Agnieszka Jankowska-Kulawy

Glucose is the principal energy substrate for the brain. Hypo- and hyperglycemic episodes frequently occur in senescent people, contributing to functional and structural impairment of brain neurons and causing cognitive deficits in this population. In this study, we investigate whether long-term changes in the extracellular concentration of glucose affect viability and transmitter functions of septum-derived SN56 cholinergic neuronal cells through alterations in acetyl-CoA availability. Cells with low cholinergic expression (NCs) and cAMP/retinoic acid-induced high cholinergic expression (DCs) were investigated. Hypoglycemia brought about similar (approximately 20–30%) decreases in pyruvate dehydrogenase complex (PDHC) and ATP-citrate lyase (ACLY) activities and a 65% decline in lactate dehydrogenase (LDH) activity in NCs and DCs. Choline acetyltransferase (ChAT) and LDH activities in DCs were about 3–8 and 1.7–2.4 times higher than in NCs over the tested glucose concentration range, respectively. DCs appeared to be more resistant than NCs to hypoglycemia, as evidenced by lower glucose IC₅₀ values for cell count and intracellular LDH activity. On the other hand, some of functional properties of DCs, such as the cholinergic phenotype and their plasma membrane functions (trypan blue exclusion, TB+), were found to be more sensitive to hypoglycemia than those of NCs, as demonstrated by the higher IC₅₀ for glucose in DCs. Acetyl-CoA levels in DCs were 40% lower than in NCs, and decreased by about 25% with increasing hypoglycemia in both cell types. The cytotoxic effects of amyloid-β_25–35 (Aβ) and sodium nitroprusside (SNP; NO generator) were also tested. In 25 mM glucose medium, these toxic compounds exerted greater detrimental effects on DCs than on NCs. In contrast, in 1 mM glucose, more evident cytotoxicity of SNP and Aβ was observed in NCs. These data suggest that the higher rate of glycolysis in differentiated cholinergic septal neurons may be a protective mechanism against hypoglycemia.

Glucose is a principal energy substrate for the brain. Hypo- and hyperglycemic episodes are frequent in senescent people thereby contributing to functional and structural impairment of brain neurons, yielding cognitive deficits in this population. In this report we investigate whether long-term changes in extracellular concentration of glucose may affect viability and transmitter functions of septum-originated SN56 cholinergic neuronal cells through alterations in their acetyl-CoA availability. Cell phenotypes with low (NC) and cAMP/retinoic acid in-duced, high expression cholinergic phenotype (DC) were investigated. Hypoglycemia brought about similar about 20-30% decreases of in pyruvate dehydrogenase complex (PDHC) and ATP-citrate lyase (ACLY) and 65% decline of lactate dehydrogenase (LDH) activities in NC and DC, respectively. Choline acetyltransferase (ChAT) and LDH activities in DC were about 3 to 8 and 1.7 to 2.4 times higher than in NC, over the entire range of glucose concen-trations, respectively. In effect DC appeared to be more resistant than NC to hypoglycemia, as evidenced by lower values of [IC50] of glucose against cell count and intracellular LDH, re-spectively. On the other hand, some of functional properties of DC such as cholinergic phe-notype (ChAT) and plasma membrane function (trypan blue exclusion, TB+) were found to be more prone to hypoglycemia than in NC, as demonstrated by higher respective [IC50] for glu-cose in DC. Acetyl-CoA levels in DC were 40% lower than those in NC, and both decreased parallelly with deepening hypoglycemia by about 25%. Cytotoxic effects of amyloid-β25-35 (Aβ) and sodium nitroprusside (NO generator-SNP) in those conditions were tested. In 25 mM glucose medium these toxic compounds in DC exerted greater detrimental effects than in NC. On the contrary, in 1 mM glucose more evident cytotoxicity of SNP and Aβ was ob-served in NC. These data may indicate that higher rate of glycolysis in differentiated cholin-ergic septal neurons may establish a protective mechanism against hypoglycemia.

T cells play an important role in the development and progression of type 1 diabetes (T1D). Checkpoint receptors regulate T cell activity, and their expression may be linked to the cells’ metabolic state. This study aims to investigate the association between T regulatory (Treg) and T conventional (Tconv) cells expressing various checkpoint inhibitors and glucose metabolism in type 1 diabetes patients and healthy controls (HCs). The study included 28 participants, with 16 of them diagnosed with type 1 diabetes, while 12 constituted a healthy control group. Multicolor flow cytometry, spectrophotometric analysis, and bead-based multiplex assays were utilized for the analyses. The study revealed that the most significant difference in T cell subsets in peripheral blood concerned TIGIT. Compared to healthy subjects, the percentages of TIGIT+ Tregs and TIGIT+ Tconvs were lower in T1D patients. Interestingly, hyperglycemia in in vitro cultures reduced percentages of TIGIT+ Tregs and TIGIT+ Tconvs, and to some extent also CTLA-4+ Tregs. A decreased percentage of these subsets was, in turn, associated with reduced glucose uptake and lower activity of the enzymes responsible for various stages of glucose metabolism. The described associations suggest a negative influence of hyperglycemia in T1D on immune regulation via a TIGIT-dependent mechanism. Hyperglycemia seems to reduce the percentage of highly regulatory TIGIT+ Tregs both in vivo and in vitro, and it is associated with reduced glucose consumption by these cells. At the same time, a reduction in the percentage of TIGIT+ Tconvs under such conditions may facilitate higher activity of Tconvs, including aberrant autoimmune reactions.

Glycolysis-derived pyruvate is the almost exclusive source of acetyl-CoA for energy production in mitochondrial compartments of all types of neuronal and glial cells. Neurons utilize several times more glucose than glial cells due to their neurotransmitter functions. Cholinergic neurons that are responsible for cognitive functions require additional amounts of acetyl-CoA for acetylcholine-transmitter synthesis in their cytoplasmic compartment. It may be assured by preferential localization of ATP-citrate lyase (ACLY) in the cytoplasm of cholinergic neurons’ perikaryons and axonal terminals. This thesis is supported by the existence of strong regional and developmental correlations of ATP-citrate lyase and choline acetyltransferase (ChAT) activities and ACh levels in the brain. Electrolytic or chemical lesions of cholinergic nuclei cause proportional loss of the above parameters in the respective cortical target areas. On the other hand, the regional activity of mitochondrial pyruvate dehydrogenase complex (PDHC), which synthesizes nearly the whole pool of neuronal acetyl-CoA, displays no correlation with cholinergic innervation. It makes cholinergic neurons highly susceptible to brain pathologies impairing energy metabolism. Therefore, the ACLY pathway, which provides acetyl units directly to the site of acetylcholine synthesis in cholinergic nerve terminals, plays a key role in the maintenance of cholinergic neurotransmission. On the other hand, in cholinergic motor neurons, various ACLY–protein complexes are involved not only in neurotransmission but also in axonal transport of cholinergic elements from the perikaryon to cholinergic neuro-muscular junctions. This review presents findings supporting this thesis.

Mild thiamine deficiency aggravates Zn accumulation in cholinergic neurons. It leads to the augmentation of Zn toxicity by its interaction with the enzymes of energy metabolism. Within this study, we tested the effect of Zn on microglial cells cultivated in a thiamine-deficient medium, containing 0.003 mmol/L of thiamine vs. 0.009 mmol/L in a control medium. In such conditions, a subtoxic 0.10 mmol/L Zn concentration caused non-significant alterations in the survival and energy metabolism of N9 microglial cells. Both activities of the tricarboxylic acid cycle and the acetyl-CoA level were not decreased in these culture conditions. Amprolium augmented thiamine pyrophosphate deficits in N9 cells. This led to an increase in the intracellular accumulation of free Zn and partially aggravated its toxicity. There was differential sensitivity of neuronal and glial cells to thiamine-deficiency–Zn-evoked toxicity. The co-culture of neuronal SN56 with microglial N9 cells reduced the thiamine-deficiency–Zn-evoked inhibition of acetyl-CoA metabolism and restored the viability of the former. The differential sensitivity of SN56 and N9 cells to borderline thiamine deficiency combined with marginal Zn excess may result from the strong inhibition of pyruvate dehydrogenase in neuronal cells and no inhibition of this enzyme in the glial ones. Therefore, ThDP supplementation can make any brain cell more resistant to Zn excess.

The human brain is characterised by the most diverse morphological, metabolic and functional structure among all body tissues. This is due to the existence of diverse neurons secreting various neurotransmitters and mutually modulating their own activity through thousands of pre- and postsynaptic interconnections in each neuron. Astroglial, microglial and oligodendroglial cells and neurons reciprocally regulate the metabolism of key energy substrates, thereby exerting several neuroprotective, neurotoxic and regulatory effects on neuronal viability and neurotransmitter functions. Maintenance of the pool of mitochondrial acetyl-CoA derived from glycolytic glucose metabolism is a key factor for neuronal survival. Thus, acetyl-CoA is regarded as a direct energy precursor through the TCA cycle and respiratory chain, thereby affecting brain cell viability. It is also used for hundreds of acetylation reactions, including N-acetyl aspartate synthesis in neuronal mitochondria, acetylcholine synthesis in cholinergic neurons, as well as divergent acetylations of several proteins, peptides, histones and low-molecular-weight species in all cellular compartments. Therefore, acetyl-CoA should be considered as the central point of metabolism maintaining equilibrium between anabolic and catabolic pathways in the brain. This review presents data supporting this thesis.

Chronic hyperglycemia contributes to vascular complications in diabetes. Resveratrol exerts anti-diabetic and anti-platelet action. This study aimed to evaluate the effects of resveratrol on metabolism and the function of blood platelets under static and in in vitro flow conditions in patients with type 2 diabetes. Blood obtained from 8 healthy volunteers and 10 patients with type 2 diabetes was incubated with resveratrol and perfused over collagen-coated capillaries. Isolated blood platelets were incubated with resveratrol and activated by collagen to assess platelet function, metabolism, ATP release, TXA₂ production, lipid peroxidation, and gluthatione content. In the type 2 diabetes group, plasma glucose and fructosamine concentrations were significantly higher than in the healthy group. In in vitro studies, collagen-induced thrombi formation in the blood of diabetic patients was 33% higher than in the healthy group. Resveratrol reduced thrombi by over 50% in the blood of healthy and diabetic patients. TXA₂ production was 47% higher in diabetic platelets than in the healthy group. Resveratrol reduced TXA₂ release by 38% in healthy platelets and by 79% in diabetic platelets. Resveratrol also reduced the activities of enzymes responsible for glycolysis and oxidative metabolism in the platelets of both groups. These data indicate that the resveratrol-induced inhibition of platelet metabolism and TXA₂ release may lead to a reduction of platelet function and thrombus formation in patients with type 2 diabetes. Therefore, resveratrol may be beneficial to prevent vascular complications as a future complementary treatment in aspirin-resistant diabetic patients.

Brain pathologies evoked by thiamine deficiency can be aggravated by mild zinc excess. Cholinergic neurons are the most susceptible to such cytotoxic signals. Sub-toxic zinc excess aggravates the injury of neuronal SN56 cholinergic cells under mild thiamine deficiency. The excessive cell loss is caused by Zn interference with acetyl-CoA metabolism. The aim of this work was to investigate whether and how astroglial C6 cells alleviated the neurotoxicity of Zn to cultured SN56 cells in thiamine-deficient media. Low Zn concentrations did not affect astroglial C6 and primary glial cell viability in thiamine-deficient conditions. Additionally, parameters of energy metabolism were not significantly changed. Amprolium (a competitive inhibitor of thiamine uptake) augmented thiamine pyrophosphate deficits in cells, while co-treatment with Zn enhanced the toxic effect on acetyl-CoA metabolism. SN56 cholinergic neuronal cells were more susceptible to these combined insults than C6 and primary glial cells, which affected pyruvate dehydrogenase activity and the acetyl-CoA level. A co-culture of SN56 neurons with astroglial cells in thiamine-deficient medium eliminated Zn-evoked neuronal loss. These data indicate that astroglial cells protect neurons against Zn and thiamine deficiency neurotoxicity by preserving the acetyl-CoA level.

Objectives: Zinc excitotoxicity and thiamine pyrophosphate deficiency (TD) are known pathogenic signals contributing to mechanism of different encephalopathies through inhibition of enzymes responsible for energy metabolism such as pyruvate dehydrogenase, aconitase or ketoglutarate dehydrogenase. The aim of this work was to investigate whether subclinical Zn excess and TD, frequent in aging brain, may combine yielding overt neuronal impairment. Results: Clonal SN56 cholinergic neuronal cells of septal origin were used as the model of brain cholinergic neurons, which are particularly susceptible to neurodegeneration in the course of Alzheimer’s disease, hypoxia and other dementia-linked brain pathologies. Neither subtoxic concentration of Zn (0.10 mM) nor mild 20–25% TD deficits alone caused significant negative changes in cultured cholinergic neurons viability and their acetyl-CoA/acetylcholine metabolism. However, cells with mild TD accumulated Zn in excess, which impaired their energy metabolism causing a loss of neurons viability and their function as neurotransmitters. These negative effects of Zn were aggravated by amprolium which is an inhibitor of thiamine intracellular transport. Conclusion: Our data indicate that TD may amplify otherwise non-harmful border-line Zn excitotoxic signals yielding progress of neurodegeneration.