Effect of low-volume exercise on hepatic steatosis in adults with obesity plus normal glucose, prediabetes or type 2 diabetes: a randomised controlled trial.

Objectives: This study aimed to evaluate the effects of a novel, low-volume combined high-intensity interval training (HIIT) and progressive resistance training (PRT) in overweight/obese adults. Methods: This randomised control trial compared the effect of regular supervised HIIT combined with PRT (Exercise) with an unsupervised stretching intervention (Control), in previously inactive adults with either normal glucose (NG), pre-diabetes or type 2 diabetes (T2DM) with body mass index of >25 kg/m2. Participants were randomly allocated (1:1) to receive low-volume exercise or control by an online randomisation tool. The primary outcome was the difference in change of hepatic steatosis between Exercise and Control. A prespecified sensitivity analysis was undertaken for weight stable participants (<5% change in bodyweight from baseline). Secondary outcomes were change in hepatic steatosis within the glucose groups, glycaemic control, cardiorespiratory fitness, muscle strength and body composition. Results: Between June 2018 and May 2021, 162 participants were randomly assigned (NG: 76, pre-diabetes: 60, T2DM: 26) and 144 were included in the final analysis. Mean absolute change in hepatic steatosis was -1.4% (4.9) in Exercise (n=73) and -0.1% (7.2) in Control (n=71)(p=0.25). By preplanned sensitivity analysis, the mean change in hepatic steatosis with Exercise (n=70) was -1.5% (5) compared with 0.7% (4.6) with Control (n=61) (p=0.017). Subgroup analysis within the glucose groups showed that exercise reduced hepatic steatosis in those with pre-diabetes but not NG or T2DM (pre-diabetes: -1.2% (4.4) in Exercise and 1.75% (5.7) in Control, p=0.019). Conclusion: These findings show that low-volume HIIT with PRT yields improvements in muscle strength and cardiorespiratory fitness and may have a small effect on hepatic steatosis. Trial registration number: The trial was prospectively registered with the ANZCTR (ACTRN12617000552381).

Deep Immune and RNA Profiling Revealed Distinct Circulating CD163+ Monocytes in Diabetes-Related Complications.

CD163, a scavenger receptor with anti-inflammatory function expressed exclusively on monocytes/macrophages, is dysregulated in cases of diabetes complications. This study aimed to characterize circulating CD163+ monocytes in the presence (D+Comps) or absence (D-Comps) of diabetes-related complications. RNA-sequencing and mass cytometry were conducted on CD163+ monocytes in adults with long-duration diabetes and D+Comps or D-Comps. Out of 10,868 differentially expressed genes identified between D+Comps and D-Comps, 885 were up-regulated and 190 were down-regulated with a ≥ 1.5-fold change. In D+Comps, 'regulation of centrosome cycle' genes were enriched 6.7-fold compared to the reference genome. MIR27A, MIR3648-1, and MIR23A, the most up-regulated and CD200R1, the most down-regulated gene, were detected in D+Comps from the list of 75 'genes of interest'. CD163+ monocytes in D+Comps had a low proportion of recruitment markers CCR5, CD11b, CD11c, CD31, and immune regulation markers CD39 and CD86. A gene-protein network identified down-regulated TLR4 and CD11b as 'hub-nodes'. In conclusion, this study reports novel insights into CD163+ monocyte dysregulation in diabetes-related complications. Enriched centrosome cycle genes and up-regulated miRNAs linked to apoptosis, coupled with down-regulated monocyte activation, recruitment, and immune regulation, suggest functionally distinct CD163+ monocytes in cases of diabetes complications. Further investigation is needed to confirm their role in diabetes-related tissue damage.

Circulating CD31+ Angiogenic T cells are reduced in prediabetes and increase with exercise training.

