Bloodletting Ameliorates Insulin Sensitivity and Secretion in Parallel to Reducing Liver Iron in Carriers of HFE Gene Mutations

ArticleinDiabetes care 31(1):3-8 · January 2008with14 Reads
Impact Factor: 8.42 · DOI: 10.2337/dc07-0939 · Source: PubMed
Abstract

To clarify the pathogenesis of diabetes associated with mutations of the hemochromatosis (HFE) gene, 17 carriers, 9 normal glucose tolerant (NGT) and 8 diabetic, were evaluated in an interventional trial. At enrollment and after a 2-year bloodletting period, euglycemic-hyperinsulinemic clamp, oral glucose tolerance test (OGTT), liver histology (nonalcoholic fatty liver disease activity score [NAS]), and liver iron content (LIC) were assessed. NGT subjects had significantly higher baseline insulin sensitivity (P <or= 0.001), secretion, and insulinogenic index (calculated from the OGTT) (P <or= 0.0001 for both) and lower LIC (P = 0.004) and NAS (P = 0.02) than diabetic patients. Baseline LIC correlated negatively with insulin secretion (NGT r(0) = -0.676, P <or= 0.0001; diabetes r(0) = -0.589, P = 0.02) and insulin sensitivity (M value) (NGT r(0) = -0.597, P = 0.009; diabetes r(0) = -0.535, P = 0.03) and positively with NAS (diabetes r(0) = 0.649, P = 0.007) and triglycerides (NGT r(0) = 0.563, P = 0.015). At month 24, circulating iron was reduced by 179 +/- 26% in NGT and 284 +/- 54% in diabetic subjects. Insulin secretion (NGT 20 +/- 4%; diabetes 33 +/- 7%) and insulin sensitivity (NGT 25 +/- 5%; diabetes 18 +/- 3%) increased. LIC decreased in both groups (NGT 126 +/- 42%; diabetes 61 +/- 13%), and NAS ameliorated (NGT 65.1 +/- 6.5 vs. 38.1 +/- 6.83; P <or= 0.0001; diabetes 2.1 +/- 10.7 vs. 69.9 +/- 10; P <or= 0.0001). Iron depletion ameliorates insulin secretion and sensitivity in NGT and diabetic carriers of HFE gene mutations. This amelioration occurs in parallel with decreased LIC and improved NAS. These results justify glucose tolerance testing and prophylactic iron depletion in asymptomatic carriers as well.

Full-text

Available from: Melania Manco, Nov 16, 2015
Bloodletting Ameliorates Insulin
Sensitivity and Secretion in Parallel to
Reducing Liver Iron in Carriers of HFE Gene
Mutations
FRANCESCO EQUITANI,
MD
1
JOS`E MANUEL FERNANDEZ-REAL,
MD
2
GIACOMO MENICHELLA,
MD
1
MAURIZIO KOCH,
MD
3
MENOTTI CALVANI,
MD
4
VALERIO NOBILI,
MD
5
GELTRUDE MINGRONE,
MD, PHD
4
MELANIA MANCO,
MD, PHD
5
OBJECTIVE To clarify the pathogenesis of diabetes associated with mutations of the
hemochromatosis (HFE) gene, 17 carriers, 9 normal glucose tolerant (NGT) and 8 diabetic, were
evaluated in an interventional trial.
RESEARCH DESIGN AND METHODS At enrollment and after a 2-year bloodletting
period, euglycemic-hyperinsulinemic clamp, oral glucose tolerance test (OGTT), liver histology
(nonalcoholic fatty liver disease activity score [NAS]), and liver iron content (LIC) were assessed.
RESULTS NGT subjects had significantly higher baseline insulin sensitivity (P 0.001),
secretion, and insulinogenic index (calculated from the OGTT) (P 0.0001 for both) and lower
LIC (P 0.004) and NAS (P 0.02) than diabetic patients. Baseline LIC correlated negatively
with insulin secretion (NGT r
0
⫽⫺0.676, P 0.0001; diabetes r
0
⫽⫺0.589, P 0.02) and
insulin sensitivity (M value) (NGT r
0
⫽⫺0.597, P 0.009; diabetes r
0
⫽⫺0.535, P 0.03)
and positively with NAS (diabetes r
0
0.649, P 0.007) and triglycerides (NGT r
0
0.563,
P 0.015). At month 24, circulating iron was reduced by 179 26% in NGT and 284 54%
in diabetic subjects. Insulin secretion (NGT 20 4%; diabetes 33 7%) and insulin sensitivity
(NGT 25 5%; diabetes 18 3%) increased. LIC decreased in both groups (NGT 126 42%;
diabetes 61 13%), and NAS ameliorated (NGT 65.1 6.5 vs. 38.1 6.83; P 0.0001;
diabetes 2.1 10.7 vs. 69.9 10; P 0.0001).
