MKR Mice Research Articles

Prevalence and impact of diabetes on survival of patients with multiple myeloma in different racial groups

AbstractMultiple myeloma (MM) is twice as common in Black individuals compared with in White individuals, and diabetes mellitus (DM) disproportionately affects Black patients. Although numerous studies have shown a correlation between DM and MM, this has not been studied in the context of race and in vivo mechanisms. We conducted a retrospective clinical study of 5383 patients with MM of which 15% had DM (White, 12% and Black, 25%). Multivariable Cox models showed reduced overall survival (OS) for patients with DM (hazard ratio, 1.27; 95% confidence interval, 1.11-1.47; P < .001). This appeared to be driven by a marked difference in OS between White patients with and without DM but not in Black patients. In contrast, obesity was associated with better OS in Black patients but not in White patients. To complement this analysis, we assessed MM growth in a genetically engineered immunocompromised nonobese diabetic (Rag1−/−/muscle creatinine kinase promoter expression of a human IGF1R [M] with a lysine [K] to arginine [R] point mutation) mouse model to evaluate the mechanisms linking DM and MM. MM.1S xenografts grew in more Rag1−/−/MKR mice and grew more rapidly in the Rag1−/−/MKR mice compared with in controls. Western blot analysis found that MM1.S xenografts from Rag1−/−/MKR mice had higher phosphorylated S6 ribosomal protein (Ser235/236) levels, indicating greater activation of the mammalian target of rapamycin pathway. Our study is, to our knowledge, the first to evaluate racial differences in DM prevalence and survival in MM, as well as the effect of DM on tumor growth in mouse models. Our results suggest that DM may contribute to the higher incidence of MM in Black patients; and to improve survival in MM, DM management cannot be ignored.

Zuogui Jiangtang Qinggan Formula improves glucolipid metabolism in type 2 diabetes mellitus complicated with non-alcoholic fatty liver disease by regulating FoxO1/MTP/APOB signaling pathway

This study aimed to investigate the effect and mechanism of Zuogui Jiangtang Qinggan Formula(ZGJTQG) on the glucolipid metabolism of type 2 diabetes mellitus(T2DM) complicated with non-alcoholic fatty liver disease(NAFLD). NAFLD was induced by a high-fat diet(HFD) in MKR mice(T2DM mice), and a model of T2DM combined with NAFLD was established. Forty mice were randomly divided into a model group, a metformin group(0.067 g·kg~(-1)), and high-and low-dose ZGJTQG groups(29.64 and 14.82 g·kg~(-1)), with 10 mice in each group. Ten FVB mice of the same age were assigned to the normal group. Serum and liver tissue specimens were collected from mice except for those in the normal and model groups after four weeks of drug administration by gavage, and fasting blood glucose(FBG) and fasting insulin(FINS) levels were measured. The levels of total cholesterol(TC), triglyceride(TG), and low-density lipoprotein(LDL) were detected by the single reagent GPO-PAP method. Very low-density lipoprotein(VLDL) was detected by enzyme-linked immunosorbent assay(ELISA). Alanine aminotransferase(ALT) and aspartate ami-notransferase(AST) were determined by the Reitman-Frankel assay. The pathological changes in the liver were observed by hematoxylin-eosin(HE) staining and oil red O staining. Real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR) and Western blot were adopted to detect the mRNA and protein expression of forkhead transcription factor O1(FoxO1), microsomal triglyceride transfer protein(MTP), and apolipoprotein B(APOB) in the liver. The results showed that high-dose ZGJTQG could signi-ficantly reduce the FBG and FINS levels(P&lt;0.05, P&lt;0.01), improve glucose tolerance and insulin resistance(P&lt;0.05, P&lt;0.01), alleviate the liver damage caused by HFD which was reflected in improving liver steatosis, and reduce the serum levels of TC, TG, LDL, VLDL, ALT, and AST(P&lt;0.05, P&lt;0.01) in T2DM mice combined with NAFLD. The findings also revealed that the mRNA and protein expression of FoxO1, MTP, and APOB in the liver was significantly down-regulated after the intervention of high-dose ZGJTQG(P&lt;0.05, P&lt;0.01). The above study showed that ZGJTQG could effectively improve glucolipid metabolism in T2DM combined with NAFLD, and the mechanism was closely related to the regulation of the FoxO1/MTP/APOB signaling pathway.

Metformin regulates bone marrow stromal cells to accelerate bone healing in diabetic mice.

