Fasting proinsulin levels are significantly associated with 20 year cancer mortality rates. The Hoorn Study
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- Walraven, I., van ’t Riet, E., Stehouwer, C.D.A. et al. Diabetologia (2013) 56: 1148. doi:10.1007/s00125-013-2864-6
Proinsulin is possibly associated with cancer through activation of insulin receptor isoform A. We sought to investigate the associations between proinsulin and 20 year cancer mortality rates.
The study was performed within the Hoorn Study, a population-based study of glucose metabolism in individuals aged 50–75 years in the Dutch population. Fasting proinsulin levels were measured twice by a double-antibody radioimmunoassay. Participants were continuously followed to register mortality; causes of death were derived from medical records. Cox survival analyses were performed to assess the 20 year risk of death from cancer in relation to proinsulin. All analyses were adjusted for age and sex, with additional adjustments for traditional risk factors. The effect modification of glucose metabolism and sex was tested.
Proinsulin levels were measured in 438 individuals (41% normal glucose tolerance, 35.7% impaired glucose metabolism, 23.3% type 2 diabetes). Of these participants, 53 died from cancer. After adjustment for age and sex, proinsulin >16.5 pmol/l (the upper tertile) was significantly associated with a twofold risk of cancer mortality (HR 2.01, 95% CI 1.16, 3.46) compared with individuals with lower proinsulin levels. Additional adjustment for glucose metabolism, BMI and smoking did not substantially change the results (HR 1.91, 95% CI 1.04, 3.52). No interaction with glucose metabolism or sex was observed.
Individuals with fasting proinsulin levels >16.5 pmol/l have a twofold risk of cancer mortality over a 20 year time span. These findings provide population-based evidence for the independent association between high proinsulin levels and cancer mortality rates.
KeywordsCancer Cancer mortality Epidemiology Glucose metabolism Proinsulin Type 2 diabetes
Fasting plasma glucose
HOMA of insulin resistance
International Classification of Diseases
Impaired glucose metabolism
Insulin receptor A
Insulin receptor B
−2 Log likelihood
Normal glucose metabolism
Individuals with impaired glucose metabolism (IGM) or type 2 diabetes might have an increased risk of cancer and cancer-related mortality [1, 2, 3, 4, 5, 6, 7]. Insulin resistance, hyperglycaemia, high levels of insulin, and production of IGF-I and IGF-II have been suggested to induce tumour cell growth, thereby possibly providing a mechanistic link between glucose metabolism and cancer [1, 3, 6, 8].
Proinsulin is a precursor of insulin and is co-secreted proportionally to insulin by the pancreatic beta cell . Increased levels of proinsulin in the circulation are considered to be a sign of defective proinsulin-to-insulin conversion and may reflect beta cell dysfunction [10, 11, 12]. Indeed, hyperproinsulinaemia in non-diabetic individuals is known to be highly predictive of type 2 diabetes [11, 13]. An in vitro study by Malaguarnera et al  recently showed that proinsulin binds with and activates insulin receptor isoforms, in particular insulin receptor A (IR-A). IR-A mediates effects on growth and survival [15, 16, 17] and is overexpressed in several types of cancer, for example thyroid cancer .
None of the studies [1, 2, 3, 4, 5, 6, 7] that reported on the association between glucose metabolism and cancer took proinsulin into account. In the light of recent evidence  for the potential link between proinsulin and cancer, proinsulin could explain part of the overall risk of developing cancer. To our knowledge, no other study has addressed the association between proinsulin and cancer mortality rates. Such data are needed, since they might contribute to knowledge of the mechanisms involved. The Hoorn Study is a population-based cohort study with a long-term and precise follow-up of cause-related death. In a subsample of the Hoorn Study cohort, proinsulin concentrations were determined on two separate days at baseline in 1989 . This paper reports on the association between proinsulin and cancer mortality.
This study was conducted within the Hoorn Study, a population-based cohort study of type 2 diabetes in the Dutch population, which started in 1989 and had 2,484 participants . More extensive measurements were performed in a stratified random sample of the Hoorn Study (n = 631) . Insulin and proinsulin levels were measured in individuals with normal glucose metabolism (NGM) and IGM, and in newly diagnosed type 2 diabetes patients not using glucose-lowering therapy. Participants with a verified history of type 2 diabetes were excluded from proinsulin measurements (n = 56) . Furthermore, we excluded participants who, for technical reasons (n = 63) or because of missing samples (n = 74), did not have proinsulin values at one or both of the oral glucose tolerance tests. This left a final cohort size of 438 individuals. Written informed consent was obtained from all participants. The Ethical Review Committee of the VU University Medical Center (Amsterdam, the Netherlands) approved the Hoorn Study.
