Diabetes and hypoglycemia
Severe hypoglycemia is considered a medical emergency as it causes organ and brain damage. The types of symptoms that depend on duration and severity of hypoglycemia includes autonomic symptoms (sweating, irritability, and tremulousness), cognitive impairment, seizures, and coma. Brain damage, trauma, cardiovascular complications, and death are major complications of severe hypoglycemia [
223]. The incidence of hypoglycemia depends on the degree of glycemic control. Threefold increase in incidences of severe hypoglycemia and coma in intensively treated group was observed when compared to conventionally treated group in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study [
224].
The risk of hypoglycemia in randomized controlled trials of glucose regulation in stroke settings has been reported ranging from 7 to 76% [
225‐
230]. The ischemic brain is particularly susceptible to hypoglycemia [
231]. In the presence of stroke, it is possible that incidents of hypoglycemia may be mistaken for progressing severity of stroke, given that symptoms of hypoglycemia include impaired cognitive functioning, hemiparesis, seizures, and coma.
Hypoglycemia is proposed to be linked with angina, myocardial infarction, and acute CVD [
232‐
234]. Hypoglycemia causes a cascade of physiologic effects and may induce oxidative stress [
235], induce cardiac arrhythmias [
236], contribute to sudden cardiac death [
236], and cause cerebral ischemic damage [
237], presenting several potential mechanisms through which acute and chronic episodes of hypoglycemia may increase CVD risk.
Increased levels of C-reactive protein (CRP), IL-6, IL-8, TNF-α, and endothelin-1 have been shown during hypoglycemia [
238,
239]. Wright et al. [
240] and Gogitidze Joy et al. [
32] confirmed that hypoglycemia induced an increase in proinflammatory mediators and platelet activation, and has an inhibitory effect on fibrinolytic mechanisms. Hypoglycemia also increases production of vascular endothelial growth factor (VEGF), increases platelet and neutrophil activation leading to endothelial dysfunction, and decreased vasodilation, resulting in increased risk for CVD events [
241]. Furthermore, IL-1 has been shown to increase the severity of hypoglycemia [
242]. Moderate hypoglycemia acutely increases circulating levels of plasminogen activator inhibitor-1 (PAI-1), VEGF, vascular adhesion molecules (VCAM, ICAM, E-selectin), IL-6, and markers of platelet activation (P-selectin) in T1D patients and healthy individuals [
32]. Thus, hypoglycemia can result in complex vascular effects including activation of prothrombotic, proinflammatory, and proatherogenic mechanisms in T1D patients and healthy individuals. In addition, a link has been made between low glucose levels and the unexpected sudden death in T1D patients without CVD, also known as “dead in bed” syndrome [
243].
Recurrent severe hypoglycemia results in brain damage [
244], with preferential vulnerability in the cerebral cortex and hippocampus [
244‐
246]. Evidence suggests that neuronal damage resulting from hypoglycemia is enhanced in diabetic compared to non-diabetic brains [
245]. Hypoglycemia causes a loss of ionic homeostasis or increase in ROS that can further lead to neuronal inflammation and death [
246].
Impact of hypoglycemia in the diabetic brain
Hypoglycemia is of major concern in diabetes as it leads to severe impairment of CNS function. Severe and/or long duration hypoglycemia may result in severe morbidity and even death. Repeated episodes of hypoglycemia are suggested to increase the risk of atherosclerosis [
247]. Acute hypoglycemia results in endothelial dysfunction, vasoconstriction, white blood cell activation, and release of inflammatory mediators including cytokines
via sympathoadrenal stimulation and release of counter-regulatory hormones [
32]. All these changes increase the risk of myocardial and cerebral ischemia [
240].
Recurrent/moderate hypoglycemia also aggravates post-ischemic brain damage in diabetic rats [
53]. In this study, rats treated with insulin and exposed to recurrent hypoglycemic episodes experienced a 44% increase in neuronal death compared with rats similarly treated with insulin but not exposed to hypoglycemia, demonstrating that prior exposure to recurrent hypoglycemia can lead to more extensive cerebral ischemic damage. Relatively severe recurrent hypoglycemia itself induces neuronal death in the CA1 hippocampus and cortex of streptozotocin-induced diabetic rats [
248,
249].