Diabetes mellitus is a group of chronic diseases characterized by high blood glucose levels. Diabetic patients have a higher risk of sustaining osteoporotic fractures than non-diabetic people. The fracture healing is usually impaired in diabetics, and our understanding of the detrimental effects of hyperglycemia on fracture healing is still inadequate. Metformin is the first-line medicine for type 2 diabetes (T2D). However, its effects on bone in T2D patients remain to be studied. To assess the impacts of metformin on fracture healing, we compared the healing process of closed-wound fixed fracture, non-fixed radial fracture, and femoral drill-hole injury models in the T2D mice with and without metformin treatment. Our results demonstrated that metformin rescued the delayed bone healing and remolding in the T2D mice in all injury models. In vitro analysis indicated that compromised proliferation, osteogenesis, chondrogenesis of the bone marrow stromal cells (BMSCs) derived from the T2D mice were rescued by metformin treatment when compared to WT controls. Furthermore, metformin could effectively rescue the impaired detrimental lineage commitment of BMSCs isolated from the T2D mice in vivo as assessed by subcutaneous ossicle formation of the BMSC implants in recipient T2D mice. Moreover, the Safranin O staining of cartilage formation in the endochondral ossification under hyperglycemic condition significantly increased at day 14 post-fracture in the T2D mice receiving metformin treatment. The chondrocyte transcript factors SOX9 and PGC1α, important to maintain chondrocyte homeostasis, were both significantly upregulated in callus tissue isolated at the fracture site of metformin-treated MKR mice on day 12 post-fracture. Metformin also rescued the chondrocyte disc formation of BMSCs isolated from the T2D mice. Taken together, our study demonstrated that metformin facilitated bone healing, more specifically bone formation and chondrogenesis in T2D mouse models.

Intestinal microbiota regulates diabetes and cancer progression by IL-1β and NOX4 dependent signaling cascades.

Diabetes changes the host microbiota, a condition known as dysbiosis. Dysbiosis is an important factor for the pathogenesis of diabetes and colorectal cancer (CRC). We aimed at identifying the microbial signature associated with diabetes and CRC; and identifying the signaling mechanism altered by dysbiosis and leading to CRC progression in diabetes. MKR mice that can spontaneously develop type 2 diabetes were used. For CRC induction, another subset of mice was treated with azoxymethane and dextran sulfate sodium. To identify the role of microbiota, microbiota-depleted mice were inoculated with fecal microbial transplant from diabetic and CRC mice. Further, a mouse group was treated with probiotics. At the end of the treatment, 16S rRNA sequencing was performed to identify microbiota in the fecal samples. Blood was collected, and colons were harvested for molecular, anatomical, and histological analysis. Our results show that diabetes is associated with a microbial signature characterized by reduction of butyrate-forming bacteria. This dysbiosis is associated with gastrointestinal complications reflected by a reduction in colon lengths. These changes are reversed upon treatment with probiotics, which rectified the observed dysbiosis. Inoculation of control mice with diabetic or cancer microbiota resulted in the development of increased number of polyps. Our data also show that inflammatory cytokines (mainly interleukin (IL)-1β) and NADPH oxidase (NOX)4 are over-expressed in the colon tissues of diabetic mice. Collectively our data suggest that diabetes is associated with dysbiosis characterized by lower abundance of butyrate-forming bacteria leading to over-expression of IL-1β and NOX4 leading to gastrointestinal complications and CRC.

Activation of 20-HETE Synthase Triggers Oxidative Injury and Peripheral Nerve Damage in Type 2 Diabetic Mice

Diabetic Peripheral Neuropathy (DPN), highly prevalent among patients with diabetes, is characterized by peripheral nerve dysfunction. Reactive Oxygen Species (ROS) overproduction has been suggested to orchestrate diabetic complications including DPN. Untargeted antioxidant therapy has exhibited limited efficacy, highlighting a critical need to explore ROS sources altered in a cell-specific manner in DPN. Cytochromes P450 (CYP) enzymes are prominent sources of ROS. Particularly, the 20-HETE synthase, CYP4A, is reported to mediate diabetes-induced renal, retinal, and cardiovascular injuries. This work investigates the role of CYP4A/20-HETE in DPN and their mechanisms of action. Non-obese type 2 Diabetic mice (MKR) were used and treated with a CYP4A-inhibitor (HET0016) or AMPK-activator (Metformin). Peripheral nerves of MKR mice reflect increased CYP4A and 20-HETE levels, concurrent with altered myelin proteins and sensorimotor deficits. This was associated with increased ROS production and altered Beclin-1 and LC3 protein levels, indicative of disrupted autophagic responses in tandem with AMPK inactivation. AMPK activation via Metformin restored nerve integrity, reduced ROS production, and regulated autophagy. Interestingly, similar outcomes were revealed upon HET0016 treatment whereby ROS production, autophagic responses, and AMPK signaling were normalized in diabetic mice. Altogether, the results highlight hyperglycemia-mediated oxidative injury in DPN through a novel CYP4A/20-HETE/AMPK pathological axis. PerspectiveTo our knowledge, this is the first study to highlight the role of CYPs/20-HETE-induced oxidative injury in the pathogenesis of diabetic peripheral neuropathy. Targeting the identified pathological axis CYP4A/20-HETE/AMPK may be of clinical potential in predicting and alleviating peripheral nerve injury in patients with Type 2 Diabetes Mellitus.

