Undenatured Type II Collagen Relieves Bone Impairment through Improving Inflammation and Oxidative Stress in Ageing db/db Mice

Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely researched. The aim was to confirm the improvement effects of undenatured type II collagen (UC II) on bone impairment in ageing db/db mice, and the ageing model was established by normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control groups.

Article

Undenatured Type II Collagen Relieves Bone Impairment

through Improving Inflammation and Oxidative Stress in

Ageing db/db Mice

Rui Fan, Yuntao Hao, Xinran Liu, Jiawei Kang, Jiani Hu , Ruixue Mao, Rui Liu, Na Zhu, Meihong Xu

and Yong Li *

Abstract: Ageing-related bone impairment due to exposure to hyperglycemic environment is scarcely

researched. The aim was to confirm the improvement effects of undenatured type II collagen

(UC II) on bone impairment in ageing db/db mice, and the ageing model was established by

normal feeding for 48-week-old. Then, the ageing db/db mice were randomly assigned to UC II

intervention, the ageing model, and the chondroitin sulfate + glucosamine hydrochloride control

groups. After 12 weeks of treatment, femoral microarchitecture and biomechanical parameters were

observed, biomarkers including bone metabolism, inflammatory cytokines, and oxidative stress were

measured, and the gastrocnemius function and expressions of interleukin (IL) 1β, receptor activator

of nuclear factor (NF)-κB ligand (RANKL), and tartrate-resistant acid phosphatase (TRAP) were

analyzed. The results showed that the mice in the UC II intervention group showed significantly

superior bone and gastrocnemius properties than those in the ageing model group, including bone

mineral density (287.65 ± 72.77 vs. 186.97 ± 32.2 mg/cm3), gastrocnemius index (0.46 ± 0.07 vs.

0.18 ± 0.01%), muscle fiber diameter (0.0415 ± 0.005 vs. 0.0330 ± 0.002 mm), and cross-sectional area

(0.0011 ± 0.00007 vs. 0.00038 ± 0.00004 mm2). The UC II intervention elevated bone mineralization

and formation and decreased bone resorption, inflammatory cytokines, and the oxidative stress. In

addition, lower protein expression of IL-1β, RANKL, and TRAP in the UC II intervention group was

observed. These findings suggested that UC II improved bones impaired by T2DM during ageing,

and the likely mechanism was partly due to inhibition of inflammation and oxidative stress.

Keywords: bone impairment; db/db mice; undenatured type II collagen (UC II); inflammation;

oxidative stress; T2DM

1. Introduction

Diabetes mellitus (DM), caused by dysfunctional glucose metabolism, is a chronic

endocrine disorder. Type 2 DM (T2DM) is a multifactorial disease, the central pathome-

chanisms of which are hyperinsulinemia, insulin resistance, and chronic inflammation,

and progressive secondary β-cell failure occurs at the later stages [1,2]. With the in-

creasing duration of DM, patients suffer from the complications including retinopathy,

nephropathy, neuropathy, and vascular disease. DM accelerates material and microstruc-

tural bone deficits, finally leading to some bone diseases, including osteoporosis and

osteoarthritis [3–5]. Now, bone damage is known as a complication of DM , which is

associated with significant morbidity, mortality, and reduction in quality of life . Hence,

the development of effective management of promoting early intervention and prevention

of bone degeneration to improve the quality of life among elderly T2DM patients is needed.

There is a close relevance between bone and glucose metabolism . Osteocalcin

is a good example; it is produced by osteoblasts and odontoblasts and also plays an im-

portant role as an endogenous insulin sensitizer . Thus, bones are both influenced by

glucose metabolism and are capable of modulating it . T2DM was reported to impair

bone health by unbalancing a series of processes including bone formation and resorption,

collagen formation and crosslinking due to hyperglycemia, high level of reactive oxygen

species (ROS), inflammation and advanced glycation end products (AGEs) [11–14]. In ad-

dition, insulinopenia and lack of IGF-1 also influenced osteoblasts [15,16]. It is well-known

that skeletal muscles are the main site for insulin-mediated glucose disposal and energy

metabolism. It has been reported that persons with DM have accelerated muscle loss . In

fact, skeletal muscles and bones have a coupled and cross-talk relationship, which shows

the secreting myokines including IGF-1 and acting for monokine communication .

The accumulation of these pathomechanisms ultimately leads to decreased bone quality

in T2DM. Therefore, what we need to improve is the understanding of the factors that

determine bone health in people with T2DM, especially in elderly patients. The current

research revealed the mechanism of influence of UC II on bone loss in ageing db/db mice

in the musculoskeletal system for the first time.

Collagen, the main component of bones and cartilages, possesses an important func-

tion. Type II collagen, the main part of cartilages, is an interconnected network of collagen

and proteoglycans that are crucial in maintaining joint flexibility and resistance to stress

and fractures [18,19]. Several studies have suggested that a diet with a high percentage

of collagen peptides improves bone collagen metabolism and the complications of hyper-

glycemia [20,21]. According to many reports, type II collagen can treat arthritis due to its

anti-inflammatory properties [22,23]. Undenatured type II collagen (UC II), showing intact

biological activity, receiving much attention , was reported to be effective in rheumatoid

arthritis and arthritis [25,26] due to reducing joint inflammation and promoting cartilage

repair [27,28]. The potential mechanism is due to the oral tolerance. When consumed, UC

II is believed to be taken up by Peyer’s patches, where it activates immune cells. When they

recognize type II collagen in joint cartilages, Treg cells secrete anti-inflammatory mediators

(cytokines). This action helps reduce joint inflammation . Unexpectedly, a prior report

found UC II could augment bone mineral density, bone volume, and trabecular number,

decrease trabecular separation, and improve intense bone resorption, which aids in the

maintenance of cancellous bone, although this was a non-significant trend .

On the basis of the role of various cytokines in T2DM coupled with the previous

findings, it is speculated that supplementation with UC II could relieve bone impairment

among T2DM patients. In order to simulate a real situation of bone impairment during

the developing process of T2DM, 48-week-old db/db mice normally fed with a basic

feed were used as the ageing model, a model established for the first time in this study.

We investigated the effects of UC II on bone quality, microstructure, biomechanics, and

metabolism properties in ageing db/db mice and explored the underlying mechanisms

with a comprehensive and systematic perspective of the musculoskeletal system for the

first time. This study could identify possible evidence of nutritional solutions for bone

impairment in the future research on elderly T2DM patients.

2. Results

2.1. General Condition and Disease State

The mice in the NC group exhibited smooth, shiny, and lustrous hair, energy, and fecal

columnar formation, whereas the mice in the AM and YM groups featured weak, coarse,

and dull hair, unresponsiveness, and decreased activity. Compared with the AM and YM

groups, the CG and UC groups showed more activity.

Figure 1 shows the body weight; the weight in the AM group was significantly larger

than in the YM and NC groups (p < 0.05), while the body weight difference between the

AM and intervention (CG and UC) groups showed no statistical significance (p> 0.05).

The gastrocnemius index in the AM group was the lowest, which presented significant

differences between the NC, YM, CG, and UC groups (p < 0.05). The gastrocnemius index

in the UC group was significantly higher than in the NC and CG groups (p < 0.05), which

indicated that UC II resulted in an obvious increase in the gastrocnemius index.

 differences between the NC, YM, CG, and UC groups (p < 0.05). The gastrocnemius index

in the UC group was significantly higher than in the NC and CG groups (p < 0.05), which

indicated that UC II resulted in an obvious increase in the gastrocnemius index.