AIMS: To investigate circulating angiogenic cells in adults with prediabetes and the effect of a structured exercise program. METHODS: A cohort of adults with overweight/obesity and either normal glucose (NG) or prediabetes were randomised to receive exercise (Exercise) (as twice weekly supervised combined high intensity aerobic exercise and progressive resistance training, and once weekly home-based aerobic exercise) or an unsupervised stretching intervention (Control) for 12 weeks. Circulating angiogenic T cells, muscle strength, and cardiovascular disease risk factors, including blood lipids, arterial stiffness, central haemodynamic responses, and cardiorespiratory fitness (VO2peak) in those with prediabetes (n = 35, 16 Control, 19 Exercise) and NG (n = 37, 17 Control, 20 Exercise) were analysed at baseline and after the 12-week intervention. RESULTS: At baseline, compared with NG those with prediabetes demonstrated reduced VO2peak, angiogenic CD31+CD8+ T cells and VEGFR2+CD4+ T cells, and increased systolic blood pressure. CD31+ T cells were negatively correlated with cardiovascular disease (CVD) risk. Compared with Control, exercise training increased muscle strength, VO2peak, and CD31+CD4+ and CD31+CD8+ T cells in NG and prediabetes. CONCLUSIONS: Circulating angiogenic CD31+ T cells are decreased in people with prediabetes and are enhanced with exercise training. Exercise increases CD31+ T cells, and through this mechanism it is proposed that it may reduce CVD risk. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry number: ACTRN12617000552381.

Cardio-metabolic health effects of CPAP treatment for sleep apnoea during weight loss: A randomised controlled pilot trial.

BACKGROUND AND AIMS: This study assessed whether the addition of continuous positive airway pressure (CPAP) during weight loss would enhance cardiometabolic health improvements in patients with obesity and Obstructive Sleep Apnoea (OSA). METHODS AND RESULTS: Patients with overweight or obesity, pre-diabetes and moderatesevere OSA were randomised to receive CPAP therapy with a weight loss programme (CPAP+WL) or a weight loss programme alone (WL alone). PRIMARY OUTCOME: 2-hour glucose assessed by an oral glucose tolerance test. SECONDARY OUTCOMES: 24 hr blood pressure, body composition (DEXA) and fasting blood markers. 17 patients completed 3-month follow-up assessments (8 CPAP+WL and 9 WL alone). Overall, participants in both groups lost ∼12 kg which reduced polysomnography determined OSA severity by ∼45 %. In the CPAP+WL group, CPAP use (compliance 5.29 hrs/night) did not improve any outcome above WL alone. There was no improvement in 2-hour glucose in either group. However, in the pooled (n = 17) analysis there were overall improvements in most outcomes including insulin sensitivity (.000965 units, p = .008), sleep systolic BP (- 16.2 mmHg, p = .0003), sleep diastolic BP (-9.8 mmHg, p = 0.02), wake diastolic BP (- 4.3 mmHg, p = .03) and sleepiness (Epworth Sleepiness Score -3.2, p = .0003). In addition, there were reductions in glucose area under the curve (-230 units, p = .009), total (-0.86 mmol/L, p = 0.006) and LDL cholesterol (-0.58 mmol/L, p = 0.007), triglycerides (-0.75 mmol/L, p = 0.004), fat mass (-7.6 kg, p < .0001) and abdominal fat (-310 cm3, p < .0001). CONCLUSION: Weight loss reduced OSA and improved sleepiness and cardiometabolic health. These improvements were not further enhanced by using CPAP. Results suggest weight loss should be the primary focus of treatment for patients with OSA and obesity.

Diabetic ketoacidosis in an adult with beta-ketothiolase deficiency (BKD) involving a novel ACAT1 variant : first report of established diabetes in BKD and a review of the literature.