CONCLUSIONS Iron depletion ameliorates insulin secretion and sensitivity in NGT and
diabetic carriers of HFE gene mutations. This amelioration occurs in parallel with decreased LIC
and improved NAS. These results justify glucose tolerance testing and prophylactic iron deple-
tion in asymptomatic carriers as well.
Diabetes Care 31:3–8, 2008
T
he most common iron overload dis-
order in the general population is
type 1 hereditary hemochromatosis
(HH). The disease occurs in 5 per 1,000
Caucasian people of northern European de-
scent (1). It is an autosomal recessive disor-
der mostly due to a homozygous mutation
within the hemochromatosis (HFE) gene on
chromosome 6, the Cys282Tyr mutation,
or, less frequently, the His63Asp mutation.
The clinical significance of heterozygosis for
these mutations is still controversial (1).
HH is associated with an increased rate
of diabetes. Iron exerts a toxic effect on
-cells, which causes cell apoptosis and
death (2,3). Iron depletion ameliorates
-cell function in patients with (4,5) and
without iron overload (6,7). In HH, iron de-
pletion has also been associated with the
amelioration of insulin sensitivity in pa-
tients who have not developed diabetes (8).
In liver, iron depot contributes to impaired
insulin metabolism by reducing the insulin-
extracting capacity (9), leading to hepatic
insulin resistance (10).
Increased insulin resistance favors the
progression from nonalcoholic fatty liver
disease (NAFLD) to nonalcoholic steato-
hepatitis, fibrosis, and cirrhosis and further
increasing both peripheral and hepatic in-
sulin resistance in a vicious cycle (11). In
muscle, iron interferes with glucose uptake
(2). Bloodletting enhances insulin sensitiv-
ity in healthy donors (4) and patients with
iron-induced insulin resistance (12–14).
Nonhomozygous HFE gene mutations are
generally associated with a mild variant of
hemochromatosis. When the disease is clin-
ically evident, it is probably due to the oc-
currence of conditions such as the
metabolic syndrome (12).
Therefore, the pathogenesis of diabetes
in homozygous or heterozygous carriers of
HFE gene mutations remains poorly under-
stood, with both decreased insulin secretion
and sensitivity being potential contributing
factors. Thus, to add new insight into the
pathogenesis of this topic, we studied pro-
spectively the effects of bloodletting on in-
sulin sensitivity and secretion, serum
biochemical parameters, liver iron content
(LIC), and histology in nine normal glucose
●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●
From
1
Transfusion Medicine, SanFilippo Neri Hospital, Rome, Italy; the
2
Section of Diabetes, Endocrinol-
ogy, and Nutrition, University Hospital, Girona, Spain; the
3
Liver Unit, SanFilippo Neri Hospital, Rome,
Italy; the
4
Department of Internal Medicine, Catholic University, Rome, Italy; and the
5
Department of
Hepato-Gastroenterology and Nutrition, Bambino Gesu` Hospital and Research Institute, Rome, Italy.
Address correspondence and reprint requests to Melania Manco, MD, PhD, Department of Hepato-
Gastroenterology and Nutrition, Bambino Gesu` Hospital and Research Institute, S. Onofrio 4 square, 00165
Rome, Italy, E-mail: melaniamanco@tiscali.it.
Received for publication 17 May 2007 and accepted in revised form 16 September 2007.
Published ahead of print at http://care.diabetesjournals.org on 24 October 2007. DOI: 10.2337/dc07-
0939. Clinical trial reg. no. NCT00440986, clinicaltrials.gov.
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; FFM, free fat mass; HH,
type 1 hereditary hemochromatosis; IGI, insulinogenic index; LIC, liver iron content; NAFLD, nonalcoholic
fatty liver disease; NAS, nonalcoholic fatty liver disease activity score; NGT, normal glucose tolerant; NMR,
nuclear magnetic resonance; OGTT, oral glucose tolerance test.
A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion
factors for many substances.
© 2008 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Clinical Care/Education/Nutrition/Psychosocial Research
ORIGINAL ARTICLE
DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008 3
Page 1
tolerant (NGT) subjects and eight subjects
with newly diagnosed diabetes with ei-
ther HH or heterozygous HFE gene mu-
tations before and after 2 years of
bloodletting procedures.
RESEARCH DESIGN AND
METHODS The study participants
were recruited from all consecutive male
newly diagnosed HFE homozygous or het-
erozygous mutation carrier referrals to the
Hemochromatosis Unit at the SanFilippo
Neri General Hospital from January 2003 to
December 2006. Healthy male subjects
(n 10), enrolled in a contemporaneous
study at Catholic University (15), and
healthy blood donors (n 23) at SanFil-
ippo Neri General Hospital served as con-
trol subjects. They were not smokers; were
matched for age, BMI, and body composi-
tion; did not have biochemical signs of iron
overload (ferritin 300 g/l and transferrin
saturation 45%); and had no evidence of
ultrasound liver brightness.
