Florin Despa

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca2+ handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Seru2009279 activates CaMKII autonomously, creating molecular memory even after Ca2+ concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca2+ release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

Neuroinflammation and neurologic deficits in diabetes linked to brain accumulation of amylin

BackgroundWe recently found that brain tissue from patients with type-2 diabetes (T2D) and cognitive impairment contains deposits of amylin, an amyloidogenic hormone synthesized and co-secreted with insulin by pancreatic β-cells. Amylin deposition is promoted by chronic hypersecretion of amylin (hyperamylinemia), which is common in humans with obesity or pre-diabetic insulin resistance. Human amylin oligomerizes quickly when oversecreted, which is toxic, induces inflammation in pancreatic islets and contributes to the development of T2D. Here, we tested the hypothesis that accumulation of oligomerized amylin affects brain function.MethodsIn contrast to amylin from humans, rodent amylin is neither amyloidogenic nor cytotoxic. We exploited this fact by comparing rats overexpressing human amylin in the pancreas (HIP rats) with their littermate rats which express only wild-type (WT) non-amyloidogenic rodent amylin. Cage activity, rotarod and novel object recognition tests were performed on animals nine months of age or older. Amylin deposition in the brain was documented by immunohistochemistry, and western blot. We also measured neuroinflammation by immunohistochemistry, quantitative real-time PCR and cytokine protein levels.ResultsCompared to WT rats, HIP rats show i) reduced exploratory drive, ii) impaired recognition memory and iii) no ability to improve the performance on the rotarod. The development of neurological deficits is associated with amylin accumulation in the brain. The level of oligomerized amylin in supernatant fractions and pellets from brain homogenates is almost double in HIP rats compared with WT littermates (Pu2009<u20090.05). Large amylin deposits (>50xa0μm diameter) were also occasionally seen in HIP rat brains. Accumulation of oligomerized amylin alters the brain structure at the molecular level. Immunohistochemistry analysis with an ED1 antibody indicates possible activated microglia/macrophages which are clustering in areas positive for amylin infiltration. Multiple inflammatory markers are expressed in HIP rat brains as opposed to WT rats, confirming that amylin deposition in the brain induces a neuroinflammatory response.ConclusionsHyperamylinemia promotes accumulation of oligomerized amylin in the brain leading to neurological deficits through an oligomerized amylin-mediated inflammatory response. Additional studies are needed to determine whether brain amylin accumulation may predispose to diabetic brain injury and cognitive decline.

Cardioprotection by Controlling Hyperamylinemia in a “Humanized” Diabetic Rat Model

Background Chronic hypersecretion of the pancreatic hormone amylin is common in humans with obesity or prediabetic insulin resistance and induces amylin aggregation and proteotoxicity in the pancreas. We recently showed that hyperamylinemia also affects the cardiovascular system. Here, we investigated whether amylin aggregates interact directly with cardiac myocytes and whether controlling hyperamylinemia protects the heart. Methods and Results By Western blot, we found abundant amylin aggregates in lysates of cardiac myocytes from obese patients, but not in controls. Aggregated amylin was elevated in failing hearts, suggesting a role in myocyte injury. Using rats overexpressing human amylin in the pancreas (HIP rats) and control myocytes incubated with human amylin, we show that amylin aggregation at the sarcolemma induces oxidative stress and Ca2+ dysregulation. In time, HIP rats developed cardiac hypertrophy and left‐ventricular dilation. We then tested whether metabolites with antiaggregation properties, such as eicosanoid acids, limit myocardial amylin deposition. Rats were treated with an inhibitor of soluble epoxide hydrolase, the enzyme that degrades endogenous eicosanoids. Treatment doubled the blood concentration of eicosanoids, which drastically reduced incorporation of aggregated amylin in cardiac myocytes and blood cells, without affecting pancreatic amylin secretion. Animals in the treated group showed reduced cardiac hypertrophy and left‐ventricular dilation. The cardioprotective mechanisms included the mitigation of amylin‐induced cardiac oxidative stress and Ca2+ dysregulation. Conclusions The results suggest blood amylin as a novel therapeutic target in diabetic heart disease and elevating blood levels of antiaggregation metabolites as a pharmacological strategy to reduce amylin aggregation and amylin‐mediated cardiotoxicity.