BACKGROUND: Diabetes presenting in young adults is often challenging to classify. Diabetic ketoacidosis is typically seen in autoimmune type 1 diabetes mellitus and more rarely in young onset type 2 diabetes mellitus. Beta-ketothiolase deficiency (BKD) is a rare autosomal recessive condition affecting isoleucine catabolism and ketone body metabolism. BKD typically manifests in childhood as recurrent episodes of ketoacidosis, the frequency of which tends to reduce with age. There is a paucity of data with respect to the co-existence of persistent dysglycemia with BKD. CASE PRESENTATION AND LITERATURE REVIEW: We present a novel case of diabetes presenting as diabetic ketoacidosis in a 34-year-old man with BKD, with genetically confirmed compound heterozygosity for variants in ACAT1, including a novel ACAT1 c.481T>C, p.(Tyr161His) variant. Diabetes in people with BKD presents unique diagnostic and management challenges. To further contextualize our findings, we conducted a comprehensive narrative review of the existing literature with respect to dysglycemia in those with BKD, especially in adulthood. There are no existing reports describing diabetes in adults with BKD. Stress hyperglycemia is not uncommon when children with BKD are acutely unwell, with several pediatric case reports describing short-lived hyperglycemia but normal HbA1c measurements during metabolic crises (indicating the absence of persistent hyperglycemia). CONCLUSIONS: This is the first report of diabetic ketoacidosis in an adult with BKD, with an elevated HbA1c consistent with persistent hyperglycemia. This case highlights the importance of checking HbA1c in people with BKD and hyperglycemia in order to uncover potential coexisting diabetes, facilitating timely management and preventing complications. Increased reporting on the longitudinal outcomes of those with rare metabolic disorders is essential for identifying potential associations with conditions like diabetes.

Australian evidence-based guidelines for the prevention and management of diabetes-related foot disease: a guideline summary.

INTRODUCTION: Diabetes-related foot disease (DFD) - foot ulcers, infection, ischaemia - is a leading cause of hospitalisation, disability, and health care costs in Australia. The previous 2011 Australian guideline for DFD was outdated. We developed new Australian evidence-based guidelines for DFD by systematically adapting suitable international guidelines to the Australian context using the ADAPTE and GRADE approaches recommended by the NHMRC. MAIN RECOMMENDATIONS: This article summarises the most relevant of the 98 recommendations made across six new guidelines for the general medical audience, including: prevention - screening, education, self-care, footwear, and treatments to prevent DFD; classification - classifications systems for ulcers, infection, ischaemia and auditing; peripheral artery disease (PAD) - examinations and imaging for diagnosis, severity classification, and treatments; infection - examinations, cultures, imaging and inflammatory markers for diagnosis, severity classification, and treatments; offloading - pressure offloading treatments for different ulcer types and locations; and wound healing - debridement, wound dressing selection principles and wound treatments for non-healing ulcers. CHANGES IN MANAGEMENT AS A RESULT OF THE GUIDELINE: For people without DFD, key changes include using a new risk stratification system for screening, categorising risk and managing people at increased risk of DFD. For those categorised at increased risk of DFD, more specific self-monitoring, footwear prescription, surgical treatments, and activity management practices to prevent DFD have been recommended. For people with DFD, key changes include using new ulcer, infection and PAD classification systems for assessing, documenting and communicating DFD severity. These systems also inform more specific PAD, infection, pressure offloading, and wound healing management recommendations to resolve DFD.

Comparison of digital and traditional skin wound closure assessment methods in mice.

BACKGROUND: Chronic skin wounds are a common complication of many diseases such as diabetes. Various traditional methods for assessing skin wound closure are used in animal studies, including wound tracing, calliper measurements and histological analysis. However, these methods have poorly defined wound closure or practical limitations. Digital image analysis of wounds is an increasingly popular, accessible alternative, but it is unclear whether digital assessment is consistent with traditional methods. This study aimed to optimise and compare digital wound closure assessment with traditional methods, using a diabetic mouse model. Diabetes was induced in male C57BL/6J mice by high-fat diet feeding combined with low dose (65 mg/kg of body weight) streptozotocin injections. Mice fed normal chow were included as controls. After 18 weeks, four circular full-thickness dorsal skin wounds of 4 mm diameter were created per mouse. The wounds were photographed and measured by callipers. Wound closure rate (WCR) was digitally assessed by two reporters using two methods: wound outline (WCR-O) and re-epithelialisation (WCR-E). Wounded skin tissues were collected at 10-days post-wounding and wound width was measured from haematoxylin and eosin-stained skin tissue. RESULTS: Between reporters, WCR-O was more consistent than WCR-E, and WCR-O correlated with calliper measurements. Histological analysis supported digital assessments, especially WCR-E, when wounds were histologically closed. CONCLUSIONS: WCR-O could replace calliper measurements to measure skin wound closure, but WCR-E assessment requires further refinement. Small animal studies of skin wound healing can greatly benefit from standardised definitions of wound closure and more consistent digital assessment protocols.

