1

Mechanisms of Glucose Homeostasis Regulation Following Bariatric Surgery

Daniel Svedberg1, Dr. Alessandro Bartolomucci2

College of Liberal Arts1, Department of Integrative Biology and Physiology2. University of Minnesota, Minneapolis, MN 55455 December 2014 Abstract Long-term restoration of normal glucose homeostasis has been robustly observed in

patients with Type 2 Diabetes receiving bariatric surgery for weight loss, including increased

insulin secretion, decreased insulin resistance, and improved glucose tolerance. The pace,

longevity, and superiority of bariatric surgery’s effects on glucose homeostasis hint at

mechanisms independent of simple caloric restriction and adiposity reduction. Increased

secretion of incretins GLP-1 and hormone PYY follows bariatric surgery, and are thought to

mediate changes to glucose homeostasis. GLP-1 promotes insulin secretion, increases satiety,

and decreases insulin resistance. GLP-1 receptor agonists mimic the effects of bariatric surgery

on glucose homeostasis, and receptor antagonists reduce improvements to glucose homeostasis

following bariatric surgery. Despite this, mouse models of GLP-1 receptor deficiency experience

the same changes in glucose homeostasis as wild-type mice following bariatric surgery,

indicating that GLP-1 signaling is not strictly necessary for improvements in glucose

homeostasis. Exclusion of the duodenum and part of the proximal jejunum from contact with

nutrients is proposed to mediate increases in GLP-1 following certain types of bariatric surgery.

Placing an endoluminal sleeve in the duodenum of rats reduces food intake, alters macronutrient

selection, improves glucose homeostasis, and increases energy expenditure. Overall, changes to

glucose homeostasis following bariatric surgery are mediated by a variety of mechanisms

including hormones, but which of these mechanisms are strictly necessary is still unclear.

2

Introduction Over 34% of adults in the United States today are obese (Ogden, Carroll, Kit, & Flegal,

2014) and over 9% of Americans have Type 2 Diabetes (T2DM) (Centers for Disease Control

and Prevention 2014). The aging population in the United States is particularly vulnerable to

metabolic diseases, and will continue to occupy more and more of our healthcare focus.

Understanding how to treat such metabolic disorders continues to be a growing priority in the

field.

Over the last few decades, various weight loss-inducing bariatric surgery techniques have

emerged, and the long-term restoration of normal glucose homeostasis has been robustly

observed in T2DM patients receiving bariatric surgery for weight loss (Pories et al., 1992).

Research using human patients and animal models of obesity, T2DM, and bariatric

surgery offers insight into the unknown mechanisms of glucose homeostasis restoration. These

mechanisms go beyond simple volume restriction, and implicate a variety of complex

physiological systems extending from the enteroendocrine regulation of hormones to central

nervous management of nutrient intake.

Pathological glucose homeostasis T2DM in itself is a poorly defined diagnosis, but a component that consistently comprises

its pathology is faulty glucose homeostasis. In healthy humans, the ingestion of food increases

blood plasma glucose levels, and the pancreatic beta-cells release insulin. Insulin binds to surface

receptors on skeletal and liver cells in order to allow for glucose absorption by these cells. Faulty

metabolic homeostasis associated with obesity, and consists of poor hepatic cell insulin

insensitivity and decreased insulin secretion.

The magnitude of insulin resistance and decreased insulin secretion can progress to a

point where the glucose cannot be removed from the blood and taken into the cells fast enough to

3

prevent blood glucose from reaching toxic levels. This phenomenon is known as diabetic glucose

intolerance. Glucose intolerance caused by faulty insulin action is the typical phenotype and

pathological mechanism of T2DM.

It is important to note diabetic glucose intolerance is correlated with the degeneration and

eventual destruction of pancreatic beta cells. Diabetic glucose intolerance tends to worsen over

time in patients; T2DM has been long regarded as a progressive, irreversible disease (Taylor,

2013) with varying phases of beta-cell degeneration (Weir & Bonner-Weir, 2004) and

widespread immunological activation affecting the function of tissues that manage glucose

homeostasis (Odegaard & Chawla, 2013).

Quantifying glucose homeostasis Increased blood glucose concentration has long been used as a diagnostic criterion of

diabetes, since impaired homeostatic function ultimately leads to elevated blood glucose levels.

In humans, a fasting plasma glucose concentration of over 126 mg/dL (7.0 mmol/L) is

considered diabetic (American Diabetes Association, 2014). For a variety of reasons, it does not

make sense to establish and use benchmarks of blood glucose concentration in animal studies, so

changes in blood glucose concentrations are often compared to values obtained from control

groups in the study.

Since diabetic impairment of glucose homeostasis directly affects insulin secretion, many

studies will use changes in blood insulin concentration to illustrate glucose homeostasis. Blood

insulin concentration, much like blood glucose concentration, is often expressed in millimolar

concentrations as a part-per-liter.

There are two general ways researchers quantify insulin resistance. The only direct

method is the hyperinsulemic-euglycemic clamp. Briefly, insulin and glucose levels are tightly

4

controlled, and the rates of glucose and insulin infusion needed to maintain stable blood glucose

concentration are used to gauge insulin resistance. In contrast to a subject with normal insulin

activity, a subject with insulin resistance require higher rates of insulin infusion and can only

tolerate lower rates of glucose infusion to maintain stable blood glucose concentrations (Ayala et

al., 2011). The hyperinsulemic-euglycemic clamp is important because data from clamp studies

have been used to create models that estimate insulin resistance from basal blood insulin and

glucose concentrations. Such indices include the Homeostatic Model Assessment (HOMA) and

the Quantitative Insulin Sensitivity Check Index (QUICKI). These indices correlate relatively

well with clamp data in humans, with correlations around .8 for HOMA and .78 for QUICKI

(Katz et al., 2000; Matthews et al., 1985).

