Changes in HbA1c during the first six years after the diagnosis of Type 2 diabetes mellitus predict long-term microvascular outcomes.

To analyze the association between change in HbA1c during the first 6 years after diagnosis of Type 2 diabetes mellitus (Type 2 DM) and incident micro- and macrovascular morbidity and mortality during 13 years thereafter. This is an observational study of the participants in the intervention arm of the randomized controlled trial Diabetes Care in General Practice (DCGP) in Denmark. 494 newly diagnosed persons with Type 2 DM aged 40 years and over with three or more measurements of HbA1c during six years of intervention were included in the analyses. Based on a regression line, fitted through the HbA1c-measurements from 1 to 6 years after diabetes diagnosis, glycaemic control was characterized by the one-year level of HbA1c after diagnosis, and the slope of the regression line. Outcomes were incident diabetes-related morbidity and mortality from 6 to 19 years after diabetes diagnosis. The association between change in HbA1c (the slope of the regression line) and clinical outcomes were assessed in adjusted Cox regression models. The median HbA1c level at year one was 60 (IQR: 52-71) mmol/mol or (7.65 (IQR: 6.91-8.62) %). Higher HbA1c levels one year after diagnosis were associated with a higher risk of later diabetes-related morbidity and mortality. An increase in HbA1c during the first 6 years after diabetes diagnosis was associated with later microvascular complications (HR per 1.1 mmol/mol or 0.1% point increase in HbA1c per year; 95% CI) = 1.14; 1.05-1.24). Change in HbA1c did not predict the aggregate outcome 'any diabetes-related endpoint, all-cause mortality, diabetes-related mortality, myocardial infarction, stroke, or peripheral vascular diseases. We conclude that suboptimal development of glycaemic control during the first 6 years after diabetes diagnosis was an independent risk factor for microvascular complications during the succeeding 13-year follow-up, but not for mortality or macrovascular complications.

RCT Entities:   Population Interventions Outcomes

Background

In general, persons with Type 2 diabetes mellitus (Type 2 DM) have an increased cardiovascular morbidity and mortality [1, 2], and an elevated haemoglobin A1c (HbA1c) increases the risk of both micro- and macrovascular complications in these persons [3-5]. Randomized controlled trials have shown that treatment with anti-diabetic medication lowers the risk of complications[6]. It is unknown if the lower risk associated with anti-diabetic treatment is fully attributable to the lowering of HbA1c levels [7], or that other pleiomorphic factors, such as the pharmaceutical properties of the anti-diabetic drugs, account for this effect.

The Diabetes Care in General Practice (DCGP) study was a cluster randomized controlled trial assessing the effect on mortality and morbidity of structured personal care compared with routine care in a population-based sample of persons newly diagnosed with clinical Type 2 DM in Denmark [8]. After six years, the structured personal care group had improved glycaemia, blood pressure, total cholesterol, and microalbuminuria. There were no differences in terms of morbidity and mortality between the two groups [8]. However, a 19-year follow-up of the DCGP study showed that participants in the intervention group had a lower incidence of myocardial infarction and ‘any diabetes-related outcome’, while mortality rate was similar between the two groups [9]. The intervention group displayed a large inter-individual variation in HbA1c [10]. On average, an initial drop in HbA1c was observed, followed by an increase during the remaining five years of the intervention period. Only age and HbA1c at time of diagnosis were predictive of the variation in HbA1c during follow-up [10].

Earlier studies have used a single HbA1c measurement [5] or an average of several HbA1c measurements [11] to predict later outcomes. In the present confirmatory study, the aim was to determine the relation between changes in HbA1c during 6 years of intervention in persons with newly diagnosed Type 2 DM and any diabetes-related endpoint, all-cause mortality, diabetes-related mortality, myocardial infarction, stroke, peripheral vascular disease and microvascular disease during the 13 years thereafter.

Participants and methods

Study design and population

This is a cohort study of the intervention group from the DCGP study, a cluster randomized controlled trial. Informed consent was obtained from all participants. The protocol was in accordance with the Helsinki declaration and was approved by the ethics committee of Copenhagen and Frederiksberg (V.100.869/87). All data were fully anonymized before they were accessed. In 1988, 474 general practitioners volunteered to take part in the study. These were randomized to structured personal care or routine care [8]. All doctors were to include all participants aged 40 years or over with newly diagnosed diabetes (Fig 1). The treatment guidelines in the intervention group included follow-up every three months and individualized goal setting for each participant supported by prompting of doctors, printed guidelines, feedback, and continuing medical education. The control group was not included in the present analysis, as yearly HbA1c measurements were performed only in the intervention group. Hence, this study is confined to the 536 participants who were alive and were examined at the end of the intervention 6 years after the diabetes diagnosis. Participants with less than three HbA1c measurements between 1 year after diagnosis and 6-year follow-up were excluded. The final study sample included 494 participants (Fig 1). As only 2.5% of the cohort started insulin treatment within 180 days after diagnosis, we consider that 97.5% had Type 2 DM [12].

Fig 1

Flow chart depicting the number of participants in the Diabetes Care in General Practice (DCGP) study included in the current study.

Definition of glycaemic control (determinants)

At the time of diabetes diagnosis and at the annual examinations, the fraction of HbA1c was determined by the same ion-exchange, high-performance liquid chromatography method with a relatively high reference interval (5.4–7.4% (36–57 mmol/mol)) at Odense University Hospital [8]. In general, participants experienced an improved glycemic control with a more or less pronounced remission of hyperglycaemia during the first year after diabetes diagnosis [10, 13]. To determine the individual development in glycaemic control, we fitted linear regression lines to the HbA1c measurements from one to (on average) six years after the diagnosis for each participant individually. Glycaemic control was then characterized for each individual participant from this regression line, firstly, by the one-year intercept of the regression line (“one-year HbA1c level”) and, secondly, as the slope of the regression line (“yearly HbA1c change”) [10] (see Fig 2).