Bree and collaborators [
245] showed that insulin-induced severe hypoglycemia in normal animals elicits brain damage in the cortex, cornus ammonis (CA)1, and CA3 hippocampal regions, and that the diabetic condition increases the vulnerability to neuronal death in these specific brain areas. These results suggest that diabetes can be a critical factor aggravating neuronal damage in hypoglycemia.
Decreased cognitive function can also lead to an increased risk of hypoglycemia and CVD events, and thus mortality [
250]. In a study examining magnetic resonance imaging of the brain in a cohort of 22 patients with T1D, brain abnormalities were more common in patients with T1D who had a history of repeated (five or more) hypoglycemic episodes [
251]. In some of the strongest evidence to date of the detrimental effects of hypoglycemia on cognitive function, Whitmer et al. [
252] investigated the association of hospitalization or emergency department visits for hypoglycemia and dementia development in older adults with T2D. They reported a dose/response relationship between the number of hypoglycemia episodes and the risk for developing dementia.
Inflammatory response in diabetes/hyperglycemia
Increased systemic and cerebrovascular inflammation is one of the key pathophysiological features in diabetes and its vascular complications [
253,
254]. Though the etiology of diabetic complications is multifactorial, chronic inflammation is thought to play a critical role [
255,
256]. Key mechanisms of hyperglycemia-induced inflammation include NFkB-dependent production of proinflammatory cytokines, TLR expression, increased oxidative stress, and inflammasome activation [
256‐
259].
Increased expression of proinflammatory cytokines has been demonstrated in diabetes (reviewed in [
260]). Proinflammatory cytokines IL-12 and IL-18 were shown to be elevated in serum of diabetic patients compared to healthy subjects and were positively associated with CRP, which is one of the most important biomarkers of chronic inflammation [
261,
262]. CRP itself exerts direct proinflammatory effects on human endothelial cells, inducing the expression of adhesion molecules [
263]. IL-12 and IL-18 have been shown to exert strong proinflammatory activity that synergize with each other, as well as with TNF-α or IL-1 [
264]. NFκB controls the induction of many inflammatory genes. During hyperglycemia, NFκB is rapidly and dramatically activated in vascular cells resulting in a subsequent increase in leukocyte adhesion and transcription of proinflammatory cytokines [
41]. A significant increase in expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), followed by activation of NFkB and signal transducer activator of transcription 3 (STAT3) inflammatory pathways, was reported in cultured astrocytes treated with high glucose [
265]. Under diabetic conditions, hyperglycemia also causes inflammatory reactions in other organs and tissues in vivo [
266,
267]. It has been reported that high glucose in vitro can cause ROS production and expression of proinflammatory cytokines and chemokines in a variety of cells [
268‐
270]. Expression of adhesion molecules on endothelial cells of both hyperglycemic and diabetic animals, and patients with diabetes, is enhanced compared to normal controls [
271].
TLRs play an important role in human and animal model of diabetes. Mice with an inactive TLR-4 gene were significantly less prone to diet-induced insulin resistance [
272,
273]. Likewise, inhibition of TLR-2 function in mice exposed to a high-fat diet led to improved sensitivity and decreased activation of proinflammatory pathways [
274]. Furthermore, polymorphisms in TLRs and in members of TLR downstream signaling pathways that encode hyper- or hypoactive responses predict the development of T1D and T2D [
275,
276]. TLR ligands activate B cell cytokine production, most significantly IL-8, in diabetes mellitus vs. non-diabetic donors [
277]. The circulating levels of danger molecules including the high-mobility group box-1 (HMGB-1), heat shock proteins, and hyaluronan that activates TLR signals [
278] are known to be increased in T2D patients [
258]. Potential roles for TLR-2 and TLR-4 in the pathology of diabetes have been demonstrated recently (reviewed in detail in [
279]).