Non-Obese MKR Mouse Model of Type 2 Diabetes Reveals Skeletal Alterations in Mineralization and Material Properties.

ABSTRACTObesity is a common comorbidity of type 2 diabetes (T2D). Therefore, increased risk of fragility fractures in T2D is often confounded by the effects of obesity. This study was conducted to elucidate the mechanistic basis by which T2D alone leads to skeletal fragility. We hypothesized that obesity independent T2D would deteriorate bone's material quality by accumulating defects in the mineral matrix and undesired modifications in its organic matrix associated with increased oxidative stress and hyperglycemia. To test this hypothesis, we used 15‐week‐old male non‐obese mice with engineered muscle creatine kinase promoter/human dominant negative insulin growth factor 1 (IGF‐I) receptor (MKR) and FVB/N wild‐type (WT) controls (n = 12/group). MKR mice exhibit reduced insulin production and loss of glycemic control leading to diabetic hyperglycemia, verified by fasting blood glucose measurements (>250 mg/dL), without an increase in body weight. MKR mice showed a significant decrease in femoral radial geometry (cortical area, moment of inertia, cortical thickness, endosteal diameter, and periosteal diameter). Bone mineral density (BMD), as assessed by micro–computed tomography (μCT), remained unchanged; however, the quality of bone mineral was altered. In contrast to controls, MKR mice had significantly increased hydroxyapatite crystal thickness, measured by small‐angle X‐ray scattering, and elongated c‐axis length of the crystals evaluated by confocal Raman spectroscopy. There was an increase in changes in the organic matrix of MKR mice, associated with enhanced glycoxidation (carboxymethyl‐lysine [CML] and pentosidine) and overall glycation (fluorescent advanced glycation end products), both of which were associated with various measures of bone fragility. Moreover, increased CML formation positively correlated with elongated mineral crystal length, supporting the role of this negatively charged side chain to attract calcium ions, promote growth of hydroxyapatite, and build a physical link between mineral and collagen. Collectively, our results show, for the first time, changes in bone matrix in a non‐obese T2D model in which skeletal fragility is attributable to alterations in the mineral quality and undesired organic matrix modifications. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Title: Genomic and Systemic Metabolism Differences Associated with Racial Disparities in Multiple Myeloma

BackgroundRacial disparities exist in the prevalence of multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS), with significantly higher prevalence in African American/ black (AA) compared to non-AA individuals. AA patients are also younger than non-AA at the time of diagnosis. We aimed to determine if differences in systemic metabolism and genetics might contribute to these racial disparities in MM. There are currently no approved anti-myeloma therapeutics that simultaneously reverse the metabolic drivers for MM tumor growth.MethodsStudies were approved by the IRB and IACUC. We used a large health system electronic medical record database to characterize the metabolic phenotype of AA and white individuals with MM. Body mass index (BMI) was calculated from height and weight measures. BMI≥30kg/m 2 defined obesity. HbA1c ≥6.5% defined diabetes. Genomic analysis was performed by extracting mutational signatures from tumor samples of MM patients as previously described (PMID: 23945592), and comparing their prevalence between AA and white patients. We used an immunodeficient non-obese mouse model of type 2 diabetes (Rag1-/-/MKR) and controls (Rag1-/-) for in vivo MM1.S xenograft studies. ON123300, a dual ARK5/CDK4 kinase inhibitor was used for preclinical studies.ResultsOf our population of 3170 people, 2128 individuals were AA (21.7%) or white (78.3%). Females comprised 53.2% (n=246) of the AA population and 40.2% (n=669) of the white population. The highest prevalence of obesity was in AA women (46%) > white men (41%) > AA men (36%) > white women (35%). The prevalence of diabetes was greater in AA men (37%) and women (34%) than white men (24%) and women (19%). BMI was a poor predictor of diabetes in the AA population with MM, where diabetes affected 1 in 5 AA individuals with normal BMI, but only 1 in 12 white individuals.Genomic characterization of AA patients, revealed a significant enrichment for the COSMIC SBS1 clock-like mutational signature in AA patients compared to white (p < 0.05), which was confirmed after adjusting for age. The SBS1 signature correlates with the age of individuals and may represent a cell division clock.The male Rag1-/-/MKR mice had hyperglycemia and insulin resistance, and were not obese relative to control mice when fed a regular chow diet. The MM1.S tumor xenografts grew much more rapidly in the Rag1-/-/MKR mice compared with controls; 60 days post injection, tumors were approximately 5 times larger than controls and ex vivo analysis of protein lysates revealed increased activation of the insulin receptor (IR) / insulin-like growth factor 1 receptor (IGF-1R) and mTOR signaling pathway. Our previous studies (Perumal et al, CCR 2016) have characterized a role for ARK5(AMPK-related protein kinase 5), which is a major regulator of glutamine uptake in tumor cells that co-express ARK5 and MYC. Preclinical testing of MM cell lines and xenograft mouse models show a robust reduction of MM tumor growth as well as inhibition of IGF-1R-downstream signaling pathways, which contribute to tumor proliferation.ConclusionsIn our large diverse cohort of individuals with MM, we found significant racial disparities in the prevalence of obesity and type 2 diabetes, in addition to enrichment of a clock-like mutational signature in MM from AA individuals. Our findings suggest a signature of accelerated aging in MM from AA patients, which is concordant with younger age at diagnosis. In our mouse model, MM xenografts grew more rapidly in the diabetic mice and had more activation of mTOR signaling, a pathway known to regulate clock and aging gene signatures. ON123300 was able to reverse the metabolic drive as well as directly reduce tumor growth in vivo xenograft models. DisclosuresParekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