Fig 2

Graphic depiction of the covariate LEVEL at year one of the 6-year intervention period after diagnosis and the SLOPE of the change in HbA1c from year 2 through 6 year of the intervention period.

Definition of outcomes

The outcomes were all-cause mortality, diabetes-related mortality (i.e. cardiovascular mortality, death due hypo- and hyperglycemia, and sudden death) myocardial infarction, stroke, peripheral vascular disease, and microvascular disease, any diabetes-related endpoint (i.e. presence of any of the aforementioned categories) (please see S1 Table for the exact definitions of the different outcomes). If a participant had an outcome at the time of diabetes diagnosis, the participant was excluded from the analyses of that specific outcome. Outcomes were assessed in the Danish registries until 31 December 2008. The vital and emigration status of all participants were ascertained through the Danish Civil Registration System [14]. Information on morbidity and causes of death were retrieved from the National Hospital Discharge Registry [15] and Danish Register of Causes of Death [8].

Covariates

Covariates were selected based on measured confounders known to affect both mortality and HbA1c. Information about covariates was collected at 6-year follow-up [10]. Age, sex, and cohabitation status (dichotomous: living alone yes/no) were registered. Body mass index (BMI, kg/m2) was calculated based on height measured at the time of diagnosis and weight measured at the end of the intervention, on average 6 years later, by the general practitioner with the scales available in the clinic. Wasting/pathological weight loss was defined as unintentional weight loss ≥ 1 kg per year (wasting yes/no) (16). Current smoking status was dichotomized (yes/no). Information about leisure time physical activity (LTPA) was assessed from the International Physical Activity Questionnaire and dichotomized as “sedentary LTPA yes/no” [16]. Hypertension was defined as systolic blood pressure ≥ 160, and/or diastolic pressure ≥ 90 mmHg, and/or antihypertensive and/or diuretic treatment. Laboratory tests included total cholesterol (mmol/l), fasting triglycerides (mmol/l), serum creatinine (μmol/l), and urinary albumin (mg/l). Type of treatment at the end of intervention was categorized as: diet only, oral antidiabetic, or insulin.

Statistical methods

The effect of one-year HbA1c levels was expressed as hazard ratios (HRs) calculated using a Cox proportional hazard regression model indicating the multiplicative change in incidence rate (hazard) per 1.1 mmol/mol or (0.1% point) increment in HbA1c. The effect of the yearly HbA1c change (the slope of the regression line) on the outcome was expressed as HRs per 1.1 mmol/mol or (0.1% point) increase in HbA1c/year. The effect of the 1-year HbA1c level and yearly change in HbA1c was assessed in unadjusted as well as in adjusted models: Model 1 included age, sex, living alone, BMI (and one-year HbA1c levels in the analyses including change); Model 2 additionally included hypertension, total cholesterol, triglycerides, LTPA, smoking status, and wasting. To assess the linearity of the effect of glycaemic control, a restricted cubic spline was used to model the effect of glycaemic control after adjustment as in Model 2. Sensitivity analyses were performed adjusted for urinary albumin, creatinine, and anti-diabetic medication (Model 3). To investigate if one-year HbA1c level, sex, and anti-diabetic medication affected the relation between the yearly HbA1c change and risk of morbidity and mortality, we repeated the aforementioned analyses, stratified on one-year HbA1c level, sex, and type of treatment. All analyses were performed in SAS (Version 9.4).

To validate the linearity assumptions of our general linear regression models, we performed non-linear spline regression analyses on the associations between yearly HbA1c change and the clinical outcomes. The incidence of microvascular disease increased approximately linearly with increasing slope of the regression line (data not shown).

Results

The median age was 69.1 year at the end of the intervention, on average six years after diagnosis, and 50% of participants were men (Table 1). At the end of follow-up, 62% received oral anti-diabetic medication and 29% was treated with diet only. One year after diagnosis, HbA1c levels were on average lower than at time of diagnosis. During the following five years the observed average HbA1c increased with 0.19% per year (95% confidence interval (95% CI): 0.01–0.43) (Table 1), (equivalent to a rise of. 2.1 mmol/mol (95% CI: 0.11–4.70)).

Table 1

Participant characteristics.

	n	Characteristics
Baseline HbA1c level (mmol/mol) 1	411	88 (72–103)
Baseline HbA1c level (%) 1		10.2 (8.7–11.6)
One-year HbA1c level (mmol/mol) 1	494	60 (52–71)
One-year HbA1c level (%) 1		7.65 (6.91–8.62)
Yearly HbA1c change (mmol/mol/year)	494	2.1 (0.11–4.70)
Yearly HbA1c change (%/ year)		0.19 (0.01–0.43)
Age at end study (years)	494	69.1 (60.1–77.4)
Sex (male) (n,%)	494	244 (49.4)
Living alone (n,%)	459	167 (36.4)
BMI (kg/m2)	483	28.3 (25.6–31.7)
Baseline systolic blood pressure (mmHg)	489	150 (132–160)
Baseline diastolic blood pressure (mmHg)	489	85 (80–90)
Systolic blood pressure (mmHg)	492	149 (132–160)
Diastolic blood pressure (mmHg)	492	80 (80–90)
Hypertension 2 (n,%)	494	361 (73.1)
Total cholesterol (mmol/l)	491	6.0 (5.2–6.8)
Fasting triglycerides (mmol/l)	463	1.73 (1.22–2.50)
Sedentary leisure time physical activity	453	128 (28.3)
Smoking (n,%)	457	146 (32.0)
Wasting 3 (n,%)	494	82 (16.6)
Anti-diabetic treatment (n,%)	494
Diet only		142 (28.7)
Oral agents		304 (61.5)
Insulin		48 (9.7)
Serum creatinine (μmol/l)	491	89 (80–103)
Urinary albumin (n,%)	468
Normal		290 (62.0)
Microalbuminuria		158 (33.8)
Proteinuria		20 (4.3)

Unless otherwise stated, participant characteristics are from six years after diabetes diagnosis. Data are numbers (%) or medians (interquartile range (IQR)). N denotes the number of participants for whom variables were available.