Inclusion criteria were male sex, age
4060 years, BMI 30 kg/m
2
, no serious
associated illness (heart failure, cirrhosis,
or panhypopituitarism), and no con-
sumption of alcohol or use of medica-
tions. Only NGT and diabetic subjects
were enrolled (16), whereas those with
impaired glucose tolerance (three sub-
jects) were excluded. All subjects received
general nutritional counseling at baseline
with prescription of a balanced diet regi-
men (carbohydrate 50 60%, fat 23–
30%, and protein 15–20%) but no extra
physical activity. A1C 7.5% at baseline
or during the follow-up was considered a
reason for dropping out of the study.
Diagnosis of HFE mutations
(Cys282Tyr, His63Asp, and Ser65Cys)
was genetically confirmed by real-time
PCR. Non-HFE gene mutations (TFR2,
c-HAMP, and HJV) were tested. Con-
versely, HFE and non-HFE gene mutations
were ruled out in healthy subjects (17).
Bloodletting was performed every 2
weeks by phlebotomy or erythrocyta-
pheresis (Hemonetics MCS-plus Blood
Cell Processor; Hemonetics, Braintree,
MA), and blood volume was restored to
normal by hemodilution using a normal
saline solution. In phlebotomy, 450 ml of
blood was removed for each procedure.
Patients were admitted to the unit for
clinical and iron balance evaluation every
2 weeks. In the beginning, treatment was
done every 2 weeks to achieve the target
value of ferritin 100 ng/ml and satura-
tion 45% in patients with nuclear mag-
netic resonance (NMR) negative for iron
depots in liver or 50 ng/ml and satura-
tion 30% in patients with NMR positive
for iron accumulation; thereafter, timing
of procedures was based on the individual
need to maintain these values. At baseline
and at 24 months, body composition, in-
sulin secretion and sensitivity, liver NMR
and heart ultrasound results, liver histol-
ogy, and iron content were evaluated.
The study protocol conformed to the
Table 1—Anthropometric and biochemical variables in healthy control subjects and subjects carrying HFE gene mutations according to glucose
tolerance
Healthy control
subjects
NGT subjects Diabetic subjects
Baseline 24 months Baseline 24 months
n 33 9 8
Cys282Tyr homozygous 0 5 2
His63Asp homozygous 0 2 3
Cys282Tyr heterozygous 0 2 1
His63Asp heterozygous 0 0 1
Cys282Tyr-His63Asp compound
heterozygous
00 1
Age (years) 48.7 0.93 53.2 4.3 55.2 4.3 51.5 4.5 53.4 4.3
BMI (kg/m
2
)
26.8 0.54 26.5 0.98 25.7 0.7
a
25.6 1.3 24.8 1.2
a
FFM (kg) 61.7 1.08 59.7 2.1 58.4 1.9 58.5 2.0 57.2 1.2
Fasting glucose (mg/dl) 96.4 8.6 95.8 4.0 85.9 4.0 136.9 5.9
fg
105.1 7.4
af
Fasting insulin (IU/ml) 12.6 0.7 14.0 1.6 12.3 0.9 13.4 1.7 14.1 1.8
Fasting C-peptide (ng/ml)) 0.71 0.09 0.6 0.3 1.11 0.3 0.7 0.2 1.25 0.2
Total cholesterol (mg/dl) 181.5 5.3 235.6 13.2
j
196.2 5.8
aj
253.4 12.3
j
206.5 22.5
b
Fasting triglycerides (mg/dl) 146.9 7.2 192.2 9.5
g
137.6 9.5
a
231.6 16.7
cj
156.6 13.8
af
ALT (IU/l) 27.1 1.5 61.8 5.0
j
46.4 4.1
ah
73.1 6.5
i
40.9 5.2
ag
AST (IU/l) 21.5 1.3 41.4 4.3
j
32.4 3.7
g
45 3.2
j
29 2.5
a
-GT (IU/l) 23.4 1.1 61.1 7.1
j
43.4 4.9
ai
65 4.9
j
39.6 6.5
a
White blood cell count (cells/l) 6,578 540 6,466 558 6,166 365 6,850 525 6,000 588
A1C (%) 3.3 0.3
f
3.0 0.4 3.0 0.2 6.5 0.6
f
3.5 0.4
a
Serum iron (g/dl) 75.0 1.3 119.7 10.7
j
42.9 2.0
b
142.2 14.0
j
38.9 2.1
b
Serum ferritin (ng/ml) 44.03 1.34 942.1 134.3
j
72.1 8.5
b
1,148.8 235.3
j
54.7 7.3
b
Transferrin saturation index (%) 25 0.1 67 3.2
j
76 8.5
j
68 4.6
j
68 4.59
j
M (mol/kg
FFM
/min)
48.5 2.1 37.9 3.3 50.6 2.4
b
24.9 2.2
ej
30.2 2.1
bej
Insulin secretion (mIU mm
2
)
2.19 0.4 1.67 0.2 2.17 0.4
b
0.24 0.06
g
0.36 0.06
adg
IGI (IU/ml mg/dl
1
)
24.7 4.8 17.4 3.4 19.5 3.4
b
3.37 0.8
f
4.43 1.0
bf
Data are means SEM. Levels of significance on Wilcoxon’s signed ranks test:
a
P 0.05;
b
P 0.01. Levels of significance on post hoc analysis between NGT and
diabetic subjects:
c
P 0.05;
d
P 0.01;
e
P 0.001;
f
P 0.0001. Levels of significance on post hoc analysis between control subjects and NGT and diabetic subjects:
g
P 0.05;
h
P 0.01;
i
P 0.001;
j
P 0.0001. To correct values for glucose to millimoles per liter, multiply by 0.05551; for insulin to picomoles per liter by 7.175;
for total cholesterol to millimoles per liter by 0.02586; for triglycerides to millimoles per liter by 0.01.