P-102: Genomic and systemic metabolism differences associated with racial disparities in Multiple Myeloma

<h3>Background</h3> Racial disparities exist in the prevalence of multiple myeloma (MM) and monoclonal gammopathy of unknown significance (MGUS), with significantly higher prevalence in African American/ black (AA) compared to non-AA individuals. AA patients are also younger than non-AA at the time of diagnosis. We aimed to determine if differences in systemic metabolism and genetics might contribute to these racial disparities in MM. <h3>Methods</h3> Studies were approved by the IRB and IACUC. We used a large health system electronic medical record database to characterize the metabolic phenotype of AA and white individuals with MM. Body mass index (BMI) was calculated from height and weight measures. BMI≥30kg/m2 defined obesity. HbA1c ≥6.5% defined diabetes. Genomic analysis was performed by extracting mutational signatures from tumor samples of MM patients as previously described, and comparing their prevalence between AA and white patients. We used an immunodeficient non-obese mouse model of type 2 diabetes (Rag1-/-/MKR) and controls (Rag1-/-) for in vivo MM1.S xenograft studies. <h3>Results</h3> Of our population of 3170 people, 2128 individuals were AA (21.7%) or white (78.3%). Females comprised 53.2% (n=246) of the AA population and 40.2% (n=669) of the white population. The highest prevalence of obesity was in AA women (46%) > white men (41%) > AA men (36%) > white women (35%). The prevalence of diabetes was greater in AA men (37%) and women (34%) than white men (24%) and women (19%). BMI was a poor predictor of diabetes in the AA population with MM, where diabetes affected 1 in 5 AA individuals with normal BMI, but only 1 in 12 white individuals. Genomic characterization of AA patients, revealed a significant enrichment for the COSMIC SBS1 clock-like mutational signature in AA patients compared to white (p < 0.05), which was confirmed after adjusting for age. The SBS1 signature correlates with the age of individuals and may represent a cell division clock. Our finding suggests accelerated aging in AA MM patients, which is concordant with younger age at diagnosis. The male Rag1-/-/MKR mice had hyperglycemia and insulin resistance, and were not obese relative to control mice when fed a regular chow diet. The MM1.S tumor xenografts grew much more rapidly in the Rag1-/-/MKR mice compared with controls; 60 days post injection, tumors were approximately 5 times larger than controls and ex vivo analysis of protein lysates revealed increased activation of the insulin receptor (IR) / insulin-like growth factor 1 receptor (IGF-1R) and mTOR signaling pathway. <h3>Conclusions</h3> In our large diverse cohort of individuals with MM, we found significant racial disparities in the prevalence of obesity and type 2 diabetes, in addition to enrichment of a clock-like mutational signature in MM from AA individuals. In our mouse model, MM xenografts grew more rapidly in the diabetic mice and had more activation of mTOR signaling, a pathway known to regulate clock and aging gene signatures.