FBG levels were less than 9.0 mmol/L in the CG and UC groups, i.e., lower than those

in the AM and YM groups (>15 mmol/L; p < 0.05), but the levels were slightly larger than

in the NC group (<7.5 mmol/L; p> 0.05). There was no significant difference in FINS be-

tween the different groups. Homeostatic model assessment of insulin resistance (HOMA-

IR) is an index that evaluates the level of insulin resistance. The NC group showed the

lowest HOMA-IR, and the HOMA-IR index of the UC group was significantly lower than

in the AM group (p < 0.05).

Figure 1. The effect of UC II on the body weight, gastrocnemius index, plasma glucose, and insulin level in the five groups.

The data are the means ± SD, (n = 5); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the

YM group, d p < 0.05 versus the CG group.

2.2. Micro-CT Femoral Analysis

Micro-CT imaging was performed to quantitatively measure the bone mass on the

basis of BMD and the most common microarchitectural parameters of cancellous bone

including: BV/TV, Tb.N, Tb.Th, Tb.Sp, which reflect bone microvolume, trabecular bone

number, thickness, and spacing, respectively. Micro-CT analysis results in Figure 2 show

that the effects of UC II on femoral BMD and histomorphometry were significant. BMD

values in the AM and NC groups were relatively small and significantly smaller than

those in the YM and UC groups (p < 0.05). BMD in the UC group was the largest among

the five groups, 2.07, 1.84, and 1.39 times higher compared with those values in the AM,

NC, and CG groups, respectively. BV/TV represents the fraction of a given volume of in-

terest, total volume, or tissue volume that is occupied by bone. The effect of UC on the

BV/TV level was similar to that on BMD, which exhibited a significantly larger level in the

YM, CG, and UC groups than in the AM and NC groups (p < 0.05). For BS/BV, the level

order was as follows: AM group> NC group> YM group> CG group> UC group, with

the BS/BV values in the AM group significantly larger than in other groups (p < 0.05); the

values in the CG and UC groups were significantly smaller than those in the AM and NC

groups (p < 0.05). In addition, the smallest values of Tb.Th and Tb.N were observed in the

AM group, and they significantly differed from those in the CG, YM, and UC groups (p <

Figure 1. The effect of UC II on the body weight, gastrocnemius index, plasma glucose, and insulin level in the five groups.

The data are the means ± SD, (n = 5); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the

YM group, d p < 0.05 versus the CG group.

FBG levels were less than 9.0 mmol/L in the CG and UC groups, i.e., lower than those

in the AM and YM groups (>15 mmol/L; p < 0.05), but the levels were slightly larger than in

the NC group (<7.5 mmol/L; p> 0.05). There was no significant difference in FINS between

the different groups. Homeostatic model assessment of insulin resistance (HOMA-IR) is

an index that evaluates the level of insulin resistance. The NC group showed the lowest

HOMA-IR, and the HOMA-IR index of the UC group was significantly lower than in the

AM group (p < 0.05).

2.2. Micro-CT Femoral Analysis

Micro-CT imaging was performed to quantitatively measure the bone mass on the

basis of BMD and the most common microarchitectural parameters of cancellous bone

including: BV/TV, Tb.N, Tb.Th, Tb.Sp, which reflect bone microvolume, trabecular bone

number, thickness, and spacing, respectively. Micro-CT analysis results in Figure 2 show

that the effects of UC II on femoral BMD and histomorphometry were significant. BMD

values in the AM and NC groups were relatively small and significantly smaller than those

in the YM and UC groups (p < 0.05). BMD in the UC group was the largest among the five

groups, 2.07, 1.84, and 1.39 times higher compared with those values in the AM, NC, and

CG groups, respectively. BV/TV represents the fraction of a given volume of interest, total

volume, or tissue volume that is occupied by bone. The effect of UC on the BV/TV level

was similar to that on BMD, which exhibited a significantly larger level in the YM, CG, and

UC groups than in the AM and NC groups (p < 0.05). For BS/BV, the level order was as

follows: AM group> NC group> YM group> CG group> UC group, with the BS/BV

values in the AM group significantly larger than in other groups (p < 0.05); the values in

the CG and UC groups were significantly smaller than those in the AM and NC groups

(p < 0.05). In addition, the smallest values of Tb.Th and Tb.N were observed in the AM

group, and they significantly differed from those in the CG, YM, and UC groups (p < 0.05).

The largest value of Tb.N was observed in the UC group, which was significantly different

from that in the other groups (p < 0.05), while the AM group showed the largest values of

Tb.Sp, which were significantly larger than those in other groups (p < 0.05).

 0.05). The largest value of Tb.N was observed in the UC group, which was significantly

different from that in the other groups (p < 0.05), while the AM group showed the largest

values of Tb.Sp, which were significantly larger than those in other groups (p < 0.05).

Figure 2. The histomorphometry of the femur in the five groups. (A) The images of the changes in bone microarchitecture.

(B) The mineral density and histomorphometry of the femur. The data are expressed as the means ± SD (n = 5 in the AM

group, n = 7 in the YM, CG, NC, and UC groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05

versus the YM group, d p < 0.05 versus the CG group.

2.3. Dynamic Histomorphometric and Biochemical Markers of Bone Turnover Analysis

Dynamic histomorphometric analyses of representative fluorescence images ob-

tained from the femur are shown in Figure 3A. The green fluorescence in the images is

calcein green, which represents the first mineralization label; the red fluorescence in the

images is alizarin complexone, which represents the second mineralization label. The

width of the gap between the red and the green fluorescence in the images could, to some

extent, represent the bone formation rate. Relatively larger gap widths were observed in

Figure 2. The histomorphometry of the femur in the five groups. (A) The images of the changes in bone microarchitecture.

(B) The mineral density and histomorphometry of the femur. The data are expressed as the means ± SD (n = 5 in the AM

group, n = 7 in the YM, CG, NC, and UC groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05

versus the YM group, d p < 0.05 versus the CG group.

2.3. Dynamic Histomorphometric and Biochemical Markers of Bone Turnover Analysis

Dynamic histomorphometric analyses of representative fluorescence images obtained

from the femur are shown in Figure 3A. The green fluorescence in the images is calcein

green, which represents the first mineralization label; the red fluorescence in the images

is alizarin complexone, which represents the second mineralization label. The width of

the gap between the red and the green fluorescence in the images could, to some extent,

represent the bone formation rate. Relatively larger gap widths were observed in the UC

group, and relatively smaller ones were, remarkably, observed in the AM and NC groups,

which indicated that UC II treatment led to bone formation, which was consistent with the

results of bone microarchitecture (Figure 3).

the UC group, and relatively smaller ones were, remarkably, observed in the AM and NC

groups, which indicated that UC II treatment led to bone formation, which was consistent

with the results of bone microarchitecture

Bone formation and bone resorption determined bone turnover . In addition, the

serological assessment of bone turnover in the five groups is shown in Figure 3B. AGEs

play an important role in the bone diseases development (such as osteoporosis), especially

among T2DM patients. Their accumulation in the bone alters osteoblasts, inducing en-

hanced osteoclastogenesis and impaired matrix mineralization (downregulation of alka-

line phosphatase and osteocalcin mRNA) . OC, mainly controlling mineralization, and

BALP, representing bone formation, were selected. TRAP, a marker of osteoclast numbers,

was selected to represent bone resorption on the basis of a previous report . As ex-

pected, the serum level of BALP in the UC group was obviously larger than in the AM,

NC, YM, and CG groups (p < 0.05), which was consistent with the histomorphometric

bone analysis. Similarly to the BALP data, the OC serum content in the OM group was

significantly lower than in the UC and CG groups (p < 0.05). Moreover, TRAP serum levels

were the lowest in the UC group (p < 0.05).

