Glycemic variability persistent oxidative stress, and diabetic complications

by A. Ceriello, Spain and Italy

Institut d’Investigacions
Biomèdiques August Pi i Sunyer
(IDIBAPS) and Spanish Biomedical
Research Centre in Diabetes and
Associated Metabolic Disorders
(CIBERDEM), Barcelona, SPAIN
Department of Cardiovascular
and Metabolic Diseases
IRCCS Multimedica
Sesto San Giovanni
Milan, ITALY

Several studies show that lowering glycated hemoglobin (HbA1c) reduces the risk of both micro- and macrovascular complications in both type 1 and type 2 diabetes. However, evidence suggests that targeting HbA1c may not always result in improved outcomes. Recent findings confirm that chronic hyperglycemia contributes to complications of diabetes, but also that variability in blood glucose levels may strongly influence the development of complications. While hyperglycemia induces diabetic complications that produce oxidative stress, in vitro and in vivo studies show that glucose excursions can induce even more free radical overgeneration than chronically high glucose levels, suggesting that oxidative stress also plays a key role in inducing the complications linked to glucose variability. Targeting of glucose variability therefore seems to be the next challenge for optimal prevention of diabetic complications.

In diabetes “dysglycemia” is defined as the cluster of glucose variability and fasting and postprandial hyperglycemia: all these 3 parameters contribute to the value of glycated hemoglobin (HbA1c).1

HbA1c is considered the “golden” biomarker of overall glycemic exposure, but significant glycemic fluctuations may occur at the same HbA1c value.2 Like postprandial hyperglycemia (PPH),3 glucose variability4 has been proposed as an independent risk factor for cardiovascular disease, in people with or without diabetes. Glucose variability is a heterogeneous concept difficult to define and measure; most often it is defined as5:
• Within-day glucose variability
• Standard deviation of glucose values (SDBG) in a certain period of time
• Between-day variability of fasting plasma glucose (FPG)
• Postprandial peaks
• Variability of HbA1c over time
• Hypoglycemic episodes

As it represents the average of all these variables, HbA1c cannot clarify past or future hypoglycemic events, recovery with or without hyperglycemia, episodes of PPH, or glucose fluctuations. Consequently, an ideal value of HbA1c might comprise glycemic swings of different amplitudes. This could explain the lack of benefit of tight versus standard glucose control in type 2 diabetes (T2D) with relatively high baseline car- diovascular risk, as well as the persistent excess mortality in type 1 diabetes (T1D) despite good glucose control.6 Hypoglycemia is a serious complication of diabetes treatment. Any episode of hypoglycemia, either isolated or recurrent, is a component of glucose variability. Hypoglycemia is a well-known risk factor for cardiovascular disease.7 Hyperglycemia following hypoglycemia has an ischemia-reperfusion−like effect and is a further cardiovascular disease risk factor.8,9

Large prospective clinical studies in T1D and T2D have reported a clear relationship between time-averaged mean levels of blood glucose (measured by HbA1c) and diabetes complications10 Also, glycemic variability might be linked to the development of diabetes complications.11

Epidemiological evidence in diabetes

The DCCT trial (Diabetes Control and Complications Trial) showed that variability of HbA1c, more than that of glucose, is a predictor of microvascular complications.<sup<12 In another study, 100 T1D patients were followed up for a period of about 11 years.13 Glucose variability (defined as SDBG) was calculated from 70 self-monitored measurements taken over a period of four weeks. This study showed that HbA1c was an independent predictor of the incidence (P=0.004) and prevalence (P=0.01) of nephropathy, while SDBG was found to be a highly significant predictor of hypoglycemia unawareness (P=0.001) and of the prevalence of peripheral neuropathy (borderline value, P=0.07). Based on these data, the authors concluded that glucose variability may be important in the development of peripheral neuropathy in T1D patients, and that the nervous system may be particularly vulnerable to glycemic variability.13

Muggeo et al found that mortality from all causes14 and from cardiovascular disease15 in elderly T2D patients was mainly correlated with the variability/instability of FPG rather than with its absolute values. This outcome has been confirmed in a large cohort of more than 5000 T2D patients,16 and, more recently, in the post-hoc analysis of the ADVANCE study (Action in Diabetes and Vascular disease: PreterAx and DiamicroN MR Controlled Evaluation).17

Basic scientific evidence

In conditions of hyperglycemia, in cells that are insulin-independent and unable to decrease the rate of glucose transport, like the endothelial cells, persistent hyperglycemia is produced18 and is followed by high production of free radicals. 19,20 It has been suggested that oxidative stress, in particular increased superoxide production in the mitochondria, is the key link between hyperglycemia and diabetic complications.11