Anthropometric measurements were obtained from all participants. Weight, height, and hip and waist circumferences were measured with participants barefooted and wearing light clothes. Blood pressure was measured on the right upper arm after 5 min of rest, using a random-zero sphygmomanometer (Hawksley-Gelman, Lancing, UK). Mean blood pressure was then calculated as the mean of two measurements .
Fasting plasma glucose (FPG) concentrations and 2 h postload plasma glucose concentrations were determined by the glucose dehydrogenase method (Merck, Darmstadt, Germany). For the present analyses, we applied the American Diabetes Association  diagnostic criteria of 2011 for  type 2 diabetes (i.e. HbA1c ≥ 6.5%/[48 mmol/mol] and/or FPG ≥ 7.0 mmol/l and/or 2 h plasma glucose ≥ 11.1 mmol/l);  pre-diabetes (non-diabetic levels for all three markers of hyperglycaemia and at least one of the following in the indicated ranges: HbA1c 5.7–6.4% [39–48 mmol/mol], FPG 5.7–6.9 mmol/l or 2 h plasma glucose 7.8–11.0 mmol/l); and  NGM (defined as HbA1c < 5.7% [39 mmol/mol] and/or FPG < 5.6 mmol/l and/or 2 h plasma glucose < 7.8 mmol/l) .
Insulin and proinsulin levels were measured twice at an interval of 2 weeks.
Immunospecific insulin was measured in serum by a double-antibody radio immunoassay (lot SP21; Linco Research, St Louis, MO, USA), in which proinsulin and 32,33 split proinsulin cross-reacts by 0.2%. Proinsulin was measured by a double-antibody radioimmunoassay (Lilly Laboratory for Clinical Research, Indianapolis, IN, USA), in which 31,32 proinsulin cross-reacts by 63%. Serum total cholesterol, HDL-cholesterol and triacylglycerol were measured by enzymatic techniques (Boehringer-Mannheim, Mannheim, Germany) .
Insulin resistance was estimated by HOMA of insulin resistance (HOMA-IR) .
Cancer mortality rates follow-up
The municipal register of the city of Hoorn supplied information on the vital status of all participants. Causes of death were derived from the medical records of general practitioners and from the local hospital. All causes of death were coded according to the International Classification of Diseases (ICD), Injuries and Causes of Death, ninth revision . Death from cancer was defined as ICD-9 codes 140 to 239 (neoplasms). The mortality rate follow-up ended on 1 January 2009.
Variables are presented as percentage, mean (±SD) or median (interquartile range) in the event of a skewed distribution. Normality was tested for and if distribution was skewed, log-transformation was performed. Since, despite performance of log-transformation, proinsulin did not exhibit a linear relationship with cancer mortality rates, the population was categorised into tertiles of proinsulin levels. Differences in baseline characteristics between different tertiles of proinsulin level were tested with Student’s t tests (continuous variables) and χ2 tests (categorical variables). Differences in participants for whom proinsulin values were missing or not missing were tested using Student’s t tests (continuous variables) and χ2 tests (categorical variables).
Cumulative incidence rates of cancer and all-cause death were investigated according to tertiles of proinsulin. The follow-up duration was calculated as the time between baseline examination and date of death, loss to follow-up or end of follow-up on 1 January 2009.
Survival curves for tertiles of proinsulin were plotted for cancer and all-cause mortality. Proportional hazards assumptions for every model were tested by interpretation of the survival plots. Cox proportional hazards analyses were performed to investigate associations between proinsulin and cancer and all-cause mortality. First, we constructed a model in which we adjusted for age and sex. Then, we adjusted for possible confounding or mediating variables. The variables included are known to be associated with cancer (BMI and smoking) and/or are closely related to proinsulin (fasting specific insulin, estimated insulin resistance [HOMA-IR], glucose metabolism, fasting glucose). We constructed two different models, one in which we adjusted for all possible confounding variables, and the second only adjusting for variables with values of p < 0.10. To assess improvement of the model, changes in −2 log likelihood (−2LL) were tested. We also tested for interaction with glucose metabolism (according to ADA 2011 criteria) and sex, by adding an interaction term to the model, where p < 0.10 was considered to indicate statistically significant interactions. In a subpopulation (n = 189) for which we had 11-year follow-up information, sensitivity analyses were performed to investigate the possible mediating effect of the development of type 2 diabetes in the future and the possible use of glucose-lowering medication. Data are presented as HR with a 95% CI. The reported HRs can be interpreted as relative risks. Values of p < 0.05 were considered statistically significant. All analyses were performed with SPSS 20 (SPSS, Chicago, IL, USA).