To quantify glucose tolerance, the oral glucose tolerance test (OGTT) and its derivatives

are used in both humans and animals. In the OGTT, subjects are first fasted for 8-10 hours, and

then consume a fixed amount of glucose solution. Blood samples are taken right before glucose

consumption and at time intervals following ingestion. Subjects that have blood glucose

concentrations above normal values over the course of the study are determined to have glucose

intolerance(Bartoli, Fra, & Carnevale Schianca, 2011). Similar to the OGTT, the Mixed Meal

Tolerance Test (MMTT) measures blood glucose concentrations following a fixed nutrient meal,

designed to mirror the complex nutrient profiles of a regular meal, rather than just glucose

(Greenbaum et al., 2008).

Bariatric surgery achieves changes to Glucose Homeostasis Bariatric surgery rapidly restores proper glucose homeostasis and reverses diabetic

glucose intolerance in the majority of patients (Stefater, Wilson-Pérez, Chambers, Sandoval, &

Seeley, 2012). This effect is robust and well studied in both humans and animal models. In the

5

days following bariatric surgery, the majority of patients with T2DM demonstrate decreased

insulin resistance, lower blood glucose levels, and better-proportioned blood insulin levels.

Remarkably, the function of existing pancreatic beta-cells is returned to normal (Camastra et al.,

2007). Even more remarkable is that all of these effects occur within a week of bariatric surgery,

at a rate independent of weight loss (Stefater et al., 2012).

There are three commonly used gastric modification procedures: Roux-en-Y Gastric

Bypass (RYGB), Adjustable gastric banding (AGB), and the Vertical Sleeve Gastrectomy

(VSG). RYGB does not necessarily restrict the size of the stomach, but instead re-routes the

passage of food around the upper intestine (duodenum) to the lower intestine to effectively

reduce the length of the small intestine, with the intention of reducing nutrient absorption.

Adjustable gastric banding consists of placing a band in the upper stomach to form a restricted

pouch, with the intention of reducing food intake. VSG cuts away a significant portion of the

stomach, and joins the remains into a slim tube with significantly less volume as compared to

before the surgery, with the intent of reducing reduced nutrient absorption (Stefater et al., 2012).

Figure 1: Three commonly used gastric procedures: A) Roux-en-Y Gastric Bypass (RYGB) B) Adjustable Gastric Banding (AGB), C) Vertical Sleeve Gastrectomy. Adapted from (Bradley, Magkos, & Klein, 2012)

without permission.

6

One study demonstrated that VSG reduced HOMA insulin resistance in diabetic patients

to 29%, serum glucose to 68%, and serum insulin to 71% of pre-surgical levels, 60 days post-

operatively. More astounding, the lowest levels of insulin resistance, serum glucose, and serum

insulin were attained within 3 days post-operatively (Rizzello et al., 2010). Another study

evaluated glucose homeostasis in RYGB patients. Similar to VSG patients, insulin resistance in

RYGB patients fell to about 30%, plasma glucose levels to 60%, and plasma insulin to 30% of

pre-surgical levels, 12 months post-operatively (Wickremesekera, Miller, Naotunne, Knowles, &

Stubbs, 2005).

All three of these gastric procedures were designed for weight loss and ultimately reduce

calorie absorption, as well as reduced calorie consumption. Pair-fed animals, which are allowed

to eat only as much food as the bariatric surgery animals, normally achieve the same amount of

weight loss(Chambers et al., 2011; Stefater et al., 2010). It was originally hypothesized that

decreasing stomach volume, leading to increased satiety was the mechanism of lowering calorie

consumption (Stefater et al., 2012). This hypothesis is an oversimplification that cannot account

for a variety of extraneous phenomena observed following bariatric surgery. Surgical procedures

like VSG and RYGB are less likely to fail than AGB; breakage of the band used in AGB leads to

weight regain, while stomach dilation in VSG does not lead to weight gain. VSG, which leaves a

larger gastric pouch than AGB, leads to more weight loss than AGB in the first place. It was

hypothesized that as a result bariatric surgery patients would eat foods with a higher calorie

density to compensate for the negative balance. AGB patients indeed do seek out more calorie

dense foods, but VSG and RYGB patients do the opposite. These patients instead seek foods

with low calorie density such as leafy vegetables, and also exhibit a decreased preference for

fatty food. Collectively, a number of factors correlate VSG and RYGB as fundamentally similar

7

weight-reducing surgeries by non-restrictive means, since patients lose weight from both of these

procedures similarly with no direct relationship to remaining stomach size. The implication is

that the act of surgically remodeling the digestive tract fundamentally changes metabolic

homeostasis through a variety of mechanisms, from the highest order of central nervous system

control down to the genetic regulation of cells in metabolic organs. The proposed mechanisms

behind these changes are numerous, and most likely a number of them contribute to the overall

effect that gastric surgery has on weight loss (Stefater et al., 2012).

Select hormones mediate changes to glucose homeostasis Amongst a number of potential mechanisms for weight loss in gastric surgery,

modulation of hormones is one of the best studied. Following bariatric surgery, there are changes

in the secretion of a variety of hormones (Stefater et al., 2012). The effects of these hormones are

multiple; some modulate hunger and satiety, while others are incretins, which modulate

pancreatic secretions. Although the full function of every hormone altered by bariatric surgery

has not been yet characterized, there are a number of hormones that are under investigation

Despite alterations in secretions, not all hormones play a role in alterations to glucose

homeostasis following bariatric surgery. The profiles of food intake-modulating hormones like

leptin, ghrelin, and cholecystokinin (CCK) change following bariatric surgery, but studies using

animal models of bariatric surgery that are insensitive to leptin, ghrelin, or CCK have

demonstrated that all of these hormones probably don’t contribute to establishing the weight loss

and behavioral changes observed following bariatric surgery (Aguirre, Stylopoulos, Grinbaum,

& Kaplan, 2008; Stefater et al., 2012).