Produced in excess, free radicals cascade down four main pathways, thus explaining the microvascular complications of diabetes: increased production of advanced glycation endproducts, activation of the protein kinase C isoforms, the polyol pathway, and the hexosamine pathway.19,20 Also, free radicals in excess activate other pathways, which in turn lead to endothelial dysfunction.20 Evidence suggests that the same phenomenon, the generation of oxidative stress, underlines the deleterious effect of oscillating glucose.11

Glycemic variability has been investigated in different cell lines and also in animal studies. It has a deleterious effect on renal cells (mesangial and tubulointerstitial), umbilical endothelial cells, and pancreatic β cells.11 Also, apoptotic cell death is increased in β cells and endothelial cells cultured in fluctuating glucose, as compared with continuous high glucose.11 Remarkably, increased expression of fibrogenesis markers in human renal cortical fibroblasts is dependent on high glucose “peaks,” but is independent of the total amount of glucose to which cells are exposed.11

Short-term glucose oscillations induce oxidative stress and, consequently, long-term pro-atherogenic, epigenetic, and gene expression changes, which persist later in conditions of normoglycemia.21,22 Short-term high glucose induces metabolic memory after glucose normalization and has a more deleterious effect than constant high glucose.23 Experimental studies using antioxidant treatments support the link between metabolic memory and oxidative stress.24,25

It should be stressed that apart from the increased production of reactive species following hyperglycemia, the antioxidant defense is of great significance in the development of diabetes complications. It has been shown that defective intracellular defense against these reactive oxygen and nitrogen species determines organ damage and therefore, specific diabetes complications.26,27 In oscillating glucose conditions, cells are unable to increase their own intracellular antioxidant defenses sufficiently, thereby promoting even more oxidative damage than in constant high glucose.28

In animal studies, it has been demonstrated that acute high glucose determines monocyte-endothelial adhesion when compared with continuous hyperglycemia.11 Also, acute oscillations in blood glucose concentrations in atherogenic-prone mice fed maltose enhanced macrophage adhesion to endothelial cells and the formation of fibrotic arteriosclerotic lesions.11 When the glucose fluctuations were reduced, monocyte-endothelial adhesion decreased.11

In another study,29 significant endothelial cell apoptosis and dysfunction were observed in the aorta of the acute blood glucose fluctuation group, in which there were reduced B cell lymphoma-2 and pro caspase-3 levels and enhanced B cell lymphoma 2−associated X protein mitochondrial translocation and caspase-3 p17 protein levels, in comparison with the constant high glucose group. Also observed in the acute blood glucose fluctuation group were increased malondialdehyde and 8-isoprostaglandin levels in plasma, oxidative stress in vascular endothelial cells, and inflammatory cytokines in plasma and vascular endothelial cells.29

Moreover, hypoglycemia, which is part of the concept of glucose variability, induces oxidative stress, endothelial dysfunction, and inflammation, and activates thrombosis.8,9,30 Severe and moderate recurrent hypoglycemic events induced by insulin treatment determine oxidative stress and mitochondrial dysfunction, which contribute to selective neuronal damage.31 In neuronal cells and in animals, however, when hypoglycemia is rescued and hyperglycemia results, this generates an ischemia- reperfusion effect, leading to overgeneration of free radicals.32

In vivo evidence

In vivo, however, the situation seems to be even more complex. Prospective studies in T1D and T2D clearly established the role of oxidative stress in the progress of diabetic complications.33-35 It is of interest that in normal healthy controls and in subjects with diabetes, hyperglycemia, mainly acute, results in endothelial dysfunction and inflammation, and oxidative stress is the common denominator.36,37

In comparison with constant high plasma glucose, recurrent fluctuations of plasma glucose increase circulating inflammatory cytokines in subjects without diabetes, and worsen endothelial dysfunction in both controls and T2D patients.38 The use of an antioxidant treatment ameliorates this phenomenon, suggesting that oxidative stress plays a key role as a causal mechanism.37 It has been reported that daily glucose fluctuations in T2D are strong predictors of increased formation of reactive species.39 In patients who have had T2D for a short time and whose metabolic control is optimal, glucose variability has been associated with markers of endothelial and cardiovascular damage.28 Another study demonstrated that oxidative stress is linked to glucose variability and is also the only independent predictor of increased left ventricular mass.40 Oscillating glucose has more harmful effects than constant high glucose on endothelial function and oxidative stress, two key factors in favoring cardiovascular complications in diabetes.21

Furthermore, there is an ischemia-reperfusion−like effect when recovery from hypoglycemia is followed by hyperglycemia in humans.8 Oxidative stress emerges as the key mechanism in all the phenomena reported above, even though the detailed mechanism through which fluctuating glucose is harmful is only partly defined.