1Reference value 36–57 mmol/mol (5.4–7.4%);

2Hypertension: systolic/diastolic blood pressure ≥ 160 and/or 90 mmHg;

3Wasting: unintentional weight loss ≥ 1 kg per year in participants without intention to lose weight.

Higher HbA1c levels one year after diagnosis were associated with a higher risk of later diabetes-related morbidity and mortality (all p value < 0.05; see Table 2).

Table 2

Relation between one-year level of haemoglobin A1c and risk of outcomes during 13 years of post-intervention follow-up.

	Events before or during intervention (n, %)	Events during post-intervention follow-up (n, %)	Model 1		Model 2		Model 3
			HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
Any diabetes-related endpoint	157 (31.9)	196 (58.3)	1.24(1.09–1.41)	0.001	1.26(1.10–1.45)	0.001	1.19(1.01–1.41)	0.03
All-cause mortality	-	295 (59.7)	1.14(1.02–1.27)	0.02	1.18(1.05–1.33)	0.006	1.14(0.98–1.33)	0.09
Diabetes-related mortality	-	186 (37.8)	1.18(1.02–1.35)	0.02	1.24(1.07–1.44)	0.005	1.3(1.08–1.55)	0.005
Myocardial infarction	46 (9.4)	121 (27.1)	1.16(0.96–1.38)	0.09	1.22(1.01–1.48)	0.04	1.34(1.05–1.70)	0.02
Stroke	47 (9.5)	78 (17.5)	1.42(1.12–1.79)	0.004	1.46(1.13–1.88)	0.004	1.37(0.98–1.91)	0.07
Peripheral vascular disease	2 (0.4)	20 (4.1)	2.05(1.44–2.91)	<0.0001	1.85(1.28–2.68)	0.001	1.43(0.93–2.21)	0.10
Microvascular disease	15 (3.1)	54 (11.3)	1.55(1.24–1.92)	0.0001	1.68(1.33–2.13)	<0.0001	1.48(1.12–1.96)	0.006

Events are given as count data with percentages (%). Other data are given as hazard ratios (HR) with 95% confidence interval (CI) and corresponding p values for outcomes during 13 years of post-intervention follow-up expressed per 1.1 mmol/mol (0.1%) increment in levels of HbA1c at 1 year after diagnosis. Model 1 is adjusted for age, sex, living alone, and BMI. Model 2: as model 1, but additionally adjusted for hypertension, cholesterol, triglycerides, sedentary leisure time physical activity, smoking, and wasting. Model 3: as model 2, but additionally adjusted for anti-diabetic medication, urinary albumin, and creatinine.

Next we assessed the relation between yearly HbA1c changes dichotomized on the median (0.188 percentage point per year during the 6-year intervention period) and the incidence of various outcomes during the 13 years thereafter. Table 3 shows the risk for various outcomes for those with a median or higher increase in HbA1 levels during the intervention period, relative to those with change in HbA1c below the median. An increase in HbA1C levels equal or higher than median was associated with a higher risk of myocardial infarction during the follow-up.

Table 3

Relation between dichotomized change in haemoglobin A1c during the 6-year intervention period and risk of outcomes during the subsequent 13 years of follow-up.

	Events before or during intervention (n, %)	Events during post-intervention follow-up (n, %)	Model 1		Model 2		Model 3
			HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
Any diabetes-related endpoint	157 (31.9)	196 (58.3)	0.91(0.65–1.28)	0.59	0.92(0.63–1.32)	0.63	0.82(0.55–1.22)	0.33
All-cause mortality	-	295 (59.7)	1.22(0.94–1.60)	0.14	1.24(0.94–1.64)	0.13	1.12(0.83–1.50)	0.46
Diabetes-related mortality	-	186 (37.8)	1.40(0.99–1.98)	0.060	1.40(0.96–2.03)	0.077	1.27(0.86–1.86)	0.23
Myocardial infarction	46 (9.4)	121 (27.1)	1.81(1.17–2.79)	0.0074	2.14(1.30–3.52)	0.0030	2.07(1.26–3.41)	0.0040
Stroke	47 (9.5)	78 (17.5)	0.95(0.52–1.73)	0.86	1.01(0.51–2.02)	0.98	0.89(0.43–1.85)	0.76
Peripheral vascular disease	2 (0.4)	20 (4.1)	2.09(0.70–6.19)	0.19	1.47(0.55–3.95)	0.45	1.02(0.40–2.63)	0.97
Microvascular disease	15 (3.1)	54 (11.3)	1.67(0.85–3.27)	0.13	1.71(0.85–3.47)	0.14	1.49(0.74–3.05)	0.27

Events are given as count data with percentages (%). Other data are given as hazard ratios (HR) with 95% confidence interval (CI). Hazard ratios denote the effect of HbA1c change equal and above median relative to HbA1c change below the median during the 6-year intervention period after diagnosis on outcomes during 13 years of post-intervention. Model 1 is adjusted for age, sex, living alone, BMI, and one-year HbA1c level. Model 2: as model 1, but additionally adjusted for hypertension, cholesterol, triglycerides, sedentary leisure time physical activity, smoking, and wasting. Model 3: as model 2, but additionally adjusted for anti-diabetic medication, urinary albumin, and creatinine.