Emerging evidence suggests that activation of the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome leads to the maturation and secretion of IL-1β and is involved in the pathogenic mechanisms of obesity-induced inflammation, insulin resistance, and diabetes development [
280]. Obesity-induced danger signals have been reported to activate the NLRP3 inflammasome and induce the production of IL-1β in adipose tissue in T2D patients and in mice fed a high-fat diet [
281]. Circulating levels of CXCL-10 and CCL-2, as well as IFN-γ mRNA (messenger ribonucleic acid) and protein levels in adipose tissue were significantly reduced in NLRP3-deficient mice, suggesting that the NLRP3 inflammasome plays a role in the macrophage-T cell interactions that are associated with sustained levels of chronic inflammation in obesity-induced metabolic diseases [
281]. Moreover, the saturated fatty acid palmitate induces activation of the NLRP3 inflammasome in hematopoietic cells, which is responsible for the impairment of insulin signaling and inhibition of glucose tolerance in mice [
282].
Inflammatory response in hypoglycemia
Recurrent/moderate hypoglycemia induces oxidative injury in hippocampal dendrites, and microglial activation in hippocampus and cerebral cortex [
248]. They observed oxidative damage, as assessed by the lipoperoxidation product 4-hidroxynonenal, in the hippocampal CA1 dendritic layer and microglial activation. The degree of microglial activation in the hippocampus of recurrent/moderate hypoglycemia-exposed diabetic rats was 194% higher than in normoglycemic rats exposed to recurrent/moderate hypoglycemia [
248]. This study confirmed that inflammatory responses are also induced after recurrent/moderate hypoglycemia. Microglial activation is induced in severe hypoglycemia and contributes to neuronal injury by releasing neurotoxic substances, including superoxide, nitric oxide, and metalloproteinases [
283‐
285]. Activation of microglia appears to play a role in the neutrophil infiltration and recruitment which in turn contributes to brain damage [
286,
287]. Increased number of infiltrating neutrophils in hypoglycemia vulnerable brain regions following hypoglycemic brain injury suggests its potential role in hypoglycemic brain injury [
288].
In another study by Cardoso et al. [
289], recurrent hypoglycemia (twice daily for 2 weeks) in streptozotocin-induced diabetic rats potentiated an increase in lipid peroxidation and a decrease in aconitase activity, used as an index of oxidative stress, in mitochondria from diabetic animals. Previous findings showed that recurrent hypoglycemia differentially alters mitochondrial bioenergetics and the antioxidant defense response in the cortex and the hippocampus, the hippocampus being most affected. Limiting ROS production and restoring blood glucose to levels not exceeding the physiological range prevents neuronal death [
31]. On the other hand, the administration of pyruvate and lactate in combination with glucose reduces the death of hippocampal neurons [
288,
290,
291]. This finding suggests the therapeutic potential of antioxidants, lactate, and pyruvate administration combined with glucose to limit the adverse consequences of glucose reperfusion. On the other hand, it has been recently shown that the administration of minocycline to rats 6 h after hypoglycemic coma and daily for a week results in reduced microglial reactivity, neuronal death, and cognitive impairment [
288]. Further investigation is needed to extrapolate these findings to clinical practice.
Cerebral ischemia-induced inflammatory response in the diabetic brain
Diabetes continues to expand rapidly in the USA. Worldwide, it is projected that diabetes will affect 439 million people by the year 2030 [
292]. As mentioned above, diabetes is a predisposing risk factor for cerebrovascular diseases and increases stroke incidence. In humans, diabetes increases the risk of stroke incidence as well as post-stroke mortality [
293‐
295]. Diabetes duration has also been shown to increase the risk of ischemic stroke. With every year of diabetes, the risk is increased by 3% and triples with diabetes of more than 10 years [
296]. Diabetes predisposes humans to stroke, and stroke-induced brain damage is known to be exacerbated by poor functional recovery in these patients [
297]. Several clinical studies indicated that patients with diabetes had poorer outcomes following stroke [
298‐
302].