Identification of Cellular Damage in Uteri From Hyperinsulinemic Mice Treated With Unopposed Estradiol

Abstract Unopposed estradiol and obesity are known risk factors for endometrial adenocarcinoma (EC). Endometrium from women with obesity was found to have an increase in mutations relative to tissue from normal weight women, indicating DNA damage may be accelerated in the setting of obesity. Since obesity is associated with high levels of insulin and anovulatory cycles, we sought to mimic these conditions in a mouse model. We previously found that hyperinsulinemic MKR mice, without the confounder of obesity, have an increased incidence of nuclear atypia in endometrial glands. We hypothesized that hyperinsulinemia and unopposed estradiol have a synergistic effect on inducing abnormal architecture and DNA damage in the endometrium, than either alone. At 8-10 weeks old, cohorts of MKR (n=20) and WT (n=20) mice underwent ovariectomy and placement of either an estradiol (E2) or placebo (P) pellet. Metabolic profiling included insulin tolerance testing and MR for body composition. At 3 months post-implantation, mice received a partial hysterectomy and second pellet replacement. At 6 months, the remaining uterus was bisected into pieces. A blinded histological analysis was conducted by a gynecology pathologist. A marker of DNA damage due to oxidative stress, 8-oxoguanine-DNA-glycosylase (8-OHdG), was quantified by ELISA. Data was analyzed using Kruskal-Wallis test with multiple test correction, or Fischer’s exact test. By 6 months, MKR-E2 treated mice had a 27% lower body weight than MKR-P mice (p&amp;lt;0.05), and 31% lower than WT-E2 mice (p&amp;lt;0.01). WT-E2 and WT-P had similar weight, and were similar to MKR-P (p=ns). Percent body fat was similar across all 4 cohorts of mice (p=ns). Since placebo-treated mice had small, atrophied uteri with minimal gland formation, E2 pellet failure was determined by the presence of small, atrophied uteri and occurred in 4 MKR and 3 WT mice at either 3 or 6 months. All other MKR and WT E2 treated mice had enlarged uteri. The frequency of endometrial gland dilation was similar in MKR-E2 and WT-E2 uteri (p=ns), but all MKR mice had moderate-severe dilation, whereas WT mice had 50% mild and 50% moderate-severe dilation (p=0.07). Focal hyperplasia was present in one MKR-E2 mouse, and nuclear atypia was present in one WT-E2 mouse. MKR-P uteri had a 7-fold higher mean 8-OHdG relative to MKR-E2 uteri (5.0±3.7 vs 0.7±1.6, p&amp;lt;0.002). WT-E2 and WT-P uteri had similar 8-OHdG (1.6±0.8 vs 1.8±0.6, p=ns), as did MKR-E2 and WT-E2 uteri (p=ns). Our findings show that hyperinsulinemia exacerbates the cystic dilation induced by chronic unopposed estradiol, indicating a synergy of insulin and estradiol in promoting abnormal glandular growth in the endometrium. Surprisingly, uterine DNA damage was highest in the setting of hyperinsulinemia alone, in a hormonal state mimicking post-menopause. Further work is needed to understand the effect of estradiol on intrauterine oxidative stress-induced damage.

Progranulin promotes bone fracture healing via TNFR pathways in mice with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) significantly increases bone fragility and fracture risk. Progranulin (PGRN) promotes bone fracture healing in both physiological and type 1 diabetic conditions. The present study aimed to investigate the role of PGRN in T2DM bone fracture healing. MKR mice (with an FVB/N genetic background) were used as the T2DM model. Drill-hole and Bonnarens and Einhorn models were used to investigate the role of PGRN in T2DM fracture healing in vivo. Primary bone marrow cells were isolated for molecular and signaling studies, and reverse transcription-polymerase chain reaction, immunohistochemical staining, and western blotting were performed to assess PGRN effects in vitro. PGRN mRNA and protein expression were upregulated in the T2DM model. Local administration of recombinant PGRN effectively promoted T2DM bone fracture healing in vivo. Additionally, PGRN could induce anabolic metabolism during endochondral ossification through the TNFR2-Akt and Erk1/2 pathways. Furthermore, PGRN showed anti-inflammatory activity in the T2DM bone regeneration process. These findings suggest that local administration of exogenous PGRN may be an alternative strategy to support bone regeneration in patients with T2DM. Additionally, PGRN might hold therapeutic potential for other TNFR-related metabolic disorders.

A GC-MS-Based Metabolomics Investigation of the Protective Effect of Liu-Wei-Di-Huang-Wan in Type 2 Diabetes Mellitus Mice.

Materials and Methods MKR mice were used for the development of diabetes with high-fat diet feeding. These mice were further injected with streptozocin (STZ) to aggravate kidney failure. Fasting blood glucose (FBG) and urinary albumin-to-creatinine ratio (ACR values) were determined to validate the successful establishment of diabetic models with desired kidney dysfunction. Metabolomics approach coupled with gas chromatography-mass spectrometry (GC-MS) and random forest (RF) algorithm was proposed to discover the metabolic differences among model group and control group as well as to examine the therapeutic efficacy of traditional Chinese medicine, Liu-Wei-Di-Huang-Wan (LWDHW), in diabetes and associated kidney failure. Results Some metabolites such as 3-hydroxybutyric acid, citric acid, hexadecanoic acid, and octadecanoic acid showed significant differences between the control group and model group. Treatment with LWDHW resulted in a significant decrease in FBG and ACR values. These results suggested that LWDHW could have beneficial effects in diabetes-associated renal failure.