Diabetic foot ulcers: weekly versus second-weekly conservative sharp wound debridement.

Diabetic foot ulcers (DFU) are a serious and costly long-term complication of diabetes, and are one of the most prevalent hard-to-heal (chronic) wound types. Conservative sharp wound debridement (CSWD) is a mainstay of care. It is performed regularly until healing is achieved (when there is adequate blood flow for healing) to support endogenous healing and improve the efficacy of advanced healing therapies. CSWD is supported by evidence-based treatment guidelines, despite a lack of prospective studies. The first prospective randomised study to compare different frequencies of CSWD-the Diabetes Debridement Study (DDS)-showed no difference in healing outcomes at 12 weeks between those ulcers debrided weekly and those debrided every second week. A DFU may require more or less frequent debridement according to individual wound characteristics; however, the new data from DDS can inform clinical decisions and service provision. The implications of weekly versus second-weekly debridement are discussed.

Circulating soluble CD163 as a potential biomarker of diabetes complications.

AIMS: To investigate whether soluble CD163 (sCD163) is altered in those with diabetes and various subtypes of complications and non-alcoholic fatty liver disease (NAFLD), and whether it can assess disease complications and severity in people with diabetes. METHODS: Adults with diabetes (n = 101) were recruited and assessed for the presence of any complications (D+Comps). Liver steatosis presence was determined by ultrasound and liver stiffness measurement (LSM) by transient elastography. Liver pathology other than non-alcoholic steatohepatitis (NASH) was excluded. Plasma sCD163 was measured by ELISA. RESULTS: sCD163 was higher in D+Comps (n = 59) compared to D-comps (n = 42) in those with microvascular complications (n = 56; 1.3-fold), including a 1.4-fold increase in chronic kidney disease (CKD) (n = 42). sCD163 correlated positively with HbA1c and urinary albumin-creatinine ratio and negatively with HDL-c in D+Comps. sCD163 was increased 1.7-fold in those with advanced NASH fibrosis (LSM ≥ 10.3 kPa, n = 19) compared to those without (LSM < 10.3 kPa, n = 80). The AUC-ROC-curve was 0.64 for sCD163 to detect CKD and 0.74 to detect advanced NASH fibrosis. CONCLUSIONS: In this study, the elevated circulating sCD163 occurred in people with diabetes who had microvascular complications or advanced NASH fibrosis, suggesting sCD163 may have clinical utility as a biomarker in certain diabetes complications and disease severity in NAFLD.

