This study showed for the first time that using an ultrasensitive method for serum C-peptide, around 60% of long-term Brazilian T1D with childhood-onset (~17 yrs of diabetes duration) had residual insulin secretion. These detectable FCP levels was associated with older age at T1D diagnosis and shorter diabetes duration. Importantly this group with even low levels of residual C-peptide had lower prevalence of microalbuminuria adjusted for diabetes duration and HbA1c levels.
In recent years, the development of an ultrasensitive assay for plasma C-peptide detection (change the lower limits of detection of approximately 50pmol/L to levels as low as 1.15pmol/L) has made it possible to detect residual secretion of β-cell function in people with T1D, even after decades of disease [9-11]. The percentage of residual FCP that we found is five times higher than the 13.2% found in a group of adults with T1D with a shorter disease time when using a regular C-peptide assay in routine clinics [12].
Studies with similar characteristics to our group, found 55-66% positivity (lower limit of 1.15pmol/L for fasting serum C-peptide) and 52% for urinary C- peptide (>30pmol/L) [10,14] while others like participants of Exchange Clinic Network found detectable C-peptide in 29% among 900 participants, suggesting that residual C-peptide secretion is present in almost one out of three T1D individuals, three or more years from diabetes diagnosis [5].
According to our analysis, we verified that one of the main factors related to the frequency of FCP considered positive (>1.15pmol/L) were age at diabetes diagnosis and the duration of the disease. These two factors, also shown in our population, are in according to other groups where age at the diagnosis was positively associated with C-peptide values [5,10,11]. The relationship between age at T1D diagnosis and residual C-peptide can be explained by different insulin profiles found at diagnosis as it has been shown in new onset teenage T1D that still retain approximately 40% of residual insulin-containing islets [13].
In our population each increase of one year of age correspond to 8% reduction (OR =0.92) in probability of having FCP+. It would be expected better values in our patients than the English and American ones which had longer disease duration, however our patients presented a marginally lower prevalence of residual C-peptide secretion [11,14] In addition, detectable C-peptide related to better A1c was found in 38% of children and adolescents after 10 years of diabetes compared to 24% of our patients from public care services. Insufficient metabolic control may have contributed to this result. However, a recent systematic review and meta-analyses of all randomized controlled trials (RCTs) to preserve β-cell function in people with newly diagnosed T1D shown there is a lack of robust evidence that interventions to improve glucose control preserve β-cell function and efforts to treatment algorithms should be a priority [15].
However, one major debate today is what C-peptide level is clinically useful. In part because some studies that correlated residual C-peptide to chronic diabetes complications or hypoglycemia sometimes measure FCP [11,16-17] sometimes use stimulated C-peptide [18] besides the sensibility limit of ultra-sensitivity C-peptide assay used [16].
In our T1D participants with a mean 17 years of disease duration, using a C-peptide assay with a limit of sensibility of 1.15 pmol/L we did not find any association between residual C-peptide and lower HbA1c. This agrees with studies with similar T1D populations, while Trialnet participants and T1D from the UNITED Team found this association only during the early course of diabetes [16,19-21]. Also, a recent work with young adult T1D with 5 years of diabetes duration demonstrated that only those with high levels of residual C-peptide (peak after stimulus test > 4.0 pmol/L or >1.2ng/mL) shown β cell responsiveness to hyperglycemia and likely contribute to glycemic control [18]. We have studied β cell responsiveness to sulfonylurea test in a sample of nine patients with FCP+ and found that the maximum peak during the test was 0.286 pmol/L (data not shown) which is approximately 93% less than shown in the study above [18]. This might help understanding why we do not get an association between C-peptide levels and HbA1c in our T1D group and why we did no find significant difference in the prevalence of hypoglycemia between our FCP+ and FCP- groups. Nevertheless, heterogeneous relationship between the glucagon response to insulin-induced hypoglycemia does exist [23] but again is most evident in T1D with high levels of residual C-peptide [18].
In relation to diabetes chronic complications and residual C-peptide in our study using FCP with limit of sensibility of the assay equal 1.15 pmol/L, we found an inverse relationship between the C-peptide reserve and albuminuria in FCP+ T1D patients.
