Introduction

Amyloid beta peptides in the blood originate not only from the brain but also from almost all peripheral cells, including platelets, vascular wall cells, muscle cells, liver cells and so forth. Despite the low correlation between plasma and brain amyloid beta peptides, the measurement of amyloid beta peptides in plasma or serum (less stable) could be the least invasive and most cost-effective biomarker assay for Alzheimer’s disease and also vascular dementia [1,2].

Pioglitazone, a peroxisome proliferator-activated receptor γ agonist, has been suggested to have potential for the treatment of Alzheimer’s disease [3-5], and a large phase 3 clinical trial is currently being undertaken to evaluate its safety and efficacy [6]. In the previous report [7], the effects of low-dose pioglitazone on several age-related biomarkers including amyloid beta peptides 1-40 and 1-42 (amyloid β1-40 and β1-42) were assessed in Japanese elderly patients with type 2 diabetes. Since serum amyloid β1-40 level tended to be increased by the add-on treatment with pioglitazone in female patients, changes in serum amyloid beta peptides after the pioglitazone treatment were reassessed by increasing the number of study patients in this report. It was unexpectedly found that high serum levels of amyloid beta peptides decreased and their low levels increased after 6-month treatment with pioglitazone.

Materials and Methods

Study Patients. Forty-three patients (21 males, 22 females) with type 2 diabetes aged ≥ 70 years, who gave their informed consent to the study in accordance with the Declaration of Helsinki, were examined at Matsumoto Medical Center Matsumoto Hospital. The effects of the 6-month treatment with low-dose pioglitazone (15 mg for males, 7.5 mg for females) on serum amyloid β levels were assessed. Twenty-four patients (12 males, 12 females) in the previous report [6] were included, and the same data from them were used. Clinical characteristics of all the patients at the baseline are shown in Table 1. Serum creatinine levels were 0.9±0.1 mg/dl in male patients and 0.7±0.2 mg/dl in female patients. Four insulin-treated patients, 5 patients who were started on pioglitazone treatment during hospitalization, and 10 patients whose antidiabetic (other than sulfonylurea) or antihypertensive agents were changed during the 6-month treatment with pioglitazone were newly included. One of the 19 patients newly included had been diagnosed with Alzheimer’s disease.

Blood Collection and Measurements. Before and after the 6-month treatment with pioglitazone, casual blood samples were collected and stored in serum at -20ºC until used. Amyloid β1-40 and β1-42 were measured with Human βAmyloid (1-40) and (1-42) ELISA kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and high-molecular-weight (HMW) adiponectin was measured by chemiluminescent enzyme immunoassay (CLEIA). The measurements were performed by a referee laboratory (SRL, Inc., Tokyo, Japan). HbA1c levels were measured at the outpatient clinic by an automated analyzer ADAMS HA-8160 (ARKRAY, Tokyo, Japan).

Statistical analysis. Results are expressed as mean±SD. Differences between data were analyzed by two-sided paired or unpaired t-test when appropriate, with a significance level at p<0.05.

Results

At the baseline, as shown in Figure 1, there were significant correlations (p<0.02) between serum HMW adiponectin level and serum amyloid β1-40 level in male and female patients, and between serum HMW adiponectin level and serum amyloid β1-42 level in male patients. After the 6-month treatment with pioglitazone, serum HMW adiponectin levels increased from 4.7±3.1 to 13.5±15.6 μg/mL in male patients and from 7.0±3.5 to 13.9±6.0 μg/mL in female patients, where the HMW adiponectin levels increased in all the patients. At the 6-month treatment with pioglitazone, no significant correlation was found between HMW adiponectin and amyloid β1-40 (r=0.287, p=0.208) or amyloid β1-42 (r=0.181, p=0.432) in male patients, or between HMW adiponectin and amyloid β1-40 (r=0.174, p=0.439) or amyloid β1-42 (r=0.200, p=0.371) in female patients.

Figure 2 shows individual changes in serum amyloid β1-40 or β1-42 levels before and after the pioglitazone treatment in each of male and female patients. There was no significant difference in the mean values of amyloid β1-40 levels (46.4±12.6 vs. 47.1±11.5 pmol/L, p=0.711) or β1-42 levels (3.0±1.2 vs. 2.9±0.9 pmol/L, p=0.687) in male patients, or in those of amyloid β1-40 levels (48.1±15.6 vs. 50.6±10.4 pmol/L, p=0.411) or β1-42 levels (3.2±1.4 vs. 3.0±1.4 pmol/L, p=0.677) in female patients. Interestingly, however, it appeared that higher levels of serum amyloid β1-40 and β1-42 decreased and their lower levels increased after the pioglitazone treatment, as shown in each of panels. In one patient with Alzheimer’s disease, serum amyloid β1-40 and β1-42 levels decreased from 55.9 to 43.4 pmol/L and from 3.1 to 1.3pmol/L.

Then, as shown in Tables 2 and 3, data were reassessed when patients were classified by the serum amyloid β1-40 level of 50 pmol/L or β1-42 level of 3 pmol/L at the baseline. Patients with serum amyloid β1-40 levels >50 pmol/L or β1-42 levels > 3 pmol/L showed a significant decrease in the mean value of amyloid β1-40 or β1-42 levels after the pioglitazone treatment in total patients, being predominant in female patients (Table 2). On the contrary, patients with serum amyloid β1-40 levels < 50 pmol/L or β1-42 levels < 3 pmol/L showed a significant increase in the mean value of amyloid β1-40 or, to a lesser extent, β1-42 levels after the pioglitazone treatment in total patients, being predominant in female patients (Table 3). There was no significant difference in the mean HbA1c values between before and after the pioglitazone treatment, or between patients with higher and lower amyloid β levels (Tables 2 and 3).