Butyrate modulates diabetes-linked gut dysbiosis: epigenetic and mechanistic modifications.

Diabetic dysbiosis has been described as a novel key player in diabetes and diabetic complications. However, the cellular/molecular alterations associated with dysbiosis remain poorly characterized. For that, control, non-obese type 2 diabetic MKR mice and MKR mice treated with butyrate were used to delineate the epigenetic, cellular and molecular mechanisms by which dysbiosis associated with diabetes induces colon shortening and inflammation attesting to gastrointestinal disturbance. Our results show that dysbiosis is associated with T2DM and characterized by reduced Bacteroid fragilis population and butyrate-forming bacteria. The reduction of butyrate-forming bacteria and inadequate butyrate secretion result in alleviating HDAC3 inhibition and altering colon permeability. The observed changes are also associated with an increase in ROS production, a rise in NOX4 proteins, and a shift in the inflammatory markers, where IL-1β is increased and IL-10 and IL-17α are reduced. Treatment with butyrate restores the homeostatic levels of NOX4 and IL-1β. In summary, our data suggest that in T2DM, dysbiosis is associated with a reduction in butyrate content leading to increased HDAC3 activity. Butyrate treatment restores the homeostatic levels of the inflammatory markers and reduces ROS production known to mediate diabetes-induced colon disturbance. Taken together, our results suggest that butyrate could be a potential treatment to attenuate diabetic complications.

1172-P: GLUT4-Overexpressing Engineered Muscle Tissue Implants as a Potential Novel Therapy for Type 2 Diabetes: Lessons from Diabetic Mice

In spite of current therapy protocols many T2DM patients fail to achieve blood glucose homeostasis goals. Insulin resistance in T2DM is associated with impaired GLUT4 function and cellular content. Thus, our aim was to improve overall glucose homeostasis by constructing engineered muscle tissue, using genetically modified skeletal muscle cells that over-express the GLUT4 transporter (OEG4). The OEG4 cells were grafted on biodegradable scaffolds and cultured to allow the formation of engineered muscle tissue constructs (EMC). Using an in-vitro 2-deoxyglucos (DOG) uptake assay, the OEG4-EMCs showed a 1.8-fold increase in DOG uptake with insulin stimulation, compared to unmodified controls. For in-vivo efficacy evaluation OEG4-EMCs and empty constructs, not seeded with cells, as control were implanted in the abdominal wall of immunodeficient recombination-activating gene 1 (Rag1)/MKR mice model of T2DM. Following i.p glucose tolerance test mice implanted with OEG4-EMCs had significantly (p&amp;lt;0.05) reduced maximal blood glucose levels (185+/-14.8 mg/dL), compared to diabetic controls group (294+/-23 mg/dL) and similar to nondiabetic mice (188+/-8.1 mg/dL). In addition, glucose levels returned to basal levels faster than in the control group. Since, EMC implants improved diabetic mice overall glucose homeostasis our data support the potential of such implants as novel therapeutic mode for T2DM therapy. Disclosure M. Beckerman: None. E. Karnieli: None. C. Harel: None. A. Klip: None. P.J. Bilan: None. E. Gallagher: None. D. LeRoith: Advisory Panel; Self; AstraZeneca, MannKind Corporation. S. Levenberg: None. Funding Rina and Avner Schneur Charitable Giving Fund

Insulin/IGF1 signalling mediates the effects of β2 -adrenergic agonist on muscle proteostasis and growth.

