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Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin-Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats

International Journal of Diabetology & Vascular Disease Research, 2013 © 29
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
International Journal of Diabetology & Vascular Disease Research(IJDVR)
ISSN 2328-353X
Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin-Mediated Metabolic and Microvascular
Responses in Skeletal Muscle of High Fat Fed Rats
Research Article
Premilovac D1, Roberts-Thomson KM1, Ng HL1, Bradley EA1, Richards SM1, Rattigan S1, Keske MA1*
Menzies Research Institute Tasmania, University of Tasmania, Hobart, Australia.
Background: The aim of the current study was to determine whether a unique blueberry tea (BT) ameliorates insulin resistance by improv-
ing metabolic and vascular actions of insulin in skeletal muscle.
Methods: Male Sprague Dawley rats were fed normal (4.8% fat wt/wt, ND) or high (22.6% fat wt/wt, HFD) fat diets for 4 weeks. A second
group of HFD rats was provided BT (4.0% wt/vol) in the drinking water during the nal 2 weeks. Animals were subjected to an intraperi-
toneal glucose tolerance test (1g glucose/kg IPGTT) or euglycaemic hyperinsulinaemic clamp (10mU/min/kg x 2hr).
Results: HFD rats displayed signicantly (p<0.05) higher plasma glucose levels at 15 and 30 mins following the IPGTT compared to ND,
and this increase was completely abolished by BT treatment. Glucose infusion rate, muscle glucose uptake, and microvascular perfusion in
muscle were signicantly (p<0.05) impaired during clamps in HFD and all markedly improved (p<0.05) with BT treatment. Insulin-mediated
changes in femoral artery blood ow were unaffected by HFD or BT treatment.
Conclusions: We conclude that BT treatment ameliorates glucose intolerance and insulin resistance by restoring both metabolic and mi-
crovascular insulin sensitivity in high fat-fed rats. Therefore, BT consumption may have therapeutic implications for insulin resistance and
type 2 diabetes.
Key Words: Insulin Sensitivity; Glucose Tolerance; High Fat Diet; Blueberry Tea; Microvascular Perfusion; Muscle Glucose Uptake.
*Corresponding Author:
Michelle A. Keske
Menzies Research Institute Tasmania, University of Tasmania, Hobart,
Tel: (03) 62 26 2669; Fax: (03) 62 26 7704
Received: November 21, 2013
Accepted: December 16, 2013
Published: December 18, 2013
Citation: Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensi-
tivity by Augmenting Insulin- Mediated Metabolic and Microvascular Re-
sponses in Skeletal Muscle of High Fat Fed Rats. Int J Diabetol Vasc Dis
Res. 1(5), 29-36. doi:
Copyright: Keske MA© 2013 This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium,
provided the original author and source are credited.
Insulin resistance plays a key role in the pathogenesis of type-2
diabetes (T2D). While the root causes of insulin resistance are
multifactorial, two of the major contributors are increased dietary
fat and a sedentary lifestyle [1-4]. A dening feature of insulin re-
sistance is impaired glucose disposal by the skeletal muscle, which
is normally responsible for ~80% of insulin-mediated glucose
uptake in the post-prandial state [5].
In addition to the classical metabolic actions of insulin to stimu-
late glucose uptake in skeletal muscle, insulin also has important
vascular actions [6-13]. Our research implicates vascular dysfunc-
tion in skeletal muscle as one major cause of muscle insulin resist-
ance. Insulin stimulates both total blood ow to skeletal muscle
[11-13] and increases microvascular perfusion of the myocytes
[6-10]. However insulin’s actions on microvascular perfusion and
total muscle blood ow are separate events and insulin-mediated
glucose uptake is altered by changes in microvascular perfusion
and not total muscle blood ow [7,14].
We have demonstrated that insulin infusion or the ingestion of a
mixed meal stimulate microvascular blood ow in skeletal muscle
in both experimental animals [6-10] and humans [15-17]. Insu-
lin stimulates microvascular recruitment to facilitate delivery of
glucose and insulin to the myocyte to enhance glucose disposal
[6-10,18]. Insulin resistant rats [3] and obese humans [15,16] also
display marked reductions in insulin-mediated microvascular re-
cruitment and insulin-mediated muscle glucose uptake suggesting
that the loss of muscle perfusion may contribute to the insulin re-
sistance. Recently, we reported that high fat-induced insulin resist-
ance can originate from impaired microvascular insulin responses
and that this microvascular defect precedes the development of
myocyte insulin resistance [14].
Current treatments for T2D are limited, have unwanted side ef-
fects, and lose effectiveness over time. Finding novel therapeutic
treatments that act by improving microvascular and metabolic in-
sulin responses in skeletal muscle may aid in the prevention of
insulin resistance and T2D or potentially enhance the action of
current treatments. There is a growing public interest in the use
of complementary and alternative approaches for treating insulin
resistance and T2D. Blueberries and blueberry leaves have been
reported to have anti-diabetic actions [19-21] and have been used
as a traditional medicine for glycaemic control [22,23]. Here we
International Journal of Diabetology & Vascular Disease Research, 2013 © 30
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
report on novel ndings demonstrating that a unique blueberry
tea blend (BT) restores metabolic and microvascular insulin sensi-
tivity in muscle of the high fat-fed rat model.