Firstly, it is of interest that serum HMW adiponectin level was found to significantly correlate with serum amyloid β1-40 and β1-42 levels at the baseline (but not with amyloid β1-42 in female patients). Adiponectin is secreted by adipocytes, and exists in 3 major forms (trimers, hexamers and HMW multimers) in plasma, influencing biological processes including energy metabolism, vascular function and immune responses. HMW adiponectin, which is predominantly upregulated by pioglitazone or caloric restriction, can activate adenosine 5’-monophosphate-activated protein kinase most potently, but does not enter the cerebrospinal fluid [8,9]. Adiponectin has been suggested to have positive effects for the prevention and treatment of Alzheimer’s disease and dementia [10,11]. Unexpectedly, however, the Framingham Heart Study demonstrated that increased plasma adiponectin levels were an independent risk factor for the development of both all-cause dementia and Alzheimer’s disease in females over a mean follow-up of 13 years [12]. A meta-analysis of plasma amyloid β levels indicated that in longitudinal studies, cognitively normal individuals who converted to Alzheimer’s disease had higher baseline amyloid β1-40 and β1-42 levels [13]. Accordingly, it may be conceivable that positive correlations were found between serum HMW adiponectin and amyloid β peptides in the present study. No direct interaction between them should exist, because their significant correlation disappeared after the pioglitazone treatment. In a similar vein, high adiponectin levels have been reported to be associated with increased cardiovascular disease and all-cause mortality in the elderly population, where weight loss and sarcopenia are presumed to be involved in their association [14,15].

 Secondly, it is noteworthy that high serum levels of amyloid beta peptides decreased and their low levels increased after the 6-month treatment with low-dose pioglitazone. The relevance of low-dose pioglitazone was discussed in the previous report [7]. When serum amyloid β1-40 or β1-42 levels were divided by the level of 50 pmol/L or 3 pmol/L at the baseline, the mean value of the higher levels significantly decreased and that of the lower levels significantly increased after the pioglitazone treatment in total patients. Although the association between plasma amyloid β peptides and Alzheimer’s disease is inconclusive, it is in general thought that plasma amyloid β peptides increase with age (production in the brain) and begin to decrease before the conversion to Alzheimer’s disease (deposition in the brain) [1,2,16-18]. Such pathophysiological changes might have been augmented by the pioglitazone treatment in the present study. This presumption is, however, inconsistent with the potential protective effects of pioglitazone for Alzheimer’s disease [3-5]. Distinct changes in amyloid β peptides during a short period as demonstrated in the present study have not been described thus far. The patients studied and their treatments were somewhat heterogeneous. If a presumption favoring the pioglitazone treatment would be made, pioglitazone could enhance clearing amyloid β peptides from the brain [18,19] and inhibit amyloid β oversynthesis in the brain [20,21]. In addition, there is a possibility that pioglitazone could modulate the production of amyloid β peptides in the peripheral cells [1].

 In conclusion, serum levels of amyloid beta peptides positively correlated with serum HMW adiponectin levels only at the baseline. High serum levels of amyloid beta peptides decreased and their low levels increased after 6-month treatment with pioglitazone in Japanese elderly patients with type 2 diabetes. The relevance of such changes would be worthy of further investigation to use peripheral amyloid β peptides as better biomarkers for Alzheimer’s disease and vascular dementia.

Conflict of interest

The author declares that there is no conflict of interest regarding the publication of this paper.

Figure 1: Correlations between serum high-molecular-weight adiponectin levels and serum amyloid β1-40 or β1-42 levels in (A) male and (B) female elderly patients with type 2 diabetes at the baseline are shown. Except between the adiponectin and amyloid β1-42 levels in females, there were significant correlations between them.

Figure 2: Individual changes in serum amyloid β1-40 or β1-42 levels before and after the pioglitazone treatment in each of male and female elderly patients with type 2 diabetes are shown. There was no significant difference in the mean values (open circles) of amyloid β1-40 levels (46.4±12.6 vs. 47.1±11.5 pmol/L, p=0.711) or β1-42 levels (3.0±1.2 vs. 2.9±0.9 pmol/L, p=0.687) in male patients, or in those of amyloid β1-40 levels (48.1±15.6 vs. 50.6±10.4 pmol/Lp=0.411) or β1-42 levels (3.2±1.4 vs. 3.0±1.4 pmol/L, p=0.677) in female patients. However, it appears that higher levels of serum amyloid β1-40 and β1-42 decrease and their lower levels increase after the pioglitazone treatment in each of panels.

 Table 1: Baseline clinical characteristics of 43 elderly patients with type 2 diabetes.

Table 2: Relevant data before and after the treatment of low-dose pioglitazone for 6 months (15 mg for males, 7.5mg for female) in patients with serum amyloid β1-40 levels > or < 50 pmol/L. 

Table 3: Relevant data before and after the treatment of low-dose pioglitazone for 6 months (15 mg for males, 7.5mg for female) in patients with serum amyloid β1-42 levels > or < 3 pmol/L. 

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