Iron overload and insulin metabolism
4 DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008
Page 2
principles of the Declaration of Helsinki
and to the recommendations of the Ethics
Committee at the SanFilippo Neri Gen-
eral Hospital. The nature and the purpose
of the study were carefully explained be-
fore informed consent was requested
from each patient.
Insulin secretion was calculated as the
corrected insulin response from 30-min
insulin and glucose levels: {insulin 30/
[glucose 30 (glucose 30 –3.9)}] (milli-
International Units millimoles squared)
(18). The insulinogenic index (IGI) was cal-
culated as (insulin 30 insulin 0)/(glucose
30 glucose 0) (19).
Insulin sensitivity was estimated by a
euglycemic-hyperinsulinemic clamp as
described previously (20). Whole-body
glucose uptake (M value in micromoles
per kilogram free fat mass [FFM] per
minute) was determined during a primed
constant infusion of insulin (at the rate of
6 pmol min
1
kg
1
). It was normalized
for kilograms of FFM.
Body composition was estimated by
the isotopic dilution method. FFM (in ki-
lograms) was obtained by dividing total
body water by 0.73 (21).
Histological features of steatosis, in-
flammation (portal and lobular), hepato-
cyte ballooning, and fibrosis were scored
by using the scoring system for NAFLD
(22). Features of steatosis, lobular inflam-
mation, and hepatocyte ballooning were
combined in a score going from 0 to 8,
named the NAFLD activity score (NAS).
NAS 5 is diagnostic of nonalcoholic ste-
atohepatitis, NAS 2 is diagnostic of sim-
ple steatosis, and values in between are
considered indeterminate.
Liver iron content (LIC) was mea-
sured in all subjects by means of atomic
absorption spectroscopy according to the
method of Barry and Sherlock (23). Liver
tissue samples of at least 0.5 mg dry
weight that had been frozen immediately
at 20°C after collection were used.
Serum was stored at 80°C for later
assays. Plasma insulin and C-peptide
were measured by a specific radioimmu-
noassay (MYRIA Technogenetics, Milan,
Italy). A1C was measured by high-
performance liquid chromatography (L-
9100; Hitachi, Rahway, NJ).
Statistics
Data are presented as means SEM. Before
statistical analysis, normal distribution and
homogeneity of the variances were tested.
Parameters that did not fulfill tests were log
transformed before analysis. To evaluate the
effect of 24-month bloodletting on contin-
uous variables, a Wilcoxon signed-ranks
test was used. To compare the effect of
bloodletting in NGT and diabetic subjects
separately at baseline and at the end of the
follow-up, a Mann-Whitney U test was per-
formed. To compare HFE gene mutation
carriers versus healthy subjects and to eval-
uate the effect of blood withdrawn through-
out the follow-up, it was necessary to
perform repeated ANOVA and post hoc
analysis (Bonferroni post hoc test), when-
ever appropriate.
Relationships between variables were
determined by linear correlation analysis
(Spearman’s r), and regression analysis
was performed by standard techniques.
Levels of statistical significance were set at
P 0.05. Data analyses were performed
with SPSS statistical software (V12.0;
SPSS, Chicago, IL).
RESULTS
Comparison of carriers of HFE gene
mutations versus control subjects
Control subjects were matched with both
diabetic and NGT groups of HFE gene
mutation carriers in age, BMI, and FFM.
Fasting glucose and insulin did not differ.
