Vitamin D and metabolic health:effects on molecular pathways of adipocyte inflammation and insulin resistance

Abstract Vitamin D is an essential micronutrient, playing a vital role in the regulation of calcium homeostasis and bone metabolism. In recent years, vitamin D has been increasingly recognized for its regulatory functions in immunomodulation, anti-inflammation, cellular proliferation and differentiation. The pandemic vitamin D deficiency has been associated with an increasing risk for several metabolic diseases. However, the effects of vitamin D deficiency and its supplementation on the underlying molecular mechanisms of these diseases are poorly understood. This thesis focuses on the effect of vitamin D on the regulation of molecular and metabolic signaling pathways involved in adipose tissue inflammation and hepatic insulin resistance. Study I examined the effect of vitamin D on the downregulation of inflammatory signaling pathways in adipose tissue. Vitamin D inhibited the translocation of the transcriptional factor NFκB as well as inhibiting the expression of inflammatory cytokines in human adipocytes differentiated from mesenchymal stromal cells (MSCs) and mature adipocytes isolated from intra-abdominal fat biopsies. In Study II, induction of vitamin D deficiency via a deficient diet impaired systemic glucose homeostasis and insulin sensitivity in lean and obese mice. Subsequent vitamin D re-supplementation restored the insulin sensitivity and ameliorated the hepatic tissue inflammation and oxidative stress in vitamin D deficient lean but not obese mice. In Study III, lower vitamin D levels were associated with a higher incidence of the metabolic syndrome in a cross-sectional cohort of older subjects living at latitude 65° North. In conclusion, vitamin D has an important role in modulating the molecular pathways involved in adipose tissue inflammation and insulin resistance in hepatic tissue. Adequate levels of vitamin D are necessary for the regulation of metabolic pathways to maintain body homeostasis. The generated data provide novel insights into the cellular mechanisms of vitamin D and represent a basis for oral vitamin D supplementation in the prevention of metabolic diseases.Tiivistelmä D-vitamiini on välttämätön mikroravinne, jolla on tärkeä tehtävä kalsiumin homeostaasin ja luun aineenvaihdunnan säätelyssä. Viime vuosina D-vitamiinin on todistettu osallistuvan yhä useampiin säätelytoimintoihin liittyen immuunivasteeseen, tulehduksen torjuntaan sekä solujen jakautumiseen ja erilaistumiseen. Maailmanlaajuisesti yleinen D-vitamiinin puutostila on yhdistetty useiden aineenvaihduntasairauksien kohonneeseen riskiin. D-vitamiinin puutoksen ja D-vitamiinilisän käytön vaikutusta näiden sairauksien molekyylimekanismeihin ei kuitenkaan tunneta vielä tarkemmin. Tämä opinnäytetyö keskittyy D-vitamiinin vaikutukseen rasvakudoksen tulehduksessa ja maksan insuliiniresistenssissä olevien molekyyli- ja metabolisten signalointireittien säätelyssä. Tutkimuksessa I tarkasteltiin D-vitamiinin vaikutusta rasvakudoksen tulehduksellisten signalointireittien hillitsemiseen. D-vitamiini inhiboi transkriptionaalisen tekijän NFκB:n siirtymistä ja tulehduksellisten sytokiinien ilmentymistä ihmisen mesenkymaalisista stroomasoluista (MSC) erilaistetuissa rasvasoluissa sekä sappirakkoleikkausten yhteydessä otetuista rasvanäytteistä eristetyissä kypsissä rasvasoluissa. Tutkimuksessa II havaittiin, että D-vitamiinin puutoksen aiheuttaminen puutteellisen ruokavalion avulla heikensi verenkierron glukoositasapainoa ja insuliiniherkkyyttä sekä hoikilla, että liikalihavilla hiirillä. Sitä vastoin D-vitamiini lisäravinteena palautti insuliiniherkkyyden ja lievitti maksakudoksen tulehdusta sekä oksidatiivista stressiä D-vitamiini puutteellisilla hoikilla hiirillä, mutta ei liikalihavilla hiirillä. Tutkimuksessa III selvisi, että kohortissa matalammat D-vitamiinitasot ovat yhteydessä suurempaan metabolisen oireyhtymän esiintyvyyteen iäkkäämmillä koehenkilöillä, jotka elävät pohjoisella pallonpuoliskolla (65° N). Yhteenvetona voidaan todeta, että D-vitamiinilla on tärkeä säätelevä rooli rasvakudostulehduksen ja insuliiniresistenssin molekyylireittien kontrolloinnissa maksakudoksessa. Riittävät D-vitamiinitasot ovat välttämättömiä aineenvaihduntareittien säätelyssä kehon homeostaasissa. Tutkimuksen tulokset tarjoavat uusia näkökulmia D-vitamiinin roolista solumekanismien säätelyssä ja perustan D-vitamiinilisien käytöstä aineenvaihduntasairauksissa

Vitamin D deficiency induces insulin resistance and re‐supplementation attenuates hepatic glucose output via the PI3K‐AKT‐FOXO1 mediated pathway