Table 4 shows the relation between continuous changes in HbA1c levels during the intervention period Change in HbA1c during the intervention period was not associated with the aggregate outcome any diabetes-related endpoint, all-cause mortality, diabetes-related mortality, myocardial infarction, and stroke in the multivariable analyses. However, yearly HbA1c change was associated with a higher incidence of microvascular complications (HR, 95% CI: 1.14, 1.05–1.24) and peripheral vascular disease (1.14, 1.00–1.30). In sensitivity analyses, when additionally adjusting for urinary albumin, creatinine, and anti-diabetic medication, the association between yearly HbA1c change and microvascular disease remained (1.11; 1.01–1.21; P = 0.024), but the association with peripheral vascular disease was attenuated (1.05; 0.89–1.24; P = 0.58).

Table 4

Relation between yearly change in haemoglobin A1c during the 6-year intervention period and risk of outcomes during the subsequent 13 years of follow-up.

	Events before or during intervention (n, %)	Events during post-intervention follow-up (n, %)	Model 1		Model 2		Model 3
			HR (95% CI)	P value	HR (95% CI)	P value	HR (95% CI)	P value
Any diabetes-related endpoint	157 (31.9)	196 (58.3)	0.99(0.95–1.04)	0.71	1(0.95–1.05)	0.94	0.98(0.92–1.03)	0.42
All-cause mortality	-	295 (59.7)	1.01(0.97–1.05)	0.66	1.02(0.98–1.07)	0.32	1.00(0.96–1.06)	0.82
Diabetes-related mortality	-	186 (37.8)	1.00(0.96–1.05)	0.86	1.01(0.96–1.07)	0.63	1.00(0.94–1.07)	0.96
Myocardial infarction	46 (9.4)	121 (27.1)	1.02(0.96–1.08)	0.57	1.04(0.97–1.11)	0.23	1.06(0.99–1.14)	0.10
Stroke	47 (9.5)	78 (17.5)	1.02(0.94–1.10)	0.65	1.04(0.95–1.14)	0.40	1.01(0.91–1.13)	0.81
Peripheral vascular disease	2 (0.4)	20 (4.1)	1.17(1.05–1.30)	0.006	1.14(1.00–1.30)	0.047	1.05(0.89–1.24)	0.59
Microvascular disease	15 (3.1)	54 (11.3)	1.11(1.03–1.20)	0.006	1.14(1.05–1.24)	0.002	1.11(1.01–1.21)	0.024

Events are given as count data with percentages (%). Other data are given as hazard ratios (HR) with 95% confidence interval (CI) and corresponding p values for outcomes during 13 years of post-intervention follow-up expressed per 1.1 mmol/mol (0.1%) per year change in HbA1c during the 6-year intervention period after diagnosis. Model 1 is adjusted for age, sex, living alone, BMI, and one-year HbA1c level. Model 2: as model 1, but additionally adjusted for hypertension, cholesterol, triglycerides, sedentary leisure time physical activity, smoking, and wasting. Model 3: as model 2, but additionally adjusted for anti-diabetic medication, urinary albumin, and creatinine.

In additional analyses, we investigated whether the effect of yearly HbA1c change on the outcomes was dependent on the participant’s characteristics, such as one-year HbA1c level, sex, and type of anti-diabetic treatment (Table 5). The relation between yearly HbA1c change and incidence of all-cause and diabetes-related mortality was stronger for women than for men (p for interaction 0.033 and 0.038 respectively). Regarding the modifying effect of diabetic medication, treatment with insulin was associated with an increased risk of diabetes-related mortality compared to participants treated with oral medication or diet only. Moreover, oral medication was a risk indicator for later myocardial infarction when compared to diet only and insulin therapy.

Table 5

Modifying effect of one-year HbA1c level, sex, and antidiabetic treatment, on the relation between yearly change in in haemoglobin A1c during the 6-year intervention period and risk for various outcomes during the subsequent 13-year post-intervention follow-up.