Some incretins have been shown to directly alter glucose homeostasis following bariatric

surgery. Glucagon-Like Peptide-1 (GLP-1) is a hormone produced by the enteroendocrine cells

8

when they are exposed to nutrients in the small intestine, and GLP-1 secretion increases

following bariatric surgery. Peptide YY (PYY) is another enteroendocrine hormone released

from the ileum; increased PYY secretion is observed in RGYB patients (Stefater et al., 2012).

GLP-1 is a peptide secreted by the enteroendocrine L-cells, and has a variety of effects on

glucose homeostasis. GLP-1 primarily works as an incretin that promotes insulin secretion from

pancreatic beta-cells, promotes somatosin secretion, and inhibits glucagon secretion (Salehi &

D’Alessio, 2014). Furthermore, GLP-1 decreases food intake, and decreases insulin resistance

(Fig 6.) (Drucker & Nauck, 2006).

Figure 6: Effects of GLP-1 on Glucose Homeostasis. Reproduced from (Drucker & Nauck, 2006)without permission

GLP-1 appears to act directly on hepatocytes, administration of GLP-1 receptor agonist

Exendin-4 (Ex-4), in vitro decreased insulin resistance by decreasing stresses induced by high

lipid content on cellular mechanisms occurring in the endoplasmic reticulum. The same study

9

administered Ex-4 in diabetic mice and reported decreased insulin resistance (Lee et al., 2014).

GLP-1 also increases energy expenditure (Day et al., 2009). Administering PYY or GLP-1

peripherally decreases food consumption in both animals and humans. The mechanism of

decreased food consumption in peripheral GLP-1 and PYY administration are thought to be

mediated by the vagal nerve; ablation of the vagal nerve in mice eliminates the satiating effect of

peripheral GLP-1 and PYY (Abbott et al., 2005). Peripheral administration of Ex-4, a GLP-1

receptor agonist, decreases food-reward motivated behavior in rats (Dickson et al., 2012).

Conversely, the GLP-1r antagonist Exendin-(9-39) (Ex-9) blocks the action of GLP-1 in

GLP-1 receptors (GLP-1r). Chronic administration of Ex-9 in RYGB and VSG rats attenuates

weight loss as compared to rats that did not receive the antagonist (Chambers et al., 2011). Ex-9

administration also resulted in decreased glucose tolerance and insulin secretion in VSG mice

(Wilson-Pérez et al., 2013). Similarly, a study using 9 human RYGB patients with T2DM

showed that peripheral Ex-9 administration increased glucose tolerance and insulin secretion

(Jørgensen et al., 2013). GLP-1r agonists are used independently of bariatric surgery improve

glucose homeostasis as well (Bode, 2011; Day et al., 2009; Wilson-Pérez et al., 2013). GLP-1r

agonist Ex-4 is commonly used in animal model experiments. In one experiment, Ex-4 decreases

hunger, induces weight loss, increases insulin secretion, and improves glucose tolerance in mice

(Ye et al., 2014).

The mechanisms by which GLP-1 secretion is increased through bariatric surgery are still

under investigation. One theory is that bariatric surgery increases gastric dumping, which would

cause the rapid delivery of un-churned food particles with low surface area to the small intestine.

This gastric dumping has been proposed to then stimulate the enteroendocrine cells to secrete

GLP-1. It seems intuitive that bariatric surgery would induce gastric dumping; RYGB destroys

10

the pylorus, leaving no control valve over how fast nutrients enter the small intestine. VSG on

the other hand, has a restricted pouch that may be less efficient at churning food particles, and

may dump food into the intestine quicker. Despite these theories, there is no conclusive evidence

correlating increased gastric dumping or poor digestion to bariatric surgery (Stefater et al., 2012).

One interesting perspective is bile acids. Plasma bile acids are increased in RYGB and

VSG patients. Certain bile acids stimulate GLP-1 secretion from enteroendocrine cells in vivo;

GLP-1 receptor knockout mice given an oral bile acid determined to stimulate GLP-1 secretion

have increased post-prandial glucose levels as compared to mice that express GLP-1 receptors

(Rafferty et al., 2011).

Caloric restriction achieves changes to glucose homeostasis It is important to note that although bariatric surgery has the ability to reverse T2DM and

pathological glucose homeostasis, simple caloric restriction can do the same. A study where 10

human subjects with T2DM underwent acute, medically supervised caloric restriction of about

600 kcal/day demonstrated that around 80% of subjects had achieved normal insulin secretion

and glucose tolerance within 7 days (Lim et al., 2011; Taylor, 2013). These patients maintained

the caloric restriction for a full month, and 60% of the subjects retained the reversal of T2DM.

The initial reversal rate and speed at which reversal of T2DM occurred in caloric restriction is

very similar to that seen in bariatric surgery. The implication is that the normalization of glucose

homeostasis following bariatric surgery does not occur by a singular, easily explained

mechanism exclusive to bariatric surgery, but rather, a number of interrelated mechanisms.