BackgroundStimulation of β2‐adrenoceptors can promote muscle hypertrophy and fibre type shift, and it can counteract atrophy and weakness. The underlying mechanisms remain elusive.MethodsFed wild type (WT), 2‐day fasted WT, muscle‐specific insulin (INS) receptor (IR) knockout (M‐IR−/−), and MKR mice were studied with regard to acute effects of the β2‐agonist formoterol (FOR) on protein metabolism and signalling events. MKR mice express a dominant negative IGF1 receptor, which blocks both INS/IGF1 signalling. All received one injection of FOR (300 μg kg−1 subcutaneously) or saline. Skeletal muscles and serum samples were analysed from 30 to 240 min. For the study of chronic effects of FOR on muscle plasticity and function as well as intracellular signalling pathways, fed WT and MKR mice were treated with formoterol (300 μg kg−1 day−1) for 30 days.ResultsIn fed and fasted mice, one injection of FOR inhibited autophagosome formation (LC3‐II content, 65%, P ≤ 0.05) that was paralleled by an increase in serum INS levels (4‐fold to 25‐fold, P ≤ 0.05) and the phosphorylation of Akt (4.4‐fold to 6.5‐fold, P ≤ 0.05) and ERK1/2 (50% to two‐fold, P ≤ 0.05). This led to the suppression (40–70%, P ≤ 0.05) of the master regulators of atrophy, FoxOs, and the mRNA levels of their target genes. FOR enhanced (41%, P ≤ 0.05) protein synthesis only in fed condition and stimulated (4.4‐fold to 35‐fold, P ≤ 0.05) the prosynthetic Akt/mTOR/p70S6K pathway in both fed and fasted states. FOR effects on Akt signalling during fasting were blunted in both M‐IR−/− and MKR mice. Inhibition of proteolysis markers by FOR was prevented only in MKR mice. Blockade of PI3K/Akt axis and mTORC1, but not ERK1/2, in fasted mice also suppressed the acute FOR effects on proteolysis and autophagy. Chronic stimulation of β2‐adrenoceptors in fed WT mice increased body (11%, P ≤ 0.05) and muscle (15%, P ≤ 0.05) growth and downregulated atrophy‐related genes (30–40%, P ≤ 0.05), but these effects were abolished in MKR mice. Increases in muscle force caused by FOR (WT, 24%, P ≤ 0.05) were only partially impaired in MKR mice (12%, P ≤ 0.05), and FOR‐induced slow‐to‐fast fibre type shift was not blocked at all in these animals. In MKR mice, FOR also restored the lower levels of muscle SDH activity to basal WT values and caused a marked reduction (57%, P ≤ 0.05) in the number of centrally nucleated fibers.ConclusionsNS/IGF1 signalling is necessary for the anti‐proteolytic and hypertrophic effects of in vivo β2‐adrenergic stimulation and appears to mediate FOR‐induced enhancement of protein synthesis. INS/IGF1 signalling only partially contributes to gain in strength and does not mediate fibre type transition induced by FOR.

Hyperinsulinemia Promotes Esophageal Cancer Development in a Surgically-Induced Duodeno-Esophageal Reflux Murine Model.

Hyperinsulinemia could have a role in the growing incidence of esophageal adenocarcinoma (EAC) and its pre-cancerous lesion, Barrett’s Esophagus, a possible consequence of Gastro-Esophageal Reflux Disease. Obesity is known to mediate esophageal carcinogenesis through different mechanisms including insulin-resistance leading to hyperinsulinemia, which may mediate cancer progression via the insulin/insulin-like growth factor axis. We used the hyperinsulinemic non-obese FVB/N (Friend leukemia virus B strain) MKR (muscle (M)-IGF1R-lysine (K)-arginine (R) mouse model to evaluate the exclusive role of hyperinsulinemia in the pathogenesis of EAC related to duodeno-esophageal reflux. FVB/N wild-type (WT) and MKR mice underwent jejunum-esophageal anastomosis side—to end with the exclusion of the stomach. Thirty weeks after surgery, the esophagus was processed for histological, immunological and insulin/Insulin-like growth factor 1 (IGF1) signal transduction analyses. Most of the WT mice (63.1%) developed dysplasia, whereas most of the MKR mice (74.3%) developed squamous cell and adenosquamous carcinomas, both expressing Human Epidermal growth factor receptor 2 (HER2). Hyperinsulinemia significantly increased esophageal cancer incidence in the presence of duodenal-reflux. Insulin receptor (IR) and IGF1 receptor (IGF1R) were overexpressed in the hyperinsulinemic condition. IGF1R, through ERK1/2 mitogenic pattern activation, seems to be involved in cancer onset. Hyperinsulinemia-induced IGF1R and HER2 up-regulation could also increase the possibility of forming of IGF1R/HER2 heterodimers to support cell growth/proliferation/progression in esophageal carcinogenesis.

Long-term treatment with the ghrelin receptor antagonist [d-Lys3]-GHRP-6 does not improve glucose homeostasis in nonobese diabetic MKR mice.