Characteristics according to different tertiles of proinsulin concentration
Tertile of proinsulin (pmol/l)
6.0 (3.8, 7.8)
12.3 (10.9, 14.2)
24.3 (19.6, 34.4)
Sex (% male)
Fasting glucose (mmol/l)
2 h postload glucose (mmol/l)
NGM (n [%])
IGM (n [%])
Type 2 diabetes (n [%])
Fasting specific insulin (pmol/l)
74.6 (55.4, 92.5)
81.6 (63.2, 112.2)*
120.6 (88.9, 159.7)*
2.6 (1.8, 3.3)
3.0 (2.2, 4.3)*
4.9 (3.5, 6.9)*
Physical activity (h per day)
1.0 (0.6, 2.0)a
1.1 (0.5, 1.8)b
1.0 (0.5, 1.8)c
Current smoker (%)
Smoking (pack years)
7.5 (0.0, 23.7)
4.0 (0.0, 23.2)*
14.4 (0.0, 34.9)*
20 year follow-up
All-cause mortality (n [%])
Cancer mortality (n [%])
Participants who, for technical reasons (n = 63) or because of missing samples (n = 74), had missing proinsulin values had significantly lower fasting specific insulin values and lower HOMA-IR. This subpopulation also had fewer individuals with type 2 diabetes. The prevalence of cancer mortality was similar to that in the group for which proinsulin levels were available (12.9% vs 13.1%).
Proinsulin and all-cause and cancer mortality rates
Cancer, all-cause and all-cause except cancer mortality rates in the group with proinsulin >16.5 pmol/l vs those with ≤16.5 pmol/l (reference category)
Mortality rates in group with proinsulin >16.5 pmol/l
All causes except cancer
HR (95% CI)
HR (95% CI)
HR (95% CI)
Model 1 (adjusted for age and sex)
2.01 (1.16, 3.46)*
1.62 (1.18, 2.24)*
1.45 (0.97, 2.17)
Model 2 (adjusted as for Model 1 + glucose metabolism, BMI and smoking)
1.91 (1.04, 3.52)*
1.44 (0.99, 2.09)
1.24 (0.78, 1.98)
Model 3 (adjusted as for Model 2 + insulin and HOMA-IR)
2.08 (1.09, 3.96)*
1.47 (0.99, 2.16)
1.23 (0.76, 2.01)
We tested for interaction of proinsulin with glucose metabolism and sex, by adding interaction terms into the crude model. Both product terms for glucose metabolism (IGM vs NGM, p = 0.11 and type 2 diabetes vs NGM, p = 0.67) and sex (p = 0.21) were not statistically significant. We also stratified the analyses to visually examine whether the effect was different for different categories of glucose metabolism, and although power was weaker due to the smaller groups, the association between proinsulin and cancer mortality rates was almost equal in every category of glucose metabolism.
The present study shows that high proinsulin (>16.5 pmol/l) levels are significantly associated with a twofold risk of cancer mortality over a 20 year period compared with proinsulin levels below 16.5 pmol/l. This association was independent of glucose metabolism, fasting glucose, specific insulin, estimated insulin resistance, BMI and smoking (Table 2). Furthermore, this study shows that proinsulin levels were more strongly associated with cancer mortality than with all-cause mortality rates.
To our knowledge, this is the first paper to report on the association between proinsulin and cancer mortality rates. The in vitro study of Malaguarnera et al  might provide a possible mechanistic link for our findings, showing as it did that proinsulin differentially binds to and activates the two insulin receptor isoforms, with a higher affinity for IR-A than for insulin receptor B (IR-B). IR-A is a low-specificity receptor with high affinity not only for insulin but also for IGF-II; it can also activate intracellular signalling and biological effects in response to IGF-I. The above study  also showed that proinsulin was almost equipotent to insulin in inducing cell proliferation and migration in cells expressing IR-A at various levels.