2.4. Bone Biomechanical Parameter Analysis

The three-point bending technique was used to assess the mechanical properties

(fracture point, stiffness, and elasticity) of the femur. A constant amount of force was ap-

plied to the midpoint of the femur diaphysis to determine the maximum load that could

Figure 3. Dynamic histomorphometric and biochemical markers of bone turnover analyses. (A) Dynamic histomorphometric

analyses of representative fluorescence images (n = 5); (B) serum levels of biochemical markers of bone turnover (n = 5, 7, 9,

9, and 10 in the AM, CG, UC, NC, and YM groups); a p < 0.05 versus the AM group.

Bone formation and bone resorption determined bone turnover . In addition, the

serological assessment of bone turnover in the five groups is shown in Figure 3B. AGEs

play an important role in the bone diseases development (such as osteoporosis), especially

among T2DM patients. Their accumulation in the bone alters osteoblasts, inducing en-

hanced osteoclastogenesis and impaired matrix mineralization (downregulation of alkaline

phosphatase and osteocalcin mRNA) . OC, mainly controlling mineralization, and

BALP, representing bone formation, were selected. TRAP, a marker of osteoclast numbers,

was selected to represent bone resorption on the basis of a previous report . As expected,

the serum level of BALP in the UC group was obviously larger than in the AM, NC, YM,

and CG groups (p < 0.05), which was consistent with the histomorphometric bone analysis.

Similarly to the BALP data, the OC serum content in the OM group was significantly lower

than in the UC and CG groups (p < 0.05). Moreover, TRAP serum levels were the lowest in

the UC group (p < 0.05).

2.4. Bone Biomechanical Parameter Analysis

The three-point bending technique was used to assess the mechanical properties

(fracture point, stiffness, and elasticity) of the femur. A constant amount of force was

applied to the midpoint of the femur diaphysis to determine the maximum load that could

be placed on the femur until fracture/breakage occurred. Table 1 shows biomechanical

femoral parameters in the five groups. The values of the maximum load in the CG and UC

groups were significantly larger than those in the AM and NC groups (p < 0.05), of which

the value in the UC group was significantly larger than that in the YM group (p < 0.05), and

biomechanical femoral properties, including the energy to ultimate load, Young’s modulus,

stiffness, and breaking energy in the UC group showed significant differences between the

AM, NC, YM, and CG groups (p < 0.05).

Table 1. The bone biomechanical parameters of the femur in the five groups.

Groups Maximum

Load (N)

Energy to Ultimate

Load (J) Young’s Modulus (MPa)

Stiffness

(N/mm)

Breaking Energy

(J/m2)

AM 7.49 ± 1.49 0.0013 ± 0.001 775.18 ± 139.11 25.02 ± 9.19 692.99 ± 127.45

NC 9.69 ± 2.11 0.0037 ± 0.001 a 1205.54 ± 371.03 a 54.85 ± 13.53 a 1605.45 ± 257.17 a

YM 9.89 ± 1.88 0.0045 ± 0.001 a 1190.34 ± 11.49 a 43.32 ± 2.77 a 1136.68 ± 22.99 a,b

CG 15.44 ± 3.84 a,b 0.0058 ± 0.002 a 1603.71 ± 102.08 a,b,c 53.11 ± 12.82 a 1572.01 ± 312.61 a,c

UC 18.01 ± 3.00 a,b,c 0.0110 ± 0.003 a,b,c,d 2875.55 ± 312.98 a,b,c,d 79.94 ± 15.04 a,b,c,d 2210.94 ± 15.09 a,b,c,d

The data are expressed as the means ± SD, n = 5; a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM

group, d p < 0.05 versus the CG group.

2.5. Inflammation and Oxidative Stress in the Serum

It was approved that increased production of adipocytes and hyperglycemia feed

the cycle of chronic inflammation by producing ROS and inflammatory cytokines, which

induce osteoblast apoptosis to bring the negative effect on bone health. In the current study,

to assess the inflammation and oxidative stress, inflammatory cytokines and the oxidative

stress reaction were investigated. The serological assessment of inflammatory cytokines

in the five groups is shown in Figure 4A. Similar change trends were observed for IL-1β,

IL-6, and tumor necrosis factor (TNF) α. The levers of IL-1β, IL-6, and TNF-α in the NC,

YM, CG and UC groups were significantly smaller in the OM group (p < 0.05). In addition,

IL-1β, IL-6, and TNF-α levels in the UC group were significantly smaller than those in the

other groups (p < 0.05).

In order to investigate the effect of UC II on oxidative stress, the activity of superoxide

dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the serum

of the mice was examined. As shown in Figure 4B, compared with the AM group, the other

groups showed obviously stronger activities of SOD and GSH-Px and a significantly lower

MDA level (p < 0.05). Regarding the MDA content, values in the UC group were the smallest

among the five groups (significantly; p < 0.05), whereas there were no significant differences

between the SOD and GSH-Px activity found in the two intervention groups (p> 0.05).

be placed on the femur until fracture/breakage occurred. Table 1 shows biomechanical

femoral parameters in the five groups. The values of the maximum load in the CG and

UC groups were significantly larger than those in the AM and NC groups (p < 0.05), of

which the value in the UC group was significantly larger than that in the YM group (p <

0.05), and biomechanical femoral properties, including the energy to ultimate load,

Young’s modulus, stiffness, and breaking energy in the UC group showed significant dif-

ferences between the AM, NC, YM, and CG groups (p < 0.05).

2.5. Inflammation and Oxidative Stress in the Serum

It was approved that increased production of adipocytes and hyperglycemia feed the

cycle of chronic inflammation by producing ROS and inflammatory cytokines, which in-

duce osteoblast apoptosis to bring the negative effect on bone health. In the current study,

to assess the inflammation and oxidative stress, inflammatory cytokines and the oxidative

stress reaction were investigated. The serological assessment of inflammatory cytokines

in the five groups is shown in Figure 4A. Similar change trends were observed for IL-1β,

IL-6, and tumor necrosis factor (TNF) α. The levers of IL-1β, IL-6, and TNF-α in the NC,

YM, CG and UC groups were significantly smaller in the OM group (p < 0.05). In addition,

IL-1β, IL-6, and TNF-α levels in the UC group were significantly smaller than those in the

other groups (p < 0.05).

In order to investigate the effect of UC II on oxidative stress, the activity of superox-

ide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in

the serum of the mice was examined. As shown in Figure 4B, compared with the AM

group, the other groups showed obviously stronger activities of SOD and GSH-Px and a

significantly lower MDA level (p < 0.05). Regarding the MDA content, values in the UC

group were the smallest among the five groups (significantly; p < 0.05), whereas there

were no significant differences between the SOD and GSH-Px activity found in the two

intervention groups (p> 0.05).

2.6. Histopathological Muscle Analysis and its Biomarker

We had known that the gastrocnemius decreased in the AM group and the UC II

intervention could increase the gastrocnemius mass. The gastrocnemius morphological

characteristics and pathology are shown in the Figure 5A,B. Muscle cells in the AM group

lost their normal morphology, the nuclear center migration phenomenon was serious, and

the cells showed remarkable differences in size and had a disordered, loose arrangement.

In addition, the intercellular substance level increased, and there was a large amount of

connective-tissue hyperplasia and inflammatory cell infiltration between muscle cells.

