Being born small increases your risk of developing Type 2 diabetes (T2D) with age. Furthermore, data even suggest that some of the diseases ("complications") in the eyes, kidneys, nerves, liver, blood vessels and heart often seen in T2D patients may not only be due to high blood sugar levels, but rather they to some extent are due to reduced growth in your mother´s womb. The Inter99 cohort included 6784 Danish citizens aged 30 to 60 years when established 20 years ago. Data from the Inter99 cohort showed a strong role of low birth weight (LBW) on T2D risk. The aim is now to reexamine risk of T2D and complications in all the alive 6004 elderly Inter99 participants. Importantly, today there are available techniques to perform detailed examinations for even the earliest signs of complications in both subjects with and without diabetes, and the results of this study will altogether provide important new insights into both the origin and classification of T2D and associated complications. It is hypothesized that being born with lower birth weights increases the adult risk of T2D and heart disease and associated complications in the large and smaller blood vessels.

BACKGROUND Type 2 Diabetes (T2D) affecting globally more than 400 million people represents one of the most significant global health challenges of our time. However, despite huge research investments, there are still limited insights into its complicated etiology and pathophysiology during the life-course. T2D is furthermore defined by arbitrary glycemic thresholds, which inadequately captures the diversity of clinical presentations and sub-phenotypes with differential complications and damages in multiple organs including small and large vessels, heart, kidney, liver and nerve tissue, often present already at the time of disease onset.

The heterogeneity of phenotypes in T2D is likely rooted in differential genetic, prenatal, and postnatal non-genetic etiologies between individuals. As for genetics, the known 568 T2D susceptibility variants are estimated to account for 18% of the putative genetic contribution to T2D. The totality of data from human famine catastrophes conclusively confirm the initial reports from Hertfordshire, UK, of an adverse intrauterine environment associated with low birth weight (LBW) playing a significant role in the development of T2D. The concept of fetal and early life developmental programming has been confirmed, validated and mechanistically examined in multiple animal studies. Studies in humans and animals have provided compelling evidence of an adverse fetal environment contributing to virtually all the known multiple organ defects influencing glucose homeostasis. Furthermore, emerging evidence consistently suggest that LBW and an adverse fetal environment play a direct and independent role in the development of important complications in T2D including cardiovascular disease, hypertension, dyslipidemia and vascular dysfunctions. Vascular stiffness reflected by elevated pulse-wave-velocity are early signs of micro- and/or macrovascular disease in patients with and without T2D. The fact that T2D patients often have a significant burden of vascular and metabolic complications already at diagnosis, indicates that factors influencing risk of T2D at the same time represent direct causes of co-morbidities that it is otherwise considered as complications to hyperglycemia. Accordingly, a distinct subgroup of T2D patients, as a result of an adverse fetal environment, may be affected by diabetic complications to a larger extent than patients with T2D due to other factors. Indeed, it was reported that LBW accounts for most of the excess cardiovascular disease (CVD) mortality in T2D. Moreover, LBW is associated with smaller kidneys with fewer nephrons and increased risk of chronic kidney disease. Another study reported increased risk of cardiac autonomic function (heart rate variability) in children, which is a known risk factor of CVD morbidity and mortality. Patients with T2D are at increased risk of developing NAFLD, which may progress to non-alcoholic stereo hepatitis (NASH), overt liver cirrhosis and liver cancer. NAFLD and NASH are clinically silent and under-diagnosed diseases with a global prevalence of 25-30% and is associated with increased risk of CVD in a non-genetic manner. Recent unpublished data from our group showed a threefold increased liver fat content in young, non-diabetic LBW men when studied before and after 4 weeks of a high carbohydrate challenge diet.

AIM To give a detailed phenotypical cardiometabolic description of the population-based Inter99-cohort, in order to examine associations of size at birth and prematurity, as proxies for the early fetal environment, concurrent lifestyle factors and genetic risk with the age-specific incidence of T2D and CVD, and its complications and co-morbidities, among Danish citizens aged 50-80 years.

