2.1.1 The Insulin Stress Response Theory-

The insulin stress response theory postulates a characteristic pattern in fasting insulin concentrations in the blood over time following life-threatening injury. This pattern has a number of features which indicate that changes in the concentration of fasting insulin over time represent a stress response to injury. The insulin stress response theory proposes that these changes in fasting insulin concentrations indicate a temporary and reversible change in glucose stimulated insulin secretion (GSIS) by the pancreas following life threatening injury. 

The theory proposes that the change in fasting insulin concentration in the blood following injury represents a specific endocrine response to life threatening injury. This endocrine response varies between individuals depending on a number of regulatory factors outlined below. However, these factors produce an underlying pattern in fasting insulin concentrations indicating that this hormone is highly regulated following life threatening injury. 

Following life threatening injury there are four principal features that indicate a specific insulin secretory stress response to life threatening injury. 

1. Following life threatening injury there is a change in fasting insulin concentration in the blood from the unstressed state to the stressed state  

2. When this change in fasting insulin concentration is measured over time there is also a corresponding correlation observed with endocrine mediators that are known to mediate a change in GSIS from the pancreas. These mediators include incretin and adipokine mediators outlined below.

3. Followiing the stressed state there is an equal and opposite change in fasting insulin concentrations over time to return back to the unstressed state as the subject recovers from injury. 

4. During this recovery period, (over the same time period), there is also a corresponding correlation with the same incretin and adipokine mediators that return back to their unstressed state concentrations.  

Fasting insulin concentrations in the unstressed state vary considerably between individuals depending on metabolic factors such as adiposity, nutritional status, endocrine diseases and exercise preconditioning. Therefore, for example, within a group of subjects with normal fasting blood glucose concentrations, the unstressed fasting insulin concentration is a baseline from which to measure the response to injury. The change in the concentration of insulin from the unstressed state to the stressed state following injury is postulated to most accurately reflect the changes in endocrine functions of insulin in response to injury. For example, this measurement will most accurately reflect the change in insulin secretion that is attributable to the injury. 

Within a group of subjects with normal fasting blood glucose concentrations unstressed fasting inuslin concentrations vary considerably between individuals. Due to the wide range of fasting insulin conentrations observed between individuals, the ability of the host to meet the metabolic demands of the stress response to injury are more accurately determined by the change in fasting insulin concentration from the unstressed to the stressed state rather than the absolute value of fasting insulin concentrations in the blood. For example a subject with high fasting insulin concentrations has a limited ability to increase fasting insulin concentration in response to injury. This is the key to understanding the insulin stress respose to injury where the change in fasting insulin concentrations over time most accurately identifies the response to injury. The reasons why this response has remained undetected within clinical medicine to date are outlined in the section  (Why has the insulin stress response remained unrecognised? ).  

Therefore the characreristic pattern of fasting insulin concentrations includes an initial change from the unstressed state to reach a point of maximal change in concentration in the stressed state. Over time this is followed by an equal and opposite change in fasting insulin concentration to return the the unstressed state following recovery from the injury, The postulated concentration time curve of fasting insulin concentration over time following life threatening injury is displayed in Fig 2.

When the change in fasting insulin concentrations from the unstressed to the stressed state is measured over time there are also corresponding correlations with changes in endocrine mediators that are known to regulate glucose stimulated insulin secretion from the pancreas. These endocrine mediators include the incretin hormone glucose dependent insulinotropic polypeptide (GIP) and the adipokine visfatin/NAMPT. These correlations indicate that the change observed in fasting insulin concentrations are in part mediated by these two endocrine mediators.

The concentration of fasting insulin in the blood then returns from the stressed state back to the pre-injury unstressed state as the subject recovers from the injury. When this pattern of response is observed in a group of subjects these changes are observed to be highly regulated where the initial change in concentration following injury is followed by an equal but opposite change in concentration back to the unstressed value. In addition, this is also accompanied by a corresponding change in fasting concentrations of both GIP and visfatin/NAMPT back to their resting unstressed concentrations. 

Therefore, the insulin stress response theory proposes that changes in the fasting concentration of insulin from the unstressed state to the stressed state following injury occur in part due to a change in GSIS mediated by both the incretin and the adipokine mediators. In addition, the nature of this response over time, where these mediators are observed to exhibit an equal but opposite change in concentration from the stressed state back to the unstressed state, further supports a response activated by life threatening injury.  This pattern of endocrine response is also observed for other stress response mediators including, for example cortisol and inflammatory cytokines.  

