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Original Research Communications |
1 From the Department of Medicine, the Division of Internal Medicine, University of Helsinki, and Downing College, University of Cambridge, United Kingdom.
2 Presented in abstract form at the 11th International Symposium on Atherosclerosis, 5–9 October 1997, Paris (Atherosclerosis 1997;134:32). 3 Supported by grants from Helsinki University Central Hospital, the Finnish Diabetes Research Association, and The Howard Foundation, Cambridge, United Kingdom. 4 Reprints not available. Address correspondence to TA Miettinen, Department of Medicine, University of Helsinki, PO Box 340, FIN-00029 Helsinki, Finland. E-mail: tatu.a.miettinen{at}helsinki.fi.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We investigated glucose, cholesterol, and lipoprotein metabolism at baseline and 2 y after weight reduction in obese patients with type 2 diabetes consuming an isoenergetic diet.
Design: Sixteen subjects were randomly chosen to consume a very-low-energy or low-energy diet for 3 mo, after which they consumed a weight-maintenance diet for up to 2 y. Cholesterol absorption and metabolism, LDL and HDL kinetics, and variables of glucose metabolism were studied at baseline and 2 y.
Results: Baseline serum sex hormone binding globulin (SHBG) was significantly related to cholesterol absorption efficiency, and serum glucose and insulin concentrations were associated with cholesterol synthesis. After 2 y, body weight was reduced by 6 ± 1 kg (P < 0.01), body mass index by 6% (P < 0.05), and blood glucose by 14% (P < 0.01); the ratio of serum SHBG to insulin increased by 66% (P < 0.05). Serum and VLDL, LDL, and HDL triacylglycerol were significantly reduced by 13–24%. Despite unchanged serum concentrations of cholesterol, cholesterol absorption efficiency and the ratio of serum plant sterols to cholesterol—indicators of cholesterol absorption—increased by 28% (P < 0.01) and 20–31% (P < 0.05 for both), respectively; the fractional removal of LDL apolipoprotein B decreased. Fecal excretion of cholesterol as neutral sterols decreased significantly by 11%. Changes in body weight were significantly negatively correlated with changes in ratios of cholesterol to serum plant sterols and cholestanol.
Conclusions: Baseline cholesterol absorption and synthesis were related to respective serum SHBG, glucose, and insulin values. Weight reduction increased cholesterol absorption and improved variables of glucose metabolism. These results suggest that low cholesterol absorption and high synthesis may be part of the insulin resistance syndrome.
Key Words: Cholesterol absorption • cholesterol synthesis • insulin resistance • sex hormone binding globulin • plant sterols • lathosterol • obesity • type 2 diabetes • weight loss
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alterations in lipoprotein metabolism in type 2 diabetes were described in detail previously (6–8), but the metabolism of cholesterol and bile acids has been less well characterized. It is known from earlier studies that cholesterol absorption can be low in persons with type 2 diabetes (9, 10), and serum plant sterols—indicators of cholesterol absorption efficiency (11)—are also low in persons with type 2 diabetes (9, 12) as well as in subjects with high-normal blood glucose concentrations (13). Bile acid and cholesterol synthesis and cholesterol excretion as neutral sterols are higher in patients with type 2 diabetes than in healthy populations (9, 10, 14–17). However, there have been no studies conducted to determine how cholesterol and lipoprotein metabolism are interrelated and how cholesterol metabolism is associated with insulin resistance in type 2 diabetes. Weight reduction is the primary means of treatment in overweight diabetic subjects. It is not known, however, whether long-term weight reduction affects cholesterol absorption and metabolism in these subjects. To this end, the aims of this study were 1) to investigate in obese type 2 diabetic subjects whether cholesterol and lipoprotein metabolism are related to serum variables of glucose metabolism at baseline, and 2) to study the effects of weight reduction on glucose, cholesterol, and lipoprotein metabolism during a prolonged follow-up.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7.0 mmol/L) with a mean (±SEM) age of 52.3 ± 1.8 y and a body mass index (BMI; in kg/m2) > 30 (Table 1
). Exclusion criteria were insulin therapy, diabetic microangiopathy, hepatic or thyroid disease, unstable angina pectoris or myocardial infarction, or invasive CAD treatment in the previous year. All women were postmenopausal and none had received hormone replacement therapy. All subjects gave informed consent and the protocol was approved by the Ethics Committee of the Department of Medicine, University of Helsinki.
