Metabolic syndrome: mechanisms, pathophysiology and laboratory assessment

Metabolic syndrome is a common pathological condition characterized by a group of risk factors that raises the risk for type 2 diabetes and cardiovascular disease (CVD). This article summarizes the pathophysiology, molecular mechanisms and animal models of metabolic syndome discussed during our 3-part webinar series, presented by Prof. Khosrow Adeli.

By Khosrow Adeli, PhD
FCACB, DABCC, Head and Professor, Clinical Biochemistry, The Hospital for Sick Children, University of Toronto, Toronto, Canada.

Metabolic syndrome

One-quarter of the world’s adults suffer from metabolic syndrome and are five times more likely to develop type 2 diabetes, and twice as likely to die from heart disease or stroke, than adults without the disorder. Diabetes is considered a worldwide epidemic with increasing incidence over time. Metabolic syndrome is defined by abdominal obesity in combination with two of the following four factors: raised triglycerides, lowered HDL cholesterol, raised blood pressure and raised fasting plasma glucose. A large number of biomarkers are used to assess patients with metabolic syndrome, such as urine albumin and C-reactive protein as indicators of albuminuria and sub-clinical inflammation respectively, both components of the disorder.

Insulin resistance and obesity are thought to be the most important risk factors for metabolic syndrome. Considered an epidemic, obesity rates are going up for both adults and children worldwide. Obesity can have genetic associations, but the high rate seen in recent times is most likely caused by environmental factors, such as inadequate physical activity and poor eating habits. The main problem in obesity is the visceral fat, which  - as opposed to subcutaneous fat - accumulates deep inside the abdomen. Visceral fat releases free fatty acids into the circulation which find their way to other tissues not designed to store fat, such as the liver, heart and skeletal muscle. Visceral fat will also wrap itself around these internal organs leading to insulin resistance.

Insulin resistance: A hallmark of the metabolic syndrome​​

Liver, muscle, intestinal and fat cells can become resistant to insulin in metabolic syndrome. In the liver, the condition results in an increase in glucose production and secretion, while in muscle and fat cells, reduced glucose uptake results in hyperglycemia. The pancreas then releases even more insulin, leading to a transient hyperinsulinemia. After years of producing excess insulin, the pancreas may fail causing a drop in insulin, an increase in glucose and development of full-blown type 2 diabetes. Typically, a person may have had insulin resistance for many years before developing overt diabetes, thereby stressing the importance of early diagnosis of the metabolic syndrome.

There is growing evidence that the small intestine plays a critical role in the development of insulin resistance, metabolic syndrome and type 2 diabetes. It is now thought of as the largest endocrine organ in the body, due to the multitude of peptide hormones it secretes. One of these is glucagon-like peptide 1 (GLP-1), a peptide hormone that stimulates the pancreatic beta cells to produce insulin in response to a meal, slows gastric emptying, increases satiety and improves insulin sensitivity. The peptide has a short half-life, as it is degraded and inactivated by dipeptidyl peptidase 4 (DPP-4). Several GLP-1 analogs and DPP-4 inhibitors have been developed by pharmaceutical companies as anti-diabetic agents that are currently used in the clinic to treat type 2 diabetic patients. DPP-4 inhibitors (such as linagliptin) have been shown to increase GLP-1 action and decrease levels of the diabetes marker hemoglobin A1c (HbA1c) within weeks, indicating improved glycemic control. The role of the small intestine in insulin resistance is seen in obese, diabetic patients who have undergone gastric bypass surgery, where the stomach is surgically connected to the jejunum. Within days of this procedure, a resolution of diabetes is observed in the majority of patients. These patients experienced normal glucose levels, increased insulin secretion and normal HbA1c levels, independent of weight loss.

Insulin signaling and molecular basis of insulin resistance

Insulin and leptin are hormones involved in key metabolic signaling pathways. The insulin receptor, located at the cell surface, is normally in the “off-switch” state. After a meal, insulin is secreted and binds to its receptor activating the insulin receptor, thereby turning on the insulin signaling pathway. This occurs through autophosphorylation of the receptor which then recruits substrates that mediate the downstream signaling cascade. The activated insulin receptor binds to adaptors, such as insulin receptor substrate-1 (IRS1), which mediate the recruitment and activation of downstream mediators. Protein tyrosine phosphatases (PTPs) are abundant regulators of the insulin signaling pathway and can turn the receptor back off by removing phosphate groups, and effectively block the signal. Insulin must inhibit PTPs just long enough for it to act on the receptor so that a signal can go through. As soon as the signal goes through, the pathway is inhibited. This tight regulation of the pathway is important to avoid issues that would result from over-activation by insulin, which is a potent growth factor.