Similarly to the AM group, the intercellular substance level in the CG group increased,

and there was a large amount of fibrous connective-tissue hyperplasia and inflammatory

cell infiltration; however, it was inconsistent because the CG group showed normal cell

morphology, uniform sizes, and a slight nuclear center migration phenomenon. Com-

pared with the AM and CG groups, the NC and YM groups showed less fibrous connec-

tive-tissue hyperplasia and inflammatory cell infiltration, and the cells were uniform in

size and tidily arranged. UC resulted in a normal polygonal cell without degenerating

necrotic cells, uniform size, tightly arranged, and with the disappearance of disorderly

connective tissue and inflammatory cells.

Impaired glucose/insulin metabolism may indirectly affect bones and muscles by al-

tering skeletal muscle signaling, such as IGF-1. It was reported that IGF-1 was associated

with increasing the muscle mass and the muscle fiber area. Figure 5B shows the effect of

UC II on muscle fiber parameters in the five groups. The AM group showed the smallest

values in muscle fiber diameter, number, and cross-sectional area, while the intervention

with UC II significantly increased the muscle fiber diameter, number, and cross-sectional

area (p < 0.05). The improvement effect of positive substances (CS + GH) on the muscle

fiber cross-sectional area was smaller compared with that of the UC II intervention.

Figure 4. Serum levels of inflammatory cytokines and oxidative stress in the five groups. (A) Serum levels of inflammatory

cytokines including IL-1β, IL-6, and TNF-α; (B) serum levels of oxidative stress indexes including MDA, SOD, and GSH-Px.

Serum data are expressed as the means ± SD (n = 5, 7, 9, 9, and 10 in the AM, CG, UC, NC, and YM groups); a p < 0.05

versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus the CG group.

2.6. Histopathological Muscle Analysis and its Biomarker

We had known that the gastrocnemius decreased in the AM group and the UC II

intervention could increase the gastrocnemius mass. The gastrocnemius morphological

characteristics and pathology are shown in the Figure 5A,B. Muscle cells in the AM group

lost their normal morphology, the nuclear center migration phenomenon was serious, and

the cells showed remarkable differences in size and had a disordered, loose arrangement.

In addition, the intercellular substance level increased, and there was a large amount

of connective-tissue hyperplasia and inflammatory cell infiltration between muscle cells.

Similarly to the AM group, the intercellular substance level in the CG group increased,

and there was a large amount of fibrous connective-tissue hyperplasia and inflammatory

cell infiltration; however, it was inconsistent because the CG group showed normal cell

morphology, uniform sizes, and a slight nuclear center migration phenomenon. Compared

with the AM and CG groups, the NC and YM groups showed less fibrous connective-tissue

hyperplasia and inflammatory cell infiltration, and the cells were uniform in size and tidily

arranged. UC resulted in a normal polygonal cell without degenerating necrotic cells,

uniform size, tightly arranged, and with the disappearance of disorderly connective tissue

and inflammatory cells.

Impaired glucose/insulin metabolism may indirectly affect bones and muscles by

altering skeletal muscle signaling, such as IGF-1. It was reported that IGF-1 was associated

with increasing the muscle mass and the muscle fiber area. Figure 5B shows the effect of

UC II on muscle fiber parameters in the five groups. The AM group showed the smallest

values in muscle fiber diameter, number, and cross-sectional area, while the intervention

with UC II significantly increased the muscle fiber diameter, number, and cross-sectional

area (p < 0.05). The improvement effect of positive substances (CS + GH) on the muscle

fiber cross-sectional area was smaller compared with that of the UC II intervention.

IGF-1 is known to activate IGF-1R which acts through the PI3K/Akt and the MAPK/ERK

pathways . It has been speculated the function of IGF-1 was partly affected by inflamma-

tion, oxidative stress, and diabetes . In order to investigate the effect of UC II on oxidative

stress in the gastrocnemius muscle of the db/db mice, the activity of SOD, GSH-Px, and

MDA in the gastrocnemius muscle was determined. According to Figure 5C, the activities of

SOD and GSH-Px in the AM group were significantly lower than those in four other groups,

and the UC group showed a significantly higher level than that in the other groups (p < 0.05),

while the MDA content in the UC group was remarkably decreased (p < 0.01), indicating

that the UC intervention exerted protection against SOD and GSH-Px depletion and MDA

accumulation in the gastrocnemius muscle tissue of the db/db mice.

IGF-1, secreted from skeletal muscles, was an important growth factor for skeletal

development . For the IGF-1 levels, the lowest content was found in the AM group,

which was obviously lower than that in the other groups (p < 0.05); an intervention with

UC II increased the IGF-1 content, which was higher than in the AM, YM, and CG groups

and lower than in the NC group (p < 0.05).

IGF-1 is known to activate IGF-1R which acts through the PI3K/Akt and the

MAPK/ERK pathways . It has been speculated the function of IGF-1 was partly af-

fected by inflammation, oxidative stress, and diabetes . In order to investigate the ef-

fect of UC II on oxidative stress in the gastrocnemius muscle of the db/db mice, the activity

of SOD, GSH-Px, and MDA in the gastrocnemius muscle was determined. According to

Figure 5C, the activities of SOD and GSH-Px in the AM group were significantly lower

than those in four other groups, and the UC group showed a significantly higher level

than that in the other groups (p < 0.05), while the MDA content in the UC group was re-

markably decreased (p < 0.01), indicating that the UC intervention exerted protection

against SOD and GSH-Px depletion and MDA accumulation in the gastrocnemius muscle

tissue of the db/db mice.

IGF-1, secreted from skeletal muscles, was an important growth factor for skeletal

development . For the IGF-1 levels, the lowest content was found in the AM group,

which was obviously lower than that in the other groups (p < 0.05); an intervention with

UC II increased the IGF-1 content, which was higher than in the AM, YM, and CG groups

and lower than in the NC group (p < 0.05)

2.7. Immunohistochemical Analysis

It was reported that some proinflammatory cytokines, including IL-1β, IL-6, and

TNF-α, also stimulate osteoclast activity by the actions of RANKL . RANKL, an essen-

tial factor for osteoclast differentiation and function, mediates bone loss by promoting the

bone-resorbing activity of osteoclasts and prolongs their survival [37,38]. The receptor ac-

tivator of (nuclear factor) kappa B ligand (RANKL) signaling pathway was analyzed. Fig-

ure 6A shows that the immunostaining of IL-1β in the AM, NC, YM, and CG groups was

stronger than in the UC group. The areal density of IL-1β in the AM group was signifi-

cantly higher than in the four other groups (p < 0.05), which indicated that IL-1β in the

AM group was remarkably more strongly expressed, while the UC II intervention signif-

icantly decreased the expression of IL-1β (p < 0.05). The RANKL expression is shown in

Figure 6B. A higher expression in the AM group was clearly detected, and there was little

expression in the NC and UC groups. Compared with the areal density in the AM group,

it was significantly decreased in the four other groups (p < 0.05). AOD in the UC group

was obviously lower than in the AM, NC, YM, and CG groups (p < 0.05). Figure 6C shows

that the immunoreactive signal for TRAP in the UC group was weaker than that in the

other groups. AOD indicated that the highest expression was in the AM group, while the

lowest expression was in the UC group; TRAP expression in the UC group was relatively

lower than in the AM, NC, and YM groups (p < 0.05).

Figure 5. H&E staining of the gastrocnemius muscle and its characteristic parameter. (A) H&E staining of the gastrocnemius.