Intracellular Na+ Concentration ([Na+]i) Is Elevated in Diabetic Hearts Due to Enhanced Na+–Glucose Cotransport

Background Intracellular Na+ concentration ([Na+]i) regulates Ca2+ cycling, contractility, metabolism, and electrical stability of the heart. [Na+]i is elevated in heart failure, leading to arrhythmias and oxidative stress. We hypothesized that myocyte [Na+]i is also increased in type 2 diabetes (T2D) due to enhanced activity of the Na+–glucose cotransporter. Methods and Results To test this hypothesis, we used myocardial tissue from humans with T2D and a rat model of late-onset T2D (HIP rat). Western blot analysis showed increased Na+–glucose cotransporter expression in failing hearts from T2D patients compared with nondiabetic persons (by 73±13%) and in HIP rat hearts versus wild-type (WT) littermates (by 61±8%). [Na+]i was elevated in HIP rat myocytes both at rest (14.7±0.9 versus 11.4±0.7 mmol/L in WT) and during electrical stimulation (17.3±0.8 versus 15.0±0.7 mmol/L); however, the Na+/K+-pump function was similar in HIP and WT cells, suggesting that higher [Na+]i is due to enhanced Na+ entry in diabetic hearts. Indeed, Na+ influx was significantly larger in myocytes from HIP versus WT rats (1.77±0.11 versus 1.29±0.06 mmol/L per minute). Na+–glucose cotransporter inhibition with phlorizin or glucose-free solution greatly reduced Na+ influx in HIP myocytes (to 1.20±0.16 mmol/L per minute), whereas it had no effect in WT cells. Phlorizin also significantly decreased glucose uptake in HIP myocytes (by 33±9%) but not in WT, indicating an increased reliance on the Na+–glucose cotransporter for glucose uptake in T2D hearts. Conclusions Myocyte Na+–glucose cotransport is enhanced in T2D, which increases Na+ influx and causes Na+ overload. Higher [Na+]i may contribute to arrhythmogenesis and oxidative stress in diabetic hearts.

Intraneuronal Amylin Deposition, Peroxidative Membrane Injury and Increased IL-1β Synthesis in Brains of Alzheimer’s Disease Patients with Type-2 Diabetes and in Diabetic HIP Rats

Amylin is a hormone synthesized and co-secreted with insulin by pancreatic β-cells that crosses the blood-brain barrier and regulates satiety. Amylin from humans (but not rodents) has an increased propensity to aggregate into pancreatic islet amyloid deposits that contribute to β-cell mass depletion and development of type-2 diabetes by inducing oxidative stress and inflammation. Recent studies demonstrated that aggregated amylin also accumulates in brains of Alzheimers disease (AD) patients, preponderantly those with type-2 diabetes. Here, we report that, in addition to amylin plaques and mixed amylin-Aβ deposits, brains of diabetic patients with AD show amylin immunoreactive deposits inside the neurons. Neuronal amylin formed adducts with 4-hydroxynonenal (4-HNE), a marker of peroxidative membrane injury, and increased synthesis of the proinflammatory cytokine interleukin (IL)-1β. These pathological changes were mirrored in rats expressing human amylin in pancreatic islets (HIP rats) and mice intravenously injected with aggregated human amylin, but not in hyperglycemic rats secreting wild-type non-amyloidogenic rat amylin. In cultured primary hippocampal rat neurons, aggregated amylin increased IL-1β synthesis via membrane destabilization and subsequent generation of 4-HNE. These effects were blocked by membrane stabilizers and lipid peroxidation inhibitors. Thus, elevated circulating levels of aggregated amylin negatively affect the neurons causing peroxidative membrane injury and aberrant inflammatory responses independent of other confounding factors of diabetes. The present results are consistent with the pathological role of aggregated amylin in the pancreas, demonstrate a novel contributing mechanism to neurodegeneration, and suggest a direct, potentially treatable link of type-2 diabetes with AD.

