Vascular complications are the primary cause of morbidity and mortality and a major contributor to health care costs in patients with diabetes (1). While the etiology of vascular disease is multifactorial, endothelial dysfunction characterized by a decrease in nitric oxide (NO) synthesis from endothelial NO synthase (eNOS) and blunted endothelium-dependent vasodilation is a seminal feature in diabetes-associated vascular disease. Given the dramatic increase in the incidence of diabetes worldwide, there is an urgent need for new therapies that targets this cellular defect in diabetes.

Although long considered a toxic waste product of heme metabolism, the yellow pigment bilirubin is now recognized as an important vasoprotective molecule. Bilirubin is formed by the action of inducible heme oxygenase-1 (HO-1) or constitutively expressed HO-2, which degrades heme into biliverdin, carbon monoxide (CO), and iron (Fig. 1). Biliverdin is rapidly reduced to bilirubin by biliverdin reductase (BVR). Bilirubin is further processed in the liver, where it is conjugated with glucuronic acid by uridine diphosphate-glucuronosyltransferase A1A (UGT1A1) to a water-soluble form for elimination. While early work identified the potent antioxidant properties of bilirubin, more recent studies found that bilirubin elicits anti-inflammatory and antiapoptotic effects in endothelial cells and improves endothelium-dependent vasodilation in murine hypercholesterolemic blood vessels (25). Clinical studies also support a protective role, as serum bilirubin concentrations show an inverse relationship with risk for a constellation of cardiovascular disorders (6). Interestingly, serum bilirubin levels are lower in patients with diabetes (7), and low serum values were identified as a crucial predictor of cardiovascular disease in specific populations with diabetes (8). Moreover, diabetic patients with Gilbert syndrome, who have moderately elevated levels of circulating bilirubin, have a lower rate of vascular complications than patients without the syndrome, further highlighting the potential importance of this pigment in diabetes (9).

Figure 1

Schematic diagram depicting the metabolism of heme and the beneficial actions of bilirubin in diabetes-induced endothelial dysfunction. Bilirubin, generated by the concerted action of HO-1 and BVR, leads to the sequential activation of Akt and eNOS and synthesis of NO, which improves endothelium-dependent vasodilation and insulin resistance. These salutary actions are further amplified by the ability of bilirubin to inhibit PKC, inflammation, and oxidative stress, which are known mediators of endothelial dysfunction in diabetes. Bilirubin is removed from the circulation by the liver and metabolized by UGT1A1 to yield water-soluble conjugated bilirubin for elimination. BR, bilirubin; BV, biliverdin; Fe2+, ferrous iron; ROS, reactive oxygen species.

Figure 1

Schematic diagram depicting the metabolism of heme and the beneficial actions of bilirubin in diabetes-induced endothelial dysfunction. Bilirubin, generated by the concerted action of HO-1 and BVR, leads to the sequential activation of Akt and eNOS and synthesis of NO, which improves endothelium-dependent vasodilation and insulin resistance. These salutary actions are further amplified by the ability of bilirubin to inhibit PKC, inflammation, and oxidative stress, which are known mediators of endothelial dysfunction in diabetes. Bilirubin is removed from the circulation by the liver and metabolized by UGT1A1 to yield water-soluble conjugated bilirubin for elimination. BR, bilirubin; BV, biliverdin; Fe2+, ferrous iron; ROS, reactive oxygen species.

In this issue, Liu et al. (10) use a highly integrated and complementary experimental approach to convincingly show that bilirubin corrects endothelial dysfunction in diabetes. In initial experiments, they demonstrate that pharmacological induction or adenoviral transfer of HO-1 improves endothelium-dependent vasorelaxation in both genetic and dietary mouse models of type 2 diabetes. Ex vivo induction of HO-1 also augments endothelium-dependent relaxation of diabetic blood vessels. However, the vascular benefit of HO-1 overexpression is lost when BVR is silenced, indicating that conversion of biliverdin to bilirubin is necessary for this response. A remarkable finding in the study is that serum concentrations of unconjugated bilirubin are reduced by nearly 80% in diabetic mice. Importantly, administration of unconjugated bilirubin to diabetic animals or isolated blood vessels from diabetic mice or patients with diabetes restores endothelial function. Finally, Liu et al. (10) identify protein kinase B (Akt) as the principal downstream target of bilirubin. They show that the activity of Akt and eNOS and levels of nitrite, a metabolite of NO, are reduced in aortas of diabetic mice, and this is corrected by bilirubin. However, this improvement is prevented by Akt inhibition. Comparable results are also obtained when cultured endothelial cells are exposed to high concentrations of glucose to simulate the in vivo conditions of diabetes. Collectively, these novel findings implicate a bilirubin deficiency in diabetes and demonstrate that raising bilirubin levels either through endogenous synthesis or exogenous application improves endothelial dysfunction in diabetes by activating Akt. These findings also extend recent work showing that bilirubin augments endothelial function in diabetic rats (11).