SGLT2 inhibitors for patients with type 2 diabetes and CKD: a narrative review.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors have recently emerged as an effective means to protect kidney function in people with type 2 diabetes and chronic kidney disease (CKD). In this review, we explore the role of SGLT2 inhibition in these individuals. SGLT2 inhibitors specifically act to inhibit sodium and glucose reabsorption in the early proximal tubule of the renal nephron. Although originally developed as glucose-lowering agents through their ability to induce glycosuria, it became apparent in cardiovascular outcome trials that the trajectory of kidney function decline was significantly slowed and the incidence of serious falls in kidney function was reduced in participants receiving an SGLT2 inhibitor. These observations have recently led to specific outcome trials in participants with CKD, including DAPA-CKD, CREDENCE and EMPA-KIDNEY, and real-world studies, like CVD-REAL-3, that have confirmed the observation of kidney benefits in this setting. In response, recent KDIGO Guidelines have recommended the use of SGLT2 inhibitors as first-line therapy in patients with CKD, alongside statins, renin-angiotensin-aldosterone system inhibitors and multifactorial risk factor management as indicated. However, SGLT2 inhibitors remain significantly underutilized in the setting of CKD. Indeed, an inertia paradox exists, with patients with more severe disease less likely to receive an SGLT2 inhibitor. Concerns regarding safety appear unfounded, as acute kidney injury, hyperkalaemia, major acute cardiovascular events and cardiac death in patients with CKD appear to be lower following SGLT2 inhibition. The first-in-class indication of dapagliflozin for CKD may begin a new approach to managing kidney disease in type 2 diabetes.

Asia-Pacific consensus recommendations for application of continuous glucose monitoring in diabetes management.

Glucose monitoring has evolved from self-monitoring of blood glucose to glycated hemoglobin, and the latest continuous glucose monitoring (CGM). A key challenge to adoption of CGM for management of diabetes in Asia is the lack of regional CGM recommendations. Hence, thirteen diabetes-specialists from eight Asia-Pacific (APAC) countries/regions convened to formulate evidence-based, APAC-specific CGM recommendations for individuals with diabetes. We defined CGM metrics/targets and developed 13 guiding-statements on use of CGM in: (1) people with diabetes on intensive insulin therapy, and (2) people with type 2 diabetes on basal insulin with/without glucose lowering drugs. Continual use of CGM is recommended in individuals with diabetes on intensive insulin therapy and suboptimal glycemic control, or at high risk of problematic hypoglycemia. Continual/intermittent CGM may also be considered in individuals with type 2 diabetes on basal insulin regimen and with suboptimal glycemic control. In this paper, we provided guidance for optimizing CGM in special populations/situations, including elderly, pregnancy, Ramadan-fasting, newly diagnosed type 1 diabetes, and comorbid renal disease. Statements on remote CGM, and stepwise interpretation of CGM data were also developed. Two Delphi surveys were conducted to rate the agreement on statements. The current APAC-specific CGM recommendations provide useful guidance for optimizing use of CGM in the region.

Do diabetes-related foot ulcer wound fluid measures have clinical utility as biomarkers for healing? A systematic review.

OBJECTIVE: This study aimed to identify potential biomarkers reported in wound fluid of diabetes-related foot ulcers (DRFUs), and their ability to reflect current and prospective wound healing. METHOD: A systematic search was executed following the PRISMA methodology across five chosen databases: MEDLINE, Embase, Scopus, Cochrane Clinical Trials and Cochrane Systematic Reviews. Using keywords and phrases, it yielded 5022 results. RESULTS: Based on predetermined inclusion and exclusion criteria, 19 papers were included in the final analysis, among which: seven reported serial temporal biomarker changes in wounds; six reported measures from baseline and related them to healing rate and/or final healing outcome; four papers reported both end-points, and two papers reported solely on baseline biomarker levels in a generalised diabetic foot ulcer group. Across the studies, a total of 46 distinct markers were described from the wound fluid of n=1141 participants. Biomarkers examined included proteases, protease inhibitors, growth factors, chemokines and cytokines, with proteases being the largest subcategory making up 16 (34.8%) of the markers investigated (n=7). Matrix metalloproteinase-9 (MMP-9) was the most frequently investigated protease and it currently holds the most biomarker promise (n=5). Wound bacterial profiles variably related to wound healing outcome (n=5). One study reported biophysical markers rather than biomarkers, including measurement of wound fluid pH. Study quality was generally good. Drawing quantitative comparisons between papers was not possible due to variability in experimental design including sampling and assessment methods. CONCLUSION: These studies collectively indicate several wound fluid measures that could identify DRFU status and outcomes, and that methodological standardisation in the field is needed to determine reliable predictive thresholds for healing.