Bariatric surgery still maintains a distinct advantage in its ability to directly alter feeding

behavior and hormone profiles, which increases the probability of maintaining normal glucose

homeostasis in the long term. Voluntary weight loss is comparatively ineffective; very few

11

individuals can voluntarily maintain weight loss for many years, while the benefits from bariatric

surgery are essentially permanent.

Study: Isolated duodenal exclusion increases energy expenditure and improves glucose homeostasis in diet-induced obese rats

In order to verify that surgical alteration of the gut mediates changes to glucose

homeostasis, it is important to isolate what manipulations lead to the same changes observed

following bariatric surgery. Muñoz, Carmody, Stylopoulos, Davis, & Kaplan, 2012 hypothesized

that excluding the duodenum from contact with nutrients mediates weight loss and improvements

to glucose homeostasis seen following RYGB. This was simulated using an endoluminal plastic

sleeve that was inserted into the gut of obese rats, starting at the pylorus and extending down into

the duodenum.

Methods Rats were first fed a high fat diet to induce obesity. There were four groups of

experimental rats: sham operated, and those implanted with an endoluminal sleeve (ELS) that

was either 1 cm (ELS 1), 4 cm (ELS 4), or 10 cm (ELS 10) long. The ELS 1 tested the effect of

the anchoring mechanism, and left most of the duodenum available for contact with nutrients.

The ELS 4 covered most of the duodenum, and the ELS 10 extended to the initial segments of

the jejunum (Fig 2).

12

Figure 2: The Endoluminal Sleeve A: Typical RYGB scheme is illustrated. B & C: The endoluminal

sleeve (ELS) and length variations implanted in rats for this study.

Following surgery, body weight was measured weekly and food intake was tracked. To

determine nutritional absorption efficiency, differences in caloric content of food consumed and

excretions were determined. During post-operative week 10, energy expenditure was determined

by placing the rats in metabolic cages for 72 hours, measuring oxygen consumption, heat

production, and spontaneous motor activity over the course of the experiment. Around this time,

feeding patterns were also determined by tracking discrete meals over the course of 72 hours. A

pair-feeding experiment was performed, where weight-matched sham-operated rats were allowed

to eat only as much food as consumed by the matched ELS rat. Between post-operative weeks

10-12, ELS and sham-operated rats underwent an oral glucose tolerance test (OGTT) to

determine glucose tolerance. Finally, blood samples were taken from the rats during fasting, and

13

then analyzed for the homeostatic model assessment (HOMA) of insulin resistance and GLP-1

content.

Results All rats implanted with an ELS device lost weight and maintained lower body weights

over the course of the experiment as compared to sham-operated controls. ELS rats consume less

calories, and smaller, more frequent meals as compared to sham-operated controls. The effects of

ELS-10 on food intake are more pronounced than ELS-1 or ELS-4 (Fig 3). Furthermore, only

ELS-10 significantly increases energy expenditure. ELS-10 rats consumed more oxygen,

consumed less food, and maintained lower body weights as compared to control rats. Pair fed

rats only lost 65% as much weight as did ELS-10 rats (Fig 4).

14

Figure 3: ELS 1,4 & 10 reduce food intake A: Rats with duodenal exclusion maintained lower body weight over eight weeks. B: Absorption of nutrients is not significantly impaired by duodenal exclusion. C, D, & E: Duodenal exclusion reduces food intake and does not significantly change meal frequency. F: Only ELS-10 significantly increases energy expenditure. Reproduced from Muñoz, Carmody, Stylopoulos, Davis, & Kaplan, 2012 without permission.

15

Figure 4: ELS-10 increases energy expenditure A: ELS-10 rats maintain lower average weight over 8 weeks as compared to sham-operated (SO) and pair fed (PFS) rats. B, C & D: ELS-10 rats consume more oxygen than SO rats over 72 hours. E: ELS-10 rats exhibited less spontaneous locomotor activity than SO rats. Reproduced from Muñoz, Carmody, Stylopoulos, Davis, & Kaplan, 2012 without permission.

Although ELS-1 and ELS-4 rats demonstrate improvements in glucose homeostasis as compared

to sham-operated rats, ELS-10 rats demonstrate the most profound changes (Fig 5). Fasting

blood glucose concentration is decreased in all ELS rats as compared to sham-operated rats,

while fasting blood insulin concentration is decreased significantly only in ELS-10 rats. Oral

glucose tolerance test (OGTT) results indicate that glucose intolerance is decreased in most ELS

rats as compared to sham-operated rats, but ELS-10 rats demonstrate the best blood glucose

concentration control. Finally, HOMA-IR data indicates that only ELS-10 rats have significantly

16

decreased insulin resistance as compared to sham-operated controls (Fig 6). Secretion of GLP-1,

an incretin (described later) that is thought to mediate effects on weight loss, decreased calorie

consumption, and improvements in glucose homeostasis, is increased in ELS-10 rats.

Figure 5: ELS-10 best improves glucose homeostasis A: Fasting blood glucose concentrations are lowest in ELS-10 rats. B: Fasting blood insulin concentrations are lowest in ELS-10 rats. C & D: OGTT indicates that ELS-10 rats have the least acute increases in blood glucose and highest insulin secretion following oral glucose administration, indicating best glucose tolerance out of ELS-10, ELS-4, ELS-1, and SO rats. Reproduced from Muñoz, Carmody, Stylopoulos, Davis, & Kaplan, 2012 without permission.

17

Figure 6: ELS-10 improves glucose homeostasis A: ELS-10 rats had lower fasting glucose blood concentrations than SO and weight-matched sham (WMS) rats. B: ELS-10 rats were less insulin resistant than SO and WMS rats as evaluated by the HOMA. C & D: ELS-10 rats had less acute blood glucose increase and higher insulin secretion in the OGTT than SO or WMS rats, indicating improved glucose tolerance. E: GLP-1 secretion is increased in ELS-10 rats as compared to SO and WMS. Reproduced from Muñoz, Carmody, Stylopoulos, Davis, & Kaplan, 2012 without permission.