Alterations in glucose metabolism in individuals with diabetes have for many years been considered as they appear at first glance, ie, simply as hyperglycemia, and its surrogate marker HbA1c is used both to estimate the risk of developing diabetic complications and to define the targets and measure the efficacy of diabetes treatments. However, over time diabetes- related glycemic alterations have been considered in more complex terms, by attempting to identify the roles of FPG, postprandial glycemia, and hypoglycemia in the overall assessment of the disease. This set of evaluations has led to the concept of glucose variability.

Much attention has recently been paid to the possibility that oscillating glucose may superimpose on HbA1c levels in determining the risk for diabetic complications. Studies in vitro and in animals confirm that oscillating glucose is more dangerous than stable high glucose, particularly in activating the pathways involved in the pathogenesis of diabetic complications. The production of free radicals, accompanied by an insufficient increase of intracellular antioxidant defenses, seems to account for this phenomenon.

Human studies also confirm that fluctuations in glucose lead to an increase in free radicals, and to endothelial dysfunction, and that these radicals have a more deleterious impact than those produced under conditions of stable high glucose. Avoiding glucose fluctuations in diabetic patients seems to be an emerging challenge in the treatment of diabetes. ■

1. Monnier L, Colette C, Owens DR. Glycemic variability: the third component of the dysglycemia in diabetes. Is it important? How to measure it? J Diabetes Sci Technol. 2008;2:1094-1100.
2. Ceriello A. The glucose triad and its role in comprehensive glycaemic control: current status, future management. Int J Clin Pract. 2010:64:1705-1711.
3. Ceriello A. Postprandial hyperglycemia and diabetes complications: is it time to treat? Diabetes. 2005;54:1-7.
4. Ceriello A, Kilpatrick ES. Glycemic variability: both sides of the story. Diabetes Care. 2013;36(Suppl 2):S272-S275.
5. Frontoni S, Di Bartolo P, Avogaro A, et al. Glucose variability: an emerging target for the treatment of diabetes mellitus. Diabetes Res Clin Pract. 2013;102:86-95.
6. Ceriello A, Genovese S, Bosi E. The evolving frontier of diabetes therapy: The renaissance of glycemology. Diabetes Res Clin Pract. 2016;118:168-171.
7. Frier BM, Schernthaner G, Heller SR. Hypoglycemia and cardiovascular risks. Diabetes Care. 2011;34(suppl 2):S132-S137.
8. Ceriello A, Novials A, Ortega E, et al. Evidence that hyperglycemia after recovery from hypoglycemia worsens endothelial function and increases oxidative stress and inflammation in healthy control subjects and subjects with type 1 diabetes. Diabetes. 2012;61:2993-2997.
9. Ceriello A, Novials A, Ortega E, et al. Hyperglycemia following recovery from hypoglycemia worsens endothelial damage and thrombosis activation in type 1 diabetes and in healthy controls. Nutr Metab Cardiovasc Dis. 2014;24:116-123.
10. Ceriello A. Hyperglycaemia and the vessel wall: the pathophysiological aspects on the atherosclerotic burden in patients with diabetes. Eur J Cardio- vasc Prev Rehabil. 2010;17(suppl 1):S15-S19.
11. Ceriello A, Ihnat MA. ‘Glycaemic variability’: a new therapeutic challenge in diabetes and the critical care setting. Diabet Med. 2010;27:862-867.
12. Kilpatrick ES, Rigby AS, Atkin SL. A1C variability and the risk of microvascular complications in type 1 diabetes: data from the Diabetes Control and Complications Trial. Diabetes Care. 2008;31:2198-2202.
13. Bragd J, Adamson U, Bäcklund LB, Lins PE, Moberg E, Oskarsson P. Can glycaemic variability, as calculated from blood glucose self-monitoring, predict the development of complications in type 1 diabetes over a decade? Diabetes Metab. 2008;34:612-616.
14. Muggeo M, Verlato G, Bonora E, et al. Long-term instability of fasting plasma glucose predicts mortality in elderly NIDDM patients: the Verona Diabetes Study. Diabetologia. 1995;38:672-679.
15. Muggeo M, Verlato G, Bonora E, et al. Long-term instability of fasting plasma glucose, a novel predictor of cardiovascular mortality in elderly patients with non-insulin-dependent diabetes mellitus: The Verona Diabetes Study. Circulation. 1997;96:1750-1754.