Control subjects had significantly lower
total cholesterol, triglycerides, liver en-
zymes, serum iron, and transferrin com-
pared with NGT and diabetic patients at
baseline and after 2 years (statistical sig-
nificance shown in Table 1). Insulin se-
cretion was significantly lower in diabetic
subjects (P 0.05, both at baseline and
after 2 years). Insulin sensitivity was
greater in control subjects than in diabetic
subjects before (P 0.0001) and after
iron depletion (P 0.0001).
Baseline values in groups of carriers
of HFE gene mutations
At baseline, NGT and diabetic subjects
differed significantly in fasting glucose
(P 0.0001), A1C (P 0.0001), triglyc-
erides (P 0.05), insulin sensitivity (P
0.001) and secretion, and IGI (P
0.0001 for both) (Table 1). No significant
differences were found in aminotrans-
ferases, whereas LIC (P 0.004) and
Figure 1—Time courses of plasma triglycerides (A) and glucose (B), serum ferritin (C), iron (D),
and levels of ALT (E) in NGT (dotted line) and diabetic (solid line) subjects with HFE gene
mutations. Levels of significance at post hoc analysis: °P 0.01; §P 0.001;
P 0.0001.
Equitani and Associates
DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008 5
Page 3
NAS (P 0.02) were significantly higher
in diabetic subjects (Table 2).
Effect of the treatment in carriers of
HFE gene mutations
No subject with diabetes dropped out the
study because of A1C 7.5%, and no
subjects discontinued the study. The goal
values of ferritin and saturation were ob-
tained in all subjects by month 5. To
achieve these values, 8 3 procedures in
NGT vs. 9 2 procedures in diabetic
subjects were needed. Throughout the
whole study, NGT subjects underwent a
total of 21 2 vs. 20 1 procedures in
diabetic subjects.
A significant improvement in several
metabolic parameters was seen as soon as
the treatment was initiated. At month 4,
ferritin (1,039.4 129.8 vs. 264.1
27.8 ng/ml; P 0.0001), iron (130.3
8.9 vs. 67.5 5.8 g/dl; P 0.0001),
and saturation (67 3 vs. 42 2%; P
0.001) significantly decreased. Choles-
terol (243.9 9.0 vs. 209.1 7.5 mg/dl;
P 0.008), triglycerides (210.8 10.2
vs. 168.35 9.1 mg/dl; P 0.005), fast-
ing glycemia (115.1 6.1 vs. 92.1 4.6
mg/dl; P 0.01), lactate dehydrogenase
(329.8 18.1 vs. 282.7 11.5 IU/l; P
0.05), aspartate aminotransferase (AST)
(43.1 2.7 vs. 32.1 2.3 IU/l; P
0.01), alanine aminotransferase (ALT)
(67.1 4.1 vs. 45.2 2.4 IU/l; P
0.001), and -glutamyltransferase (-GT)
(62.9 4.3 vs. 39.8 4.1 IU/l; P
0.0001) significantly ameliorated. Time
courses of the most representative vari-
ables are depicted separately for NGT and
diabetic subjects in Fig. 1.
At the end of the study, bloodletting
was associated with an improved meta-
bolic pathway. Compared with baseline,
ferritin decreased to 58.1 4.2 ng/ml
(P 0.0001) and serum iron to 41.0
1.5 g/dl (P 0.0001). Cholesterol
(201.2 4.3 mg/dl; P 0.0001), lactate
dehydrogenase (295.8 13.5 IU/l; P
0.004), AST (30.8 2.0 IU/l; P
0.0001), ALT (43.8 3.2 IU/l; P
0.0001), -GT (41.6 3.9 IU/l; P
0.0001), triglycerides (146.5 8.3 mg/dl
P 0.0001), and fasting glycemia
(94.9 4.6 mg/dl; P 0.01) ameliorated
in all subjects. Table 1 shows mean
SEM values separately for NGT and dia-
betic subjects.
For insulin metabolism (Table 1), af-
ter 2 years, insulin secretion increased by
20 4% in NGT and 33 7% in diabetic
subjects (NS); IGI increased by 16 5
and 47 19%, respectively (NS); and
glucose uptake increased by 25 5 and
18 3% (NS). Diabetic subjects had sig-
nificantly lower insulin secretion, IGI,
and insulin sensitivity than NGT subjects
(P 0.0001 for all parameters before and
after treatment). LIC decreased by 46 2
in NGT and by 35 2% in diabetic sub-
jects. Figure 2 shows changes in insulin
sensitivity, secretion, IGI, and LIC for
each subject from both groups.
Table 2 shows histological findings,
LIC, and NAS in NGT and diabetic sub-
jects at baseline and after 24 months.
No subject had biopsy-proven cirrho-
sis. In diabetic subjects, liver steatosis
(P 0.02) and inflammation (P 0.03)
were significantly reduced with respect
to baseline.
