Cholesterol Metabolism in Type 1 Diabetes

From among the type 1 diabetic patients of our endocrine outpatient department, four men and three women, 20–53 years of age (30.4 ± 4.4 years [mean ± SE]), with BMI 23 ± 1 kg/m², good glycemic control (HbA_1c 6–7%), and a constant insulin dose volunteered to the study. Age- and BMI-matched control subjects (three men and two women, age 24–54 years, mean 32.8 ± 5.7, BMI 23 ± 1 kg/m²) were obtained from our earlier control population. The control subjects were recommended to begin a low–saturated fat/low-cholesterol diet similar to the one that the diabetic subjects were consuming at least 3 weeks before baseline. Fasting blood samples were obtained in the morning, at ∼8:00 a.m., before the morning insulin dose in the diabetic subjects. The research protocol was accepted by the ethics committee of our department (University of Helsinki). The investigation was conducted according to the principles of the Declaration of Helsinki.

Serum cholesterol and triglycerides were measured with commercial kits (CHOD-PAP and GPO-PAP, respectively; Roche Diagnostics, Mannheim, Germany). Blood glucose and HbA_1c were assayed by routine hospital methods. VLDL, IDL, LDL, and HDL were separated with ultracentrifugation. Noncholesterol sterols (including cholestenol and lathosterol, which reliably reflect cholesterol synthesis [1], and cholestanol, a metabolite of cholesterol, and campesterol and sitosterol, the plant sterols, which are markers of cholesterol absorption [1,11]) were measured with gas-liquid chromatography (GLC) (12) from nonsaponifiable material of serum and lipoprotein fractions. For determination of free and esterified sterols, chloroform/methanol extracts of serum and lipoproteins were applied on thin-layer chromatoplate and the free and esterified sterol fractions were separated. Cholesterol and noncholesterol sterols were quantitated with GLC from the free sterol fraction, after saponification from the esterified fraction. The results of the noncholesterol sterols are given either as concentrations (micrograms per deciliter), so as to see the distribution of sterols in different lipoproteins, or as ratios of cholesterol (millimoles per mole of cholesterol) obtained from the same GLC run. The term ratio is frequently used in the text for the noncholesterol sterol–to–cholesterol ratio.

Means ± SEs were calculated, and the statistical differences between the diabetic and control groups were calculated by Student’s t test, using P < 0.05 as the limit for statistical significance. Logarithmic transformations were performed with skewed distribution.

Serum lipid levels were similar, except that IDL and LDL triglycerides were higher in the diabetic than in the control subjects (Table 1). From among the concentrations of the noncholesterol sterols in serum, those reflecting cholesterol synthesis (cholestenol and lathosterol) were lower, and the absorption marker sterols (campesterol, sitosterol, and cholestanol) were higher in type 1 diabetic compared with control subjects for nonesterified, esterified, and total cholestanol and cholestenol (Fig. 1). Additional data for lathosterol and cholestanol (Fig. 2) actually show that the serum concentrations were reflected mainly by LDL and HDL. Namely, >50% of total serum noncholesterol sterols were transported by LDL and ∼30–40% by HDL. The latter one transported relatively more esterified than nonesterified sterols, especially the absorption markers, whereas VLDL, IDL, and LDL preferentially transported free sterols (Fig. 2).

The esterification percentage of cholesterol was significantly lower in the VLDL and IDL of type 1 diabetic than in control subjects, whereas the respective values were higher for the synthesis markers, significantly so for lathosterol (Table 2). The esterification percentages were markedly low in the VLDL in both groups and increased gradually toward HDL, in which the highest percentages of esterification were seen for cholestanol (∼83%) and the lowest ones for lathosterol (63%).

The ratios of the absorption markers in serum were higher in type 1 diabetic than in control subjects, significantly so for all cholestanol ratios and esterified campesterol. On the other hand, the ratios of esterified and total cholestenol and free and total lathosterol were significantly lower in diabetic versus control subjects (Table 3).

The ratios of free, esterified, and total cholestanol were higher (almost twofold for free VLDL sterols) in all of the lipoprotein fractions of diabetic compared with control subjects. The esterified and total campesterol and sitosterol ratios were higher only in LDL in diabetic than in control subjects. The ratios of absorption sterols in both groups were usually lowest in VLDL, surprisingly high in IDL, and usually highest in HDL. Thus, measurement of the mean ratios of the absorption marker sterols to cholesterol in serum seems to overestimate those in VLDL and frequently underestimate those in HDL (Table 4).