In addition, the pattern of the change in fasting insulin concentration following injury from the unstressed to the stressed state includes a range of values from a large increase to a large decrease. This is an important feature of the insulin stress response where within a group of subjects described above, some subjects may exhibit a fall in fasting insulin concentrations. The change in fasting insulin concentration from the unstressed to the stressed state is determined by changes in the concentration of both GIP and visfatin/NAMPT that regulate changes in insulin secretion following life threatening injury. In subjects that experience a fall in fasting insulin concentrations this is accompanied by a corresponding fall in GIP concentrations in the blood which indicates a decrease in pancreatic GSIS in response to injury. In these subjects the underlying pattern of the response to injury remains where the change from the unstressed to the stressed state (ie a fall in fasting insulin concentrations) is accompanied by an equal and opposite change from the stressed state back to the unstressed state (ie a rise in fasting insulin concentrations) . Therefore, despite the clearly dysregulated nature of the insulin stress response in these subjects, the underlying pattern of response to life threatening injury measured by temporary and reversible changes in fasting insulin concentrations remains. The results of the preliminary research that identified this pattern of response is outlined further in the section  Preliminary Clinical Research  .     

2.1.2 The research conditions necessary to demonstrate the insulin stress response to injury-

To identify the stress response pattern in fasting insulin concentrations over time described above requires the following experimental preconditions-

1. In order to demonstrate the insulin stress response it is necessary to study a life threatening tissue injury of sufficient severity to produce a measurable endocrine stress response. The severity of the injury must result in a change in stress response mediators that are released into the blood following injury. Minor tissue injuries may not result in a measurable stress response to injury.   

2. The life threatening tissue injury must take place within a controlled setting where pre- and post-injury measurements of stress response mediators within the blood can be performed. These conditions can be achieved if elective surgery is chosen as the tissue injury to be studied. When the setting of elective surgery is chosen to investigate this response the surgical procedure must result in a systemic endocrine response. The systemic endocrine stress response will be most easily demonstrated in surgical procedures that are associated with the greatest response. Therefore major surgical procedures represent the most appropriate clinical settting to study this response.  

3. If a specific surgical procedure is chosen such as elective joint replacement orthopaedic surgery (eg unilateral total knee arthroplasty)  this allows comparisons between subjects assuming the severity and mode of tissue injury is constant between subjects.

4. To demonstrate this stress response pattern requires an analysis that measures the change in fasting insulin concentration in the blood from the resting "unstressed" state to the post-injury "stressed" state. This is important as the unstressed fasting insulin concentration serves as a comparator with the equivalent fasting concentration post-injury stressed state. When these values are compared it is the change in fasting insulin concentration from the unstressed to the stressed state that is assumed to represent the insulin stress response to injury. 

5. Over time Following injury the change in fasting insulin concentration in the blood described above is postulated to reach a point of maximal change from the unstressed state. Following the point of maximal change there is then a equal but opposite change in concentration back to the unstressed state as the subject recovers from the injury. When this highly regulated pattern of response over time observed in a group of subjects indicates a sequential relationship to life threatening injury which highlights a potential causal effect.

For elective joint replacement surgery, it is postulated that maximal stressed values occur at approxiamtely 24 hours post surgery which then return to their resting unstressed values over the following 48 hours. The insulin stress response following elective joint replacement surgery therefore postulates a characteristic stress response pattern in fasting insulin concentrations reaching a maximal change at 24 houts (the stressed state) and returning to baseline (unstressed state) at 72 hours following life-threatening injury. This can be represented graphically as shown in Fig. 2.This provides the basis of the preliminary clinical research study which is outlined the section Preliminary Clinical Research   

2.1.3 Insulin responders and insulin not-responders follwoing injury-

Based on the nature of the insulin stress response to injury described above subjects may be grouped into two categories depending on whether they exhibit either a rise or a fall in fasting insulin concentrations from the unstressed to the stressed state. These two  groups of subjects maybe defined as insulin responders and insulin non-responders. It is postulated that insulin non-responders represent a dysregulated response to injury and this group of subjects will be at increased risk of stress hyperglycaemia, impaired wound healing or other related complications during recovery from the injury. The insulin stress response theory postulates that an increase in GSIS represents the physiological response to mediate the beneficial functions of insulin following injury.  

In subjects where fasting insulin levels fall in response to injury this represents a paradoxical insulin stress response. In these subjects there is also a corresponding fall in the fasting concentrations of the incretin mediator glucose-dependent insulinotrpic polypeptide (GIP). For these subjects the fasting unstressed concentrations of GIP, insulin and leptin are all high indicating that a paradoxical insulin stress response occurs in subjects that exhibit features of GIP, insulin and leptin resistance. The paradoxical insulin stress response is postulated to represent a dysregulated insulin stress response in subjects with insulin, GIP and leptin resistance and represents an underlying mechanism resulting in stress hyperglycaemia in non-diabetic individuals in response to injury. 