Animal Care
Male Sprague-Dawley rats (4 weeks of age) were obtained from
the University of Tasmania Central Animal Facility. On arrival,
the rats were divided into two groups and provided either a nor-
mal (4.8% fat wt/wt; ND, Specialty Feeds, Glen Forrest, WA,
Australia) or high (22.6% fat wt/wt; HFD, Specialty Feeds) fat
diet ad libitum for 4 weeks. The macronutrients in these diets are
shown in Table 1. A second group of HFD rats were supplement-
ed with blueberry tea (HFD+BT; 4% wt/vol) during the nal two
weeks of HFD feeding. The blueberry tea, Blueberry Boost™,
was a kind donation from Dromana Blueberries (Oyster Cove,
Tasmania, Australia) which contains a propriety blend of dried
blueberries, blueberry leaves, spearmint leaves, raspberry leaves
and cinnamon. BT was brewed at the rate of 4g of loose leaves
per 100ml of boiling water, allowed to infuse for 10 minutes, then
strained through a 0.75mm mesh to remove particulate matter
and cooled. Animals in the HFD+BT group were provided with
both normal drinking water and BT in separate drinking bottles
ad libitum. The tea was replaced every two days. All animals were
housed at 21±2°C with a 12h-12h light-dark cycle. All experi-
mental procedures were approved by the University of Tasmania
Animal Ethics Committee and performed in accordance with the
Australian Code of Practice for the Care and Use of Animals for
Scientic Purposes – 2013, 8th Edition.
Surgical Preparation
Rats were anaesthetised with an intraperitoneal injection of
aqueous pentobarbital sodium (50mg/kg body wt.) and surgery
performed as previously described [6-10]. Animals were main-
tained under anaesthesia for the duration of the experiment by
an intravenous infusion of pentobarbital sodium (0.6 mg/min/
kg body wt.), and mean arterial blood pressure (MAP) and heart
rate continuously recorded. Following surgical preparation, 1 hr
was allowed for the animal’s cardiovascular parameters to stabilise.
Subsequently, animals were then subjected to either an intraperi-
toneal glucose tolerance test (protocol 1), or a euglycaemic hyper-
insulinaemic clamp (protocol 2).
Protocol 1 - Intraperitoneal glucose tolerance test
Animals were subjected to a 90 min glucose tolerance test (1g
glucose/kg IPGTT). Arterial blood was sampled from the carotid
artery at baseline (0 min) and at 15, 30, 45, 60 and 90 min fol-
lowing the glucose injection. Blood samples were centrifuged and
plasma glucose levels were assessed using a glucose analyser (YSI
2300, Yellow Springs Instruments, OH, USA).
Protocol 2- Euglycaemic hyperinsulinaemic clamp
Following an overnight fast a 2 hr infusion of insulin (10mU/
min/kg; Humulin, Eli Lilly, West Ryde, NSW, Australia) was initi-
ated through the jugular vein. A 30% glucose solution (wt/vol)
was infused at a variable rate to maintain fasting blood glucose
concentrations over the course of the experiment. Arterial blood
glucose levels were assessed from the carotid artery every 10-15
min using a glucose analyser (YSI 2300) and the glucose infusion
rate (GIR) was adjusted accordingly.
Fasting and clamp plasma insulin concentrations were determined
by ELISA (Mercodia AB, Uppsala, Sweden). Fasting plasma non
esteried fatty acid (NEFA) levels were determined using an en-
zymatic assay kit (Wako Pure Chemical Industries, Osaka, Japan).
Muscle-specic glucose uptake was assessed by uptake of 2-de-
oxy-D-[1-14C]glucose (2DG; 0.1mCi/mL; American Radiolabeled
Chemicals, Inc., MO, USA) as described previously [3,14]. Briey,
45 min prior to the conclusion of the experiment an i.v. bolus
of 2DG was given (20μCi). Arterial plasma samples (25μL) were
collected 5, 10, 15, 30 and 45 min after the 2DG bolus to assess
plasma specic activity of 2DG. At the end of the experiment the
calf muscle group (gastrocnemius, plantaris, soleus) was excised,
immediately freeze clamped and stored at -80°C until analysed.
The frozen muscle was ground under liquid nitrogen and ~100mg
was homogenised with 1.5mL of distilled water. The homogenate
was centrifuged and 1mL of supernatant was separated for free
and phosphorylated 2DG using an anion exchange column (AG-
1X8; Bio-Rad Laboratories, CA, USA). Biodegradable counting
scintillant (Amersham, Arlington Heights, IL, USA) was added
to each sample and radioactivity was measured using a scintilla-
tion counter (Tri-Carb 2800TR, Perkin Elmer, IL, USA). From
this measurement and the specic activity of 2DG in plasma, the
rate of muscle glucose uptake (R’g) was calculated as previously
described [24].