	Effectmodifier	Events before or during intervention (n, %)	Events during post-intervention follow-up (n, %)	HR (95% CI)	P value	P for interaction
	HbA1c level, mmol/mol (%)
Any diabetes-related endpoint	≤57.4 (7.4)	59 (28.5)	74 (50.0)	0.95 (0.87–1.04)	0.24	0.14
	>57.4 (7.4)	98 (34.3)	122 (64.9)	1.01 (0.96–1.06)	0.77
All-cause mortality	≤57.4 (7.4)	0	118 (57.0)	1.04 (0.98–1.11)	0.22	0.49
	>57.4 (7.4)	0	177 (61.7)	1.01 (0.97–1.06)	0.58
Diabetes-related mortality	≤57.4 (7.4)	0	72 (34.8)	1.01 (0.93–1.10)	0.78	0.97
	>57.4 (7.4)	0	114 (40.0)	1.01 (0.95–1.08)	0.66
Myocardial infarction	≤57.4 (7.4)	18 (8.7)	49 (25.2)	1.07 (0.97–1.18)	0.15	0.46
	>57.4 (7.4)	29 (9.8)	72 (28.0)	1.03 (0.95–1.11)	0.45
Stroke	≤57.4 (7.4)	17 (8.2)	23 (12.1)	0.93 (0.77–1.13)	0.47	0.16
	>57.4 (7.4)	30 (10.5)	55 (21.5)	1.05 (0.96–1.15)	0.25
Peripheral vascular disease	≤57.4 (7.4)	1 (0.5)	4 (1.9)	1.14 (0.92–1.41)	0.24	0.99
	>57.4 (7.4)	1(0.4)	16 (5.6)	1.14 (1.00–1.30)	0.048
Microvascular disease	≤57.4 (7.4)	5 (2.4)	15 (7.4)	1.10 (0.95–1.27)	0.21	0.49
	>57.4 (7.4)	10 (3.5)	39 (14.2)	1.15 (1.06–1.25)	0.001
	Sex
Any diabetes related endpoint	Women	78 (31.3)	103 (60.2)	1.02 (0.96–1.09)	0.47	0.18
	Men	79 (32.4)	93 (56.4)	0.98 (0.92–1.03)	0.41
All-cause mortality	Women	0	133 (53.2)	1.07 (1.01–1.13)	0.017	0.033
	Men	0	162 (66.4)	0.99 (0.94–1.04)	0.67
Diabetes related mortality	Women	0	86 (34.5)	1.07 (1.00–1.15)	0.049	0.038
	Men	0	100 (41.2)	0.97 (0.91–1.04)	0.46
Myocardial infarction	Women	19 (7.6)	55 (23.9)	1.09 (1.01–1.18)	0.027	0.15
	Men	27 (11.1)	66 (30.9)	1.01 (0.92–1.10)	0.89
Stroke	Women	22 (8.8)	35 (15.4)	1.08 (0.96–1.21)	0.20	0.35
	Men	25 (10.3)	43 (19.6)	1.02 (0.92–1.13)	0.72
Peripheral vascular disease	Women	1 (0.4)	5 (2.0)	1.01 (0.74–1.37)	0.95	0.32
	Men	1 (0.4)	15 (6.2)	1.17 (1.03–1.33)	0.012
Microvascular disease	Women	6 (2.4)	26 (10.7)	1.15 (1.03–1.28)	0.010	0.78
	Men	9 (3.7)	28 (12.0)	1.13 (1.02–1.25)	0.017
	Antidiabetic treatment
Any diabetes-related endpoint	Diet only	42 (29.6)	50 (50.0)	0.91 (0.84–1.00)	0.046	0.030
	Oral	99 (32.7)	124 (60.8)	1.02 (0.96–1.09)	0.43
	Insulin	16 (33.3)	22 (68.8)	0.93 (0.83–1.04)	0.22
All-cause mortality	Diet only	0	84 (59.2)	0.93 (0.86–1.01)	0.074	0.021
	Oral	0	181 (59.5)	1.04 (0.99–1.10)	0.11
	Insulin	0	30 (62.5)	1.04 (0.96–1.12)	0.33
Diabetes-related mortality	Diet only	0	60 (42.3)	0.95 (0.86–1.04)	0.26	0.023
	Oral	0	105 (34.8)	1.04 (0.97–1.11)	0.29
	Insulin	0	21 (43.8)	1.11 (1.01–1.22)	0.030
Myocardial infarction	Diet only	14 (9.9)	36 (28.1)	0.95 (0.86–1.06)	0.36	0.036
	Oral	28 (9.3)	74 (27.0)	1.10 (1.02–1.19)	0.019
	Insulin	4 (8.3)	11 (25.0)	1.05 (0.93–1.19)	0.46
Stroke	Diet only	19 (13.4)	19 (15.5)	0.97 (0.84–1.13)	0.71	0.57
	Oral	23 (7.6)	47 (16.8)	1.05 (0.94–1.17)	0.37
	Insulin	5 (10.4)	12 (27.9)	1.04 (0.89–1.21)	0.66
Peripheral vascular disease	Diet only	0 (0.0)	1 (0.7)	1.48 (1.17–1.86)	0.001	0.053
	Oral	2 (0.7)	14 (4.7)	1.07 (0.90–1.28)	0.45
	Insulin	0 (0.0)	5 (10.4)	1.12 (0.90–1.39)	0.30
Microvascular disease	Diet only	4 (2.8)	6 (4.4)	1.03 (0.90–1.19)	0.67	0.29
	Oral	10 (3.3)	41 (14.0)	1.15 (1.05–1.26)	0.002
	Insulin	1 (2.1)	7 (14.9)	1.06 (0.83–1.36)	0.64

Events are given as count data (n) with percentages (%). Other data are given as hazard ratios (HR) with 95% confidence interval (CI) and corresponding p values for outcomes during 13 year post-intervention follow-up expressed per 1.1 mmol/mol (0.1%) per year change in HbA1c during 6-year intervention period after diagnosis adjusted for age, sex, living alone, BMI, hypertension, total cholesterols, triglycerides, leisure time physical activity, smoking, and wasting.

Discussion

In this Danish population-based sample of persons with newly diagnosed Type 2 DM, a higher yearly increase in HbA1c was an independent risk factor for microvascular disease, but not for macrovascular disease or mortality during the subsequent 13-years in participants receiving structured personal care in general practice. The relation between yearly HbA1c change and the incidence of microvascular outcomes appeared to be linear. Although increasing HbA1c was associated with a higher incidence of peripheral vascular disease, this association was no longer significant after correction for urinary albumin, creatinine, and anti-diabetic medication. These findings were independent of the level of HbA1c after one year of treatment. We found no association between an increase in HbA1c during the intervention period and mortality or incident cardiovascular events during 13 years of follow-up. In the DCGP trial the intervention reduced the incidence of myocardial infarction after 19 years of follow-up compared with the control group (9). This time point coincides with the end of the 13-year follow-up in the present study. Thus, the present observational study suggest that the reduction in macrovascular disease may not attributable to quality of HbA1c control during the intervention period. but may bedue to other elements of the intervention like blood pressure control. Alternatively, the lower macrovascular morbidity in the DCGP study at 19 years may be the results of the legacy effect, the long-term health benefits of early glycemic control.

A recent study investigated the relation between the difference in HbA1c levels before and after 6 months of treatment with metformin and the subsequent risk of cardiovascular events and mortality. This study found that a decrease in HbA1c during 6 months of treatment was associated with a lower risk of cardiovascular events and mortality during a median follow-up of 2.8 years in participants [17]. Admittedly, the statistical precision of these results was limited and, since all participants used metformin, it is possible that the effect was due to the pharmacological properties of metformin instead of the decrease in HbA1c. Yet in line with these earlier findings, we also found that increasing HbA1c levels during the six-year intervention period were associated with an increased risk of microvascular complications and a tendency towards higher risk of peripheral vascular morbidity. Increasing levels of HbA1c during the intervention period may have been caused by a less intensive treatment of diabetes, or may be ascribed to diabetes subtype, weight gain, or other factors. A meta-analysis demonstrated that intensive treatment of Type 2 DM lowers the risk of microvascular complications as well as the risk of amputation of lower extremities. [18]. However, this meta-analysis failed to show an effect on macro-vascular morbidity. A follow-up study of the Veteran Affair Diabetes Trial (VADT) concluded that intensive treatment may reduce the risk of major cardiovascular events, but that the intervention did not lower mortality risk [19]. Thus, overall taken, results regarding the risk of increasing HbA1c levels are not consistent between studies. This may in part be due to methodological heterogeneity, e.g. differences in treatment regimens, duration of follow-up, and definition of quality of glycaemic control as well as participant characteristics and compliance issues.