Statistical correlations
Levels of circulating iron correlated sig-
nificantly with ALT (r
0
0.703; P
0.0001), AST (r
0
0.765; P 0.0001),
-GT (r
0
0.771; P 0.0001), C-
peptide (r
0
⫽⫺0.361; P 0.015), LIC
(r
0
0.484; P 0.004), glucose uptake
(r
0
⫽⫺0.360; P 0.004), NAS (r
0
0.364; P 0.03), cholesterol (r
0
0.573;
P 0.0001), triglycerides (r
0
0.679;
P 0.0001), IGI (r
0
0.337; P 0.002),
and HbA
2
(r
0
0.301; P 0.03). Signif
-
icant correlations were found between
ferritin and AST (r
0
0.778; P
0.0001), ALT (r
0
0.592; P 0.0001),
-GT (r
0
0.735; P 0.0001), C-
peptide (r
0
⫽⫺0.314; P 0.04), LIC
(r
0
0.40; P 0.02), NAS (r
0
0.393;
P 0.02), cholesterol (r
0
0.418; P
0.0001), triglycerides (r
0
0.629; P
0.0001), IGI (r
0
0.255; P 0.02), and
HbA
2
(r
0
0.289; P 0.04).
In NGT (y ⫽⫺0.2376x 50.286; R
2
0.40, P 0.0001) and diabetic sub-
jects (y ⫽⫺0.2905x 55.425; R
2
0.41, P 0.0001), LIC correlated with
glucose uptake. In NGT subjects, LIC cor-
related also with triglycerides (r
0
0.563; P 0.015) and negatively with
insulin secretion (r
0
⫽⫺0.676; P
0.0001).
Table 2—Histological findings, NAS, and LIC before and 24 months after bloodletting in NGT
and diabetic subjects carrying HFE gene mutations
NGT subjects Diabetic subjects
Baseline 24 months Baseline 24 months
Steatosis
a
Grade 0
Grade 1 3 (33.3) 6 (66.6)
Grade 2 3 (33.3) 2 (22.2) 4 (50)
Grade 3 3 (33.3) 1 (11.1) 8 (100) 4 (50)
Grade 4
Inflammation
a
Grade 0 1 (11.1) 3 (33.3) 1 (12.5) 5 (50)
Grade 1 7 (77.8) 6 (66.6) 6 (75) 3 (37.5)
Grade 2 1 (11.1) 1 (12.5)
Grade 3
Grade 4
Ballooning
Grade 0 5 (55.5) 5 (55.5) 1 (12.5) 2 (25)
Grade 1 2 (22.2) 1 (11.1) 1 (12.5) 5 (62.5)
Grade 2 2 (22.2) 2 (22.2) 5 (62.5) 1 (12.5)
Grade 3
Grade 4
Fibrosis
Grade 0 6 (66.6) 6 (66.6) 3 (37.5)
Grade 1 2 (22.2) 2 (22.2) 3 (37.5) 5 (62.5)
Grade 2 1 (11.1) 3 (37.5)
Grade 3 1 (11.1) 3 (37.5)
Grade 4
NAS 3.4 0.6 2.8 0.5
b
5.4 0.4
c
3.7 0.5
b
LIC (mol/g
dw
)
65.1 6.5 38.1 6.83
b
92.1 10.7
c
69.9 10
b
Data are means SEM or n (proportion of subjects affected). Levels of significance at Wilcoxon’s signed
ranks test are reported as
a
P 0.05 in diabetic subjects;
b
P 0.01 in both NGT and diabetic subjects. Levels
of significance at the intergroup comparison:
c
P 0.001.
Iron overload and insulin metabolism
6 DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008
Page 4
In diabetic subjects, LIC correlated
with NAS (r
0
0.649; P 0.007). IGI (r
0
⫽⫺0.589; P 0.016) and triglycerides
(r
0
0.775; P 0.0001) were related to
ALT levels.
In a multistep linear regression anal-
ysis, the best predictors of whole-body
glucose uptake in HFE gene mutation car-
riers (R
2
0.55; P 0.0001) were LIC
(␤⫽⫺0.549; P 0.0001) and triglycer-
ides (␤⫽⫺0.307; P 0.03). BMI, fer-
ritin, iron, ALT, insulin secretion, IGI,
and NAS were excluded variables.
CONCLUSIONS In patients with
phenotypic appearance of HFE gene mu-
tations, iron overload is associated with
impaired insulin metabolism and features
of metabolic syndrome. -Cell function
deteriorates to a larger extent than insulin
sensitivity in the early development of di-
abetes related to the HFE gene mutations.
Insulin resistance occurs when glucose
tolerance is still preserved and becomes
more severe later when glucose tolerance
is impaired. Thus, both aspects contrib-
ute significantly to the pathogenesis of di-
abetes in these patients. Constant iron
depletion ameliorates all biochemical and
histological features associated with met-
abolic syndrome. -Cell activity amelio-
rates as well as glucose uptake. Subjects
with the latter keep showing lower levels
of secretion and sensitivity compared
with NGT patients even after achieving a
normalization of iron balance.