Femoral artery blood ow (FBF) was continuously measured by
an ultrasonic ow probe positioned around the femoral artery
(Transonic, Ithica, NY, USA). Microvascular perfusion in muscle
was assessed from metabolism of exogenously infused 1-methyl
xanthine (1-MX; Sigma Aldrich, St. Louis, MO, USA) as previ-
ously published [3,6,8,14,25-29]. Briey, a bolus of allopurinol
Table 1. Macronutrient composition of the ND and HFD diets expressed as % total weight.
Protein 19.4% 19.0%
Carbohydrate 70.7% 49.0%
Fat 4.8% 22.6%
Monounsaturated (% total fat) 39% 39%
Polyunsaturated (% total fat) 44% 9%
Saturated (% total fat) 17% 52%
International Journal of Diabetology & Vascular Disease Research, 2013 © 31
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
(10μmol/kg; Sigma Aldrich) was given 5 min prior to 1-MX infu-
sion to partially inhibit the activity of xanthine oxidase and ensure
a constant saturating arterial level of 1-MX. 1-MX (0.4mg/min/
kg) was infused intravenously during the nal 60min of the ex-
periment. At the end of the experiment 100μL of arterial plasma
was added to 20μL of perchloric acid (2M) to precipitate pro-
teins. Hindleg venous plasma, obtained from the femoral vein us-
ing a 29G needle, was collected and 100μL was mixed with 20μL
of perchloric acid (2M). The arterial and venous samples were
centrifuged and the supernatant assessed for 1-MX and oxypuri-
nol concentrations using reverse-phase high-performance liquid
chromatography as previously described [6]. The metabolism of
1-MX was calculated from the plasma arterio-venous 1-MX dif-
ference multiplied by FBF and expressed as nmol/min.
Statistical Methods
Data are presented as the means ± SEM and statistics were per-
formed using SigmaStat (Systat Software, San Jose, CA, USA).
Comparisons between groups were made using One-way ANO-
VA. Comparison of time-series measurements in each group was
performed by two-way repeated measures ANOVA. When a sig-
nicant difference of p<0.05 was detected, pairwise comparisons
by Student-Newman-Keuls test was used to assess treatment dif-
Physical and biochemical characteristics of rats
At the conclusion of the dietary intervention, there were no
differences in body weight (Figure 1A) or energy intake from
food (Table 2) between ND, HFD or HFD+BT groups. Water
intake was the same between ND and HFD groups. However
the HFD+BT group consumed more uids as they were given
both water and BT ad libitum (Table 2). At the end of the dietary
intervention, there were no differences in plasma glucose con-
centrations between ND, HFD and HFD+BT rats (Figure 2A
and B). As expected, HFD rats had signicantly (p<0.05) elevated
epididymal fat pad weights (Figure 1B), fasting plasma insulin
(Figure 3A), and fasting plasma NEFA (Figure 3B) concentra-
tions when compared to ND. These outcomes were unaffected
by BT treatment, however there was a trend (p=0.07) for fasting
plasma NEFA concentrations to be lower in the HFD+BT when
compared to HFD.
Protocol 1 - Intraperitoneal glucose tolerance test:
Table 2. Food, water and BT intake in ND (4.8% fat wt/wt), HFD (22.6% fat wt/wt) and HFD+BT (4% wt/vol blueberry
tea) rats.
Food intake (kJ/day/kg body wt) 1410 ± 46 1339 ± 42 1418 ± 38
Water intake (mL/day/kg body wt) 94 ± 3 90 ± 3 61 ± 8*
BT intake (mL/day/kg body wt) - - 68 ± 4
Data are means ± SEM for n=5 measurements in each group. Comparisons between groups were made using One Way ANOVA and
Student-Newman-Keuls Post-hoc test.*P<0.05 versus ND and HFD.
Figure 1: Body weight and epididymal adiposity.
Final body weight (A) and epididymal fat pad weight (B) of ND ( ), HFD ( ) and HFD+BT ( ) rats. Data are means ± SEM for
n=14-15 rats in each group. *p<0.05 versus ND; One way ANOVA and Student-Newman-Keuls Post-hoc test.
International Journal of Diabetology & Vascular Disease Research, 2013 © 32
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
Figure 4 shows the time course of changes in plasma glucose
following the glucose tolerance test. ND, HFD and HFD+BT
groups had similar basal (non-fasting) plasma glucose levels. HFD
had signicantly higher plasma glucose at 15 and 30 min follow-
ing the glucose challenge. Interestingly, the HFD+BT group had
markedly (p<0.05) reduced plasma glucose levels at 15 and 30
min when compared to HFD. However, there were no statistical
differences in plasma glucose levels between groups at 0, 45, 60
or 90 min.
Protocol 2 - Euglycaemic Hyperinsulinaemic Clamp
Figure 5A shows the GIR required to maintain glycaemia during
insulin infusion for ND, HFD and HFD+BT groups. As expect-
ed, the HFD group had signicantly lower (p<0.05) GIR when
compared to the ND group conrming that the HFD rats were
insulin resistant. Importantly, the HFD+BT group had signi-
cantly (p<0.05) elevated GIR when compared to HFD, indicating
improved whole body insulin sensitivity. Insulin-mediated uptake
of radiolabeled glucose in skeletal muscle (R’g, Figure 5B) was
signicantly (p<0.05) impaired in HFD rats (18.6 ± 1.2 vs. 28.9 ±
2.9 μg/g/min, p<0.05). However, insulin-stimulated R’g in HFD
rats was markedly improved with BT treatment (24.4 ± 0.9 μg/g/
min, p<0.05).