2.1.4 The role of the incretin glucose-dependent insulinotropic polypeptide in the insulin stress response-

IIn addition to the characteristic stress response in fasting insulin concentrations described above, this  theory also postulates a similar pattern in the change in fasting concentrations of the incretin hormone glucose-dependent insulinotropic polypeptide hormone (GIP). These changes in concentration of GIP exhibit a strong correlation with changes in insulin concentration and support a role for this incretin hormone in the regulation of the insulin stress response to injury. This theory postulates that the incretin hormone GIP forms an endocrine pathway that regulates the insulin stress response following injury through changes in GSIS. However, further research is required to determine the mechanisms that control the release of this incretin hormone into the blood following injury.  

Similarly to the change in fasting insulin concentration following injury described above, the change in fasting  GIP concentration may also range from a large increase to a large decrease. When the strength of correlation is calculated between both these endocrine mediators, the highest correlation is observed between the changes in GIP and insulin concentrations between the unstressed and stressed states (see Preliminary Clinical Research ). This indicates that the change in incretin hormone is most closely associated with the insulin stress response rather than absolute concentrations of this endocrine mediator. Importantly, these results indicate a mechanism for the host to increase insulin secretion above fasting levels in response to physical stress. Consequently, the insulin secretory response attributable to injury is more accurately measured as the change in fasting concentration from the unstressed to stressed state rather than the absolute fasting stressed concentration.  

The incretins GIP (glucose dependent insulinotropic peptide) and GLP (glucagon like peptide) are hormones secreted by the enteroendocrine cells within the small intestine in response to ingestion of food [39–42] . These hormones enter the blood stream and enhance insulin secretion from the pancreas during the post prandial state. The discovery of the incretin hormones is an important development in our understanding of the mechanisms regulating insulin secretion from the pancreas and blood glucose concentrations. 

In addition to their glucose lowering effects the incretin hormones have a wide range of physiological functions at different sites within the body. These hormones also play an important role in diseases linked to metabolism including obesity and T2DM[40,43–45]. In obese subjects both fasting GIP and GLP concentrations in the blood are elevated and GIP is thought to have an important role in the pathophysiology of obesity . In T2DM there are characteristic abnormalities of both these hormones resulting in a diminished incretin effect. However, whilst the incretin effect of GIP cannot be pharmacologically restored, the actions of GLP can be augmented with GLP analogues. The actions of the incretin hormones during the stress response to acute tissue injury are not well investigated and their relationship to insulin secretion and blood glucose concentrations in these conditions are not known

2.1.5 GIP, Insulin and leptin resistance- 

In subjects with high concentrations of unstressed fasting GIP  there is paradoxical fall in GIP concentration in response to injury. It is this response that is associated with a fall in insulin concentrations over the same time period following injury. In these subjects fasting unstressed insulin concentrations are also high. however, the highest correlations measured between these two mediators is observed to be in the change in concentration over time rather than absolute concentrations. In addition these subjects are also observed to display high fasting unstressed leptin concentrations indicating leptin resistance. This highlights a potential adverse effects of chronically elevated unstressed insulin, GIP and leptin concentrations where there is an impaired ability of the host to respond to the metabolic requirements of life-threatening injury. 

2.1.6 The role of the adipokine visfatin/NAMPT in the insulin stress response-

As outlined in the section Preliminary Clinical Research ,  fasting insulin concentrations in the stressed state correlate with the concentration of the adipokine visfatin/NAMPT (nicontinamide phosphoribosyl transferase) . In contrast to the incretin hormone GIP, this correlation with fasting insulin concentrations  is observed between absolute concentrations of these two mediators and is only observed in the stressed state.  This correlation supports a role for this adipokine in the regulation of insulin secretion by the pancreas following injury (ie during the stressed state) . 

The association between this adipokine mediator and insulin secretion has been investigated in both human and animal studies in a wide range of different research conditions  [46–52]. Interest was first generated in human studies linking these two mediators, however, the results of this research could not be reproduced by other investigators, leading to the retraction of this initial study [53,54].  In vitro research studies investigating this mediator have demonstrated that visfatin/NAMPT regulates insulin secretion from the pancreas [55–58]. However, to date this regulation has not been demonstrated in in vivo humans studies. This association with insulin secretion has also not been studied in the setting of the stress response to physical injury. The role of visfatin/NAMPT also links the important cellular redox agent NAD+/NADH with the stress response to injury.

