The Endocrine Properties of the insulin stress response

2.2 The Endocrine Properties of the Insulin Stress Response to 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 a change in fasting serum insulin concentration can be measured in subjects between the pre-injury (unstressed) to the post-injury (stressed) states. To measure this change requires fasting serum insulin measurements to be performed both prior to and following life threatening injury Fig 1 & 2. What is important is the change in fasting insulin concentration between these two states which is considered to represent the insulin stress response to injury.    

2. The change in fasting insulin concentration from the unstressed to the stressed state is accompanied by corresponding changes in the concentration of endocrine mediators that are known to regulate GSIS by the pancreas. Strong correlations are observed between the endocrine mediators firstly the incretin hormone glucose dependent insulinotropic peptide (GIP) and secondly the adipokine hormone visfatin/ eNAMPT ( extracellular nicotinamidephosphoribosyltransferase) Fig 4

3. Following recovery from  life threatening injury, (during the transition from the stressed to the unstressed state), an equal but opposing change in fasting insulin concentration over time is observed during the return to the unstressed state. This feature of the changes in fasting serum insulin concentrations over time is suggestive of a stress response to the injury. Fig 2

4. During this recovery period there are also corresponding changes in the concentrations of the same incretin and adipokine mediators to return back to their unstressed state concentrations. The transition from the stressed state to the unstressed state shows a reverse of these concentration changes. The changes in fasting insulin concentrations are similarly correlated with changes in these endocrine mediators.  Fig 4

When these measurements are performed in a group of subjects a highly variable response is observed in terms of the change in fasting insulin concentration between the unstressed and stressed states. In some subjects fasting insulin concentrations will increase whilst in others a fall is observed. However, despite this variable response, a specific pattern is observed in the change in serum insulin concentration which has a number of features indicating that this represents an endocrine stress response regulated by endocrine mediators that control insulin secretion from the pancreas. 

The highly variable response outlined above within a group of subjects is accompanied by a similarly variable response as subjects transition from the stressed state back to the unstressed state. However, when these changes in fasting insulin concentration are compared as the host transitions between these two states and returns back to the unstressed state, a strong inverse correlation is observed between these measurements Fig 3 (see preliminary research). This is postulated to represent a characterisitc stress response pattern in the secretion of insulin into the blood.

Fasting serum insulin concentrations measured during unstressed (resting) conditions are known to vary considerably between subjects depending on metabolic factors such as adiposity, nutritional and diabetic status. The insulin stress response theory postulates that due to this variation in fasting insulin concentrations, the ability of the host to meet the metabolic demands of the stress response to injury are more accurately reflected by the change in fasting insulin concentration from the unstressed to the stressed state. 

This change in fasting insulin concentration (delta value ΔV) represents a more clinically significant measurement compared to absolute values of fasting serum insulin concentrations in the stressed state. For example, a subject with high fasting insulin concentrations under unstressed conditions has a limited ability to increase fasting insulin concentration in response to injury. This contrasts with a subject with low unstressed fasting insulin concentrations who has a greater ability to increase insulin secretion in response to life threatening injury. This is the key to understanding the insulin stress response to injury, where the change in fasting insulin concentrations over time more accurately identifies the response to injury compared to absolute fasting insulin concentrations. 

The characreristic pattern of fasting insulin concentrations over time following life threatening injury 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 concentrations to return to the unstressed state following recovery from the injury, The postulated concentration time curve of fasting insulin concentration over time following life threatening injury Fig 1.

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 GIP and the adipokine hormone visfatin/NAMPT. These correlations indicate that the change observed in fasting insulin concentrations are in part mediated by these two endocrine mediators which themselves are both stress response endocrine mediators with key metabolic and immune functions following life threatening injury.

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 stress response pattern of these mediators activated by life threatening injury.  

2.3 Insulin responders and insulin not-responders following injury-

Within a group subjects who experience life threatening tissue injuy an important and hitherto unrecognised feature of the change in fasting insulin concentration includes a range of values from a large increase to a large decrease. During the transition from the unstressed to the stressed state, some subjects exhibit a fall in fasting insulin concentrations whilst others may exhibit an increase; at the extreme range of these changes in fasting insulin, values may change by many multiples of their respective unstressed 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 are postulated to regulate insulin secretion following life threatening injury Fig 4. 

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.

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  are defined as insulin responders and insulin non-responders Fig 3. 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. 

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

IIn addition to the characteristic stress response pattern 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.  

Following life threatening injury 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 insulin and GIP concentrations in the blood, the highest correlations are observed between the changes in these endocrine mediators between the unstressed and stressed states (see Preliminary Clinical Research). This indicates that the change in incretin hormone (GIP) from unstressed concnetrations 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. This preliminary research indicates that the incretin hormones GIP and GLP have important endocrine functions within the stress response to injury.

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

2.5 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. 

2.6 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 immediate (post-injury) 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 which 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. 

2.7 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 enhance glucose entry into activated leucocytes during the acute inflammatory response.

These contradictory metabolic actions of visfatin/NAMPT released into the circulation following life threatening injury supports the insulin stress response theory where increases in GSIS are mediated by changes in mitochondial function within the beta cells of pancreas. Under 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 life threatening injury these actions are compatible with a survival response. 

The functions of eNAMPT and iNAMPT will have a number of crtically important metabolic functions following life threatening injury. These include firstly to increase NAD+ levels within leucocytes and cells involved in the inflammatory response, secondly to mediate increases in GSIS and thirdly to mediate peripheral insulin resistance within insulin sensitive tissues (adipose tissue, muscle and the liver). These actions will enhance leucocyte transmembrane glucose flux and increase ATP production through 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.  

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.  

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

 Following injury the inflammatory cells within the wound undergo a fundamental transformation in their metabolism. These cells switch their metabolism from mitochondrial oxidative phosphorylation to aerobic or anaerobic glycolysis to supply their ATP needs. This switch to glycolysis as the principal source of ATP production occurs often despite sufficient oxygen availability for mitochondrial oxidative phosphorylation. In these circumstances this is termed aerobic glycolysis and is also referred to as the Warburg effect (named after the Nobel Prize winning scientist who first discovered this cellular response). 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 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 ATP production . 

Following injury the requirements of the inflammatory cells within the wound for large quantities of glucose are necessary to generate an inflammatory response. Increases in glucose transmembrane flux for these cells is required to achieve wound healing and ultimately host survival. A combination of an increase in glucose production by the liver, increases in peripheral insulin resistance within insulin sensitive tissues and increases in insulin secretion provide three different host mechanisms to enhance 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 ATP production and wound healing but simultaneously risk potentially deleterious impairement in host glucose regulation. During the stress response to injury the host metabolic reserves that regulate blood glucose concentrations are impaired . The functions of insulin to regulate blood glucose by increasing glucose storage within insulin sensitive tissues are diminished following injury. Although blood glucose regulation following life threatening injury is similarly achieved by increases in GSIS, in contrast, this has the effect of increasing glucose delivery to the immune system rather than insulin sensitive tissues.

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 also postulates that increases in insulin secretion following injury 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. 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.