Insulin increased FBF in all groups to a similar extent in ND,
Figure 2: Plasma glucose levels.
Non-fasting (A) and fasting (B) plasma glucose levels in ND ( ), HFD ( ) and HFD+BT ( ) rats. Data are means ± SEM for n=7-9
rats in each group. Data not signicant. One way ANOVA.
Figure 3: Fasting insulin and NEFA levels.
Fasting plasma insulin (A) and NEFA (B) levels of ND ( ), HFD ( ) and HFD+BT ( ) rats. Data are means ± SEM for n=7-9 rats
in each group. *p<0.05 versus ND; # p=0.07 versus HFD. One way ANOVA and Student-Newman-Keuls Post-hoc test.
International Journal of Diabetology & Vascular Disease Research, 2013 © 33
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
HFD and HFD+BT groups (p= NS, Figure 6A). Femoral artery
vascular resistance (MAP divided by FBF) in response to insulin
was also similar between groups (107 ± 15 vs. 120 ± 21 vs. 109
± 15 mmHg.min/ml, ND, HFD and HFD+BT respectively, p=
NS). Hindlimb 1-MX metabolism, an indicator of microvascu-
lar perfusion, was signicantly (p<0.05) impaired in HFD when
compared to ND during insulin infusion (4.87± 0.63 vs. 8.43 ±
1.13 nmole/min; p<0.05, Figure 6B). BT treatment reversed the
impairment in insulin-mediated 1-MX metabolism induced by
HFD (7.87 ± 0.63 nmole/min).
The main ndings from this study were that BT treatment ame-
liorated HFD-induced glucose intolerance and insulin resistance.
BT treatment improved insulin-stimulated glucose uptake and mi-
crovascular perfusion in skeletal muscle. These effects occurred
without any changes in body weight, epididymal pad weight, fast-
ing clinical chemistries or femoral artery blood ow. These data
indicate that BT is an insulin sensitiser in skeletal muscle and acts
by improving both the metabolic and microvascular actions of
Figure 4: Glucose tolerance test.
Plasma glucose levels over time following the ip injection of 1g glucose/kg body weight in ND ( ), HFD ( ) and HFD+BT
( ) rats. Data are means ± SEM for n=8 rats in each group. *p<0.05 versus ND; # p<0.05 versus HFD. Two way repeated measures
ANOVA and Student-Newman-Keuls post-hoc test.
Figure 5: Glucose metabolism.
Glucose infusion rate (GIR) required to maintain euglycaemia of insulin clamp (A) and muscle glucose uptake (R’g) at end of clamp (B)
of ND ( ), HFD ( ) and HFD+BT ( ) rats during insulin clamp (10mU/min/kg). Data are means ± SEM for n=7-9 rats in each
group. *p<0.05 versus ND; # p<0.05 versus HFD. Two way repeated measures ANOVA, or One way ANOVA where appropriate, and
Student-Newman-Keuls post-hoc test.
International Journal of Diabetology & Vascular Disease Research, 2013 © 34
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
Blueberry Boost™ is a proprietary blend of dried blueber-
ries (37% wt/wt), blueberry leaves, raspberry leaves, spearmint
leaves and cinnamon. Blueberries [19-21], blueberry leaves [20]
and cinnamon [30-32] have each been reported to improve insu-
lin sensitivity or insulin action; however there are no reports of
raspberry leaves or spearmint on these measures. High fat fed
mice supplemented with freeze-dried blueberry powder (4% wt/
wt provided in the food x 8 weeks) prevented diet-induced obe-
sity and improved whole body insulin sensitivity in response to
an insulin tolerance test [19]. Blueberry fruit and blueberry leaf
extracts have been shown to stimulate insulin-dependent and
independent glucose uptake in cultured C2C12 muscle cells but
not in 3T3-LI adipocytes [20], suggesting an insulin-mimetic and
insulin-sensitizing effect in muscle. More recently, freeze-dried
blueberry fruit supplementation (45g per day x 6 weeks) to obese
insulin resistant individuals improved whole body insulin sen-
sitivity (assessed by euglycaemic hyperinsulinaemic clamp tech-
nique) when compared to placebo [21]. Conversely high fat-fed
mice provided freeze-dried whole blueberry powder (10% wt/
wt in diet x 92 days) increased body weight and obesity, and did
not alter glucose tolerance (assessed by IPGTT) [33]. There are
a number of studies reporting on the effects of cinnamon on
insulin sensitivity and glucose homeostasis (see review [30] and
references therein). Cinnamon (20g/kg diet x 12 weeks) enhances
whole body insulin sensitivity in high fat/sucrose fed rats without
altering body weight or adiposity [31]. Similarly, cinnamon extract
(300mg/kg/day in drinking water x 3 weeks) prevents insulin re-
sistance in high fructose fed rats [32]. Numerous clinical studies
have reported that cinnamon or cinnamon extract can improve
glucose tolerance and/or whole body insulin sensitivity in healthy
lean [34,35] and insulin resistant [36] individuals. However the ef-
fect of these nutritional agents on insulin’s vascular and metabolic
actions in muscle has not been previously explored.