Diabetes-related foot disease: new insights with an antipodean focus.

Diabetes-related foot disease (DFD), defined as ulceration, infection or destruction of tissues of the foot in a person with current or previously diagnosed diabetes mellitus, is associated with a heavy burden for both patients and the healthcare system with high morbidity, mortality and costs. Improved outcomes for people with DFD are achieved with an interdisciplinary approach and adherence to best practice clinical guidelines; however, in the Australian context, the vastness of the country presents unique challenges in achieving optimal outcomes for all people with DFD, with variation in service delivery, availability and accessibility between metropolitan, rural and remote areas. Aboriginal and Torres Strait Islander Australians and people with diabetes living in rural and remote areas experience higher rates of lower-extremity amputation, and further efforts and resources are required to improve outcomes for these high-risk groups. In recent years, there have been advances in knowledge, including the understanding of the pathogenesis of diabetes-related peripheral neuropathy, genetic polymorphisms and mechanisms of disease associated with acute Charcot neuroarthropathy, biomarkers and potential mediators of diabetes-related foot ulcer (DFU) healing, the microbiology and microbiome profile of DFUs, pressure assessment and management as well as an expanded understanding of DFU sequelae and comorbidities. In this review, we describe new insights into pathophysiology, sequelae and comorbidities of DFD with a focus on basic and translational aspects and contributions to the field from Australian and New Zealand DFD researchers.

Dose-dependent response to long-term clomiphene citrate in male functional hypogonadotropic hypogonadism.

Summary: Functional hypogonadotropic hypogonadism is a relatively common condition in middle-aged to elderly men that can significantly impair quality of life. Besides lifestyle optimisation, androgen replacement remains the mainstay of treatment; however, its adverse effects on spermatogenesis and testicular atrophy are undesirable. Clomiphene citrate is a selective oestrogen receptor modulator that acts centrally to increase endogenous testosterone without affecting fertility. Although it has demonstrated effectiveness in shorter-duration studies, its longer-term outcomes are less well-documented. In this study, we report the case of a 42-year-old male with functional hypogonadotropic hypogonadism who sustained an excellent dose-dependent, titratable clinical and biochemical response to clomiphene citrate with no known adverse effects for 7 years to date. This case highlights that clomiphene citrate has potential as a safe and titratable longer-term treatment option, and the need for further randomised control trials in therapy options to normalise androgen status. Learning points: Functional hypogonadotropic hypogonadism is a relatively common, but likely underdiagnosed, condition in middle-aged to older males. Testosterone replacement is the current mainstay of endocrine therapy but can cause sub-fertility and testicular atrophy. Clomiphene citrate is a serum oestrogen receptor modulator that acts centrally to increase endogenous testosterone production without affecting fertility. It has potential as a safe and efficacious longer-term treatment option that can be titrated to increase testosterone and relieve clinical symptoms in a dose-dependent manner. Longitudinal prospective studies as randomised control trials evaluating alternatives to exogenous testosterone are required.

Timing of minor and major amputation in patients with diabetes-related foot ulceration admitted to two public tertiary referral hospitals in Australia.

BACKGROUND: There is limited information regarding the number of patients with diabetes-related foot ulceration (DFU) who receive minor or major amputation, and how quickly these amputations occur. This study aimed to identify the incidence of index minor and major amputation among inpatients with DFU over 4 years, and where amputation occurred during the patient's index DFU-related admission, investigate prognostic factors. METHODS: The incidence of index minor and major amputation, and the admission sequence during which amputation occurred were identified from DFU-related admissions to two public hospitals during 2014-2018. Where minor or major amputation occurred during the patient's index DFU-related admission, prognostic factors were investigated using logistic regression. RESULTS: DFU-related hospital admissions were required by 564 patients. The incidence of minor amputation over 4 years was 34% (n = 193). The incidence of minor amputation during the patient's index DFU-related admission was 28% (n = 155), which was associated with requiring revascularisation (odds ratio [OR] 2.33, 95% CI 1.53-3.55, P < 0.001). The incidence of major amputation over 4 years was 8% (n = 45). The incidence of major amputation during the patient's index DFU-related admission was 6% (n = 31), which was associated with having more comorbidities (OR 1.58, 95% CI 1.10-2.26, P = 0.01) and receiving care for a mental health condition (OR 3.85, 95% CI 1.48-10.01, P = 0.006). CONCLUSION: Most amputations occurred during the patient's index DFU-related hospital admission. Major amputation during a patient's index admission was associated with more comorbidities and mental health conditions.