These findings confirm recent data
from McClain’s group (7,8), but they also
add significant insights to our knowledge
of the pathophysiology of diabetes associ-
ated with HFE gene mutations. In a small
cohort of selected homozygous patients,
McClain et al. found NGT subjects having
normal insulin secretion and sensitivity,
whereas subjects with impaired glucose
tolerance compensated for diminished in-
sulin secretion by increased insulin sensi-
tivity. Subjects in whom this
compensatory mechanism failed seemed
to be those who developed overt diabetes,
showing lower insulin secretion rates but
normal glucose uptake (8).
In the present cohort of homozygous
and heterozygous subjects, we observed
values of glucose uptake in NGT subjects
overlapping with those of healthy volun-
teers, thus confirming a previous report in
nondiabetic, noncirrhotic carriers of het-
erogeneous HFE gene mutations (24). In
diabetic subjects both insulin sensitivity
and secretion are significantly impaired. It
is conceivable that the lower value of glu-
cose uptake that we observed in our dia-
betic subjects might be due to the genetic
heterogeneity of our cohort compared
with that of Klein’s series.
Furthermore, values of glucose up-
take, apparently within a normal range in
NGT subjects, are further enhanced after
iron depletion, as already described in
healthy frequent blood donors (4), sug-
gesting that high levels of circulating iron
can reduce the potential insulin sensitiv-
ity of a subject. In healthy volunteers,
75% of glucose uptake occurs in muscle
and the remaining 25% in liver. It has
been hypothesized that these proportions
may be modified in presence of iron over-
load (25). Accordingly, we found that the
liver iron depot correlates with systemic
glucose uptake and its reduction is asso-
ciated with the amelioration of histologi-
cal grading and NAS (Table 2). In this
regard, NGT carriers of HFE gene muta-
Figure 2—Individual changes in insulin secretion (A), IGI (B), whole-body insulin uptake (M value) (C), and LIC (D) in NGT (dotted line) and
diabetic (solid line) subjects carrying HFE gene mutations. Levels of significance at post hoc analysis: P 0.05; °P 0.01; §P 0.001;
P
0.0001.
Equitani and Associates
DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008 7
Page 5
tions have a mean value of liver iron con-
tent that is below the threshold
commonly considered as diagnostic for
hemochromatosis, and this value is sig-
nificantly reduced after treatment. In dia-
betic subjects, who have higher levels of
LIC than NGT subjects, iron depletion is
associated with the improvement of all
parameters related to insulin metabolism,
liver function, and histology. The de-
crease in liver iron content is associated
with the significant amelioration of ste-
atosis and inflammation, perhaps because
iron is able to participate in the formation
of powerful oxidant species (26). These
differences in outcomes may be due to the
multifactorial nature of the transition
from normal glucose tolerance to im-
paired glucose tolerance and diabetes in
carriers of HFE gene mutations. Over-
weight/obesity-related steatosis, high lev-
els of triglycerides, or the presence of
obesity-related morbidities may play a
role as cofactors.
In summary, we have demonstrated
that the loss of insulin secretory capacity
can be the primary event leading to HFE
gene mutation–related diabetes, but the
role for decreased insulin sensitivity can-
not be disregarded, with liver acting as a
primary site of insulin resistance and con-
tributing to the ultimate expression of
diabetes. Increased body fatness, environ-
mental factors, and risk factors for meta-
bolic syndrome can work together. Thus,
obesity and related morbidities should be
actively addressed in the management of
carriers of HFE gene mutations. Especially
important for the prevention of diabetes-
related morbidities that can occur very
early, this would justify glucose tolerance
testing of carriers of HFE gene mutations
as well as prophylactic iron depletion in
unaffected individuals with diagnosed
disease. The reversibility of iron-related
abnormalities in insulin metabolism war-
rants further investigation.
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Iron overload and insulin metabolism
8 DIABETES CARE, VOLUME 31, NUMBER 1, JANUARY 2008
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    • "Bloodletting has been shown to increase the insulin sensitivity [33], which is a major risk factor for the development of metabolic syndrome if impaired. In a constant line, Ranaei-siadat and his colleagues measured the venous blood concentrations of several molecules before and after five times of cupping (one time per month) in young healthy males (20-27 years). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Wet cupping (Hejamah) has been used as alternative treatment for several diseases. Objectives: Materials and methods: 16 participants were treated with hejamah for 2 consecutive months. Blood pressure was measure before and 30 minutes after the treatment. Blood samples were collected from all participants before and 48 hours after hejamah and all participants were fasting for 12 hours before sample collection. Results: Fasting blood glucose was significantly decreased before the treatment in the second month only. The levels of serum triglycerides significantly decreased after the first treatment and remained low in the 2nd month. There was no significant difference between the different time points in total cholesterol except for the 48 hours of the second month compared to the samples collected before the procedure of the same month. There was a significant decrease in LDL and significant increase in HDL following hejamah therapy (P<0.05). Significant decrease in sodium and significant increase in potassium 48 hours following treatment with hejamah was observed in the 2 months (P<0.05). Conclusions: The performance of hejamah during fasting state could represent a useful complementary method for the regulation of diastolic blood pressure and prevention/treatment of risk factors associated with cardiovascular diseases. Further studies are required to explore the role of hejamah in controlling blood pressure and prevention of cardiovascular diseases.