Discussion Muñoz, Carmody, Stylopoulos, Davis, & Kaplan determined that isolated duodenal

exclusion through an endoluminal sleeve (ELS) in an animal model decreases weight, decreases

caloric consumption, increases energy expenditure, and improves glucose homeostasis.

Furthermore, most of these changes are only significant only when full duodenal exclusion,

extending to the proximal sections of the jejunum, is employed. The authors express that these

18

studies add to existing data generated by the group on improvements in glucose homeostasis

resulting from ELS implantation in rats.

Since ELS-1 and ELS-4 still produced reductions in weight and improvements to glucose

homeostasis, the authors hypothesized that the crown of the ELS device slowed gastric

emptying, which would contribute to increased satiety by increasing stomach distention

following a meal. The authors believe that data on RYGB surgery in animals indicates that

RYGB delays gastric emptying, and that in this sense ELS and RYGB are analogues.

A number of possible mechanisms of were proposed to explain the weight loss and

improvements in glucose homeostasis seen following ELS implantation. First, the exclusion of

the duodenum from contact with nutrients, and the rapid delivery of nutrients to the jejunum,

could stimulate changes in neuroendocrine signaling, to mediate changes in glucose homeostasis

and central nervous system control of food intake. Despite evidence that this response to

duodenal exclusion occurs, the authors noted that VSG, which does not divert nutrients from the

duodenum, still produces similar improvements in glucose homeostasis. This study found that

secretion of GLP-1, a hormone that stimulates insulin secretion and increases satiety, is increased

in ELS-10 rats. The authors proposed that changes in glucose homeostasis resulting from ELS

implantation could be mediated by GLP-1, although it was also noted that postprandial insulin

secretion was not significantly increased in ELS-10 rats as compared to sham-operated rats,

despite increased GLP-1 secretion in ELS-10 rats. The functions of GLP-1 are further explored

later in this paper, but briefly, it is not absolutely clear that GLP-1 mediates the changes to

glucose homeostasis following bariatric surgery. Nonetheless, a more complex profile of

neuroendocrine signaling alterations could produce the effects on glucose homeostasis as seen in

this study. Finally, it was proposed that duodenal exclusion could increase interactions between

19

intestinal secretions and the endoluminal membrane. This has been shown to increase plasma

bile acids, which in turn has been shown to mediate improvements in glucose homeostasis.

The authors note that it is unknown if an endoluminal membrane that extends further into

the jejunum than the ELS-10 device would provide stronger changes in glucose homeostasis and

weight loss. Furthermore, it is noted that the ELS-10 has significant differences from RYGB, and

should not be regarded as equivalent. Most importantly, the duodenum could still subject

mechanical manipulation of passing nutrients through the walls of the ELS device.

Overall, the authors concluded that duodenal exclusion mimics the effects of RYGB

surgery on weight loss and glucose homeostasis. Furthermore, the authors asserted that duodenal

exclusion studies implicated the duodenum and the proximal jejunum as mediators of enteric

signals mediating the effects of bariatric surgery.

It is important to consider that VSG, a surgery that does not bypass the duodenum, causes

similar changes to food intake as the ELS device and duodenal-exclusion surgeries. This is

confounding, because pure restriction, as seen in AGB, does not mediate the same changes as

VSG does. Conversely, the ELS studies, which model varying degrees of duodenal exclusion in

the absence of restriction, confirm that restriction is not necessary for weight loss, but indicate

that duodenal exclusion is essential. The only conclusion that can be drawn is that surgical

manipulation of the digestive tract provides a distinct advantage over caloric restriction in its

ability to induce changes to glucose homeostasis.

Study: Vertical Sleeve Gastrectomy Is Effective in Two Genetic Mouse Models of Glucagon-Like Peptide 1 Receptor Deficiency Increased GLP-1 activity has been robustly observed following bariatric surgery, but

GLP-1 receptor antagonist/agonist studies present a variety of ambiguities. Genetic models of

20

GLP-1 receptor deficiency presented an opportunity to cement the GLP-1 receptor as a key

mediator of changes to glucose homeostasis following bariatric surgery. Wilson-Pérez et al.,

2013 hypothesized that the GLP-1 receptor mediates the effects of VSG, that the effects of

bariatric surgery on glucose homeostasis would be attenuated in two models of GLP-1 receptor

deficient mice.

Methods A group of wild-type mice underwent a high fat diet to induce obesity, and then

underwent VSG or sham surgery. 12 weeks post-operatively, the control mice were killed and

trunk blood was collected for analysis. The second group of mice were GLP-1 receptor knockout

(GLP-1r KO), meaning these mice lacked GLP-1 receptors through development. GLP-1r KO

mice were fed a high fat diet, and then underwent either VSG or a sham surgery. Following

surgery, Wild-type and GLP-1r KO mice underwent a battery of tests.

Mice were tested for nutrient preference. Whole body composition was examined through

magnetic resonance imaging. A mixed meal tolerance test was performed to examine glucose

tolerance. Finally mice were tested for an anorectic response to GLP-1 receptor agonist Ex-4.

The third group of mice had GLP-1 receptor depletion through Cre-Lox recombination, where

genes coding for GLP-1 receptors were deleted later in development to generate GLP-1r flΔCMV

mice. Following the same protocol as the first two groups, GLP-1r flΔCMV mice underwent the

mixed-meal test and Ex-4 infusion. Throughout the study, food consumption and mouse weight

was tracked carefully for all mice.