The bar indicates 50 µm. Magnification: 40×. (B) Diameter, number, and cross-sectional area of muscle fibers. (C) Biomarkers

including oxidative stress and monokines IGF-1. The data are expressed as the means ± SD, n = 5; a p < 0.05 versus the AM

group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus the CG group.

2.7. Immunohistochemical Analysis

It was reported that some proinflammatory cytokines, including IL-1β, IL-6, and TNF-

α, also stimulate osteoclast activity by the actions of RANKL . RANKL, an essential

factor for osteoclast differentiation and function, mediates bone loss by promoting the

bone-resorbing activity of osteoclasts and prolongs their survival [37,38]. The receptor

activator of (nuclear factor) kappa B ligand (RANKL) signaling pathway was analyzed.

Figure 6A shows that the immunostaining of IL-1β in the AM, NC, YM, and CG groups

was stronger than in the UC group. The areal density of IL-1β in the AM group was

significantly higher than in the four other groups (p < 0.05), which indicated that IL-1β

in the AM group was remarkably more strongly expressed, while the UC II intervention

significantly decreased the expression of IL-1β (p < 0.05). The RANKL expression is shown

in Figure 6B. A higher expression in the AM group was clearly detected, and there was

little expression in the NC and UC groups. Compared with the areal density in the AM

group, it was significantly decreased in the four other groups (p < 0.05). AOD in the UC

group was obviously lower than in the AM, NC, YM, and CG groups (p < 0.05). Figure 6C

shows that the immunoreactive signal for TRAP in the UC group was weaker than that

in the other groups. AOD indicated that the highest expression was in the AM group,

while the lowest expression was in the UC group; TRAP expression in the UC group was

relatively lower than in the AM, NC, and YM groups (p < 0.05).

3. Materials and Methods

3.1. Materials

Undenatured type II collagen (UC II), with the conformational integrity of the triple-

helical structure remaining intact, was purchased from SEMNL Biotechnology Co. Ltd.

(Beijing, China). UC II, prepared from a chicken sternum at a low temperature, showed

the molecular weight of 300 kDa, and its content was as follows: collagen, 263.0 mg/g;

hydroxyproline, 32.9 mg/g. The triple-helical structure and the second structure (17.2% of

the α helix, 21.1% of the β sheet, 44.0% of the β turn, and 17.1% of the nonregular coil) are

shown in the Supplementary Materials. The amino acid composition is shown in Table 2.

Chondroitin sulfate (CS) and glucosamine hydrochloride (GH) were obtained from Wilke

Resources (Lenexa, KS, USA).

Figure 6. Immunohistochemistry analysis of the five groups. Magnification: ×400. IHC quantification is shown in the

bar graphs. (A) Protein expressions of IL-1β in the femur; (B) protein expressions of RANKL in the femur; (C) protein

expressions of TRAP in the femur. The data are the means ± SD (n = 5, 7, 9, 9, and 10 in the AM, CG, UC, NC, and YM

groups); a p < 0.05 versus the AM group, b p < 0.05 versus the NC group, c p < 0.05 versus the YM group, d p < 0.05 versus

the CG group.

3. Materials and Methods

3.1. Materials

Undenatured type II collagen (UC II), with the conformational integrity of the triple-

helical structure remaining intact, was purchased from SEMNL Biotechnology Co. Ltd.

(Beijing, China). UC II, prepared from a chicken sternum at a low temperature, showed

the molecular weight of 300 kDa, and its content was as follows: collagen, 263.0 mg/g;

hydroxyproline, 32.9 mg/g. The triple-helical structure and the second structure (17.2% of

the α helix, 21.1% of the β sheet, 44.0% of the β turn, and 17.1% of the nonregular coil) are

shown in the Supplementary Materials. The amino acid composition is shown in Table 2.

Chondroitin sulfate (CS) and glucosamine hydrochloride (GH) were obtained from Wilke

Resources (Lenexa, KS, USA).

The detection kits of superoxide dismutase (SOD), glutathione peroxidase (GSH-

Px), malondialdehyde (MDA), tumor necrosis factor α (TNF-α), interleukin (IL) 6 and

IL-1β were purchased from Shanghai Lengton Biotechnology Co., Ltd. (Shanghai, China).

Insulin was bought from the Beyotime Institute of Biotechnology (Beijing, China). The

detection kits of alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP),

and osteocalcin (OC) were bought from USCN Life Science Inc. IL-1β, IL-6, TRAP, and

receptor activator of nuclear factor (NF)-κB ligand (RANKL) antigens (mouse monoclonal

antibody) were purchased from R&D Systems, USA. The basic feed, which met the national

standard (GB 14924.3-2010), was bought from Beijing Keao Xieli Co. Ltd. (Beijing, China).

All the other reagents were analytically pure.

3.2. Animals and Experiment Design

3.2.1. Animal Feeding Conditions and Ageing db/db Mice Model Establishment

Male C57BL/KsJ-leprdb/leprdb diabetic (db/db, 10-week-old) mice and male non-

diabetic littermate (db/m) mice were obtained from the Animal Service of the Health

Science Center (Peking University, Beijing, China), production certificate No. SCXK (Bei-

jing, China) 2016–0010, use license No. SYXK (Beijing, China) 2016-0041. The environment

conditions involved constant temperature (21–25 ◦C), relative air humidity (50–60%), and

12 h light/dark cycles.

All the mice had free access to the standard food (American Institute of Nutrition Ro-

dent Diet 93G) and water. The protocols were reviewed and approved by the Institutional

Animal Care and Use Committee of Peking University (approval No. LA2015094).

Considering bone impairment associated with T2DM and ageing, we used, for the first

time, 48-week-old db/db mice normally fed with a basic feed representing the approach of

ageing as the ageing model to explore the effect of the UC II intervention begun in their

later life on the ageing-related bone impairment, meanwhile, 25-week-old db/db (the age

considered to represent the end of growth and development) mice normally fed with a

basic feed as the young model to explore the alterations of bone impairment during the

process of T2DM and ageing, which could lead to a deeper understanding of the occurrence

and development of bone impairment in T2DM. Consequently, the ageing and the young

models of db/db mice is the highlight of this work (the timepoints were selected on the

basis of previous research coupled with the observation of the general condition and the

disease state including intake, drinking, weights and fasting blood glucose provided in

Figures S3–S5 from the Supplementary Materials) [39,40].

3.2.2. Study Design

Forty-five 48-week-old db/db mice (72 ± 3 g) with plasma glucose above 11.1 mmol/L

were randomly subdivided into the ageing model group (AM; n = 15), the UC II intervention

group (UC; n = 15), and the chondroitin sulfate + glucosamine hydrochloride (CS + GH)

control group (CG; n = 15). Meanwhile, 48-week-old db/m and 25-week-old db/db mice

were used as the normal control (NC; n = 15) and the young model (YM; n = 15) groups,

respectively. The UC group was administered UC II through drinking at the dose of

6 mg/kg body weight, whereas the CG group was treated with 180 mg CS + 225 mg

GH/kg body weight through drinking. The AM, NC, and YM groups were administered

distilled water. The drinking feeding mode was a gentle feeding mode for elderly mice,

whose strict quality control and accuracy were confirmed by our team with solid results in

earlier reports [41,42].

After dividing the group, the general condition, including the coat color, mental state,

and daily activities, was monitored daily (at 10:00) till the end stage of the experiment, and

food intake, water intake, and body weight were regularly recorded each week.

All the mice were exposed to the intervention for 12 weeks. In the later stage of

the intervention period, weight loss showing a sharp decrease (approximately>20% loss

of body weight) was an indication of the dying stage, and if a mouse appeared to be

dying due to weight loss or injury, it was euthanized with CO2 to minimize suffering.