Insulin-mediated microvascular recruitment in skeletal muscle
plays an integral role in regulating muscle glucose uptake. We
have shown that acute defects in insulin-mediated microvascular
recruitment can contribute to the development of muscle insulin
resistance in vivo. Infusion of α-methyl serotonin [26], endothe-
lin-1 [27], L-NG-Nitroarginine methyl ester [7,29,37], TNFα [38],
Intralipid® plus heparin [39] and glucosamine [40] in healthy
animals attenuate insulin-mediated microvascular recruitment
and insulin-mediated muscle glucose uptake. In addition, chronic
models of insulin resistance including the high fat-fed rat (36%
fat wt/wt) [3], and obese Zucker rat [41] display marked attenu-
ation in insulin-mediated microvascular recruitment and muscle
glucose uptake. More recently we reported [14] that moderate in-
creases in dietary fat cause microvascular and not myocyte-derived
insulin resistance, which for the rst time positioned defects in
microvascular insulin action as an early event that contributes sig-
nicantly to fat-induced muscle insulin resistance. In the present
study, animals given BT for two weeks restored insulin-mediated
microvascular recruitment and this was associated with improved
insulin-stimulated muscle glucose uptake, potentially due to im-
proved delivery of insulin and glucose to myocytes.
This is the rst report of an agent that reverses microvascular
insulin resistance in muscle of the HFD rat model. Given that
the HFD model we used in this study displays both myocyte and
microvascular insulin resistance [14], the improvement in muscle
glucose uptake following BT treatment could be due to improve-
ments in both myocyte and microvascular insulin responsiveness.
However, given that BT treatment reversed the impairment in
microvascular insulin responsiveness and only ~40% of insulin-
stimulated glucose uptake in muscle, it can be speculated that BT
treatment has improved predominantly vascular and not myocyte
insulin action. The current study also demonstrates that BT treat-
ment markedly improved whole body, muscle and microvascular
insulin sensitivity without changing adiposity or fasting insulin
levels, two hallmarks of insulin resistance. This is suggestive of
BT treatment having an insulin-sensitising action in this animal
model, however whether these markers would be altered during
longer term supplementation is unknown.
As detailed previously, BT is a blend of ve plant based ingre-
dients. The present study does not indicate which ingredient(s)
Figure 6: Musclebloodow.
Femoral artery blood ow (A; FBF) and muscle microvascular recruitment (B) during insulin clamp in ND ( ), HFD ( ) and
HFD+BT ( ) rats. Data are means ± SEM for n=7-9 rats in each group. One way ANOVA and Student-Newman-Keuls post-hoc test.
*p<0.05 Versus ND; #p<0.05 Versus HFD
International Journal of Diabetology & Vascular Disease Research, 2013 © 35
Keske MA et al,. (2013) Blueberry Tea Enhances Insulin Sensitivity by Augmenting Insulin- Mediated Metabolic and Microvascular Responses in Skeletal Muscle of High Fat Fed Rats.
Int J Diabetol Vasc Dis Res. 1(5), 29-36
or bioactive components of BT are responsible for this insulin
sensitizing action, or whether in combination they have additive
or synergistic actions. Although dried blueberries are the main
constituent of this product, further studies using individual ingre-
dients would be required to fully resolve these issues.
This is the rst study to demonstrate that BT treatment markedly
improves microvascular and metabolic insulin sensitivity in skel-
etal muscle of HFD rats. These effects were apparent indepen-
dently of changes in adiposity, or clinical chemistries such as fast-
ing plasma glucose and insulin. Our study suggests that regular
consumption of BT may have important therapeutic implications
for insulin resistance and T2D, however its efcacy in humans
population remains to be determined.
Competing Interests
No conicts of interest, nancial or otherwise, are declared by
the authors.
Authors’ Contributions
D.P., K.M.R-T, M.A.K and were involved in design, conduct/
data collection, analysis and writing of the manuscript. E.A.B.,
H.L.H.N, S.M.R. and S. R. were involved in design, analysis and
writing of the manuscript. All authors read and approved the nal
This work was funded by the National Health & Medical Research
Council of Australia (grant number 1009962 to M.A.K. and S.R.).
Professor Stephen Rattigan is a Senior Research Fellow of the
National Health & Medical Research Council of Australia (grant
number 490034 to S.R). We thank Carl Sykes and Nadja Brearley
of Dromana Blueberries for donating Blueberry Boost™ for this
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... In the present study, we demonstrate a novel method that allows for targeted and sustained induction type 2 diabetes in rats by combining a HFD with osmotic mini-pump-infused STZ. In our model, HFD feeding led to obesity, increased circulating NEFA and insulin concentrations and glucose intolerance, typical of insulin resistance/pre-diabetes in both rodents 7,22 and humans 23 . The addition of STZ after three weeks of HFD feeding caused a dose-dependent increase in circulating glucose and further decline in glucose tolerance while preserving an obese, dyslipidemic phenotype. ...