The UKPDS study is comparable to the DCGP study, in that it included persons with newly diagnosed Type 2 DM, while the ADVANCE and ACCORD studies included persons already diagnosed with diabetes with comorbid cardiovascular disease and a mean diabetes duration of eight and ten years, respectively [20, 21]. In UKPDS, the measure of HbA1c was an updated mean of HbA1c based on the mean of the annual measurements during follow-up, which mathematically is essentially the same analysis we conducted for the current study. The UKPDS found that a 1% lower updated mean HbA1c was associated with a 37% lower risk for microvascular complications, and a 43% lower risk of the combined endpoint of amputation or death by peripheral vascular disease [11]. These results by and large concord with our findings. However, unlike the UKPDS study, which reported a lower all-cause and diabetes-related mortality of respectively 14% and 21% per 1% lower updated mean HbA1c [11, 21], we only found an association between HbA1c and mortality in women. A sex-specific difference in diabetes outcomes is reminiscent of earlier findings in the DCGP study of a post-hoc analysis between structured personal care compared with routine care [12]. Women receiving structured personal care showed a lower all-cause mortality, incidence of diabetes-related mortality, and any diabetes-related endpoint than women receiving routine care, while this difference was absent in men. This sex-related difference was ascribed to complex social and cultural issues involving gender [12]. Regarding microvascular complications, the UKPDS concluded that the rate of increase of the risk for microvascular disease with hyperglycaemia was larger than that for macrovascular disease [11].

A major strength of this study is the prospective design and the long-term post-trial follow up. Use of Danish registry data ensured that follow-up was virtually complete [10]. The included sample is likely to be representative of community-dwelling persons with Type 2 DM that have survived for some years with this diagnosis and have followed a structured disease management program, which nowadays is included in the clinical guidelines for diabetes. The change in HbA1c over time is a highly clinically relevant parameter since decisions on treatment adjustments are typically based on HbA1c measurements. The inclusion of repeated HbA1c measurements is anticipated to take into account a considerable proportion of the variation in the disease progression in persons with Type 2 DM [15].

Microvascular disease was treated as a surrogate measure of disease status. The outcome is a composite outcome including renal complications and eye-related complications, and the effect sizes in our study were expectedly attenuated when results were adjusted for urinary albumin and antidiabetic treatment.

Our study has a number of shortcomings. The analyses in this study are based on information of the disease progression during the intervention of the trial, and no information was available on treatment or HbA1c levels during the succeeding 13 years of follow-up. However, it has been shown that although glucose regulation deteriorates in treatment groups after the end of the intervention, a beneficial “legacy effect” on cardiovascular end-points may persist [22] [23]. A second shortcoming is large number of significance tests performed. We had no predefined ideas on how changes in HbA1c would affect later diabetes-related outcomes. The study is therefore hypothesis generating, and the found associations should therefore be confirmed in subsequent, hypothesis confirming studies. This implicates though that the typical p-value threshold of 5% should be interpreted with caution.

Conclusions

In conclusion, increasing HbA1c levels during the first six years after the diagnosis of Type 2 DM were associated with a higher incidence of microvascular complications, but not macrovascular complications and mortality during the following 13 years. These findings should further encourage clinicians to pursue optimal glycaemic control to prevent long-term microvascular complications in persons with Type 2 DM.

Definition of clinical outcomes in the Diabetes Care in General Practice (DCGP) 19-year registry-based monitoring used to classify cause of death or morbidity.

a The Danish National Death Registry and the National Hospital Discharge Registry changed coding from International Classification of Diseases 8 (ICD-8) to ICD-10 on 1 January 1994. The Danish National Death Registry contains only the first 4 characters of the ICD codes, while the National Hospital Discharge Registry contains all 5 characters. b The National Hospital Discharge Registry changed coding of surgical procedures from the 3rd edition of The Danish Classification of Surgical Procedures to the Nordic Classification of Surgical Procedures on 1 January 1996.

(DOCX)

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31 Jul 2019

PONE-D-19-16655

Changes in HbA1c during the first six years after the diagnosis of Type 2 diabetes mellitus predict long-term microvascular outcomes

PLOS ONE

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Reviewer #1: It has already been well-known that higher level of HbA1c is significantly associated with diabetic (especially, microvascular) complications. The observational period and number of subjects in this study are too few to get any conclusive results. Therefore, there is virtually very few novelty anyway. Trajectory of HbA1c is also really depend upon inclusion criteria and backgrounds of the patients, so that extrapolation of their results and their clinical relevance are also very limited.

Reviewer #2: Rozing et al examined the impact of annual changes of HbA1c during intervention period in DCGP study on diabetes-related outcomes. In general, although many of the data is confirmatory, it is still of importance with regard to regular clinical care for newly diagnosed diabetes. However, there are several points to be addressed to improve the manuscript, and to make it more comprehensive.

1. The authors stated that one year HbA1c level during intervention period strongly predicts macro- and microvascular diabetic outcomes (supplemental table 1), although the data is confirmatory of their previous publication (ref 9) as understood as “legacy effect”. In contrast, impacts of annual changes in HbA1c after 1-year treatment appear not so impressive. The reviewer is wondering what the result would be, if the annual changes in HbA1c after 1-year treatment is categorized into 2 categories, such as maintained and worse based on the median. If this categorization predicts major diabetic outcomes, it highlights the significance of keeping glycemic control for several years following newly onset of diabetes.