The free cholestenol ratio in VLDL and those of esterified and total in LDL were significantly lower in type 1 diabetic than in control subjects. The total lathosterol ratio in VLDL and those of free and total lathosterol in IDL and LDL were lower in diabetic than in control subjects. The lathosterol ratio gradually decreased from VLDL to HDL in control subjects in the free, but not in the ester, fraction and less consistently in type 1 diabetes. Thus, the ratio of total lathosterol in serum was lower than that in VLDL and IDL, but quite similar to that in LDL and HDL. The lower cholesterol synthesis in type 1 diabetic than control subjects was best revealed by the finding that the free cholestenol and lathosterol ratios in type 1 diabetes were only ∼18–50% in VLDL and IDL from those in the control subjects. The respective values for the total sterol ratios in serum were 38–58%. In both groups, the ratios of the free lathosterols were significantly higher than the esterified ones in every lipoprotein fraction. The negative correlation between absorption and synthesis sterols for the ratios of free cholestenol and cholestanol in LDL is shown in Fig. 3.

The major novel findings of the present study were the increased ratios of sterol markers for cholesterol absorption and decreased ratios of sterol markers for cholesterol synthesis in type 1 diabetic compared with control subjects. These findings can be interpreted to indicate high absorption and low synthesis of cholesterol in patients with type 1 diabetes compared with nondiabetic control subjects. Other findings showed that the difference between the two groups in free, esterified, and total noncholesterol sterols was also present in different lipoproteins. The sterol ratios in serum were only modestly different from those in different lipoproteins.

In the literature there is experimental diabetes evidence that cholesterol metabolism is altered in insulin deficiency. Namely, streptozotocin-induced (insulin-deficient) diabetes in rats hypertrophies intestinal mucosal function, enhancing fat and cholesterol absorption and reducing cholesterol synthesis (9). Serum plant sterols, campesterol and sitosterol, have been measured in these animals, and they were increased (13). The values were improved with insulin treatment. However, the gut hypertrophy in this animal model may also play a role in altering cholesterol metabolism, not only insulin deficiency per se. In humans, significantly increased campesterol and sitosterol ratios have been found in the serum of poorly controlled patients with type 1 diabetes, and the values were normalized with intensified insulin treatment (8). Unfortunately, cholesterol precursors were not measured, so changes in cholesterol synthesis could not be evaluated. One study (14) with mainly type 2 diabetic subjects also included a small group of type 1 diabetic subjects who exhibited actually slightly reduced cholesterol synthesis and fecal output of neutral sterols when compared with control subjects. In two brothers with type 1 diabetes, the percentage of cholesterol absorption was exceptionally high, ∼60% for both of them, despite marked bile acid malabsorption (15). High absorption efficiency was associated with high cholestanol and plant sterol ratios in serum. It is also known that increased amounts of dietary cholesterol increase LDL cholesterol more effectively in type 1 diabetic than in matched control subjects (16). On the other hand, reduction of dietary cholesterol with modification of fat intake has been observed (17) to effectively reduce serum cholesterol in type 1 diabetic subjects.

The differences in noncholesterol sterols between type 1 diabetic and control subjects were seen with variable statistical significance in concentrations and ratios, in free and ester fractions, and in serum and different lipoproteins. However, even though the ratios of different sterols in serum were not exactly the same as those in different lipoproteins, the mean serum ratios seem to uncover the difference between the two groups in both absorption and synthesis markers. For instance, despite a nonsignificantly higher esterified lathosterol ratio in any lipoprotein of the control subjects compared with the diabetic subjects and a nonsignificantly higher free lathosterol ratio in HDL and markedly higher ratios in the LDL, IDL, and VLDL (up to three-fold higher in VLDL) of the control subjects versus those with diabetes, the mean lathosterol ratio in serum was ∼50% higher in the control subjects than in the diabetic subjects. However, analysis of the lipoproteins could reveal more detailed information of the metabolic differences. That cholesterol synthesis in diabetic patients was reduced mainly in liver could be interpreted by the marked lowering of lathosterol release into VLDL compared with that of the control subjects. The higher ester percentages of the synthesis marker sterols than of cholesterol in the triglyceride-rich lipoproteins could contribute to the high precursor sterol ratios in diabetes. A basic reason for this high esterification could be the prolonged half-life of the precursors compared with cholesterol rather than a sterol-specific difference in lecithin cholesteryl acyl transferase activity.