Visfatin/NAMPT is a pleiotropic peptide mediator found in all tissues within the human body and is essential for life. This peptide has a wide range of functions both  within the host response to injury as well as during resting physiological states. These functions are classified as extra- and intra-cellular categories reflecting the diverse range of endocrine, immune and metabolic actions of this mediator. Gene expression studies have found this peptide to be expressed strongly within both visceral adipose tissue and leucocytes. 

This mediator was initially named pre-B cell colony enhancing factor (PBEF) reflecting its actions on the immune system [59,60]. Subsequently, Visfatin/NAMPT has been classified as an adipokine and named visfatin in view of its high concentration in visceral adipose tissue [61,62]. The enzymatic actions of visfain/NAMPT (nicotinamide phosphoribosyl transferase) catalyses the rate limiting step in the salvage synthesis pathway for NAD+[56,63,64]. This enzyme is essential for life due to the importance of NAD+/NADH in cellular metabolism. Whilst the discovery of this peptide mediator was first reported at least a quarter of a century ago, many of the functions of visfatin/NAMPT in human health and disease remain unclear[54]. There remain large gaps in our knowledge of this peptide despite many reports describing its importance in the pathophysiology of different diseases. For example, its role in the aetiology of metabolic diseases including obesity and T2DM has been debated for many years.[48,52]

The enzymatic actions of visfatin/NAMPT catalyse the formation of nicotinamide mononucleotide (NMN) which is converted to NAD+ by nicotinamide mononucleotide adenylyl transferases (NMNAT). NAD+ and NADH concentrations are highly regulated by cells depending on factors such as cellular metabolic demands, nutrient supply or the activation of cellular stress pathways. NAD+ is the oxidised form of this redox coenzyme that is an essential cofactor for glycolysis. The increased production of NAD+ by visfatin/NAMPT will thus promote glycolysis within the cytoplasm of leucocytes and within other cells involved in the acute inflammatory response. The extra-cellular functions of visfatin/NAMPT include the activation of cellular inflammatory pathways. For example, the metabolic actions of visfatin/NAMPT mediate tissue insulin resistance in adipocytes through serine phosphorylation of the insulin receptor substrate[46,47,62,65–68].

The critical role of NAD+/NADH within the cell results in this cofactor functioning as a regulator for many key intra-cellular metabolic and inflammatory pathways[69,70]. Within the nucleus of the cell NAD+ is a cofactor for many important reactions including NAD+ dependent deacetylation reactions. Sirtuins are a group of proteins that function as epigenetic regulators linking gene expression with the metabolic and inflammatory environment within the cell . The synthesis of sirtuins are NAD+ dependent and are produced in response to cellular stress signals enabling the cell to adapt to these potentially harmful environments. Through the regulation of surtuin synthesis NAD+ is able to control gene expression and cell function in response to cellular stress.

In laboratory isolated pancreas models visfatin/NAMPT has been demonstrated to mediate insulin secretion through increased concentrations of NAD+ within the cytoplasm of the B cells of the islets of Langerhans [71]. NAD+ regulates insulin secretion through the actions of sirtuin (Sirt 1) which represses gene synthesis of the mitochondrial uncoupling protein UCP-2[58]. ATP levels within the β cell regulate insulin secretion through the actions of ATP coupled K channels. This action is controlled by NAD+ where the actions of the UCP-2 control insulin secretion in response to glucose stimulus.

The secretion of visfatin/NAMPT following acute tissue injury coordinates a diverse group of functions that will support the host to generate an acute inflammatory response. These functions indicate that this mediator plays a potentially critical role in coordinating metabolic and inflammatory components of the stress response to acute tissue injury. This includes the combined actions of increasing tissue insulin resistance and increasing GSIS by the pancreas. This is achieved through increased glucose entry into leucocytes, enhanced glycolysis and increases in NAD+/NADH within the cell. Through these actions this mediator controls the pathways involved in coordinating metabolic and inflammatory mechanisms of the host response to acute tissue injury. 

2.1.7 The contradictory metabolic mechanisms of visfatin/NAMPT during the stress response-

The insulin stress response theory postulates that the metabolic functions of the adipokine visfatin/NAMPT during the stress response to injury simultaneously mediate both peripheral  insulin resistance and increases in pancreatic GSIS. These opposing endocrine functions should be considered as stress mechanisms where the combination of these mechanisms enhances glucose entry into activated leucocytes during the acute inflammatory response.