As in other studies [12, 24], proinsulin levels were associated with specific insulin levels and with insulin resistance (Table 1). Therefore, fasting specific insulin could have mediated the association between proinsulin and cancer mortality rates. However, adjustment for specific insulin in the Cox regression analysis did not diminish the statistically significant association between proinsulin and cancer mortality rates.
The association between proinsulin levels and cancer mortality rates did not significantly differ across different categories of glucose metabolism (Fig. 1). It is possible that individuals with NGM and high proinsulin levels may have developed type 2 diabetes in the future (i.e. post baseline), which might then have mediated the association between high proinsulin and cancer mortality rates in individuals with NGM. For almost half (n = 189) of our study population, we had access to information about glucose metabolism 11 years later. Of that group, ten (11.2%) of 89 individuals with NGM had developed type 2 diabetes and only one of these ten died from cancer. Of the 69 persons with IGM for whom we had follow-up information, 37 (53.6%) developed type 2 diabetes 11 years later, none of whom died from cancer during the 20 year follow-up period. Therefore, it is unlikely that the development of type 2 diabetes in the future (i.e. post baseline) would explain our results. At baseline, none of the participants with type 2 diabetes in our study population was using glucose-lowering medication, which made it possible to research the association between proinsulin and cancer mortality rates. It is also possible that the use of glucose-lowering medication post baseline could have biased our results. Therefore sensitivity analyses were performed and showed that in the group for whom we had follow-up information (n = 189), 36 participants used glucose-lowering medication 11 years later, of whom three died from cancer.
The strengths of this study include the long and complete follow-up duration (none of the participants in this subsample were lost to follow-up) with a precise cause-specific mortality registry. This enabled us to study a more precise association between proinsulin and cancer mortality rates. Furthermore, the Hoorn Study consists of a randomly selected cohort population, which limits the possibility of selection bias. Another strength of our study is that fasting proinsulin and specific insulin were measured at baseline, in duplicate, which made it possible to evaluate whether the association between proinsulin and cancer mortality rates was independent of specific insulin. A high correlation between proinsulin and specific insulin could cause multicollinearity within the regression model. We observed a Pearson correlation coefficient between proinsulin and specific insulin of 0.48 and the regression model did not substantially change when specific insulin was added to the model (Table 2).
A limitation of our study is that the radioimmunoassay used was not able to differentiate between intact proinsulin and des 31,32 proinsulin. The inhibition of the conversion from des 31,32 proinsulin to insulin by hyperglycaemia  could therefore, to some extent, lead to an overestimation of the contribution of proinsulin levels. Proinsulin concentrations are highly variable. We therefore measured fasting proinsulin levels on two separate days, at an interval of 2 weeks, and used the mean to improve precision. Furthermore, proinsulin levels were only measured at baseline and there is no indication that proinsulin levels remained in the same tertile during the complete follow-up period. Therefore we can only draw conclusions on the association between proinsulin measured at baseline and cancer mortality rates in a 20 year follow-up period. Another restriction is the relatively small sample size of this study. However, the small sample size did not result in wide confidence intervals, indicating that an apparent association between proinsulin and cancer mortality rates does indeed exist. Due to the small study population, we lacked power to differentiate between site-specific cancer mortality rates, so we can only draw conclusions on the association between proinsulin and overall cancer mortality rates. In addition, confounding might have affected our analysis. Although we adjusted for several confounders at baseline (including specific insulin), 20 years elapsed between measurements and outcomes. Since we did not adjust for these potential confounders during the follow-up period, it remains possible that other variables at follow-up might explain part of our observations.
To conclude, high proinsulin levels were found to be significantly associated with 20 year cancer mortality, independently of glucose metabolism and fasting specific insulin. These findings need to be reproduced in other populations, but are in line with existing in vitro findings . Additional studies with site-specific cancer information are warranted.
I. Walraven is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis
Duality of interest
The authors declare that there is no duality of interest associated with the manuscript.
Author contributions were as follows: JMD, GN and CDAS were responsible for the conception and design of the study; IW and GN analysed the data and wrote the paper. All authors contributed to the interpretation of data and critically reviewed the manuscript. All authors approved the final version submitted for publication.