... Indeed, individuals with type 2 diabetes progress through a far longer stage of insulin resistance-associated hyperinsulinemia, which ultimately leads to beta cell dysfunction 5,6 . While our model involves a background of HFD-induced insulin resistance 7,22 , the administration of STZ does not replicate the exact cause of beta cell death as seen in humans. While this imposes some limitations on this model, the use of STZ greatly accelerates the time-frame for development of hyperglycemia and glucose intolerance that may not otherwise occur in rodents with relatively short life-spans. ...
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Type 2 diabetes is a chronic metabolic disorder that is becoming a leading cause of morbidity and mortality. The prolonged time-course of human type 2 diabetes makes modelling of the disease difficult and additional animal models and methodologies are needed. The goal of this study was to develop and characterise a new method that allows controlled, targeted and sustained induction of discrete stages of type 2 diabetes in rodents. Using adult, male rats, we employed a three-week high fat-diet regimen and confirmed development of obesity-associated glucose intolerance, a key feature of human type 2 diabetes. Next, we utilised osmotic mini-pumps to infuse streptozotocin (STZ; doses ranging 80–200 mg/kg) over the course of 14-days to decrease insulin-producing capacity thus promoting hyperglycemia. Using this new approach, we demonstrate a dose-dependent effect of STZ on circulating glucose and insulin levels as well as glucose tolerance, while retaining a state of obesity. Importantly, we found that insulin secretion in response to a glucose load was present, but reduced in a dose-dependent manner by increasing STZ. In conclusion, we demonstrate a novel method that enables induction of discrete stages of type 2 diabetes in rodents that closely mirrors the different stages of type 2 diabetes in humans.
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To study the effect of organic and biological fertilizers on the yield and the yield components of Salvia leriifolia Benth, a factorial experiment was carried out based on a randomized complete block design with 10 treatments [five organic fertilizers: control (C0), Municipal solid waste compost of 10 (C10), 20 (C20) tons ha⁻¹, vermicompost of 3 (V3), 6 (V6) tons ha⁻¹, and Pseudomonas putida: without inoculation (S0), with inoculation (S)] with three replications. The experiment was performed at Khorasan-e-Razavi Agricultural and Natural Resources Research and Educational Center during 2015 and 2016. The results in the first growing season revealed that organic fertilizers had a significant effect on the number of leaves per plant, leaf area index (LAI) and leaves dry weight (LDW), however, the inoculation of P. putida and its interaction with organic fertilizers were not significant. In the second season, all vegetative and reproductive traits were significantly affected by different levels of organic fertilizers, but P. putida effect was significant on LAI, number and leaves dry weight and biological yield (BY). The highest leaves dry weight (1470 kg a⁻¹), LAI (0.99) and the biological yield (2558 kg ha') in the second growing season was obtained in C20S19 indicating an increase of 47,46 and 53% compared to C0S0, respectively. Similarly, the results showed that the highest number of seeds m² (129.6) and the seed yield (79 kg ha¹) were obtained in C20S1 treatment indicating two fold increase compared to control.
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Insulin stimulates microvascular recruitment in skeletal muscle and this vascular action augments muscle glucose disposal by ~40%. The aim of the current study was to determine the contribution of local nitric oxide synthase (NOS) to the vascular actions of insulin in muscle. Hooded Wistar rats were infused with the NOS inhibitor Nω-L-nitro-arginine-methyl ester (L-NAME, 10µM) retrogradely via the epigastric artery into one leg during a systemic hyperinsulinemic euglycaemic clamp ( 60 min) or saline infusion. Femoral artery blood flow, microvascular blood flow (assessed from 1-methyl xanthine metabolism) and muscle glucose uptake (2-deoxyglucose uptake) was measured in both legs. Local L-NAME infusion did not have any systemic actions on blood pressure or heart rate. Local L-NAME blocked insulin-stimulated changes in femoral artery blood flow (84%, P<0.05) and microvascular recruitment (98%, P<0.05), and partially blocked insulin-mediated glucose uptake in muscle (reduced by 34%, P< 0.05). L-NAME alone did not have any metabolic effects in the hindleg. We conclude that insulin-mediated microvascular recruitment is dependent on local activation of NOS in muscle, and that this action is important for insulin's metabolic actions.