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Methods
The 2-y study consisted of 3 periods: the run-in, weight-reduction, and weight-maintenance periods. During the run-in period, which lasted 6 wk, baseline metabolic studies were completed while the subjects consumed an ad libitum diet at home. During the weight-reduction period, which lasted for 3 mo, the VLED group ingested daily 3 servings of a VLED (97 kJ/d, Cambridge Diet; Howard Foundation, Cambridge, United Kingdom). One serving provided 14.2 g protein, 15.0 g carbohydrates, 2.7 g fat, and essential minerals, trace nutrients, and vitamins. Hypoglycemia treatment was discontinued in the VLED group. The LED group was advised to consume a low-fat, low-cholesterol diet. In the LED group, the dose of glibenclamide was adjusted so that blood glucose concentrations were <7.0 mmol/L and biguanide use was discontinued.
The weight-maintenance period continued from 4 mo until 2 y in both groups. The diets were individually tailored by the dietitian so that the daily energy balance was zero. Hemoglobin and blood pressure measurements were made and an electrocardiogram and liver, thyroid, and kidney function tests were performed.
During the isoenergetic phases at the beginning and end of the study, blood glucose, serum insulin, and SHBG—an indicator of insulin resistance (18)—were measured and metabolic and kinetic studies were performed. The subjects kept a food record for 7 d, from which the proportions of the dietary constituents were calculated (19). Additionally, subjects were given a capsule containing (
± SEM) 76.5 ± 0.323 Bq (4500 ± 19 decays/min) 4-[14C]cholesterol, 196.99 ± 0.714 Bq (11588 ± 42 decays/min) 22,23-[3H]ß-sitosterol, and 200 mg Cr2O3 3 times daily with their regular meals during this 7-d period. Stool was collected on the last 3 d of the week. Turnover studies of serum LDL and HDL were performed for 2 wk after the stool collection was completed. During this time, serum lipids; lipoproteins; apolipoprotein (apo) A-I, A-II, and B; and noncholesterol sterols were analyzed 4 times from serum samples collected after a 12-h fast.
Serum total and free cholesterol, triacylglycerol, phospholipids, and apo A-I, A-II, and B were measured with commercial kits (Boehringer Mannheim, Mannheim, Germany; Wako Chemicals, Neuss, Germany; and Orion Diagnostica, Espoo, Finland). Serum lipoproteins were separated by ultracentrifugation into the following density classes: VLDL, <1.006 kg/L; IDL, 1.006–1.019 kg/L; LDL, 1.019–1.063 kg/L; and HDL, 1.063–1.210 kg/L (20). Serum insulin was analyzed by radioimmunoassay and serum SHBG by fluoroimmunoassay with commercial kits (Pharmacia & Upjohn, Uppsala, Sweden; and Wallac, Turku, Finland).
Serum sterols were determined by gas-liquid chromatography from nonsaponifiable serum on a 50-m long SE-30 capillary column (21) (Ultra 1; Hewlett-Packard, Wilmington, DE). The procedure measures total cholesterol and noncholesterol sterols, including cholesterol precursors (
8-cholestenol, desmosterol, and lathosterol), reflecting cholesterol synthesis (11, 22, 23), and plant sterols (campesterol and sitosterol) and cholestanol, reflecting cholesterol absorption (11, 24).
Elimination of cholesterol from the body and cholesterol absorption efficiency were determined from the 3-d stool samples. Cholesterol absorption efficiency was calculated by subtracting the ratio of 14C to 3H in the stool samples from that in the diet (25), and Cr2O3 measurements (26) were applied to the measurement of fecal flow. Fecal cholesterol as fecal neutral sterols (cholesterol, coprostanol, and coprostanone), bile acids, and plant sterols were quantitated by gas-liquid chromatography (27–29).
For the kinetic studies, 50 mL plasma was drawn and autologous LDL and HDL were separated by serial preparative ultracentrifugation (for 44 h at 10°C and 321 984 x g). Apo A-I was isolated from HDL as described previously (30). Apo A-I was iodinated with 125I and LDL apo B was iodinated with 131I by using a modification of the iodine-monochloride method (31, 32). Three days before injection, the subjects started to take peroral potassium iodide. Approximately 1 mg of the labeled autologous LDL apo B and apo A-I was mixed with 5% human serum albumin, filtered, and injected simultaneously. The total amount of radioactivity did not exceed 2220 Bq (60 µCi).