16. Lin CC, Li CI, Yang SY, et al. of fasting plasma glucose: a predictor of mortality in patients with type 2 diabetes. Am J Med. 2012;125:416.e9-e18.
17. Hirakawa Y, Arima H, Zoungas S, et al. Impact of Visit-to-Visit Glycemic Variability on the Risks of Macrovascular and Microvascular Events and All-Cause Mortality in Type 2 Diabetes: TheADVANCETrial. Diabetes Care. 2014;37:2359-2365.
18. Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler Thromb Vasc Biol. 2004;24:816-823.
19. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414:813-820.
20. Ceriello A. New insights on oxidative stress and diabetic complications may lead to a “causal” antioxidant therapy. Diabetes Care. 2003;26:1589-1596.
21. Ceriello A, Esposito K, Piconi L, et al. Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients. Diabetes. 2008;57:1349-1354.
22. El-Osta A, Brasacchio D, Yao D, et al. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia. J Exp Med. 2008;205:2409-2417.
23. Schisano B, Tripathi G, McGee K, McTernan PG, Ceriello A. Glucose oscillations, more than constant high glucose, induce p53 activation and a metabolic memory in human endothelial cells. Diabetologia. 2011;54:1219-1226.
24. Inhat MA, Thorpe JE, Kamat CD, et al. Reactive oxygen species mediate a cellular « memory » of high glucose stress signalling. Diabetologia. 2007;50:1523- 1531.
25. Corgnali M, Piconi L, Inhat M, Ceriello A. Evaluation of gliclazide ability to attenuate the hyperglycaemic “memory” induced by high glucose in isolated human endothelial cells. Diabetes Metab Res Rev. 2008;24:301-309.
26. Ceriello A, Morocutti A, Mercuri F, et al. Defective intracellular antioxidant enzyme production in type 1 diabetic patients with nephropathy. Diabetes. 2000; 49:2170-2177.
27. Hovnik T, Dolzan V, Bratina NU, et al. Genetic polymorphisms in genes encoding antioxidant enzymes are associated with diabetic retinopathy in type 1 diabetes. Diabetes Care. 2009;32:2258-2262.
28. La Sala L, Cattaneo M, De Nigris V, et al. Oscillating glucose induces microRNA- 185 and impairs an efficient antioxidant response in human endothelial cells. Cardiovasc Diabetol. 2016;15:71.
29. Wu N, Shen H, Liu H, Wang Y, Bai Y, Han P. Acute blood glucose fluctuation enhances rat aorta endothelial cell apoptosis, oxidative stress and pro-inflammatory cytokine expression in vivo. Cardiovasc Diabetol. 2016;15:109.
30. Desouza CV, Bolli GB, Fonseca V. Hypoglycemia, diabetes, and cardiovascular events. Diabetes Care. 2010;33:1389-1394.
31. Languren G, Montiel T, Julio-Amilpas A, Massieu L. Neuronal damage and cognitive impairment associated with hypoglycemia: An integrated view. Neurochem Int. 2013;63:331-343.
32. Suh SW, Gum ET, Hamby AM, Chan PH, Swanson RA. Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase. J Clin Invest. 2007;117:910-918.
33. Costacou T, Evans RW, Schafer GL, et al. Oxidative stress and response in relation to coronary artery disease in type 1 diabetes. Diabetes Care. 2013;36: 3503-3509.
34. Broedbaek K, Siersma V, Henriksen T. Urinary markers of nucleic acid oxidation and long term mortality of newly diagnosed Type 2 diabetic patients. Diabetes Care. 2011;34:2594-2596.
35. Broedbaek K, Siersma V, Henriksen T, et al. Association between urinary markers of nucleic acid oxidation and mortality in type 2 diabetes: a populationbased cohort study. Diabetes Care. 2013;36:669-676.
36. Ceriello A. Acute hyperglycaemia and oxidative stress generation. Diabet Med. 1997;14:S45-S49.
37. Ceriello A, Testa R. Antioxidant anti-inflammatory treatment in Type 2 diabetes. Diabetes Care. 2009;32:S232-S236.
38. Esposito K, Nappo F, Marfella R, et al. Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. Circulation. 2002;106:2067-2072.
39. Monnier L, Mas E, Ginet C, et al. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA. 2006;295:1681-1687.
40. Di Flaviani A, Picconi F, Di Stefano P, et al. Impact of glycemic and blood pressure variability on surrogate measures of cardiovascular outcomes in type 2 diabetic patients. Diabetes Care. 2011;34:1605-1609.

Keywords: diabetes; diabetes complication; glucose variability; oxidative stress