    Full-text · Article · Jan 2014
    0Comments 0Citations
    • "They might be unable to compensate for hypovolemia as healthy donors do. Indeed, most previous evidence were from cohort and cross-sectional studies of healthy donors [20], and from high-ferritin T2DM patients and carriers of hereditary hemocromatosis in whom blood volume was restored to normal at each procedure [7,10]. "
    [Show abstract] [Hide abstract] ABSTRACT: In patients with metabolic syndrome, body iron overload exacerbates insulin resistance, impairment of glucose metabolism, endothelium dysfunction and coronary artery responses. Conversely, iron depletion is effective to ameliorate glucose metabolism and dysfunctional endothelium. Most of its effectiveness seems to occur through the amelioration of systemic and hepatic insulin resistance. In a study published by BMC Medicine, Michalsen et al. demonstrated a dramatic improvement of blood pressure, serum glucose and lipids after removing 550 to 800 ml of blood in subjects with metabolic syndrome. This effect was apparently independent of changes in insulin resistance, in contrast to previous cross-sectional and cohort studies investigating the association between iron overload, insulin resistance and cardiovascular disease. Despite drawbacks in the study design, its findings may lead the way to investigations aimed at exploring iron-dependent regulatory mechanisms of vascular tone in healthy individuals and patients with metabolic disease, thus providing a rationale for novel preventive and therapeutic strategies to counteract hypertension. Please see related article: http://www.biomedcentral.com/1741-7015/10/54
    Full-text · Article · May 2012 · BMC Medicine
    0Comments 3Citations
    • "In a small safety study on blood donation, phlebotomy resulted ina significant decrease in serum glucose and blood lipids in patients with diabetes [22]. Iron reduction by phlebotomy also enhanced insulin sensitivity in patients with ironinduced insulin resistance and in carriers of the hemochromatosis gene [20]. Notably, in these studies, the amount of removed blood was larger than in our study and the study period was longer. "
    [Show abstract] [Hide abstract] ABSTRACT: Metabolic syndrome (METS) is an increasingly prevalent but poorly understood clinical condition characterized by insulin resistance, glucose intolerance, dyslipidemia, hypertension, and obesity. Increased oxidative stress catalyzed by accumulation of iron in excess of physiologic requirements has been implicated in the pathogenesis of METS, but the relationships between cause and effect remain uncertain. We tested the hypothesis that phlebotomy-induced reduction of body iron stores would alter the clinical presentation of METS, using a randomized trial. In a randomized, controlled, single-blind clinical trial, 64 patients with METS were randomly assigned to iron reduction by phlebotomy (n = 33) or to a control group (n = 31), which was offered phlebotomy at the end of the study (waiting-list design). The iron-reduction patients had 300 ml of blood removed at entry and between 250 and 500 ml removed after 4 weeks, depending on ferritin levels at study entry. Primary outcomes were change in systolic blood pressure (SBP) and insulin sensitivity as measured by Homeostatic Model Assessment (HOMA) index after 6 weeks. Secondary outcomes included HbA1c, plasma glucose, blood lipids, and heart rate (HR). SBP decreased from 148.5 ± 12.3 mmHg to 130.5 ± 11.8 mmHg in the phlebotomy group, and from 144.7 ± 14.4 mmHg to 143.8 ± 11.9 mmHg in the control group (difference -16.6 mmHg; 95% CI -20.7 to -12.5; P < 0.001). No significant effect on HOMA index was seen. With regard to secondary outcomes, blood glucose, HbA1c, low-density lipoprotein/high-density lipoprotein ratio, and HR were significantly decreased by phlebotomy. Changes in BP and HOMA index correlated with ferritin reduction. In patients with METS, phlebotomy, with consecutive reduction of body iron stores, lowered BP and resulted in improvements in markers of cardiovascular risk and glycemic control. Blood donation may have beneficial effects for blood donors with METS. ClinicalTrials.gov: NCT01328210 Please see related article: http://www.biomedcentral.com/1741-7015/10/53.
    Full-text · Article · May 2012 · BMC Medicine
    0Comments 49Citations
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