One-quarter of all the mice or one-third of one group approaching death were considered

to signify the end of the experiment (here, the intervention lasted 12 weeks). At the end

of the experiment, the surviving mice (n = 10, 13, 14, 14, and 15 in the AM, CG, UC, NC,

and YM groups, respectively) were euthanized with CO2, and blood was obtained from

the retrobulbar plexus using heparinized anticoagulant tubes for further measurements.

After euthanasia, the main organs were weighed to calculate organ coefficients. Some

separated right femurs from the mice injected with a dye (n = 5 in each group) were fixed

in 10% neutral buffered formalin for dynamic histomorphometry; the left femurs of these

mice were packed in a gauze soaked with phosphate-buffered saline and stored at −20 ◦C

to carry out the three-point test; the right gastrocnemius of these mice was separated

and stored at –80 ◦C to perform biomarker measurements, including oxidative stress and

myokines; and the left gastrocnemius of these mice was fixed in 10% neutral buffered

formalin for histopathological analysis. The mice without an injection in each group

provided serum and femurs to measure biomarkers, and for microcomputed tomography

(CT) and histopathological analysis; histopathological analysis for the right femur also

required decalcification with 10% EDTA (changed each week). The schematic diagram of

the research protocol is shown in Figure 7.

  due to weight loss or injury, it was euthanized with CO2 to minimize suffering. One-quar-

ter of all the mice or one-third of one group approaching death were considered to signify

the end of the experiment (here, the intervention lasted 12 weeks). At the end of the ex-

periment, the surviving mice (n = 10, 13, 14, 14, and 15 in the AM, CG, UC, NC, and YM

groups, respectively) were euthanized with CO2, and blood was obtained from the

retrobulbar plexus using heparinized anticoagulant tubes for further measurements. After

euthanasia, the main organs were weighed to calculate organ coefficients. Some separated

right femurs from the mice injected with a dye (n = 5 in each group) were fixed in 10%

neutral buffered formalin for dynamic histomorphometry; the left femurs of these mice

were packed in a gauze soaked with phosphate-buffered saline and stored at –20 °C to

carry out the three-point test; the right gastrocnemius of these mice was separated and

stored at –80 °C to perform biomarker measurements, including oxidative stress and my-

okines; and the left gastrocnemius of these mice was fixed in 10% neutral buffered forma-

lin for histopathological analysis. The mice without an injection in each group provided

serum and femurs to measure biomarkers, and for microcomputed tomography (CT) and

histopathological analysis; histopathological analysis for the right femur also required de-

calcification with 10% EDTA (changed each week). The schematic diagram of the research

protocol is shown in Figure 7.

Figure 7. Schematic plot and the timeline of the research protocol.

Normal deaths were marked with blue. Injection of alizarin-3-methyliminodiacetic

acid and calcein was performed in one-week intervals (n = 5 mice per group per timepoint)

(marked with yellow). Fasting blood glucose and insulin measurements were performed

each week (in all the survived mice per group per timepoint) and at the end stage (n = 5

injected mice per group).

3.3. Fasting Plasma Glucose and HOMA-IR Measurements

Fasting plasma glucose (FBG) and insulin (FINS) in the tail vein were measured using

an Accu-Check glucometer (Roche Diagnostics) and an ELISA kit after fasting for 5 h (n =

5 injected mice of each group). The level of insulin resistance was calculated using the

following formula: (fasting glucose (mmol/L) × FINS (uIU/mL))/22.5 and expressed

as HOMA-IR.

3.4. Micro-CT Analysis

The mass and microarchitecture of undecalcified femurs were measured with micro-

CT (Inveon MM system, Siemens, Munich, Germany), and the parameters were calculated

using an Inveon Research Workplace. The parameters, including 8.82 μm for pixel size,

80 kV for voltage, 500 μA for current, and 1500 ms for exposure time were set. The trabec-

ular region of about 1–2 mm distal to the proximal epiphysis was selected. Under these

conditions, the parameters including bone volume/total volume (BV/TV), bone surface

area/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N), tra-

becular separation (Tb.Sp), and bone mineral density (BMD) were obtained.

Figure 7. Schematic plot and the timeline of the research protocol.

Normal deaths were marked with blue. Injection of alizarin-3-methyliminodiacetic

acid and calcein was performed in one-week intervals (n = 5 mice per group per timepoint)

(marked with yellow). Fasting blood glucose and insulin measurements were performed

each week (in all the survived mice per group per timepoint) and at the end stage (n = 5

injected mice per group).

3.3. Fasting Plasma Glucose and HOMA-IR Measurements

Fasting plasma glucose (FBG) and insulin (FINS) in the tail vein were measured using

an Accu-Check glucometer (Roche Diagnostics) and an ELISA kit after fasting for 5 h

(n = 5 injected mice of each group). The level of insulin resistance was calculated using the

following formula: (fasting glucose (mmol/L) × FINS (uIU/mL))/22.5 and expressed

as HOMA-IR.

3.4. Micro-CT Analysis

The mass and microarchitecture of undecalcified femurs were measured with micro-

CT (Inveon MM system, Siemens, Munich, Germany), and the parameters were calculated

using an Inveon Research Workplace. The parameters, including 8.82 µm for pixel size,

80 kV for voltage, 500 µA for current, and 1500 ms for exposure time were set. The

trabecular region of about 1–2 mm distal to the proximal epiphysis was selected. Under

these conditions, the parameters including bone volume/total volume (BV/TV), bone

surface area/bone volume (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N),

trabecular separation (Tb.Sp), and bone mineral density (BMD) were obtained.

3.5. Biomechanical Bone Parameters

In order to evaluate the biomechanical properties of the femurs, three-point bend-

ing tests were performed. The left femurs of the injected mice in each group were cen-

trally loaded with a speed of 1 mm/s using a universal testing machine (Instron 4501,

Instron, Canton, MA, USA). The parameters of the bone samples, namely, stiffness, ultimate

strength, Young’s modulus, and ultimate stress were determined and calculated based on

the deformation curve .

3.6. Slicing of Undecalcified Bones and Dynamic Histomorphometric Analysis

Seven days before euthanasia, five mice per group were intraperitoneally injected with

alizarin red (30 mg/kg body weight); 4 days later, an intraperitoneal injection of 20 mg/kg

body weight calcein was carried out . After euthanasia, the right femurs were fixed

and dehydrated and embedded in destabilized methyl methacrylate resin. The samples

were ground and polished to 40–60 µm and H&E stained. The samples were then observed

under a laser scanning confocal microscope (Leica TCS SP-2, Frankfurt, Germany) set on

534 nm (calcein) and 357 nm (alizarin red) .

3.7. Biochemical Marker Assay

TRAP, a marker of the osteoclast number, represented bone resorption. OC, mainly

controlling for mineralization, and BALP, representing bone formation, were selected. The

levels of BALP, TRAP, OC, common inflammatory cytokines (TNF-α, IL-1b, IL-6), and

common oxidative stress markers (SOD, GSH-Px, MDA) in the serum of the mice without

a dye injection were measured using relevant enzyme-linked immunoassay (ELISA) kits

according to the product instructions.

SOD, MDA, GSH-Px, and IGF-1 activities in the right gastrocnemius muscle (n = 5 in each

group) were measured using the relevant ELISA kits according to the product instructions.

3.8. Histopathological Analysis

The left gastrocnemius of the five injected mice in each group was fixed in 10% forma-

lin. The samples embedded in paraffin were stained with hematoxylin and eosin (H&E).