Consistent with earlier reports (12,13), Liu et al. (10) note that HO-1 induction moderately improves insulin sensitivity in diabetic mice. Although bilirubin was not tested, it likely mediates the metabolic action of HO-1 as bilirubin has been shown to reduce insulin resistance in the same mouse model (14). Intriguingly, CO fails to restore endothelial function in diabetic blood vessels, even though this gas is known to activate Akt and eNOS in endothelial cells (15). This finding also contrasts with previous studies demonstrating that CO restores endothelium-dependent relaxation in diabetic rats (11,16), indicating the need for further investigation in this area.

The study by Liu et al. (10) represents an important advance in the field, and similar to many good studies, it raises some interesting questions. In particular, how does bilirubin activate Akt? It is speculated that bilirubin may indirectly activate the kinase by suppressing oxidative stress and protein kinase C (PKC) activity, which are elevated in diabetes (17). This notion is reasonable but will require testing. Furthermore, what causes the precipitous decline in circulating unconjugated bilirubin in diabetes? Although vascular HO-1 levels are similar between control and diabetic animals, global induction of HO-1 in diabetic animals largely restores serum bilirubin, suggesting possible deficits in HO-1 in other tissue compartments. Alterations in HO-2, BVR, and/or UGT1A1 activity may also contribute to lower bilirubin levels in diabetes. While bilirubin ameliorates endothelial function in large conduit arteries, it is essential to extend this finding and confirm that endothelial dysfunction is also resolved in the microcirculation, as defects in these vessels contribute to diabetes-related organ pathologies. Finally, in order to translate these findings to the clinic, studies are needed to define an optimal range of the serum unconjugated bilirubin required to maintain endothelial function while averting possible toxic effects (18).

A schematic diagram depicting the beneficial actions of bilirubin in diabetes-induced endothelial dysfunction is shown in Fig. 1. In this model, bilirubin, generated via the concerted action of HO-1 and BVR, leads to the sequential activation of Akt and eNOS and synthesis of NO, which improves endothelium-dependent vasodilation and insulin resistance (19). Bilirubin also inhibits PKC activation, inflammation, and oxidative stress, which are known mediators of endothelial dysfunction. Several strategies can be used to target bilirubin in diabetes. Endogenous circulating levels of bilirubin may be elevated using pharmacological or molecular approaches that induce HO-1 expression or suppress UGT1A1 activity. The feasibility of targeting UGT1A1 is supported by a report showing that atazanavir, a UGT1A1 inhibitor, raises unconjugated bilirubin levels and improves endothelial function in patients with diabetes (20). Alternatively, bilirubin or its precursor biliverdin may be directly administered. Future translational studies mining these approaches will reveal whether Liu et al. (10) have struck gold in managing diabetic vasculopathy.

See accompanying article, p. 1564.

Funding. W.D. is supported by grants from the National Institutes of Healthhttp://dx.doi.org/10.13039/100000002 National Heart, Lung, and Blood Institute (R01 HL-59976) and the American Heart Associationhttp://dx.doi.org/10.13039/100000968 Midwest Affiliate (13GRNT16710017).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