HYPOTHESIS

1. An adverse fetal environment (LBW and/or prematurity) is associated with increased risk of developing a more severe T2D sub-phenotype with increased morbidity and mortality from micro- and macro vascular complications as well as cardiometabolic co-morbidities including NAFLD, compared with T2D patients born at term with a normal birth weight.
2. An adverse fetal environment is associated with increased morbidity and mortality from a range of micro- and macrovascular manifestations, as well as NAFLD, typically referred to as diabetes complications, even among elderly non- diabetic people with or without prediabetes (impaired glucose tolerance or impaired fasting glucose).

METHODS

The Inter99 study:

The population based Inter99 study was initiated in 1999 as a collaboration between Steno Diabetes Center Copenhagen (SDCC) and the Center for Clinical Research and Prevention (CCRP). From a population-based random sample of 61,301 30-60-year-old individuals living in the Western part of Copenhagen, Denmark, 13,016 people was invited to a health examination. The remaining control group (n=47,987) was followed in registries. The cohort was after baseline examination of 6,784 participants (52,5% acceptance rate), established as a randomized, non- pharmacological, lifestyle-counseling intervention study for prevention of diabetes and ischemic heart disease.

Clinical follow-up examinations after 5 years including glucose tolerance status had a participation rate of 66%, and the 10 years follow-up was based on registry data. While the lifestyle intervention had no effects on 5- or 10-year incidence rates of diabetes or cardiovascular disease, the in-depth clinical examinations of the cohort have contributed substantially to our knowledge of the etiology of T2D and associated traits. Original midwife records have been collected from 4.744 participants. Despite the relatively low average age of the cohort of only 46 years in 1999, a strong inverse relationship between birth weight and risk of T2D in this contemporary Danish population was confirmed. The prevalence of T2D associated retinal changes in the Inter99 cohort was studied in a subgroup of 970 participants. Interestingly, diabetic retinopathy was present in 7,5% of the 490 subjects with completely normal glucose tolerance, supporting the notion that factors other than elevated glucose contributes to the risk of diabetic complications.

Data collection:

The planned register-based study will include morbidity and mortality data from the newly established Danish Diabetes Register at SDCC. Biochemical data to estimate clinical trajectories following T2D diagnosis will be obtained from the Danish Clinical Laboratory Information System Research Database). Information on cardiovascular outcomes is based on the Danish National Patient Register. CVD is defined as ischaemic heart disease, heart failure, atherosclerotic macro-vascular disease, hypertensive disease, atrial fibrillation and cerebrovascular disease.

The 20-year cardiometabolic outcome follow-up study will include clinical examinations tailored to capture and expand our growing understanding of T2D subgroups, and with increased focus on T2D associated vascular and cardiometabolic co-morbidities and complications. The totality of data already available in the Inter99 cohort range from prenatal health information on birth weights and prematurity, glucose tolerance at baseline and 5 years follow-up, lifestyle and general health information including comprehensive Food Frequency Questionnaire data, numerous biomarkers and unparalleled genetic (GWAS) data. When subsequently combined with detailed Danish registries and our innovative clinical cardiometabolic deep phenotyping follow-up study, the cohort will constitute a hitherto unparalleled research platform to delineate the role of the fetal environment, as well as lifestyle and genetic determinants, for the development of T2D, its co-morbidities, as well as its clinical sub-phenotypes.

Examinations:

All individuals will be invited for a clinical examination at CCRP, Rigshospitalet, Glostrup, including:

1. Fasting blood samples for analysis of glucose, insulin, C-peptide, lipids, HbA1c, electrolytes, e-GFR, liver function test, extra plasma and serum for micronutrient status, metabolomics, lipidomics, transcriptomics, DNA and GAD-antibodies.
2. Urine for albumin, metabolomic and proteomic profiling.
3. Anthropometrics and body composition (bioelectrical impedance)
4. Liver stiffness by transient elastography (TE) will be used for noninvasive diagnosis of fibrosis and cirrhosis (FibroScan 502 Touch; Echosens, France).
5. Arterial stiffness and central (carotis-femoralis) pulse wave velocity (cfPWV) using the gold standard SphygmoCor© Pulse Wave method (AtCor Medical, Sydney, Australia).
6. Retinal fundus photo and optical coherence tomography (OCT) will be taken of both eyes with the Optos Ultra Widefield apparatus (Optos Monaco, Optos PLC, Dunfermline, UK) and images graded according to a modified version of the 'Proposed International Clinical DR severity scale' applying a deep learning (DL) algorithm by convolutional neural networks.
7. As a measure of cardiac autonomic neuropathy, simple bedside tests using heart rate variability (HRV) indices or response in heart rate to standing, slow breathing, or the Valsalva maneuver (cardiovascular autonomic reflex tests [CARTs]) will be used with a Vagus device (Medicus Engineering, Aarhus, Denmark).
8. Measurement of the electrical conduction system of the heart by ECG (electrocardiogram), using 12 electrodes.
9. Spirometry to measure pulmonary function i.e. expiratory forced vital capacity (FVC) and forced expiratory volume in one second (FEV1).
10. Handgrip strength will be used to measure maximum hand muscle strength. Handgrip strength is a strong predictor of both morbidity and mortality in the older population.
11. A 30 second sit to stand test will examine physical performance of participants, according to the number of times participants get to a full stand in 30 seconds.
12. Blood pressure (systolic and diastolic) is measured thrice with an electronic blood pressure monitor and fitting cuff after five minutes rest in sitting position.
13. Oxygen saturation will be measured

Participants will be asked to fill in validated questionnaires on general health, occurrence of chronic diseases, lifestyle factors and dietary intake.

On a separate occasion, participants will be invited for a coronary artery CT- calcium-scan (CCTA) at Dept. of Cardiology, Rigshospitalet (RH). CT-angiography will provide data on structural heart disease, coronary artery calcium score as well as subclinical obstructive coronary atherosclerosis, which all are associated with adverse long-term outcome. CT imaging will be performed using a 320- multidetector scanner (Aquilion One, Toshiba Medical Systems, Japan). A cardio- selective beta-blocker (metoprolol 25-150 mg) can be administered orally 1 h before scanning in participants with a heart rate of 60 bpm and no contraindications. Intravenous contrast media (Visipaque) will be used. A fixed- target protocol using one rotation acquisition with a prospective exposure window fixed at 350 ms centered at the 75% phase of the RR cycle will be used to restrict radiation dose.

A subset of participants will be asked:

• To wear a continuous glucose monitoring system (CGM) for two weeks to provide information about variability in glucose levels.
• To wear a physical activity monitor by Axivity to objectively measure physical activity and sedentary behavior by continuous 24-hour*7 days measurement using Axivity® AX3 accelerometers (www.axivity.com) skin-taped to the thigh.
• To participate in a dietary assessment study using a web-based dietary assessment software for seven consecutive days. The dietary assessment software is structured according to a typical Danish meal pattern covering breakfast, lunch, dinner and three in-between meals (morning, afternoon and evening).
• To participate in a 24-hour urine collection to estimate sodium and potassium intake using 24-hour urinary sodium excretion. Participants will also be invited to collect 24-hour urine 3 days in a row.
• To collect ons single fecal collection for the determination of the gut microbiota
• To participate in an intervention trial examining the effects of Vitamin K supplementation versus placebo on cardiovascular risk markers.

Inclusion Criteria:

• Have participated in the Inter99 Baseline examination.

Exclusion Criteria:

• Physically or cognitively unable to participate in the 3-hour clinical follow-up study.

Former participants of the Inter99 cohort, now aged 50-80 years, will be re-invited to participate in the Inter99-20-year follow-up study. A search of the Danish nationwide civil register (CPR register) in December 2019 showed that 6,004 (88.5%) participants reside in Denmark are currently alive. Based on our experience, we expect that at least 4,000 incl. 500 or more T2D patients will participate in the follow-up study. All participants are invited to a follow-up health examination in CCRP located in Glostrup.