Downstream mediators of the insulin receptor cascade can regulate carbohydrate and lipid metabolism, which become dysregulated in obesity and diabetes. IRS proteins activate PI-3 kinase, which goes on to phosphorylate lipids on the plasma membrane to create the lipid species PIP3. PIP3 enables activation of downstream kinases leading eventually to activation of Akt, a key kinase that regulates many key regulators of carbohydrate and lipid metabolism, such as FOXO1, a hepatic glucose production regulator, AS160, a glucose uptake regulator, and SREBP1c, a lipid metabolism regulator.

Protein tyrosine phosphatase 1B (PTP1B) is referred to as a Master Switch since it can block insulin action through dephosphorylation of the receptor. Insulin signal blockage due to high PTP1B levels in the cell can result in reduced activation of downstream effectors, such as IRS1, PI-3 kinase and Akt, leading to an increase in loss of insulin-mediated control of hepatic glucose production and a decrease in insulin-induced peripheral glucose uptake. Therefore, PTP1B overactivation has been implicated in the development of insulin resistance and type 2 diabetes. High levels of PTP1B have been observed in an obese mouse model as well as in obese and diabetic humans. Knocking out PTP1B protects mice from high fat-induced obesity and diabetes, due to the inability to block the insulin signal. PTP1B inhibition has been considered as a potential drug target for treatment of obesity and type 2 diabetes, however, these studies have not progressed to phase III clinical trials as drug specificity for this particular phosphatase has been an issue.

Leptin signaling and induction of leptin resistance in the metabolic syndrome

Leptin is a small, 167 amino acid long peptide, similar in structure to other cytokines and hormones. It is largely secreted by the adipose tissue, where we store triglycerides and acts on the central nervous system (CNS). Leptin was first discovered at the Jackson Labs in the US (Bar Harbour, Maine) in the 1950s. Through breeding experiments, a “fat mouse” model was discovered that became a genetic model for obesity, termed the ob/ob mouse. It turned out that these mice have a mutation in the leptin gene resulting in a state of leptin-deficiency. These mice are constantly hungry resulting in increased food consumption, weight gain and insulin resistance.

Leptin binds to the long-form of the leptin receptor mainly in the hypothalamic region of the brain, lead to receptor activation. This causes activation of JAK2 kinase, which in turn recruits the transcription factor STAT3, leading to the leptin signaling cascade. STAT3 activates and inhibits the appetite-regulating neurons POMC and NPY, respectively, leading to satiety and reduced appetite.  This is thought to occur through the potential involvement of the potassium ATP channel (KATP). Leptin resistance has been seen in obese and diabetic individuals. This can occur due to leptin receptor mutations or inhibition of the downstream signal. In fact, the absence of the leptin receptor is the molecular defect in the diabetic db/db mouse model. Leptin resistance can occur with increased activity of PTP1B or SOCS-3. PTP1B dephosphorylates JAK2, thereby preventing STAT3 activation and inhibiting the leptin signaling cascade. Leptin resistance can also occur when SOCS3 triggers recruitment of the receptor and downstream molecules to the proteasome for degradation, thereby interfering with the ability of leptin to bind and initiate the signal.

Animal models of obesity, metabolic syndrome and type 2 diabetes

Animal models that are representative of human disease are extremely valuable in understanding disease mechanisms, and in testing emerging therapeutics. Advantages of using animals include sequenced genomes, short reproduction times and large numbers of animals that can be produced and handled in the lab. Human studies are limited to measuring biomarkers in body fluids or direct physical studies, whereas with animal models, a large number of genetically-similar subjects can be studied from birth under extremely controlled conditions. Experimental animal models used for research on obesity, metabolic syndrome and type 2 diabetes include both genetic and diet-induced models.