2. Supplemental figure 2 should be mistyping, which should be indeed figure 2. This figure is not easily understood for regular clinicians. The reviewer cannot judge whether this figure is appropriately analyzed and demonstrated. This should be reviewed by an appropriate biological statistician selected by the Journal.

3. The reviewer feels that all supplemental data should be incorporated into the main text as regular Figures and Tables. Is the format including references is in line with the PLoS ONE? It should be carefully checked by the authors and the editorial technical staff.

4. page 3, line 3: The sentence of “anti-diabetic medication lowers the risk of complications (ref 6)” is not appropriate, since the research compares 2 types of anti-diabetic medications.

5. page 4, line 18: Does this mean that patients with the use of insulin within 180 days (2.5%) were not regarded as type 2 diabetes? This classification is clearly not appropriate.

6. page 5, line 2: Is the sentence “honey moon phase” properly used?

7. page 5, definition of outcomes line 1: Please clarify Diabetes-related endpoint and Diabetes-related mortality in this Section, although they are listed in Supplemental Table 2.

8. page 7, line 5: What does it mean by “HbA1c was, on average, normalized”? Please clarify.

9. page 8, line 5, Supplemental table 2: The authors only stated the impact of insulin therapy on diabetes-related death. The effect of oral therapy on myocardial infarction is also significant, with significant interaction. This should also be described and discussed.

10. page 8, last line: The reviewer is against the idea that “the reduction in macrovascular disease was not attributable to poor HbA1c control,” since this may be the result of “legacy effect”. Please clarify.

11. page 9, line 14, ref 19: This reference dealt with microvascular complications besides amputation. Both of these findings should be discussed.

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8 Oct 2019

Dear editor,

We thank you and two anonymous reviewers for the comments and suggestions regarding the previous version of our manuscript Changes in HbA1c during the first six years after the diagnosis of Type 2 diabetes mellitus predict long-term microvascular outcomes (ONE-D-19-16655). We have revised the manuscript accordingly. Our point-to-point responses to the comments are given below and are marked with an hash sign (#). Any changes in the text are marked with an asterisk (*). The letter “l” followed by a number, denotes the line number where changes in the text were made in the marked version of the manuscript.

Comments to the Author

5. Review Comments to the Author

1. Reviewer #1: It has already been well-known that higher level of HbA1c is significantly associated with diabetic (especially, microvascular) complications.

# We agree that this is a confirmatory rather than an exploratory study. We make this more explicit in the current version of our manuscript:

*“In the present confirmatory study, the aim was to determine the relation between changes in HbA1c during 6 years of intervention in persons with newly diagnosed Type 2 DM and any diabetes-related endpoint, all-cause mortality, diabetes-related mortality, myocardial infarction, stroke, peripheral vascular disease and microvascular disease during the 13 years thereafter.” (l. 49)

2. The observational period and number of subjects in this study are too few to get any conclusive results. Therefore, there is virtually very few novelty anyway.

#The intervention period and the follow-up period encompassed 6 and 13 years respectively. When studying longer-term complications of diabetes in humans this time-frame should be sufficient. Our sample included 494 patients of which approximately 60% experienced a diabetes-related event. A larger sample size would have allowed for higher accuracy, and would merely have yielded more statistically significant results, yet not necessarily more clinically significant results.

3. Trajectory of HbA1c is also really depend upon inclusion criteria and backgrounds of the patients, so that extrapolation of their results and their clinical relevance are also very limited.

#We fully agree with your concern regarding external validity when studying HbA1c trajectories. Especially in randomized controlled trials, preferential inclusion of distinct patient characteristics limits the applicability of their findings to the general population. However, in our study with virtually no exclusion criteria, the study sample represents a population, which clinicians typically encounter in their everyday clinical practice.

Reviewer #2: Rozing et al examined the impact of annual changes of HbA1c during intervention period in DCGP study on diabetes-related outcomes. In general, although many of the data is confirmatory, it is still of importance with regard to regular clinical care for newly diagnosed diabetes. However, there are several points to be addressed to improve the manuscript, and to make it more comprehensive.

1. The authors stated that one year HbA1c level during intervention period strongly predicts macro- and microvascular diabetic outcomes (supplemental table 1), although the data is confirmatory of their previous publication (ref 9) as understood as “legacy effect”. In contrast, impacts of annual changes in HbA1c after 1-year treatment appear not so impressive. The reviewer is wondering what the result would be, if the annual changes in HbA1c after 1-year treatment is categorized into 2 categories, such as maintained and worse based on the median. If this categorization predicts major diabetic outcomes, it highlights the significance of keeping glycemic control for several years following newly onset of diabetes.

#Thank you for your suggestion. We included an additional Table 3 accordingly, showing the results for those with “worsened HbA1c” levels and “stable/improved HbA1c” levels. We included the following sentence in the results section:

*“Next we assessed the relation between yearly HbA1c changes during the intervention period and the incidence of various outcomes during the 13 years thereafter. Table 3 shows the risk for various outcomes for those with a median (0.188 percentage point per year during the 6-year intervention period) or higher increase in HbA1 levels during the intervention period, relative to those with change in HbA1c below the median. An increase in HbA1C levels equal or higher than median was associated with a higher risk of myocardial infarction during the follow-up.” (l.149-155)

2. Supplemental figure 2 should be mistyping, which should be indeed figure 2. This figure is not easily understood for regular clinicians. The reviewer cannot judge whether this figure is appropriately analyzed and demonstrated. This should be reviewed by an appropriate biological statistician selected by the Journal.