Diabetic patients have a higher risk of stroke compared with non-diabetic patients [
294,
295]. Although >30% of stroke sufferers are known to be diabetic, the mechanisms that are responsible for the increased post-ischemic brain damage in this population are understudied. Oxidative stress and inflammation play a central role in tissue damage in streptozotocin-induced diabetes [
303,
304]. In addition, diabetic patients had significantly increased levels of acute phase proteins and proinflammatory cytokines such as TNF-α and IL-1, compared to non-diabetic controls [
305]. More recently, Hwang et al. [
306] demonstrated microglial activation and expression of proinflammatory cytokines, such as IFN-γ and IL-1β in the hippocampus of diabetic rats.
The experimental studies have evaluated the effect of diabetes on stroke outcome in T1D and T2D models. The post-ischemic brain damage was exacerbated in T1D rodents following global or focal ischemia [
52,
297,
307‐
310]. The exacerbated edema and infarction, worsened neurological status, and increased mortality have also been observed in T2D models following ischemia [
311‐
314]. A study by Yeung et al. showed that exacerbated post-ischemic pathological symptoms observed in db/db mice are alleviated by knocking out the enzyme of polyol pathway (aldose reductase) that converts glucose to sorbitol and further metabolizes to fructose [
315]. Uncontrolled inflammation during the acute period after stroke is a major mediator of cerebrovascular failure and brain damage [
316]. Increased expression of cell adhesion molecules enabling the extravasation of white blood cells, and further induction of proinflammatory transcription factors and other inflammatory genes are thought to be major mediators of post-ischemic inflammation [
74]. Previously published literature demonstrated the increased expression of ICAM and proinflammatory cytokines in diabetic animals after cerebral ischemia/reperfusion [
317‐
320]. At post-translational levels, IL-1β and cyclooxygenase-2 (COX-2) expressions were significantly higher following hyperglycemic ischemia than hyperglycemic shams [
321]. Lin et al. demonstrated that hyperglycemia triggered early, massive deposition of neutrophils in the post-ischemic brain, which exacerbated injury [
322]. It has been reported that the expression of ICAM-1 and the infiltration of neutrophils into ischemic tissue are closely correlated with the severity of ischemic brain damage [
323]. The gene expression of IL-1β, IL-6, MIP-1α, MCP-1, P-selectin, and E-selectin was much higher in the diabetic mouse brain compared to normoglycemic mouse brain at 12 h of reperfusion following transient MCAO [
52]. In another study, diabetic rats had an increased basal level of IL-1β and TNF-α, and inflammatory mediators COX-2 and inducible nitric oxide synthase (iNOS) expressions as compared to that of non-diabetic rats. Transient MCAO increased the gene expression of these cytokines and enzymes, which was remarkably accelerated and augmented by diabetes [
324]. Furthermore, this group showed increased expression of MPO and ICAM-1, which are hallmarks of neutrophil, and macrophage/microglia activation and exacerbation in the diabetic rat brain, indicating exacerbation of inflammatory responses in ischemic injury [
324]. Enhanced activation of NFκB in the diabetic brain mediated this increased production of proinflammatory cytokines and enzymes [
324]. NFκB is a potent inducer of inflammatory processes through its upregulation of the gene expression of proinflammatory cytokines and chemokines such as IL-1β, IL-6, interleukin-17 (IL-17), TNF-α, CRPs, MCP-1, CCL-2, and CXC [
325]. The transcription factor NFκB assumes a key role in cerebral ischemia and regulates apoptosis and inflammation [
326]. Thus, activation of NFκB is crucial for the inflammatory responses leading to gene expression of proinflammatory cytokines and mediators in immunocytes [
326]. Inhibition of NFκB represents a treatment strategy in ischemic stroke [
327].
Thus, the exacerbated inflammation might be a contributing factor to the increased post-stroke brain damage observed in the diabetic brain (Figs.