Results Wild-type mice that underwent VSG had higher plasma GLP-1 levels than sham-operated

control mice following surgery. GLP-1r KO mice weighed less than wild-type mice GLP-1r KO

mice, but lost a similar proportion of weight as wild-type mice following VSG surgery. MRI

21

analysis of body composition revealed that GLP-1r KO mice lost a larger proportion of fat mass

than did wild type mice following VSG surgery. GLP-1r KO mice did not express an anorectic

response to GLP-1 receptor agonist Ex-4, while Ex-4 induced hypophagia in wild-type mice,

indicating that there was indeed a loss of GLP-1 receptors function in GLP-1r KO mice (Fig 7).

Figure 7: Weight Changes following VSG in GLP-1r KO vs. Wild-type mice A: Wild-type VSG mice demonstrated more GLP-1 secretion than sham-operated mice. B & C: GLP-1r KO mice lost just as much weight as wild-type mice following VSG surgery. D & E: GLP-1r KO mice lost more fat mass than wild-type mice, but there was no difference in lean mass lost. F: GLP-1r KO mice did not demonstrate an anorectic response to Ex-4 when wild-type mice did. Reproduced from Wilson-Pérez et al., 2013 without permission.

VSG operated GLP-1r KO mice and wild-type mice had significantly better glucose

tolerance following a mixed-meal test than did sham operated GLP-1r KO mice and wild-type

mice. Furthermore, GLP-1 KO mice and wild-type VSG-operated mice consumed fewer calories

than sham operated GLP-1 KO and wild-type mice, and VSG operated mice ate fewer calories

from fat as compared to sham operated controls (Fig 8).

22

Figure 8: Metabolic effects of VSG in GLP-1r KO mice vs. Wild-type A: Both GLP-1r KO and Wild-type VSG operated mice had better glucose tolerance than sham-operated controls. B & C: Wild type and GLP-1r KO mice demonstrated increased insulin secretion following the mixed-meal as compared to sham-controls. D & E: Both Wild type and GLP-1r KO VSG operated mice demonstrated decreased caloric intake and decreased fat intake as compared to sham-operated controls. Reproduced from Wilson-Pérez et al., 2013 without permission.

The second models of GLP-1 receptor deficiency, GLP-1r flΔCMV mice, were confirmed

to have lost GLP-1 receptor function, and did not exhibit an anorectic response to Ex-4, unlike

wild-type mice. Following VSG, GLP-1r flΔCMV mice lost a similar proportion of weight as wild-

type VSG operated mice. Similar to GLP-1r KO mice, GLP-1r flΔCMV mice demonstrated

improvements in glucose tolerance following VSG surgery equal to improvements seen in wild-

type mice (Fig. 9)

23

Figure 9: Changes in GLP-1r flΔCMV mice following VSG surgery A & B: GLP-1r flΔCMV mice were determined to lack GLP-1 receptor function following tissue analysis and lack of hypophagia in response to Ex-4. C: GLP-1r flΔCMV mice lost as much weight following VSG surgery, as did wild type mice, in contrast to sham-operated controls. D: GLP-1r flΔCMV mice demonstrated improved glucose tolerance, as did wild-type mice, following VSG surgery, in contrast to sham-operated controls. Reproduced from Wilson-Pérez et al., 2013 without permission. Discussion

Given the data from GLP-1 receptor depletion studies, Wilson-Pérez et al. determined

that GLP-1 receptor signaling is not required for weight loss or improvements in glucose

homeostasis following VSG. This conclusion goes against evidence from studies showing that

increased GLP-1 following RYGB and VSG surgery in humans predicts weight loss, and directly

lowers food intake when compared to adjustable gastric banding, which does not increase GLP-1

secretion. Furthermore, this new hypothesis contradicts a number of studies, including one led by

Wilson-Pérez et al., deomonstrating that GLP-1 receptor antagonism with Ex-9 limits weight loss

24

and improvements to glucose homeostasis in rats, mice, and humans. The authors expressed

uncertainty regarding using a pharmacological GLP-1 receptor antagonist like Ex-9 to determine

causality of benefits resulting from GLP-1 signaling following bariatric surgery. Noted

disadvantages of pharmacological antagonists include dubious specificity and efficacy of

antagonism. It is entirely possible that Ex-9 could inadvertently activate another pathway apart

from those mediated by the GLP-1 receptor that would atteunate the benefits of bariatric surgery.

In contrast, a genetic GLP-1 receptor knockout ensures complete disruption of the GLP-1

signaling cascade. One noted possible disadvantage of genetic models of GLP-1 receptor

depletion is that the mice could somehow compensate for the loss of GLP-1 signaling function

throughout developent. Compensation has been preivously reported, but only resulted in

increased seceretion of GIP or GLP-2, which by themselves would not be able to explain the

equivocal changes seen in GLP-1 receptor depleted mice and wild type mice, following bariatric

surgery. GIP secretion promotes fat storage, but GLP-1r depleted mice have lower body fat

percentages than wild type mice, and GLP-2 has not been shown to improve glucose

homeostasis. Furthermore, GLP-1r flΔCMV experienced the same degree of weight loss and

improvements to glucose homeostasis, as did GLP-1r KO and wild type mice, despite having

experienced GLP-1 receptor depletion postnatally. The authors concluded that although

increased GLP-1 signaling may help mediate weight loss and improvements to glucose

homeostasis following VSG, it is not required, and that additional factors must mediate these

changes as well.