After the injection, 10-mL blood samples were collected and counted for 14 d. Die-away curves were constructed for [131I]LDL and [125I]HDL. The fractional catabolic rates (FCRs) for LDL apo B and HDL apo A-I were determined by using a 2-pool model. The transport rate was calculated by multiplying the FCR by the pool size. Pool size was calculated by multiplying the plasma apo concentrations by plasma volume, which was calculated to be 4.5% of body weight. LDL apo B and HDL apo A-I were measured twice in postinjection samples and their mean values were used to calculate the transport rate.
Cholesterol synthesis was calculated as the difference between the fecal steroids (neutral and acidic) of cholesterol origin and dietary cholesterol. Biliary secretion was calculated as the total intestinal cholesterol pool minus dietary cholesterol. To eliminate the effect of changing lipoprotein cholesterol concentrations during the entire study period, noncholesterol sterol values were standardized and expressed as 102 x mmol/mol cholesterol, ie, in proportions or ratios to serum total cholesterol.
Statistics
The hypothesis was tested with paired t tests, Spearman's rank correlation coefficients, and analysis of variance by using BMDP statistical software (SPSS Inc, Chicago). Logarithmic transformations were used with skewed distributions. Assuming that the obtained reduction in weight would be 5 kg and the 
level was <0.05, a sample size of 16 would give a statistical power of 0.80 (33).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Serum and metabolic variables were not significantly different between men and women or between treatment groups; therefore, the 16 subjects were analyzed in aggregate. For example, LDL-cholesterol, triacylglycerol, blood glucose, serum SHBG, and cholesterol synthesis values in women and men were 2.82 ± 0.72 and 3.28 ± 0.19 mmol/L, 3.58 ± 2.07 and 3.59 ± 0.54 mmol/L, 7.7 ± 0.9 and 8.5 ± 0.7 mmol/L, 18.7 ± 5.5 and 26.1 ± 3.7 nmol/L, and 21.1 ± 2.3 and 16.4 ± 0.9 mg•kg-1•d-1, respectively.
Two-year follow-up
All 16 subjects completed the study without side effects. After the follow-up, the mean body weight was 6 ± 1 kg lower than that at baseline (Table 1), the changes ranging from -18 to 1 kg. The daily intakes of energy, cholesterol, and plant sterols (in mg/kg body wt) did not differ significantly from baseline, but fat intake was significantly lower than baseline (Table 2). Blood glucose was reduced by 14% (P < 0.01), serum insulin decreased only slightly, and serum SHBG concentrations tended to increase, but not significantly so. The ratio of serum SHBG to insulin increased significantly, by 66% (Table 1). Free and esterified cholesterol and phospholipids were practically unchanged in serum and lipoproteins. However, triacylglycerol concentrations decreased significantly in serum and VLDL, LDL, and HDL decreased by 25%, 27%, 15%, and 13%, respectively, resulting in decreased triacylglycerol-cholesterol ratios: from 5.1 to 4.5 (P < 0.01) in VLDL, from 9.8 to 8.0 (P < 0.001) in LDL, and from 21.4 to 16.8 (P < 0.01) in HDL. The ratio of triacylglycerol to apo B in LDL decreased by 16 ± 3% (P < 0.001). The kinetic studies showed that the FCR for LDL apo B decreased significantly, whereas the transport rate for LDL apo B and HDL apo A-I kinetics did not change significantly (Table 3).
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). Cholesterol absorption efficiency increased in 13 subjects, did not change in 1 subject, and decreased in 2 subjects. Bile acid and cholesterol synthesis did not change significantly, despite significantly decreased fecal neutral steroid excretion. The proportions of plant sterols in serum increased significantly, by 20–31%; cholesterol precursor–sterol ratios tended to decrease correspondingly.
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) and with serum insulin (r = 0.558, P < 0.05). Serum insulin also correlated with fecal neutral steroids and cholesterol synthesis. Serum SHBG was associated with basal body weight and cholesterol absorption efficiency, but not with blood glucose or serum insulin.