1 and
2). Furthermore, the macrophages and neutrophils release oxygen and nitrogen free radicals which are extremely toxic to neurons. Studies indicate that the extent of stroke-induced brain injury is influenced by the systemic inflammation. It has been shown that increased peripheral inflammation, at the time of stroke, aggravates ischemic injury [
328]. Diabetic mice are known to manifest systemic inflammation as well as impaired ability to curtail inflammation [
329]. Several proinflammatory proteins including MCP-1 and IL-6 are elevated in the plasma of diabetic patients [
330,
331]. The critical role of MCP-1 in the diabetic condition has been demonstrated in studies showing that its overexpression in adipocytes leads to tissue inflammation and insulin resistance, while mice deficient in MCP-1 or its receptor C-C motif chemokine receptor-2 (CCR-2) reverse the condition [
332‐
334]. More recently, Kim et al. [
335] demonstrated that in the diabetic condition, acute inflammatory responses are perturbed in the brain following stroke and in the macrophages after lipopolysaccharide stimulation, and these alterations are associated with the exacerbation of stroke-induced injury [
335]. Interestingly, diabetic mice were found to display reduced inflammatory cytokine expression and microglial activation, and delayed wound healing [
312]. Microglial activation and the release of chemokines and cytokines are critical steps in eliciting inflammatory responses. The inability to mount a proper host immune response immediately after cerebral ischemia in diabetic microglia causes an extended inflammatory phase, which leads to a prolonged infiltration of peripheral immune cells and worsened ischemic injury [
335]. The early blunted inflammatory response of MCP-1, IL-6, and CCR-2 in the diabetic mouse brain was reported at 6 h post ischemia [
335]. Collectively, the data from this study suggest that early inflammatory responses in the diabetic brain are deregulated, and the alteration is associated with the exacerbation of stroke-induced injury.
An attenuated stroke-induced inflammatory response has been demonstrated in diabetic conditions [
312,
313]. Treatment of obese diabetic mice with the peroxisome proliferator-activated receptor γ (PPARγ) agonist darglitazone, for 7 days before induction of hypoxia–ischemia, reduced infarct size and suppressed inflammatory response at 8 and 24 h after ischemia onset [
312,
313]. Animal studies have shown that MMP plays an important role in cerebrovascular damage following permanent focal stroke in diabetic rats [
336,
337]. A greater MMP-9 activity was found in diabetic rats following stroke [
307,
336].
HMGB-1 is a novel player in the ischemic brain [
215]. Diabetes significantly increased serum HMGB level and induced worse functional outcome after stroke compared to non-diabetic rats [
338]. Diabetes exacerbates systemic inflammation as evidenced by higher serum HMGB-1 in the rat systemic inflammation model [
339]. HMGB-1 signaling promotes chemotaxis and production of cytokines in a process that involves the activation of NFκB [
340]. Moreover, it has been reported that extracellular HMGB-1 is involved in BBB disruption during the early phase of ischemic stroke [
341]. Downregulation of HMGB-1 and NFκB expression protected rat brains against focal ischemia. Suppression of the release of HMGB-1 in astrocytes leads to the attenuation of neuroinflammation, preventing the necrosis of ischemic astrocytes and NFκB expression [
342]. Inhibition of the upregulation of HMGB-1 and NFκB at the early stage brings great benefits to cerebral ischemia.
Dysregulated expression of stromal cell-derived factor (SDF)-1α and CXCR-4 has been reported in the diabetic mice brain at baseline and following ischemic stroke [
343]. The SDF-1α/CXCR-4 axis is believed to play an important role in recruiting progenitor cells into ischemic tissue. It triggers many intracellular proliferation and anti-apoptosis signals, such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and the serine/threonine Kinase Akt [
344]. Therefore, SDF-1α/CXCR-4 is a potential target for promoting repair in wound and ischemic injury.
Overall, diabetes and hypoglycemia aggravates brain damage after ischemic stroke through enhancement of the neuroinflammatory signaling cascade, particularly by the activation of microglia/macrophages, leukocytes, adhesion molecules, upregulation/accumulation of some specific proinflammatory cytokines, MMPs, TLRs, and other immune mediators at the site of injury. All these immune mediators directly or indirectly contribute to further activation of cell death pathways (Figs.
1 and
2).