Long-term treatment with the ghrelin receptor antagonist [d-Lys3]-GHRP-6 does not improve glucose homeostasis in nonobese diabetic MKR mice. Am J Physiol Regul Integr Comp Physiol 314: R71-R83, 2018. First published September 13, 2017; doi: 10.1152/ajpregu.00157.2017 .-Ghrelin secretion has been associated with increased caloric intake and adiposity. The expressions of ghrelin and its receptor (GHS-R1a) in the pancreas has raised the interest about the role of ghrelin in glucose homeostasis. Most of the studies showed that ghrelin promoted hyperglycemia and inhibited insulin secretion. This raised the interest in using GHS-R1a antagonists as therapeutic targets for type 2 diabetes. Available data of GHS-R antagonists are on a short-term basis. Moreover, the complexity of GHS-R1a signaling makes it difficult to understand the mechanism of action of GHS-R1a antagonists. This study examined the possible effects of long-term treatment with a GHS-R1a antagonist, [d-Lys3]-growth hormone-releasing peptide (GHRP)-6, on glucose homeostasis, food intake, and indirect calorimetric parameters in nonobese diabetic MKR mice. Our results showed that [d-Lys3]-GHRP-6 (200 nmol/mouse) reduced pulsatile growth hormone secretion and body fat mass as expected but worsened glucose and insulin intolerances and increased cumulative food intake unexpectedly. In addition, a significant increase in blood glucose and decreases in plasma insulin and C-peptide levels were observed in MKR mice following long-term [d-Lys3]-GHRP-6 treatment, suggesting a direct inhibition of insulin secretion. Immunofluorescence staining of pancreatic islets showed a proportional increase in somatostatin-positive cells and a decrease in insulin-positive cells in [d-Lys3]-GHRP-6-treated mice. Furthermore, [d-Lys3]-GHRP-6 stimulated food intake on long-term treatment via reduction of proopiomelanocortin gene expression and antagonized GH secretion via reduced growth hormone-releasing hormone gene expression in hypothalamus. These results demonstrate that [d-Lys3]-GHRP-6 is not completely opposite to ghrelin and may not be a treatment option for type 2 diabetes.

Hexarelin, a Growth Hormone Secretagogue, Improves Lipid Metabolic Aberrations in Nonobese Insulin-Resistant Male MKR Mice.

Despite the occurrence of dyslipidemia and its contribution to the development of insulin resistance in obese subjects, a growing number of studies have described abnormal lipid profiles among leaner persons. For example, individuals with an abnormal paucity or distribution of fat (lipodystrophy) develop severe insulin resistance, dyslipidemia, and hepatic steatosis. Deranged adipocyte metabolism and differentiation contribute to ectopic fat deposition and consequent development of insulin resistance. Growth hormone (GH) therapy has been shown to correct body composition abnormalities in some lipodystrophy patients. However, little is known about the effects of GH-releasing peptides in this regard. Hexarelin, a GH secretagogue, has recently been shown to have beneficial effects on fat metabolism via the CD36 receptor. In this study, the effects of twice daily intraperitoneal injections of hexarelin (200 μg/kg body weight) were examined in nonobese insulin-resistant MKR mice and corresponding wild-type FVB mice for 12 days. Hexarelin treatment significantly improved glucose and insulin intolerance and decreased plasma and liver triglycerides in MKR mice. These beneficial metabolic effects could be due to the improved lipid metabolism and enhanced adipocyte differentiation of white adipose tissue with hexarelin treatment. Interestingly, although food intake of hexarelin-treated MKR mice was significantly increased, this did not change total body weight. Moreover, hexarelin treatment corrected the abnormal body composition of MKR mice, as demonstrated by a decrease in fat mass and an increase in lean mass. Our results suggest a possible application of hexarelin in treatment of lipid disorders associated with the metabolic syndrome.

Erratum to: Non-metabolisable insulin glargine does not promote breast cancer growth in a mouse model of type 2 diabetes

Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25 U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.

Silencing vimentin expression decreases pulmonary metastases in a pre-diabetic mouse model of mammary tumor progression.

Increased breast cancer risk and mortality has been associated with obesity and Type 2 diabetes (T2D). Hyperinsulinemia, a key factor in obesity, pre-diabetes and T2D, has been associated with decreased breast cancer survival. In the current study, a mouse model of pre-diabetes (MKR mouse) was used to investigate the mechanisms through which endogenous hyperinsulinemia promotes mammary tumor metastases. The MKR mice developed larger primary tumors and greater number of pulmonary metastases compared to wild type (WT) mice after injection with c-Myc/Vegf overexpressing MVT-1 cells. Analysis of the primary tumors showed significant increase in Vimentin protein expression in the MKR mice compared to WT. We hypothesized that Vimentin was an important mediator in the effect of hyperinsulinemia on breast cancer metastasis. Lentiviral shRNA knockdown of Vimentin led to a significant decrease in invasion of the MVT-1 cells and abrogated the increase in cell invasion in response to insulin. In the pre-diabetic MKR mouse, Vimentin knockdown led to a decrease in pulmonary metastases. In vitro, we found that insulin increased pAKT, prevented Caspase 3 activation, and increased Vimentin. Inhibiting the PI3K/AKT pathway, using NVP-BKM120, increased active Caspase 3 and decreased Vimentin levels. This study is the first to show that Vimentin plays an important role in tumor metastasis in vivo in the setting of pre-diabetes and endogenous hyperinsulinemia. Vimentin targeting may be an important therapeutic strategy to reduce metastases in patients with obesity, pre-diabetes or T2D.

Non-metabolisable insulin glargine does not promote breast cancer growth in a mouse model of type 2 diabetes.

Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.