Brain microvascular injury and white matter disease provoked by diabetes-associated hyperamylinemia

The brain blood vessels of patients with type 2 diabetes and dementia have deposition of amylin, an amyloidogenic hormone cosecreted with insulin. It is not known whether vascular amylin deposition is a consequence or a trigger of vascular injury. We tested the hypothesis that the vascular amylin deposits cause endothelial dysfunction and microvascular injury and are modulated by amylin transport in the brain via plasma apolipoproteins.

Amylin: what might be its role in Alzheimer’s disease and how could this affect therapy?

Amylin, an amyloidogenic hormone synthesized and co-secreted with insulin by pancreatic β-cells, has binding sites in the brain possibly regulating satiety and gastric emptying. It is elevated in obesity and pre-diabetic insulin resistance (hyper-amylinemia) leading to amylin amyloid deposition in the pancreas. Moreover, amylin deposition in pancreatic islets is an important source of oxidative and inflammatory stress leading to atrophy of pancreatic islets and development of type 2 diabetes (T2D). One possible mechanism of amylin accumulation in peripheral organs is through deposition of circulating amylin oligomers, which were found in both blood vessels and parenchyma of kidneys, heart and – as we have recently shown – brain. Hence, amylin amyloid infiltration in the brain may be an important contributor to cerebrovascular injury and neurodegeneration observed in demented humans. Treatment of hyperamylinemia or the consequent formation of circulating amylin oligomers, therefore, could be a feasible therapeutic target to protect the aging brain or slow neurodegenerative processes. n nNumerous epidemiological studies show significant associations between presumed T2D and risk for Alzheimer’s disease (AD) [1]. This increased risk extends to both obesity, the major risk factor for insulin resistance, and T2D [2]. Pathological studies indicate that dementia risk associated with T2D is independent of Alzheimer’s pathology and suggest that the increased dementia risk is likely due to vascular brain injury [3,4]. An independent study also showed increased AD pathology in T2D [5]. Brain imaging studies, however, demonstrate that the risk associated with dementia and T2D is independent of vascular disease [6]. At least one study found significant hippocampal atrophy suggesting that the association between diabetes and AD may involve shared pathophysiological processes [7]. Recent reviews point to possible pathological pathways whereby hyperinsulinemia may lead to increased AD pathology through altered cerebral clearance of amyloid β protein and hyperphosphorylation of τ [1,8]. As hypothesized by de la Monte, peripheral hyperinsulinemia is associated with impairments in cerebral glucose utilization through brain insulin and IGF resistance [8]. Impaired insulin and IGF signaling lead to increased amyloid precursor protein expression, increased amyloid β production and hyperphosphorylation of microtubule τ protein, which are the two hallmark pathologic features of AD. Similarly, increased insulin levels in the brain may saturate the brain insulin degrading enzyme system or reduce LRP-1 levels which are also mechanisms for amyloid β clearance [9,10]. Accumulation of amyloid β is hypothesized to be further enhanced by the presence of advanced glycation end-products that further impair amyloid β clearance from the brain. Intriguingly, there is no evidence that amyloid plaque is increased in the brain of diabetic patients [4,11]. Moreover, induced hyperinsulinemia in AD patients demonstrated memory improvement [12], suggesting that not the elevated insulin levels per se, but conditions secondary to hyperinsulinemia may play a significant role in the AD pathology in diabetics. One possible mechanism may involve elevated secretion of amylin (hyperamylinemia). Hyperamylinemia coincides with hyperinsulinemia [13] and induces amylin amyloid deposition and proteotoxicity not only in the pancreas (a hallmark of T2D) but also in other organs, including kidneys [14] and, as we have recently shown, in heart and brain [15,16]. n nAmylin, a 37-amino acid peptide, is synthesized and co-secreted with insulin by pancreatic β-cells [13]. Within the pancreas, amylin restrains insulin and glucagon secretion [13]. It also has binding sites in the brain, possibly regulating satiety and gastric emptying [13]. Human amylin hormone may lose its function by oligomerization. Amylin oligomization and deposition is common in patients with obesity and pre-diabetic insulin resistance who have an increased secretion of this hormone [13]. Over 95% of humans with T2D stain positive for amylin amyloid deposition in pancreatic islets, where it is believed to be cytotoxic [13]. Oligomerized amylin and amylin amyloid are also detected in vasculature and tissue parenchyma of failing hearts and kidneys from obese and T2D patients suggesting the possibility of a systemic effect [15]. In the brain, we identified amylin deposits in the blood vessels and parenchyma of AD patients [16]. Moreover, we found that amylin formed the core protein deposit of some amyloid plaques or co-localized with amyloid β as part of a combined plaque. Importantly, these findings occurred in AD patients who did not suffer with T2D. These preliminary findings suggest that amylin oligomerization may be a second form of amyloid involved in AD pathophysiology. In this article we hypothesize potential mechanisms whereby amylin may interact with the AD process to increase the likelihood of expressed dementia. These hypotheses also suggest potential new avenues for AD treatment that will be discussed.