In order to evaluate the status of the muscles, gastrocnemius slides were microscopically

observed with an inverted microscope (Olympus IX70, Olympus, Tokyo, Japan). Then, the

cross-sectional area of muscle fiber diameters was measured and calculated with Image-Pro

Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Immunohistochemistry (IHC) was used to deduce the involvement of the related key

pathways, performed on the right femur of the mice without a dye injection. IL-1β, IL-6,

tartrate-resistant acid phosphatase (TRAP), and receptor activator of (nuclear factor) NF-κB

ligand (RANKL) were applied with the following antibodies: IL-1β and IL-6 antigens

(both 1:100 dilution) for proinflammatory cytokine expression; TRAP (1:100 dilution) and

RAKNL (1:100 dilution) antigens for osteoclast formation. Immunohistochemistry was

carried out on 3–5 µm-thick paraffin sections, followed by overnight incubation with the

primary antibody at 4 ◦C. After staining with DAB, the slides were counterstained with

Mayer’s hematoxylin and dehydrated through a graded ethanol series.

Quantitative image analysis was performed with the Image-Pro Plus 6.0 software

(Media Cybernetics, Inc., Rockville, MD, USA). Cumulative optical density (IOD) and

pixel area (area) values of the tissue of each positive image were obtained. Statistical

analysis of the results was expressed as the average optical density (AOD) (areal density,

AOD = IOD/area).

3.9. Statistical Analysis

The data are expressed as the means ± SD. One-way ANOVA with post-hoc LSD

(equal variances assumed) or Dunnett’s T3 test (equal variances not assumed) were per-

formed with SPSS 18.0, and p-values less than 0.05 were considered to be significant.

4. Discussion

Diabetes-associated bone disease leads to impaired bone quality and increased fracture

risk. This study addressed the alterations of bone quality-impaired T2DM and ageing,

including bone microarchitecture, remodelling, and biomechanical quality. In order to

evaluate the effect of UC II on bone quality impairment in the ageing db/db mice and its

mechanism of action, we comprehensively explored bone metabolism alterations on the

basis of inflammation and oxidative stress in the serum, bone, and skeletal muscle for the

first time. In addition, the T2DM status was examined.

BMD values in the AM group were similar to those in the NC group, which was

consistent with the previous research finding normal or even mildly elevated BMD in

T2DM patients compared to that in those without T2DM . In fact, fracture risk is

increased; although there is normal and even elevated BMD in T2DM, the evidence led to

the hypothesis that there are diabetes-associated alterations in skeletal properties . In

this study, alterations including bone microarchitecture, metabolism, and biomechanical

quality caused by T2DM were investigated. The lowest levels of BV/TV, Tb.N, and Tb.Th

were in the ageing db/db mice (AM group), which was consistent with the previous results

obtained in diabetic rats and humans with T2DM [48,49]; this deteriorated morphological

structure was sharply improved by UC II. Moreover, after the UC II intervention, the thick-

ness of the cortical bone was observed, and cortical porosity and trabecular heterogeneity

were improved. There were morphological and histological changes of bone tissue in

this study, which led to significant worsening of the mechanical properties shown in the

three-point bending test (Table 1). The mice in the AM group showed the smallest values in

the maximum load, the energy to ultimate load, Young’s modulus, stiffness, and breaking

energy, which were similar to those in another report [50,51].

In addition, the alterations of the bone metabolism were analyzed. Serum levels of

BALP and OC in the AM group were the lowest, whereas its TRAP levels were significantly

higher compared to those in the other groups. Similar conclusions were reported in

previous studies, both in rodents and people with T2DM [52–54], while the increased value

in TRAP observed in the AM group was not consistent with other reports . In fact, the

studies examining the effect of diabetes mellitus on bone resorption were not conclusive,

including unaltered, inhibited, and increased in vitro and vivo studies on animals and

human patients [55–58].

The abovementioned alterations in microarchitecture, mechanical properties, and

bone metabolism were easy to understand on the basis of both indirect and direct T2DM

effects on bone impairment. A disorder in glucose and insulin metabolism decreases the

activity of osteoblasts and osteoclasts. Hyperglycemia changes gene expression associated

with osteoblast activity, and insulin resistance may impact bone health . Inflammation

and oxidative stress induced by T2DM have negative effects on bone metabolism. These

accumulation pathomechanisms ultimately result in decreased bone formation or bone

resorption, leading to decreased bone quality [8,35]. In this study, the UC II interven-

tion relieved bone impairment in ageing db/db mice, reflected in improving the bone

microarchitecture, increasing bone formation, and decreasing bone resorption. UC II

also improved inflammation, oxidative stress, and muscle properties, which might have

elevated the disease status of T2DM.

What is the possible reason for UC II improving bone impairment induced by T2DM?

A possible mechanism was the decrease in inflammation and oxidative stress, which could

directly regulate bone metabolism through bones, and indirectly through muscles and

blood glucose.

From a comprehensive and systematic point of view, skeletal muscles and bones have

a coupled and cross-talk relationship, where bones act as a lever and muscles act as a

pulley to move the organism and for monokine communication. The mice in the AM

group exhibited lower values of BV/TV, Tb.N, and Tb.Th and showed smaller values in the

muscle mass, muscle fiber diameter, and cross-sectional area (Figures 3 and 6). In addition,

impaired glucose or insulin metabolism may impact bones by changing skeletal muscle

signaling. IGF-1, secreted from skeletal muscles, is an important growth factor for bone

development. Some investigations provided evidence that muscle IGF-1 can modulate

bone formation and maintain bone structure, which was correspondingly shown in this

study; we found higher content of IGF-1 in the CP group, which showed a higher level

of BV/TV and trabecular number (Tb.N) [28,60–63]. IGF-1 is also an important growth

factor for muscles . There are some reports that elaborated that IGF-1 is associated with

an increase in the muscle mass and muscle fiber area, even inhibiting osteopenia [65,66].

The same finding can be observed in Figures 2 and 6. It was speculated the IGF-1 function

is partly affected by inflammation, oxidative stress, and diabetes [34,35]. In this study,

treating the ageing db/db mice with UC II decreased oxidative stress (Figure 4), leading to

an increased IGF-1 content (Figure 5). This finding was first reported in this study. This

pathway might be the mechanism for an improvement of UC II in bone impairment.

The indirect effect of muscles on bone health was carried out through glucose metab-

olism. Skeletal muscles are recognized as the main site for insulin-mediated glucose

disposal and energy metabolism. Muscle wasting can exacerbate insulin resistance, and

higher muscle mass and strength are associated with a lower level of insulin resistance .

On the molecular level, skeletal homeostasis is linked to insulin sensitivity through nu-

clear receptor peroxisome proliferator-activated receptor (PPAR)γ . IR increases the

FOXO1 expression through PI3-K/MAPK, which could regulate osteoblast proliferation

through ATF4 and p53 signaling . Prolonged inflammation due to IR also stimulates

the expression of proapoptotic genes such as the bcl-2-like protein (Bax). This reduces the

expression of genes that stimulate osteoblast formation, such as the Fos-related antigen

(FRA-1) and the Runt-related transcription factor (RUNX2), resulting in decreased bone

formation. In T2DM, other proteins such as AGEs, proinflammatory cytokines, and ROS

are increased . UC II decreased oxidative stress both in the muscles and the serum

(Figures 5 and 6), indicating partly improved muscle IR (Figure 1), which was speculated

to stimulate membrane translocation of GLUT4 through skeletal muscle phosphorylated

PI3K protein expression, thereby increasing skeletal muscle glucose intake, leading to IR

improvement . UC II increased IGF-1 production by decreasing oxidative stress in the

muscle to improve muscle foundation, glucose metabolism, and insulin signaling, thereby

leading to bone formation.