1.
American Diabetes Association
.
Standards of medical care in diabetes—2014
.
Diabetes Care
2014
;
37
(
Suppl. 1
):
S14
S80
[PubMed]
2.
Stocker
R
,
Yamamoto
Y
,
McDonagh
AF
,
Glazer
AN
,
Ames
BN
.
Bilirubin is an antioxidant of possible physiological importance
.
Science
1987
;
235
:
1043
1046
[PubMed]
3.
Soares
MP
,
Seldon
MP
,
Gregoire
IP
, et al
.
Heme oxygenase-1 modulates the expression of adhesion molecules associated with endothelial cell activation
.
J Immunol
2004
;
172
:
3553
3563
[PubMed]
4.
Wei
Y
,
Liu
XM
,
Peyton
KJ
, et al
.
Hypochlorous acid-induced heme oxygenase-1 gene expression promotes human endothelial cell survival
.
Am J Physiol Cell Physiol
2009
;
297
:
C907
C915
[PubMed]
5.
Kawamura
K
,
Ishikawa
K
,
Wada
Y
, et al
.
Bilirubin from heme oxygenase-1 attenuates vascular endothelial activation and dysfunction
.
Arterioscler Thromb Vasc Biol
2005
;
25
:
155
160
[PubMed]
6.
Lin
JP
,
Vitek
L
,
Schwertner
HA
.
Serum bilirubin and genes controlling bilirubin concentrations as biomarkers for cardiovascular disease
.
Clin Chem
2010
;
56
:
1535
1543
[PubMed]
7.
Dullaart
RP
,
de Vries
R
,
Lefrandt
JD
.
Increased large VLDL and small LDL particles are related to lower bilirubin in Type 2 diabetes mellitus
.
Clin Biochem
2014
;
47
:
170
175
[PubMed]
8.
Fukui
M
,
Tanaka
M
,
Yamazaki
M
, et al
.
Low serum bilirubin concentration in haemodialysis patients with type 2 diabetes
.
Diabet Med
2011
;
28
:
96
99
[PubMed]
9.
Inoguchi
T
,
Sasaki
S
,
Kobayashi
K
,
Takayanagi
R
,
Yamada
T
.
Relationship between Gilbert syndrome and prevalence of vascular complications in patients with diabetes
.
JAMA
2007
;
298
:
1398
1400
[PubMed]
10.
Liu
J
,
Wang
L
,
Tian
XY
, et al
.
Unconjugated bilirubin mediates heme oxygenase-1–induced vascular benefits in diabetic mice
.
Diabetes
2015
;
64
:
1564
1575
[PubMed]
11.
Wang
Y
,
Ying
L
,
Chen
YY
, et al
.
Induction of heme oxygenase-1 ameliorates vascular dysfunction in streptozotocin-induced type 2 diabetic rats
.
Vascul Pharmacol
2014
;61: 
16–24
12.
Nicolai
A
,
Li
M
,
Kim
DH
, et al
.
Heme oxygenase-1 induction remodels adipose tissue and improves insulin sensitivity in obesity-induced diabetic rats
.
Hypertension
2009
;
53
:
508
515
[PubMed]
13.
Ndisang
JF
,
Jadhav
A
.
Up-regulating the hemeoxygenase system enhances insulin sensitivity and improves glucose metabolism in insulin-resistant diabetes in Goto-Kakizaki rats
.
Endocrinology
2009
;
150
:
2627
2636
[PubMed]
14.
Dong
H
,
Huang
H
,
Yun
X
, et al
.
Bilirubin increases insulin sensitivity in leptin-receptor deficient and diet-induced obese mice through suppression of ER stress and chronic inflammation
.
Endocrinology
2014
;
155
:
818
828
[PubMed]
15.
Wegiel
B
,
Gallo
DJ
,
Raman
KG
, et al
.
Nitric oxide-dependent bone marrow progenitor mobilization by carbon monoxide enhances endothelial repair after vascular injury
.
Circulation
2010
;
121
:
537
548
[PubMed]
16.
Di Pascoli
M
,
Rodella
L
,
Sacerdoti
D
,
Bolognesi
M
,
Turkseven
S
,
Abraham
NG
.
Chronic CO levels have [corrected] a beneficial effect on vascular relaxation in diabetes
.
Biochem Biophys Res Commun
2006
;
340
:
935
943
[PubMed]
17.
Ahmad
FK
,
He
Z
,
King
GL
.
Molecular targets of diabetic cardiovascular complications
.
Curr Drug Targets
2005
;
6
:
487
494
[PubMed]
18.
Gourley
GR
.
Bilirubin metabolism and kernicterus
.
Adv Pediatr
1997
;
44
:
173
229
[PubMed]
19.
Duplain
H
,
Burcelin
R
,
Sartori
C
, et al
.
Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase
.
Circulation
2001
;
104
:
342
345
[PubMed]
20.
Dekker
D
,
Dorresteijn
MJ
,
Pijnenburg
M
, et al
.
The bilirubin-increasing drug atazanavir improves endothelial function in patients with type 2 diabetes mellitus
.
Arterioscler Thromb Vasc Biol
2011
;
31
:
458
463
[PubMed]