The frequency of albuminuria found in our participants was like people with T1D with the same time of disease described in the literature (19.6% vs 14.8%) where 65.2% of microalbuminuric patients had no detectable C-peptide. However, unlike predicted, we found no correlation of C-peptide and retinopathy, perhaps because of the low prevalence found in our participants (3.9% vs 9-40%) [22]. Also, maybe the low cut-off limit we used to define FCP+ patients.
The classical work that found C-peptide protection from complications as nephropathy, neuropathy and retinopathy considered FCP levels >10pmol/L (with the same C-peptide assay that we used) almost 9 times higher than our C-peptide cut-off. Other study [16] using the same cut-off value of ours (1.15pmol/L) also did not find relation among residual C-peptide and retinopathy, neuropathy but marginally lower macroalbuminuria in those with detectable levels (23.4% vs 0%, p=0.07). A larger sample size might have allowed a bigger difference [16].
One point for discussion is why these low levels of C-peptide can decrease the prevalence of nephropathy, regardless to HbA1c level but not the two others microangiopathies (retinopathy and neuropathy).
Diabetic nephropathy (DN) is one of the major microvascular complications, present in 20 to 40% of T1D people and is one of the most important causes of kidney failure (KF), but the rate of renal decline varies widely among them. On the other hand, in addition to its well-known role as a biomarker of functional beta-cell mass, the C-peptide is a bioactive molecule with physiological effects on peripheral cell targets and with antioxidant protection on vascular endothelial. Small trials in which C-peptide was given to subjects with T1D with nephropathy or neuropathy showed that C-peptide mitigates renal and neuronal complications [24], while others had shown that C-peptide can suppress various molecular mechanisms involved in the pathophysiology of DN and therefore could prevent the onset and progression of KF [25]. Interestingly almost two decades ago, a double-blind randomized study had demonstrated that administration of biosynthetic human C-peptide plus insulin reduced glomerular permeability and urinary excretion of albumin [26].
In relation to the lower prevalence of DN in our FCP+ T1D regardless of the same HbA1c as FCP- ones, we can speculate that the former could have less glycemic variability. Since recent works have been shown that for every 100 pmol/L increases in C-peptide peak the percentage of time spent in the range (70 -180 mg/dl) increased by 2.4% with a reduction in time spent at level 1 hyperglycemia (>180 mg-dl) and level 2 hyperglycemia (250 mg-dl) by 2.6% and 1.3% respectively [27]. Is important to mention that lower time in range was associated with presence of composite microvascular complications in recent study with a group of adults with long T1D duration [28].
When we studied the relationship between C-peptide levels and nephropathy in our T1D group we found out that all patients with fasting C-peptide > 46.9 pmol/L (0.14 ng/ml) were negative for this microangiopathic complication. This number is close to cut-off used in most studies (50 pmol/L but one that considered 10pmol/) [17] for identify patients at higher risk for complications, more frequent and great glycemic excursions and low 1,5 AG levels.
Finally, at this level of glycemic control, we did not find difference on lipid profile between T1D patients with and without residual C-peptide. However, results in this area are heterogenous, some works [29] showing better lipid profile in C-peptide positive and others showing no relationship between unstimulated C-peptide values and lipid parameters in either remitters or non-remitters T1D adult [32].
The present study has some limitations as the small sample size, the cross-sectional data, using FCP although the literature has already shown a good correlation with stimulated C-peptide. Another limitation was the low prevalence of chronic diabetes complications that may have impaired the association studies. The strengths were the assay sensitivity, the heterogenous genetic background of our T1D population and the real-world data from their routine treatment.
We can conclude that most of our T1D participants, like American and European data, have residual beta-cell function demonstrated with the use of an ultrasensitive assay after a decade of disease, and this minimal detectable C-peptide appears to protect against albuminuria regardless of HbA1c.
Finally, the importance of persistent beta-cell residual function reinforces strategies for its preservation since diagnosis and suggests that a significant percentage of patients with T1D, even after decades of diagnosis, may have benefits in slowing the development of diabetes nephropathy.