On the other hand, hyperglycemia leads to the accelerated formation of AGEs and

inflammation cytokines, inhibiting osteoblasts . Therefore, inflammation and oxidative

stress were speculated to be trigger factors of aggravating bone quality impairment. This

study also found that UC II improved oxidative stress in the serum, which likely led to

decreased AGE formation and cross-linking with collagen fibers. Interfering with the

development and function of osteoblasts by upregulating the cell surface receptor for

advanced glycation end products (RAGE) , these receptors increase the production

of proinflammatory cytokines, which may feed a cycle of increased bone resorption and

chronic inflammation .

Figure 4 shows that TNF-α, IL-6, and IL-1β levels in the serum were significantly

decreased in the UC group. In previous cases of osteoarthritis, UC II was documented

to promote a significant reduction in inflammation . Figures 4, 5 and 7 show the re-

lationship between inflammation and bone resorption (TRAP). Therefore, the osteoclasts

overactivated by inflammation play a vital role in imbalanced bone metabolism . Thus,

inhibiting osteoclastogenesis through suppressing inflammation is an important strategy

for improving bone degeneration . Correspondingly, the positive expression of Il-1β in

the CP group was decreased compared with that in the AM group (Figure 6A). IL-1β, IL-6,

and TNF-α, common proinflammatory cytokines, can stimulate osteoclast activity with the

macrophage colony-stimulating factor (M-CSF) and its receptor, c-Fms, which modulate

the pool of available precursor cells for differentiation via the actions of RANKL . These

osteoclast-activating factors interact with a proven final common mediator of osteoclast

differentiation and activation, receptor activator of nuclear factor-kB (RANK) and its func-

tional ligand (RANKL). RANK is a membrane-bound TNF receptor expressed on osteoblast

precursor cells that recognizes RANKL through direct cell–cell interactions, an essential

process for the differentiation of osteoclasts from their precursor cells. RANKL, an essential

factor for osteoclast differentiation and function, is also expressed by lymphocytes and syn-

ovial fibroblasts and may mediate bone loss associated with inflammatory conditions ,

which promotes the bone-resorbing activity of osteoclasts and prolongs their survival .

Similarly, the immunohistochemical analysis showed RANKL had a higher expression in

the AM group, and low expression was observed in the CG and UC groups. The same

tendency was shown in TRAP expression, which means that UC II decreased RANKL

and TRAP expression. In this study, UC II decreased bone resorption by inhibiting serum

cytokines, as well as IL-1 and IL-6 expression, and then inhibited the expression of RANKL

and TRAP. In this study, it was speculated that UC II could improve bone impairment due

to increasing bone mineralization and formation, decreasing bone resorption, which partly

resulted from muscle function and IR improvement through decreasing oxidative stress

and inflammation (Figure 8). This conclusion was partly proved in other studies [28,77].

and its functional ligand (RANKL). RANK is a membrane-bound TNF receptor expressed

on osteoblast precursor cells that recognizes RANKL through direct cell–cell interactions,

an essential process for the differentiation of osteoclasts from their precursor cells.

RANKL, an essential factor for osteoclast differentiation and function, is also expressed

by lymphocytes and synovial fibroblasts and may mediate bone loss associated with in-

flammatory conditions , which promotes the bone-resorbing activity of osteoclasts and

prolongs their survival . Similarly, the immunohistochemical analysis showed

RANKL had a higher expression in the AM group, and low expression was observed in

the CG and UC groups. The same tendency was shown in TRAP expression, which means

that UC II decreased RANKL and TRAP expression. In this study, UC II decreased bone

resorption by inhibiting serum cytokines, as well as IL-1 and IL-6 expression, and then

inhibited the expression of RANKL and TRAP. In this study, it was speculated that UC II

could improve bone impairment due to increasing bone mineralization and formation,

decreasing bone resorption, which partly resulted from muscle function and IR improve-

ment through decreasing oxidative stress and inflammation (Figure 8). This conclusion

was partly proved in other studies [28,77].

This study has some limitations. First, the exact mechanisms of action of UC II on the

bones impaired by T2DM will be explored in future in vitro and in vivo studies. Second,

senescence-accelerated mice (SAM) will be adopted as the experimental model, especially

to demonstrate ageing characteristics from a certain aspect, such as SAMP6 and SAMP10,

and to explore the possible mechanisms of UC II in delaying and improving degenerative

bone diseases.

Figure 8. Graphical summation of the effect of UC II on impaired bone improvement and its likely mechanism.

5. Conclusions

In order to discover effective preventive measures for bone impairment in ageing

coupled with T2DM, UC II was administered in the ageing db/db mice during a period of

Figure 8. Graphical summation of the effect of UC II on impaired bone improvement and its likely mechanism.

This study has some limitations. First, the exact mechanisms of action of UC II on the

bones impaired by T2DM will be explored in future in vitro and in vivo studies. Second,

senescence-accelerated mice (SAM) will be adopted as the experimental model, especially

to demonstrate ageing characteristics from a certain aspect, such as SAMP6 and SAMP10,

and to explore the possible mechanisms of UC II in delaying and improving degenerative

bone diseases.

5. Conclusions

In order to discover effective preventive measures for bone impairment in ageing

coupled with T2DM, UC II was administered in the ageing db/db mice during a period

of 3 months. The microarchitecture, mechanical properties, and bone metabolism were

evaluated. In order to explore the likely mechanism of action of UC II, the T2DM disease

status, biomarkers, and the gastrocnemius function were analyzed. The results showed

that the UC II intervention elevated BMD and the biomechanical parameters. Our results

also demonstrated that UC II increased bone mineralization and formation and decreased

bone resorption. On the one hand, this function was partly due to decreasing inflamma-

tion, leading to inhibited RANKL and TRAP expression. On the other hand, UC II also

improved oxidative stress in both the serum and the gastrocnemius muscle, leading to

IGF-1 secretion, which facilitated bone formation and muscle growth, ultimately leading

to improved IR. Of course, the improvement of hyperglycemia and IR due to UC II could

decrease inflammation and the oxidative stress status, which feed an indirect cycle of

bone impairment.

Supplementary Materials: The following are available online, Figure S1: The image of Undenatured

type II collagen with transmission electron microscope; Figure S2: The infrared spectroscopy of

Undenatured type II collagen; Figure S3: The food intake of db/db mice and db/m mice during the

whole life in the experiments; Figure S4: The weight of db/db mice and db/m mice during the whole

life in the experiments; Figure S5: The FBG of db/db mice and db/m mice during the whole life in

the experiments.

Author Contributions: Conceptualization, Y.L. and R.F.; writing—review and editing, Y.L. and R.F.;

project administration, Y.L.; supervision, Y.L.; investigation, R.F., Y.H., X.L., J.K., J.H., R.M., R.L., N.Z.,

M.X.; writing—original draft preparation, R.F. and Y.H.; methodology, R.F. and Y.H. All authors have

read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: The protocols were reviewed and approved by the Institu-

tional Animal Care and Use Committee of Peking University (approval No. LA2015094).

Informed Consent Statement: This research article described a study exclude humans.

Data Availability Statement: The data presented in this study are available on request from the

corresponding author. The data are not publicly available due to graduation thesis based on this

relevant research results is still in the confidentiality period.

Conflicts of Interest: The authors declare no conflict of interest.

Sample Availability: No Samples of the compounds are available from the authors. All the com-

pounds were bought.

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