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Insulin produces capillary recruitment in skeletal muscle through a nitric oxide (NO)-dependent mechanism. Capillary recruitment is blunted in obese and diabetic subjects and contributes to impaired glucose uptake. This study's objective was to define whether inactivity, in the absence of obesity, leads to impaired capillary recruitment and contributes to insulin resistance (IR). A comprehensive metabolic and vascular assessment was performed on 19 adult male rhesus macaques (Macaca mulatta) after sedation with ketamine and during maintenance anesthesia with isoflurane. Thirteen normal-activity (NA) and six activity-restricted (AR) primates underwent contrast-enhanced ultrasound to determine skeletal muscle capillary blood volume (CBV) during an intravenous glucose tolerance test (IVGTT) and during contractile exercise. NO bioactivity was assessed by flow-mediated vasodilation. Although there were no differences in weight, basal glucose, basal insulin, or truncal fat, AR primates were insulin resistant compared with NA primates during an IVGTT (2,225 ± 734 vs. 5,171 ± 3,431 μg·ml(-1)·min(-1), P < 0.05). Peak CBV was lower in AR compared with NA primates during IVGTT (0.06 ± 0.01 vs. 0.12 ± 0.02 ml/g, P < 0.01) and exercise (0.10 ± 0.02 vs. 0.20 ± 0.02 ml/g, P < 0.01), resulting in a lower peak skeletal muscle blood flow in both circumstances. The insulin-mediated changes in CBV correlated inversely with the degree of IR and directly with activity. Flow-mediated dilation was lower in the AR primates (4.6 ± 1.0 vs. 9.8 ± 2.3%, P = 0.01). Thus, activity restriction produces impaired skeletal muscle capillary recruitment during a carbohydrate challenge and contributes to IR in the absence of obesity. Reduced NO bioactivity may be a pathological link between inactivity and impaired capillary function.
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Dietary fat composition can interfere in the development of obesity due to the specific roles of some fatty acids that have different metabolic activities, which can alter both fat oxidation and deposition rates, resulting in changes in body weight and/or composition. High-fat diets in general are associated with hyperphagia, but the type of dietary fat seems to be more important since saturated fats are linked to a positive fat balance and omental adipose tissue accumulation when compared to other types of fat, while polyunsaturated fats, omega-3 and omega-6, seem to increase energy expenditure and decrease energy intake by specific mechanisms involving hormone-sensitive lipase, activation of peroxisome proliferator-activated receptor α (PPARα) and others. Saturated fat intake can also impair insulin sensitivity compared to omega-3 fat, which has the opposite effect due to alterations in cell membranes. Obesity is also associated with impaired mitochondrial function. Fat excess favors the production of malonyl-CoA, which reduces GLUT4 efficiency. The tricarboxylic acid cycle and beta-oxidation are temporarily uncoupled, forming metabolite byproducts that augment reactive oxygen species production. Exercise can restore mitochondrial function and insulin sensitivity, which may be crucial for a better prognosis in treating or preventing obesity.
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Dietary supplementation with whole blueberries in a preclinical study resulted in a reduction in glucose concentrations over time. We sought to evaluate the effect of daily dietary supplementation with bioactives from blueberries on whole-body insulin sensitivity in men and women. A double-blinded, randomized, and placebo-controlled clinical study design was used. After screening to resolve study eligibility, baseline (wk 0) insulin sensitivity was measured on 32 obese, nondiabetic, and insulin-resistant subjects using a high-dose hyperinsulinemic-euglycemic clamp (insulin infusion of 120 mU(861 pmol)⋅m(-2)⋅min(-1)). Serum inflammatory biomarkers and adiposity were measured at baseline. At the end of the study, insulin sensitivity, inflammatory biomarkers, and adiposity were reassessed. Participants were randomized to consume either a smoothie containing 22.5 g blueberry bioactives (blueberry group, n = 15) or a smoothie of equal nutritional value without added blueberry bioactives (placebo group, n = 17) twice daily for 6 wk. Both groups were instructed to maintain their body weight by reducing ad libitum intake by an amount equal to the energy intake of the smoothies. Participants' body weights were evaluated weekly and 3-d food records were collected at baseline, the middle, and end of the study. The mean change in insulin sensitivity improved more in the blueberry group (1.7 ± 0.5 mg⋅kg FFM(-1)⋅min(-1)) than in the placebo group (0.4 ± 0.4 mg⋅kg FFM(-1)⋅min(-1)) (P = 0.04). Insulin sensitivity was enhanced in the blueberry group at the end of the study without significant changes in adiposity, energy intake, and inflammatory biomarkers. In conclusion, daily dietary supplementation with bioactives from whole blueberries improved insulin sensitivity in obese, nondiabetic, and insulin-resistant participants.
It is now well established that insulin, apart from its effect on carbohydrate metabolism and ion flux, may exert cardiovascular actions and, more recently, a growing body of work has emerged suggesting that marked physiological and specific effects are also present in humans. The cardiovascular responses associated with acute administration of insulin in man, in the absence of hypoglycemia, include a rise in heart rate, cardiac output and forearm blood flow and a substancial decrease in perpheral vascular resistance. Moreover, hyperinsulinemia is associated with a dose dependent increment in the circulating levels of norepinephrine and with a marked stimulation of muscle nerve simpathetic activity. Recent data have also established that the cardiovascular effects of insulin are not merely pharmacological being present during elevation of plasma insulin to within post-prandial physiological range. These findings would suggest that in normal humans, increases in plasma insulin produce both pressor (increases in simpathetic nerve acivity and norepinephrine) and depressor (vasodilation) actions. With post-prandial hyperinsulinemia these opposing actions appear relatively balanced so that arterial blood pressure does not change, presumably because the pressor acions are offset by vasodilator actions. In contrast, in subjects with autonomic failure the vasodilator effect prevails and blood pressure decreases while, experimental data suggest, that in insulin resistance state, pressor effect might predominate thus supporting the role of hyperinsulinemia in promoting hypertension. The interaction between insulin and the sympathetic nervous system as well as its role in stimulating blood flow have been subjects of renewed interest and recent controversy. Furthermore, since the degree of skeletal muscle perfusion might be an important determinant of insulin-induced glucose uptake, insulin-mediated vasodilation could act as an amplifier of insulin action in peripheral tissue, thus providing evidence of a tight coupling between hemodynamic and metabolic actions of insulin.
