidly obese patients with T2DM.58
The foregut and “hindgut” hypotheses have been proposed to explain the effects of postsurgical RYGB intestinal anatomy on insulin resistance and T2DM.7
The foregut hypothesis suggests bypass of the duodenum and proximal jejunum alone improves T2DM.59
The hindgut hypothesis suggests enhanced delivery of undigested nutrients to the ileum stimulates gut peptide secretion by mucosal L cells (eg, GLP-1, PYY) which act to inhibit appetite and improve glucose homeostasis. Several studies have examined fasting and postprandial gut peptides in morbidly obese patients after RYGB.57,60,61
Korner et al noted an exaggerated postprandial insulin response and changes in ghrelin and PYY consistent with increased satiety as a mechanism of weight loss after RYGB.56
Similar changes in postprandial glucose tolerance, insulin, GLP-1, PYY, and GIP levels were recently described as early as 1 month after RYGB in morbidly obese patients.60
However, the current study is the first to characterize fasting and postprandial incretins after RYGB in the obese Zucker rat model and directly assess insulin action using the euglycemic hyperinsulinemic clamp. Although fasting levels of total GLP-1, GIP, and PYY were similar in the experimental groups, the postprandial increase in GLP-1, GIP, PYY, and insulin in the RYGB group seems to correlate with improvements in insulin sensitivity and glucose homeostasis. Importantly, the changes in glucose homeostasis and gut peptide secretion in the Zucker rat model resemble those observed in post-RYGB patients confirming its utility as an experimental model for metabolic research on obesity and T2DM.