#We agree that interpretation of the figure is cumbersome and may therefore not be informative to regular clinicians. The main message of the figure was to show the linear relationship between the change (“slope”) of the 1%/year increment in hemoglobin A1c during the 6-year intervention period and the risk microvascular disease during 13-year post-intervention follow-up. We removed the figure from the current version of the manuscript and moved the following sentence from the results section to the statistics section:

*“To validate the linearity assumptions of our general linear regression models, we performed non-linear spline regression analyses on the associations between yearly HbA1c change and the clinical outcomes. The incidence of microvascular disease increased approximately linearly with increasing slope of the regression line (data not shown).” (l.133-136)

3. The reviewer feels that all supplemental data should be incorporated into the main text as regular Figures and Tables. Is the format including references is in line with the PLoS ONE? It should be carefully checked by the authors and the editorial technical staff.

#*We incorporated all supplemental tables and figures in the main text, except for table S1 (previously table S2).

4. page 3, line 3: The sentence of “anti-diabetic medication lowers the risk of complications (ref 6)” is not appropriate, since the research compares 2 types of anti-diabetic medications.

#Thank you pointing this out. The reference was indeed incorrect. We removed this reference and instead refer to: Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet. 1998 Sep 12;352(9131):837-53.

5. page 4, line 18: Does this mean that patients with the use of insulin within 180 days (2.5%) were not regarded as type 2 diabetes? This classification is clearly not appropriate.

#Your interpretation of the sentence is correct. Various methods exist to exclude insulin-dependent diabetes. One method (which was used) in this study, is to exclude those patients who start insulin within a certain period after diagnosis (here: 180 days) and who continue insulin treatment during a distinct period after diagnosis (here: the observation period). In our study eighteen of the 649 patients (2.5%) in the intervention group started insulin within 180 days after diagnosis. Insulin was discontinued for two of these patients during the observation period. Thus, at least 633 (97.5%) patients were considered to have type 2 diabetes. Olivarius NF, Beck-Nielsen H, Andreasen AH, Hørder M, Pedersen PA. Randomised controlled trial of structured personal care of type 2 diabetes mellitus. BMJ.2001 Oct 27;323(7319):970-5.

6. page 5, line 2: Is the sentence “honeymoon phase” properly used?

#In our manuscript, we intended to describe the early improvement of glycemic control in our study population with newly diagnosed type 2 Diabetes, irrespective of the treatment modality. The term honeymoon phase is usually reserved for type 1 diabetes, indicating a transient restoration of the beta cell function following the initiation of insulin therapy in type 1 diabetes. Whether an analogous phenomenon exists in type II DM is debated (see reference 14: Retnakaran R. Novel Strategies for Inducing Glycemic Remission during the Honeymoon Phase of Type 2 Diabetes. CanJDiabetes. 2015;39 Suppl 5:S142-S7. doi: S1499-2671(15)00563-8). If the term is appropriate here is open to discussion: we therefore decided to remove the term (and reference 14) and rephrased the sentence as follows:

* “In general, participants experienced an improved glycemic control with a more or less pronounced remission of hyperglycaemia during the first year after diabetes diagnosis.”( l.79-80)

7. page 5, definition of outcomes line 1: Please clarify Diabetes-related endpoint and Diabetes-related mortality in this Section, although they are listed in Supplemental Table 2.

#We briefly explain the definition of the diabetes-related end-points and diabetes-related mortality in the revised version of the text and refer to table S1 for a more extended definition.

* “The outcomes were all-cause mortality, diabetes-related mortality (i.e. cardiovascular mortality, death due hypo- and hyperglycemia, and sudden death) myocardial infarction, stroke, peripheral vascular disease, and microvascular disease, any diabetes-related endpoint (i.e. presence of any of the aforementioned categories) (please see table S1 for the exact definitions of the different outcomes).” (l. 89 - 93)

8. page 7, line 5: What does it mean by “HbA1c was, on average, normalized”? Please clarify.

#We meant to convey that the average HbA1c value was below the critical threshold of 7%. We decided to rephrase this sentence as follows:

* “One year after diagnosis, HbA1c levels were on average lower than at time of diagnosis.” (l.142-143)

9. page 8, line 5, Supplemental table 2: The authors only stated the impact of insulin therapy on diabetes-related death. The effect of oral therapy on myocardial infarction is also significant, with significant interaction. This should also be described and discussed.

#We included the following sentence in the results section of the manuscript:

* “Moreover, oral medication was a risk indicator for later myocardial infarction when compared to diet only and insulin therapy.”(l.177-179)

10. page 8, last line: The reviewer is against the idea that “the reduction in macrovascular disease was not attributable to poor HbA1c control,” since this may be the result of “legacy effect”. Please clarify.

#We agree with your suggestion. We included the legacy effect as a possible explanation for the long-term reduction of macrovascular morbidity. We rewrote the section as follows:

* “Thus, the present observational study suggest that the reduction in macrovascular disease may not attributable to quality of HbA1c control during the intervention period, but may be due to other elements of the intervention like blood pressure control. Alternatively, the lower macro-vascular morbidity in the DCGP study at 19 years may the results of the legacy effect, the long-term health benefits of early glycemic control.” (l.194-199)

11. page 9, line 14, ref 19: This reference dealt with microvascular complications besides amputation. Both of these findings should be discussed. (p.10; l. 211-212)

#Thank you pointing that out. We have included all the end-points from the meta-analysis in the current version of the manuscript: micro-, macrovascular morbidity and amputations. “A meta-analysis demonstrated that intensive treatment of Type 2 DM lowers the risk of microvascular complications as well as the risk of amputation of lower extremities. However, this meta-analysis failed to show an effect on macro-vascular morbidity.”

31 Oct 2019

Changes in HbA1c during the first six years after the diagnosis of Type 2 diabetes mellitus predict long-term microvascular outcomes

PONE-D-19-16655R1

Dear Dr. Rozing,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Reviewer #3: Yes

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19 Nov 2019

PONE-D-19-16655R1

Changes in HbA1c during the first six years after the diagnosis of Type 2 diabetes mellitus predict long-term microvascular outcomes

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