Constant-moderate versus high-intensity interval training on heart adiponectin levels in high-fat fed mice: a preventive and treatment approach.

CONTEXT: Research has described that adiponectin plays a key role in cardiomyocytes metabolism, however, the effects of exercise during obesity on cardiac adiponectin levels is unclear. OBJECTIVE: To investigate the effects of constant-moderate endurance (END) and high-intensity interval training (HIIT), on heart adiponectin levels in mice. MATERIAL AND METHODS: Two experiments were conducted: (1) preventive (EX1): 10 week-old male mice were fed standard (CHOW) or high-fat diet (HFD;45% fat) and simultaneously trained with END and HIIT for 10 weeks; (2) Treatment (EX2): after 10 weeks of dietary intervention, another cohort of 10 week-old mice were trained by both programmes for 10 weeks. RESULTS: In EX1, END and HIIT decreased low-molecular weight adiponectin (∼0.5-fold; p < 0.05) and increased GLUT4 levels (∼2-fold; p < .05). In EX2, HFD significantly decreased high-molecular weight adiponectin (∼0.7-fold; p < .05), and END reversed this change.Discussion and conclusion: HFD and exercise influence heart adiponectin isoforms and therefore might impact cardiomyocyte metabolism.

Regulation of CCN2 and Its Bioactivity by Advanced Glycation End Products.

Advanced glycation end products (AGEs) have been implicated in the tissue fibrosis and extracellular matrix (ECM) expansion in organ complications of diabetes mellitus and in other diseases. CCN2, also known as cellular communication factor 2 and earlier as connective tissue growth factor, is a matrix-associated protein that acts as a pro-fibrotic cytokine to cause fibrosis in tissues in many diseases. We were the first to report that AGEs regulate CCN2, which itself can then affect ECM synthesis. In this chapter, we describe the methods of preparation of soluble AGEs and matrix-bound AGEs that can be used to study AGE effect on CCN2 and ECM expansion.

The Effect of TGFß1 in Adipocyte on Inflammatory and Fibrotic Markers at Different Stages of Adipocyte Differentiation.

Transforming growth factor beta (TGFß) is a versatile cytokine. Although a profibrotic role of TGFß is well established, its effect on tissue inhibitor of metalloproteinase (TIMPs) and inflammatory mediators are incompletely described. This study investigates the profibrotic and pro-inflammatory role of TGFß1 during adipocyte differentiation. NIH3T3L1 cells were used for the in vitro study and were differentiated by adding a standard differentiation mix either with rosiglitazone (R-Diff) or without (S-Diff). Recombinant TGFß1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFß1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFß1 upregulated collagen VI mRNA and its regulators connective tissue growth factor (CCN2/CTGF), TIMP1 and TIMP3 mRNA levels in undifferentiated preadipocytes and adipocytes at commitment stage. But in the terminal differentiated adipocytes, changes in mRNA and protein of collagen VI and TIMP3 mRNA were not observed despite an increase in CCN2/CTGF, TIMP1 mRNA. Although TGFß1 upregulated interleukin-6 (IL6) and monocyte chemoattractant protein-1 (MCP1) mRNA at all stages of differentiation, decreased tumor necrosis factor-α (TNFα) mRNA was observed early in adipocyte differentiation. This study highlights the complex role of TGFß1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation.