Increased cellular protein modification by methylglyoxal activates endoplasmic reticulum-based sensors of the unfolded protein response.

The unfolded protein response (UPR) detects increased misfolded proteins and activates protein refolding, protein degradation and inflammatory responses. UPR sensors in the endoplasmic reticulum, IRE1α and PERK, bind and are activated by proteins with unexpected surface hydrophobicity, whereas sensor ATF6 is activated by proteolytic cleavage when released from complexation with protein disulfide isomerases (PDIs). Metabolic dysfunction leading to the formation of misfolded proteins with surface hydrophobicity and disruption of ATF6-PDI complexes leading to activation of UPR sensors remains unclear. The cellular concentration of reactive dicarbonyl metabolite, methylglyoxal (MG), is increased in impaired metabolic health, producing increased MG-modified cellular proteins. Herein we assessed the effect of high glucose concentration and related increased cellular MG on activation status of IRE1α, PERK and ATF6. Human aortal endothelial cells and HMEC-1 microvascular endothelial cells were incubated in low and high glucose concentration to model blood glucose control, with increase or decrease of MG by silencing or increasing expression of glyoxalase 1 (Glo1), which metabolizes MG. Increased MG induced by high glucose concentration activated IRE1α, PERK and ATF6 and related downstream signalling leading to increased chaperone, apoptotic and inflammatory gene expression. Correction of increased MG by increasing Glo1 expression prevented UPR activation. MG modification of proteins produces surface hydrophobicity through arginine-derived hydroimidazolone MG-H1 formation, with related protein unfolding and preferentially targets PDIs and chaperone pathways for modification. It thereby poses a major challenge to proteostasis and activates UPR sensors. Pharmacological decrease of MG with Glo1 inducer, trans-resveratrol and hesperetin in combination, offers a novel treatment strategy to counter UPR-related cell dysfunction, particularly in hyperglycemia associated with diabetes.

The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Fatigue: It Is Not Always in the Head.

Fatigue is a common presenting complaint in patients seen in clinics and same-day emergency care. Although it has a simple presentation, it can be challenging to diagnose and manage, particularly when an underlying medical condition presents atypically as fatigue. Here we present an interesting case of giant cell arteritis (GCA) with only fatigue as the presenting complaint. GCA is the inflammation of medium and large vessels in the body, including the aortic arch and its branches. It typically manifests above the age of 50 with headaches, jaw claudication, temporal tenderness, arthralgia, night sweats, and unintentional weight loss. Early diagnosis and treatment are of paramount importance to prevent complications, particularly permanent blindness.

Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal.

Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20 mM versus 5 mM control) in vitro for 3-6 days. Flux of glucose metabolism, MG formation and glycation and changes in cytosolic protein abundances, MG modification and proteotoxic responses were assessed. Similar studies were performed with human microvascular endothelial HMEC-1 cells where similar outcomes were observed. HAECs exposed to high glucose concentration showed increased cellular concentration of MG (2.27 ± 0.21 versus 1.28 ± 0.03 pmol/106 cells, P < 0.01) and formation of MG-modified proteins (24.0 ± 3.7 versus 14.1 ± 3.2 pmol/106 cells/day; P < 0.001). In proteomics analysis, high glucose concentration increased proteins of the heat shock response - indicating activation of the unfolded protein response (UPR) with downstream inflammatory and pro-thrombotic responses. Proteins susceptible to MG modification were enriched in protein folding, protein synthesis, serine/threonine kinase signalling, glycolysis and gluconeogenesis. MG was increased in high glucose by increased flux of MG formation linked to increased glucose metabolism mediated by proteolytic stabilisation and increase of hexokinase-2 (HK-2); later potentiated by proteolytic down regulation of glyoxalase 1 (Glo1) - the major enzyme of MG metabolism. Silencing of Glo1, selectively increasing MG, activated the UPR similarly. Silencing of HK-2 prevented increased glucose metabolism and MG formation. trans-Resveratrol and hesperetin combination (tRES-HESP) corrected increased MG and glucose metabolism by increasing expression of Glo1 and decreasing expression of HK-2. Increased MG glycation activates the UPR in endothelial cells and thereby may contribute to endothelial cell dysfunction in diabetic vascular disease where tRES-HESP may provide effective therapy.

Hepatic VLDL secretion: DGAT1 determines particle size but not particle number, which can be supported entirely by DGAT2.

We investigated whether, in view of its activity being expressed on both aspects of the endoplasmic reticulum (ER; dual membrane topology), diacylglycerol acyltransferase 1 (DGAT1) plays a distinctive role in determining the triglyceride (TAG) content of VLDL particles secreted by the liver. Mice in which the DGAT1 gene was specifically ablated in hepatocytes (DGAT1-LKO mice) had the same number of VLDL particles (apoB concentration) in the plasma 1 h after Triton 1339 treatment, but these particles were approximately half the size of VLDL particles secreted by control mice and had a proportionately decreased content of TAG, with normal cholesterol and cholesteryl ester contents. Analyses of purified microsomal fractions prepared from 16 h fasted control and DAGT1-LKO mice showed that the TAG/protein ratio in the ER was significantly lower in the latter. Electron micrographs of these livers showed that those from DGAT1-LKO mice did not show the increased lipid content of the smooth ER shown by control livers. The effects of DGAT1- and DGAT2-specific inhibitors on apoB secretion by HepG2 cells showed that DGAT1 is not indispensable for apoB secretion and demonstrated redundancy in the ability of the two enzymes to support apoB secretion. Therefore, our findings show that DGAT1 is essential for the complete lipidation and maturation of VLDL particles within the lumen of the ER, consistent with its dual topology within the ER membrane. In the mouse, DGAT2 can support apoB secretion (particle number) even when TAG availability for full VLDL lipidation is restricted in the absence of DGAT1.

Diacylglycerol acyltransferase 2 links glucose utilization to fatty acid oxidation in the brown adipocytes.

Brown adipose tissue uptake of glucose and fatty acids is very high during nonshivering thermogenesis. Adrenergic stimulation markedly increases glucose uptake, de novo lipogenesis, and FA oxidation simultaneously. The mechanism that enables this concerted response has hitherto been unknown. Here, we find that in primary brown adipocytes and brown adipocyte-derived cell line (IMBAT-1), acute inhibition and longer-term knockdown of DGAT2 links the increased de novo synthesis of fatty acids from glucose to a pool of TAG that is simultaneously hydrolyzed, providing FA for mitochondrial oxidation. DGAT1 does not contribute to this pathway, but uses exogenous FA and glycerol to synthesize a functionally distinct pool of TAG to which DGAT2 also contributes. The DGAT2-dependent channelling of 14C from glucose into TAG and CO2 was reproduced in ß3-agonist-stimulated primary brown adipocytes. Knockdown of DGAT2 in IMBAT-1 affected the mRNA levels of UCP1 and genes important in FA activation and esterification. Therefore, in ß3-agonist activated brown adipocytes, DGAT2 specifically enables channelling of de novo synthesized FA into a rapidly mobilized pool of TAG, which is simultaneously hydrolyzed to provide substrates for mitochondrial fatty acid oxidation.

Differential effects of glyoxalase 1 overexpression on diabetic atherosclerosis and renal dysfunction in streptozotocin-treated, apolipoprotein E-deficient mice.

The reactive dicarbonyls, glyoxal and methylglyoxal (MG), increase in diabetes and may participate in the development of diabetic complications. Glyoxal and MG are detoxified by the sequential activities of glyoxalase 1 (GLO1) and glyoxalase 2. To determine the contribution of these dicarbonyls to the etiology of complications, we have genetically manipulated GLO1 levels in apolipoprotein E-null (Apoe(-/-)) mice. Male Apoe(-/-) mice, hemizygous for a human GLO1 transgene (GLO1TGApoe(-/-) mice) or male nontransgenic Apoe(-/-) litter mates were injected with streptozotocin or vehicle and 6 or 20 weeks later, aortic atherosclerosis was quantified. The GLO1 transgene lessened streptozotocin (STZ)-induced increases in immunoreactive hydroimidazolone (MG-H1). Compared to nondiabetic mice, STZ-treated GLO1TGApoe(-/-) and Apoe(-/-) mice had increased serum cholesterol and triglycerides and increased atherosclerosis at both times after diabetes induction. While the increased GLO1 activity in the GLO1TGApoe(-/-) mice failed to protect against diabetic atherosclerosis, it lessened glomerular mesangial expansion, prevented albuminuria and lowered renal levels of dicarbonyls and protein glycation adducts. Aortic atherosclerosis was also quantified in 22-week-old, male normoglycemic Glo1 knockdown mice on an Apoe(-/-) background (Glo1KDApoe(-/-) mice), an age at which Glo1KD mice exhibit albuminuria and renal pathology similar to that of diabetic mice. In spite of ~75% decrease in GLO1 activity and increased aortic MG-H1, the Glo1KDApoe(-/-) mice did not show increased atherosclerosis compared to age-matched Apoe(-/-) mice. Thus, manipulation of GLO1 activity does not affect the development of early aortic atherosclerosis in Apoe(-/-) mice but can dictate the onset of kidney disease independently of blood glucose levels.