Aims Enhanced microvascular perfusion of skeletal muscle is important for nutrient exchange and contributes ∼40 insulin-mediated muscle glucose disposal. High fat-fed (36 fat wt./wt.) rats are a commonly used model of insulin-resistance that exhibit impairment of insulin-mediated microvascular recruitment and muscle glucose uptake, which is accompanied by myocyte insulin-resistance. Distinguishing the contribution of impaired microvascular recruitment and impaired insulin action in the myocyte to decreased muscle glucose uptake in these high-fat models is difficult. It is unclear whether microvascular and myocyte insulin-resistance develop simultaneously. To assess this, we used a rat diet model with a moderate increase (two-fold) in dietary fat.Methods and resultsSprague Dawley rats fed normal (4.8 fat wt./wt., 5FD) or high (9.0 fat wt./wt., 9FD) fat diets for 4 weeks were subject to euglycaemic hyperinsulinemic clamp (10 mU/min/kg insulin or saline) or isolated hindlimb perfusion (1.5 or 15 nM insulin or saline). Body weight, epididymal fat mass, and fasting plasma glucose were unaffected by diet. Fasting plasma insulin and non-esterified fatty acid concentrations were significantly elevated in 9FD. Glucose infusion rate and muscle glucose uptake were significantly impaired during insulin clamps in 9FD. Insulin-stimulated microvascular recruitment was significantly blunted in 9FD. Insulin-mediated muscle glucose uptake between 5FD and 9FD were not different during hindlimb perfusion.Conclusions Impaired insulin-mediated muscle glucose uptake in vivo can be the direct result of reduced microvascular blood flow responses to insulin, and can result from small (two-fold) increases in dietary fat. Thus, microvascular insulin-resistance can occur independently to the development of myocyte insulin-resistance.
The aetiology of the development of type 2 diabetes remains unresolved. In the present study, we assessed whether an impairment of insulin-mediated microvascular perfusion occurs early in the onset of insulin resistance. Hooded Wistar rats were fed either a normal diet (ND) or a high-fat diet (HFD) for 4 weeks. Anaesthetized animals were subjected to an isoglycaemic hyperinsulinaemic clamp (3 or 10 mU/min/kg x 2 h), and measurements were made of glucose infusion rate (GIR), hindleg glucose uptake, muscle glucose uptake by 2-deoxy-d-glucose (R'g), glucose appearance (Ra), glucose disappearance (Rd), femoral blood flow (FBF) and hindleg 1-methylxanthine disappearance (1-MXD, an index of microvascular perfusion). Compared with ND-fed animal, HFD feeding led to a mild increase in fasting plasma glucose and plasma insulin, without an increase in total body weight. During the clamps, HFD rats showed an impairment of insulin-mediated action on GIR, hindleg glucose uptake, R'g, Ra, Rd and FBF, with a greater loss of insulin responsiveness at 3 mU/min/kg than at 10 mU/min/kg. The HFD also impaired insulin-mediated microvascular perfusion as assessed by 1-MXD. Interestingly, 1-MXD was the only measurement that remained unresponsive to the higher dose of 10 mU/min/kg insulin. We conclude that the early stage of insulin resistance is characterized by an impairment of the insulin-mediated microvascular responses in skeletal muscle. This is likely to cause greater whole body insulin resistance by limiting the delivery of hormones and nutrients to muscle.
Metabolic syndrome is associated with insulin resistance, elevated glucose and lipids, inflammation, decreased antioxidant activity, increased weight gain, and increased glycation of proteins. Cinnamon has been shown to improve all of these variables in in vitro, animal, and/or human studies. In addition, cinnamon has been shown to alleviate factors associated with Alzheimer's disease by blocking and reversing tau formation in vitro and in ischemic stroke by blocking cell swelling. In vitro studies also show that components of cinnamon control angiogenesis associated with the proliferation of cancer cells. Human studies involving control subjects and subjects with metabolic syndrome, type 2 diabetes mellitus, and polycystic ovary syndrome all show beneficial effects of whole cinnamon and/or aqueous extracts of cinnamon on glucose, insulin, insulin sensitivity, lipids, antioxidant status, blood pressure, lean body mass, and gastric emptying. However, not all studies have shown positive effects of cinnamon, and type and amount of cinnamon, as well as the type of subjects and drugs subjects are taking, are likely to affect the response to cinnamon. In summary, components of cinnamon may be important in the alleviation and prevention of the signs and symptoms of metabolic syndrome, type 2 diabetes, and cardiovascular and related diseases.