Hyperamylinemia Increases IL-1β Synthesis in the Heart via Peroxidative Sarcolemmal Injury.

Hypersecretion of amylin is common in individuals with prediabetes, causes amylin deposition and proteotoxicity in pancreatic islets, and contributes to the development of type 2 diabetes. Recent studies also identified amylin deposits in failing hearts from patients with obesity or type 2 diabetes and demonstrated that hyperamylinemia accelerates the development of heart dysfunction in rats expressing human amylin in pancreatic β-cells (HIP rats). To further determine the impact of hyperamylinemia on cardiac myocytes, we investigated human myocardium, compared diabetic HIP rats with diabetic rats expressing endogenous (nonamyloidogenic) rat amylin, studied normal mice injected with aggregated human amylin, and developed in vitro cell models. We found that amylin deposition negatively affects cardiac myocytes by inducing sarcolemmal injury, generating reactive aldehydes, forming amylin-based adducts with reactive aldehydes, and increasing synthesis of the proinflammatory cytokine interleukin-1β (IL-1β) independently of hyperglycemia. These results are consistent with the pathological role of amylin deposition in the pancreas, uncover a novel contributing mechanism to cardiac myocyte injury in type 2 diabetes, and suggest a potentially treatable link of type 2 diabetes with diabetic heart disease. Although further studies are necessary, these data also suggest that IL-1β might function as a sensor of myocyte amylin uptake and a potential mediator of myocyte injury.

Hyperamylinemia as a risk factor for accelerated cognitive decline in diabetes.

Type II diabetes increases the risk for cognitive decline via multiple traits. Amylin is a pancreatic hormone that has amyloidogenic and cytotoxic properties similar to the amyloid-β peptide. The amylin hormone is overexpressed in individuals with pre-diabetic insulin resistance or obesity leading to amylin oligomerization and deposition in pancreatic islets. Amylin oligomerization was implicated in the apoptosis of the insulin-producing β-cells. Recent studies showed that brain tissue from diabetic patients with cerebrovascular dementia or Alzheimer’s disease contains significant deposits of oligomerized amylin. It has also been reported that the brain amylin deposition reduced exploratory drive, recognition memory and vestibulomotor function in a rat model that overexpresses human amylin in the pancreas. These novel findings are reviewed here and the hypothesis that type II diabetes is linked with cognitive decline by amylin accumulation in the brain is proposed. Deciphering the impact of hyperamylinemia on the brain is critical for both etiology and treatment of dementia.

The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells

Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.