A similar study using RYGB surgery on GLP-1 receptor deficient mice had similar

findings, showing that GLP-1 receptor deficient RYGB mice demonstrated equal decreases in

food consumption and body weight as wild type mice (Ye et al., 2014).

25

Ultimately, increased GLP-1 signaling has been shown to be present following bariatric

surgery, and that GLP-1 signaling mediates the same changes to glucose homeostasis does

bariatric surgery, but GLP-1 signaling is not an essential mediator of improvements to glucose

homeostasis following bariatric surgery.

Conclusion Although a bit of progress has been made in clarifying the mechanisms of glucose

homeostasis normalization in bariatric surgery patients, there are still many questions left.

Despite this, there are a variety of conclusions that the field has arrived to. Firstly, bariatric

surgery mediates effects on homoeostasis in ways that purely restrictive procedures do not;

bariatric surgery maintains a distinct advantage over caloric restriction in its ability to induce

long-term caloric restriction and improvements to glucose homeostasis. This advantage is

mediated by surgical manipulation of the digestive tract. Second, bariatric surgery induces

alterations to the profiles of hormones that affect glucose homeostasis, and that these alterations

clearly have an effect on mediating the changes to glucose homeostasis. It is still unclear which

hormones are essential to mediate these changes, though.

Most importantly, it is clear that bariatric surgery can reverse type II diabetes, and the

recent understanding of the mechanisms provide causal evidence for bariatric surgery as the

mediator of this reversal. Voluntary weight loss has long failed miserably in helping T2DM

patients achieve better health, simply because maintaining this weight loss is not possible in the

majority of patients. On the other hand, bariatric surgery has reproducibly demonstrated its

potential for a safe, durable, and reliable cure for T2DM.

26

References

Abbott, C. R., Monteiro, M., Small, C. J., Sajedi, A., Smith, K. L., Parkinson, J. R. C., … Bloom, S. R. (2005). The inhibitory effects of peripheral administration of peptide YY(3-36) and glucagon-like peptide-1 on food intake are attenuated by ablation of the vagal-brainstem-hypothalamic pathway. Brain Research, 1044(1), 127–31. doi:10.1016/j.brainres.2005.03.011

Aguirre, V., Stylopoulos, N., Grinbaum, R., & Kaplan, L. M. (2008). An endoluminal sleeve induces substantial weight loss and normalizes glucose homeostasis in rats with diet-induced obesity. Obesity (Silver Spring, Md.), 16(12), 2585–92. doi:10.1038/oby.2008.502

American Diabetes Association. (2014). Diagnosis and classification of diabetes mellitus. Diabetes Care, 37 Suppl 1(Supplement_1), S81–90. doi:10.2337/dc14-S081

Ayala, J. E., Bracy, D. P., Malabanan, C., James, F. D., Ansari, T., Fueger, P. T., … Wasserman, D. H. (2011). Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice. Journal of Visualized Experiments  : JoVE, (57), e3188. doi:10.3791/3188

Bartoli, E., Fra, G. P., & Carnevale Schianca, G. P. (2011). The oral glucose tolerance test (OGTT) revisited. European Journal of Internal Medicine, 22(1), 8–12. doi:10.1016/j.ejim.2010.07.008

Bode, B. (2011). Liraglutide: a review of the first once-daily GLP-1 receptor agonist. The American Journal of Managed Care, 17(2 Suppl), S59–70. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21517658

Bradley, D., Magkos, F., & Klein, S. (2012). Effects of bariatric surgery on glucose homeostasis and type 2 diabetes. Gastroenterology, 143(4), 897–912. doi:10.1053/j.gastro.2012.07.114

Camastra, S., Manco, M., Mari, A., Greco, A. V, Frascerra, S., Mingrone, G., & Ferrannini, E. (2007). Beta-cell function in severely obese type 2 diabetic patients: long-term effects of bariatric surgery. Diabetes Care, 30(4), 1002–4. doi:10.2337/dc06-1845

Chambers, A. P., Jessen, L., Ryan, K. K., Sisley, S., Wilson-Pérez, H. E., Stefater, M. A., … Sandoval, D. A. (2011). Weight-independent changes in blood glucose homeostasis after gastric bypass or vertical sleeve gastrectomy in rats. Gastroenterology, 141(3), 950–8. doi:10.1053/j.gastro.2011.05.050

Day, J. W., Ottaway, N., Patterson, J. T., Gelfanov, V., Smiley, D., Gidda, J., … Tschöp, M. H. (2009). A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nature Chemical Biology, 5(10), 749–57. doi:10.1038/nchembio.209

Dickson, S. L., Shirazi, R. H., Hansson, C., Bergquist, F., Nissbrandt, H., & Skibicka, K. P. (2012). The glucagon-like peptide 1 (GLP-1) analogue, exendin-4, decreases the rewarding value of food: a new role for mesolimbic GLP-1 receptors. The Journal of Neuroscience  :

27

The Official Journal of the Society for Neuroscience, 32(14), 4812–20. doi:10.1523/JNEUROSCI.6326-11.2012

Drucker, D. J., & Nauck, M. A. (2006). The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet, 368(9548), 1696–705. doi:10.1016/S0140-6736(06)69705-5

Greenbaum, C. J., Mandrup-Poulsen, T., McGee, P. F., Battelino, T., Haastert, B., Ludvigsson, J., … Kolb, H. (2008). Mixed-meal tolerance test versus glucagon stimulation test for the assessment of beta-cell function in therapeutic trials in type 1 diabetes. Diabetes Care, 31(10), 1966–71. doi:10.2337/dc07-2451