Genetic animal models

Mouse and rat are the most common genetic models used for these disorders. Mice with a mutation in the leptin gene, which regulates appetite and food intake, develop obesity very early after birth. This mouse was termed the ob/ob mouse (ob standing for obesity) and was found to have uncontrolled appetite, become morbidly obese and insulin resistant. A mouse with a mutation in its leptin receptor gene becomes obese and develops diabetes. This mouse was termed the db/db mouse (db standing for diabetes) and is a model for obesity and type 2 diabetes. Another monogenic mouse model is the yellow obese mouse. Polygenic mouse models employed include the Japanese KK mouse and the New Zealand Obese (NZO) mouse.

The most widely used rat model for obesity is the Zucker fatty rat which has a missense mutation in the leptin receptor. These rats develop obesity a few weeks after birth and can become diabetic, although not overtly. The Zucker diabetic fatty (ZDF) rat is a model for severe insulin resistance, hyperglycemia, obesity and frank diabetes. They develop many complications of diabetes that are seen in humans such as nephropathy, kidney disease and CVD. These rats have a defect in the pancreatic β-cells in the presence of normal leptin signaling. The Goto-Kakizaki (GK) rat is a lean model of type 2 diabetes, since it is not obese.

Diet-induced animal models

Diet-induced models better represent the garden-variety obesity seen in humans, which is mostly environmental, and for this reason have become more popular. Some models use a high-fat diet, alone or in combination with high cholesterol and/or high carbohydrate. High-sucrose and high-fructose diets are also used. The diet-induced obese (DIO) mouse model is generated by feeding the mouse a high-fat diet where more than 60% of their calories come from fat. This mouse becomes hyperphagic, with reduced energy utilization, and exhibits systemic and tissue insulin resistance in adipose, liver, brain and even small intestine.

Hamsters are a good representative model of obesity and diabetes because they have several similarities to human with respect to lipid metabolism and physiology. Our group has employed the fructose-fed Syrian golden hamster as an experimental model for both basic research as well as mechanism of action studies of new pharmaceutical agents. These hamsters provide an excellent model of insulin resistance. As in humans, they develop obesity, become hypertriglyceridemic, hyperinsulinemic and insulin resistant. Fructose is used in these studies since research has implicated its increased consumption in the current high prevalence of obesity. The reason fructose and not glucose has this effect, lies in its unique metabolism. Fructose in the liver is converted to glycerol-3-phosphate, a good substrate for triglyceride synthesis. These triglycerides can be stored in the liver or secreted as very low-density lipoproteins (VLDL) and can lead to fatty liver, hyperlipidemia and insulin resistance. The fructose-fed hamster model of insulin resistance was found to be sensitive to rosiglitazone, an insulin sensitizer agent, and to rosuvastatin, an HMG-CoA reductase inhibitor, indicating its potential usefulness as an experimental animal model for studies of pharmaceutical agents.

The fat, fructose and cholesterol (FFC)-fed hamster is a newer model our group has been working on that closely mimics atherogenic diet consumed in the Western diet. This model exhibits evidence of severe insulin resistance with progression to type 2 diabetes, dyslipidemia, fatty liver. Evidence of dyslipidemia and insulin resistance was seen within few days of this diet, well before the eventual weight gain.

Concluding remarks

Obesity has emerged as one of the greatest concerns to human health in the modern world and its incidence has led to a major rise in the incidence of metabolic syndrome and type 2 diabetes. Obesity is the primary risk factor for developing the metabolic syndrome, a cluster of metabolic abnormalities which include insulin resistance, fatty liver disease, dyslipidemia, and hypertension. Research over the past decade has shed considerable light on the molecular mechanisms of insulin resistance and metabolic syndrome, highlighting the key role of critical components within the insulin and leptin signaling cascades. Multiple genetic and diet-induced animal models have been employed to delineate the molecular mechanisms involved. A more recent focus has been the key role of the brain and its neural control of metabolic pathways that regulate many aspects of carbohydrate and lipid metabolism critical to development of the metabolic syndrome. A close communication between key foci within the CNS, multiple endocrine and neural pathways in the small intestine, and metabolic pathways within the liver appears to be essential for maintaining metabolic health. Further research is underway to delineate these complex metabolic networks and understand the mechanisms underlying progression to metabolic disease.