In contrast to lathosterol, both the ratios of free and esterified absorption sterols were lowest in VLDL and highest in HDL in both groups. The high ratios in HDL might point to reversed transport of these sterols from tissues to the liver by HDL itself or after transfer to LDL by cholesteryl ester transfer protein. Assuming that the absorption sterols have impaired biliary secretion in diabetes, their hepatic contents could be increased, which in association with possible low hepatic cholesterol synthesis could have produced VLDL with high absorption sterol ratio to cholesterol. The mean ratio of cholestanol in VLDL was ∼84% higher in diabetic than in control subjects, but the ratio increased more in other lipoproteins and was less marked for VLDL campesterol or sitosterol.

The findings of high cholesterol absorption and low cholesterol synthesis, as indicated by low ratios of synthesis markers to absorption markers (Fig. 3) in type 1 diabetic patients suggest that the variables of cholesterol metabolism are opposite to those in control subjects and especially in type 2 diabetic patients (10). In the latter patients, cholesterol absorption efficiency is low (2,18), whereas the synthesis of cholesterol is high (2,4,5). The findings are found also in obese subjects without diabetes (19) and in nonobese type 2 diabetic patients (20). What then are the factors regulating cholesterol metabolism in opposite directions in the two forms of diabetes? Our hypothesis is that the ABCG-ATP cassette transport system regulates cholesterol absorption and synthesis in the two types of diabetes. Briefly, current research indicates that ABC G/5 G/8 genes are almost exclusively expressed in intestinal enterocytes and liver (21). In transgenic mice or in overexpression of the two genes in mice, serum plant sterol levels and sterol absorption are reduced and cholesterol synthesis and biliary and fecal secretion of cholesterol are increased (22). Thus, the changes in cholesterol metabolism in these mice are identical to those in type 2 diabetic subjects. Accordingly, a resemblance of cholesterol metabolism in obesity (19) and type 2 diabetes (2,18,20) to that found in overexpression of ABC G/5 G/8 genes has been emphasized.

On the other hand, knockout of ABC G/5 G/8 genes in mice changes the serum sterol pattern (22) to resemble that seen in hereditary sitosterolemia (23), a metabolic disease caused by mutations of the ABC G/5 G/8 genes (21,24). In fact, downregulated expression of the two genes increases cholesterol and plant sterol absorption and prevents their biliary secretion, resulting in enhanced serum plant sterol and cholesterol levels. However, subsequently increased hepatic cholesterol inhibits cholesterol synthesis, preventing extensive increase of serum cholesterol and lowers biliary and fecal secretion of cholesterol (25). We propose that in type 1 diabetes, expression of ABC G/5 G/8 genes is downregulated, probably due to lack of effective insulin action, resulting in increased intestinal cholesterol and plant sterol absorption and their reduced biliary secretion. Consequently, serum cholestanol and plant sterol ratios are increased. Owing to enhanced accumulation of cholesterol, hepatic cholesterol synthesis is downregulated, precursor sterol ratios are reduced, and LDL cholesterol is normal or slightly increased, with extensively increased values occurring only during the ketotic state (26). These are actually the findings recorded in our present small study population. In fact, in rats with streptozotocin-induced diabetes, the expression of hepatic and intestinal mRNA of ABC G/5 G/8 was reduced compared with control subjects (27).

The present findings suggest that increased cholesterol absorption in type 1 diabetes could be a primary factor in modifying cholesterol metabolism, such that homeostatic mechanisms decrease cholesterol synthesis, a factor that could keep the LDL cholesterol concentration quite normal. These findings are explained by altered expression of ABC G/5 G/8 gene expression or the specific occurrence of their polymorphisms. The clinical importance of whether increased serum plant sterols in general and in type 1 diabetic patients are independent risk factors of atherosclerosis has recently been discussed (28–30).

The study was supported by grants from the Finnish Heart Research Foundation, the Yrjö Jahnsson Foundation, and the Kuopio University Hospital.