Jørgensen, N. B., Dirksen, C., Bojsen-Møller, K. N., Jacobsen, S. H., Worm, D., Hansen, D. L., … Holst, J. J. (2013). Exaggerated glucagon-like peptide 1 response is important for improved β-cell function and glucose tolerance after Roux-en-Y gastric bypass in patients with type 2 diabetes. Diabetes, 62(9), 3044–52. doi:10.2337/db13-0022

Katz, A., Nambi, S. S., Mather, K., Baron, A. D., Follmann, D. A., Sullivan, G., & Quon, M. J. (2000). Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. The Journal of Clinical Endocrinology and Metabolism, 85(7), 2402–10. doi:10.1210/jcem.85.7.6661

Lee, J., Hong, S.-W., Park, S. E., Rhee, E.-J., Park, C.-Y., Oh, K.-W., … Lee, W.-Y. (2014). Exendin-4 attenuates endoplasmic reticulum stress through a SIRT1-dependent mechanism. Cell Stress & Chaperones, 19(5), 649–56. doi:10.1007/s12192-013-0490-3

Lim, E. L., Hollingsworth, K. G., Aribisala, B. S., Chen, M. J., Mathers, J. C., & Taylor, R. (2011). Reversal of type 2 diabetes: normalisation of beta cell function in association with decreased pancreas and liver triacylglycerol. Diabetologia, 54(10), 2506–14. doi:10.1007/s00125-011-2204-7

Matthews, D. R., Hosker, J. P., Rudenski, A. S., Naylor, B. A., Treacher, D. F., & Turner, R. C. (1985). Homeostasis model assessment: insulin resistance and cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia, 28(7), 412–419. doi:10.1007/BF00280883

Muñoz, R., Carmody, J. S., Stylopoulos, N., Davis, P., & Kaplan, L. M. (2012). Isolated duodenal exclusion increases energy expenditure and improves glucose homeostasis in diet-induced obese rats. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 303(10), R985–93. doi:10.1152/ajpregu.00262.2012

Odegaard, J. I., & Chawla, A. (2013). Pleiotropic actions of insulin resistance and inflammation in metabolic homeostasis. Science (New York, N.Y.), 339(6116), 172–7. doi:10.1126/science.1230721

28

Ogden, C. L., Carroll, M. D., Kit, B. K., & Flegal, K. M. (2014). Prevalence of childhood and adult obesity in the United States, 2011-2012. JAMA  : The Journal of the American Medical Association, 311(8), 806–14. doi:10.1001/jama.2014.732

Pories, W. J., MacDonald, K. G., Morgan, E. J., Sinha, M. K., Dohm, G. L., Swanson, M. S., … Long, S. D. (1992). Surgical treatment of obesity and its effect on diabetes: 10-y follow-up. The American Journal of Clinical Nutrition, 55(2 Suppl), 582S–585S. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1733132

Rafferty, E. P., Wylie, A. R., Hand, K. H., Elliott, C. E., Grieve, D. J., & Green, B. D. (2011). Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice. Biological Chemistry, 392(6), 539–46. doi:10.1515/BC.2011.050

Rizzello, M., Abbatini, F., Casella, G., Alessandri, G., Fantini, A., Leonetti, F., & Basso, N. (2010). Early postoperative insulin-resistance changes after sleeve gastrectomy. Obesity Surgery, 20(1), 50–5. doi:10.1007/s11695-009-0017-2

Salehi, M., & D’Alessio, D. A. (2014). Effects of glucagon like peptide-1 to mediate glycemic effects of weight loss surgery. Reviews in Endocrine & Metabolic Disorders, 15(3), 171–9. doi:10.1007/s11154-014-9291-y

Stefater, M. A., Pérez-Tilve, D., Chambers, A. P., Wilson-Pérez, H. E., Sandoval, D. A., Berger, J., … Seeley, R. J. (2010). Sleeve gastrectomy induces loss of weight and fat mass in obese rats, but does not affect leptin sensitivity. Gastroenterology, 138(7), 2426–36, 2436.e1–3. doi:10.1053/j.gastro.2010.02.059

Stefater, M. A., Wilson-Pérez, H. E., Chambers, A. P., Sandoval, D. A., & Seeley, R. J. (2012). All bariatric surgeries are not created equal: insights from mechanistic comparisons. Endocrine Reviews, 33(4), 595–622. doi:10.1210/er.2011-1044

Taylor, R. (2013). Type 2 diabetes: etiology and reversibility. Diabetes Care, 36(4), 1047–55. doi:10.2337/dc12-1805

Weir, G. C., & Bonner-Weir, S. (2004). Five Stages of Evolving Beta-Cell Dysfunction During Progression to Diabetes. Diabetes, 53(Supplement 3), S16–S21. doi:10.2337/diabetes.53.suppl_3.S16

Wickremesekera, K., Miller, G., Naotunne, T. D., Knowles, G., & Stubbs, R. S. (2005). Loss of insulin resistance after Roux-en-Y gastric bypass surgery: a time course study. Obesity Surgery, 15(4), 474–81. doi:10.1381/0960892053723402

Wilson-Pérez, H. E., Chambers, A. P., Ryan, K. K., Li, B., Sandoval, D. A., Stoffers, D., … Seeley, R. J. (2013). Vertical sleeve gastrectomy is effective in two genetic mouse models of glucagon-like Peptide 1 receptor deficiency. Diabetes, 62(7), 2380–5. doi:10.2337/db12-1498

29

Ye, J., Hao, Z., Mumphrey, M. B., Townsend, R. L., Patterson, L. M., Stylopoulos, N., … Berthoud, H.-R. (2014). GLP-1 receptor signaling is not required for reduced body weight after RYGB in rodents. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 306(5), R352–62. doi:10.1152/ajpregu.00491.2013