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) and inversely to serum triacylglycerol. Blood glucose was inversely related to the proportion of cholestanol in serum. In addition, changes from baseline in campesterol, cholestanol, and sitosterol were related to reductions in body weight (Figure 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum total and LDL-cholesterol concentrations did not change significantly after the 2-y follow-up, probably because increased cholesterol absorption provided more cholesterol to the liver from the intestine, reducing slightly the removal (FCR) of LDL apo B. It can be assumed that VLDL production, which is elevated in type 2 diabetes (15), decreased slightly after weight reduction, which probably contributed to the lower triacylglycerol content in the VLDL, LDL, and HDL fractions.
Weight reduction in type 2 diabetes improves hyperinsulinemia and insulin resistance (34), which was observed in the present study on the basis of serum glucose, insulin, and SHBG concentrations, the latter of which is assumed to be a valid indicator of insulin resistance (18). The serum SHBG concentration was not related to glucose and insulin concentrations, but it was significantly positively correlated with cholesterol absorption efficiency and with serum plant sterol and cholestanol concentrations after weight reduction, reflecting cholesterol absorption. The inverse associations between SHBG and variables of cholesterol synthesis were not significant (r = -0.364, P < 0.1). Weight reduction was inversely correlated with the proportion of sitosterol, campesterol, and cholestanol in serum and with the absorption efficiency of cholesterol, but not with changes in blood glucose or variables of cholesterol synthesis. These associations suggest that cholesterol absorption efficiency and insulin resistance were interrelated and that the efficacy of cholesterol absorption was an inverse indicator of insulin resistance.
It can be assumed that elimination of overweight could normalize cholesterol absorption and, accordingly, normalize cholesterol metabolism in obese patients with diabetes. It is not known, however, whether decreased absorption of cholesterol is exclusively due to obesity or whether diabetes itself is a contributing factor. We showed earlier that cholesterol absorption was lower and cholesterol synthesis was higher in moderately overweight (BMI: 26.5) persons with type 2 diabetes than in nondiabetic control subjects of similar ages and BMIs (9). In addition, our preliminary results in obese subjects with and without diabetes suggest that cholesterol absorption is lower in diabetic persons (35). This suggests that diabetes or insulin resistance might be more important than obesity.
A lower cholesterol absorption efficiency was described previously in hypertriglyceridemic subjects with type 1 and 2 diabetes than in normal control subjects (10), in a mildly overweight type 2 diabetic population than in control subjects (9), and even in subjects with a high-normal glucose concentration than in those with low-normal glucose concentrations (13). It appears that increasing glucose concentrations and increasing insulin resistance are continuous variables with decreasing cholesterol absorption and increasing cholesterol synthesis. In a more recent study, plasma insulin, C-peptide, and glucagon were related to absolute but not to relative cholesterol absorption (36).
Increasing insulin resistance and obesity could change the intestinal cholesterol pool or the absorption mechanism of intestinal mucosa. Obese subjects effectively synthesize cholesterol, which increases cholesterol secretion through the biliary tract into the intestine, expanding the intestinal cholesterol pool (37). The large intestinal cholesterol pool dilutes dietary cholesterol and the marker sterols used to measure absorption and could reduce absorption efficiency. In the present study, cholesterol absorption efficiency increased after weight reduction, with no change in the intestinal cholesterol pool (Table 4), and serum plant sterols and cholestanol increased proportionately with weight loss (Figure 2), suggesting that improved insulin resistance, in combination with only slightly decreased body weight, enhanced mucosal capacity to absorb cholesterol and sterols. When cholesterol absorption is low, hepatic cholesterol synthesis is up-regulated, and when more cholesterol is absorbed from the intestine and transported to the liver, cholesterol synthesis and LDL receptor activity are diminished (reduced FCR for LDL apo B). Cholesterol synthesis was high in most (9, 10, 14–17) but not all (38) studies of type 2 diabetes including nonobese and obese subjects, and decreased when patients became euglycemic (14, 39). On the other hand, low absorption efficiency and high cholesterol synthesis was observed in overweight nondiabetic subjects (40) and BMI was inversely related to cholesterol absorption efficiency (41), suggesting that obesity itself results in a hyperinsulinemic state and alters cholesterol metabolism. Accordingly, the present study showed that low cholesterol absorption efficiency may be part of the insulin resistance syndrome.
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