The net effect of visfatin/NAMPT release into the circulation supports these metabolic components of the acute inflammatory response to acute tissue injury. Under resting unstressed conditions these opposing metabolic actions appear to be contradictory and have led to confusion in our understanding of the role of visfatin/NAMPT within the body [46,72,73] . However, under the conditions of acute stress these actions are compatible with a survival response. These actions namely increased GSIS, peripheral insulin resistance and increases in NAD+ will increase leucocyte transmembrane glucose flux and increase  ATP production from glycolysis. In addition, the effects of this adipokine will also increase the synthesis of SIRT 1 leading to altered gene expression to support the activation of inflammatory pathways such as NF Kβ, RAS/RAK/IRK and mTOR [56,64,74].

The metabolic functions of visfatin/NAMPT secretion in response to life threatening injury may at first appear to be counterintuitive, they support both counter regulatory endocrine actions, (including mediating tissue inflammation and insulin resistance), whilst also increasing insulin secretion from the pancreas. Herein lies a key paradox of the host metabolic response to injury- the metabolic needs of the host and the wound are in competition for fuel substrates and have differing metabolic priorities; this concept is explained in more detail below. 

These endocrine actions of the adipokine mediator visfatin/NAMPT support the role of insulin as a stress response hormone that is secreted in response to life threatening injury. The actions of insulin under unstressed conditions regulate blood glucose concentrations by increasing glucose flux across the cell membrane into insulin sensitive tissues. By contrast, the actions of insulin under stressed conditions are also to regulate blood glucose concentrations by increasing glucose flux across the cell membrane into the immune system as these cells increase their metabolic requirements.  

2.1.8 The conflicting metabolic requirements of the wound and the host-

 Following injury the inflammatory cells within the wound switch their metabolism from the unstressed to the stressed state by inhibiting mitochondrial oxidative phosphorylation and relying on aerobic or anaerobic glycolysis for ATP production. When cells switch to aerobic glycolysis as their principal source of ATP production despite sufficient oxygen availability for mitochondrial oxidative phosphorylation this is termed the Warburg effect . This metabolic switch to glycolysis is determined by the availability of NAD+ within the cytoplasm. If NAD+ levels fall, the enzyme pyruvate dehydrogenase is inhibited and the cell switches to glycolysis. 

Following life threatening injury Immune and inflammatory cells within the wound are therefore dependent on glycolysis to generate ATP. As this is an inefficient mode of ATP generation compared to oxidative phosphorylation these cells require large quantities of glucose following injury. These cells also require large quantities of NAD+, a cofactor for glycolysis, which is augmented by the actions of visfatin/NAMPT. The metabolic actions of the stress response to injury including the actions of insulin act to increase glucose transmembrane flux across the leucocyte cell membrane to enhance the inflammatory response. 

Following injury the requirements of the wound for glucose are paramount to generate an inflammatory response to achieve  both wound healing and ultimately  host survival. A combination of an increase in glucose production, increases in peripheral insulin resistance and increases in insulin secretion all assist the delivery of glucose to cells of the immune system. It is the latter of these metabolic adaptations to physical stress that  has remained unrecognised within clinical medicine. 

These metabolic adaptations to injury provide glucose for wound healing but simultaneously risk potentially deleterious consequences in glucose regulation for the host. During the stress response to injury the metabolic reserves of the host in terms of glucose regulation are diminished. To meet the metabolic needs of the wound the host metabolic response to injury risks dysglycaemia with its attendant adverse effects on the body tissues and vital organs.  The insulin stress response provides an additional component of the metabolic response to injury which functions to increase glucose delivery to the wound and immune cells as well as to regulate blood glucose concentrations in the blood. The insulin stress response theory postulates an additional function of insulin following injury which is to stimulate cell growth and new blood vessels formation within the wound. 

Stress dysglycaemia following life threatening injury represents a failure of these metabolic stress mechanisms to regulate blood glucose. As explained in the knowledge gap sections current knowledge relating to the stress response to injury does not consider insulin to be a stress hormone. In addition, current best knowledge holds that increases in insulin secretion will have a limited effect when there is both an increase in tissue insulin resistance as well as increased glucose production mediated by counter regulatory hormones of the stress response. However, the insulin stress response theory postulates that the metabolic needs of the wound and the immune system are prioritised. These cells show increases in expression of both GLUT 4 as well as non-insulin glucose transporters  following activation by cytokines in response to injury [75] . In these circumstances insulin functions both to lower blood glucose concentrations but also to increase glucose flux across the cell membrane of cells involved in the inflammatory response.