Abstract Background: Pandemic vitamin D deficiency is associated with insulin resistance and type 2 diabetes. Vitamin D supplementation has been reported to have improved glucose homeostasis. However, its mechanism to improve insulin sensitivity remains unclear. Methods and results: Male C57BL/6J mice are fed with/without vitamin D control (CD) or Western (WD) diets for 15 weeks. The vitamin‐D‐deficient lean (CDVDD) and obese (WDVDD) mice are further subdivided into two groups. One group is re‐supplemented with vitamin D for 6 weeks and hepatic insulin signaling is examined. Both CD and WD mice with vitamin D deficiency developed insulin resistance. Vitamin D supplementation in CDVDD mice significantly improved insulin sensitivity, hepatic inflammation, and antioxidative capacity. The hepatic insulin signals like pAKT, pFOXO1, and pGSK3β are increased and the downstream Pepck, G6pase, and Pgc1α are reduced. Furthermore, the lipogenic genes Srebp1c, Acc, and Fasn are decreased, indicating that hepatic lipid accumulation is inhibited. Conclusion: The results demonstrate that vitamin D deficiency induces insulin resistance. Its supplementation has significant beneficial effects on pathophysiological mechanisms in type 2 diabetes but only in lean and not in the obese phenotype. The increased subacute inflammation and insulin resistance in obesity cannot be significantly alleviated by vitamin D supplementation. This needs to be taken into consideration in the design of new clinical trials

Vitamin D status and components of metabolic syndrome in older subjects from northern Finland (latitude 65° north)

Abstract Introduction: Vitamin D deficiency has been linked to the increased risk of several chronic diseases, especially in people living in the Northern Latitudes. The aim of this study was to assess the vitamin D status in older subjects born in 1945 in Northern Finland (latitude 65°North), and to examine its associations to components of metabolic syndrome (MetS). Methods: In this cross-sectional study, we invited 904 subjects born in 1945 from the Oulu region (Oulu45 cohort), out of an original cohort of 1332 subjects. In the cohort, plasma 25 hydroxyvitamin D (25OHD) levels were determined by an enzyme immunoassay of 263 men and 373 women, with a mean age baseline of 69±0.5 years old. We assessed the participants’ usage of vitamin D supplements, as well as their lifestyle factors, using a questionnaire. Results: Nearly 80% of the subjects had low vitamin D levels [either vitamin D deficient (<50 nmol/L) or insufficient (50–75 nmol/L)], and only 20% of the participants had sufficient vitamin D levels (>75 nmol/L) (based on the American Endocrine Society guidelines). The low vitamin D status was associated with a high prevalence of MetS; a significantly higher number of subjects with MetS (41%) had low vitamin D levels in comparison to the non-MetS subjects (38%) (p ≤ 0.05). The subjects under vitamin D supplementation had a significantly lower incidence of MetS (42.6% vs 57.4%) and its components in comparison to the non-supplemented subjects (p ≤ 0.05). Conclusions: Low vitamin D levels are a risk factor for MetS amongst other lifestyle factors, such as dietary habits and physical inactivity, among older subjects in the Northern Latitudes (65°North). Optimal supplementation of vitamin D, along with rich dietary sources of vitamin D, are highly recommended for older subjects as a means to positively affect, e.g., hypertension, insulin resistance, and obesity, as components of the MetS

Multi-day prolonged low- to moderate-intensity endurance exercise mimics training improvements in metabolic and oxidative profiles without concurrent chromosomal changes in healthy adults

Abstract Background: Oxidative stress results in lipid, protein, and DNA oxidation, resulting in telomere erosion, chromosomal damage, and accelerated cellular aging. Training promotes healthy metabolic and oxidative profiles whereas the effects of multi-day, prolonged, and continuous exercise are unknown. This study investigated the effects of multi-day prolonged exercise on metabolic and oxidative stress as well as telomere integrity in healthy adults. Methods: Fifteen participants performed a 14-day, 260-km, wilderness canoeing expedition (12 males) (EXP) (24 ± 7 years, 72 ± 6 kg, 178 ± 8.0 cm, 18.4 ± 8.4% BF, 47.5 ± 9.3 mlO₂ kg⁻¹ min⁻¹), requiring 6–9 h of low- to moderate-intensity exercise daily. Ten controls participated locally (seven males) (CON) (31 ± 11 years, 72 ± 15 kg, 174 ± 10 cm, 22.8 ± 10.0% BF, 47.1 ± 9.0 mlO₂ kg⁻¹ min⁻¹). Blood plasma, serum, and mononuclear cells were sampled before and after the expedition to assess hormonal, metabolic, and oxidative changes. Results: Serum cholesterol, high- and low-density lipoprotein, testosterone, insulin, sodium, potassium, urea, and chloride concentrations were not different between groups, whereas triglycerides, glucose, and creatinine levels were lower following the expedition (p < 0.001). Malondialdehyde and relative telomere length (TL) were unaffected (EXP: 4.2 ± 1.3 vs. CON: 4.1 ± 0.7 μM; p > 0.05; EXP: 1.00 ± 0.48 vs. CON: 0.89 ± 0.28 TS ratio; p = 0.77, respectively); however, superoxidase dismutase activity was greater in the expedition group (3.1 ± 0.4 vs. 0.8 ± 0.5 U ml⁻¹; p < 0.001). Conclusion: These results indicate a modest improvement in metabolic and oxidative profiles with increased superoxidase dismutase levels, suggesting an antioxidative response to counteract the exercise-associated production of free radicals and reactive oxygen species during prolonged exercise, mimicking the effects from long-term training. Although improved antioxidant activity may lead to increased TL, the present exercise stimulus was insufficient to promote a positive cellular aging profile with concordant chromosomal changes in our healthy and young participants

Association of physical activity with telomere length among elderly adults:the Oulu cohort 1945

Abstract Introduction: Physical activity (PA) has been associated with telomere shortening. The association of PA intensity or volume with telomere length (TL) is nonetheless unclear. The aim of our study was to investigate the associations of exercise intensity and volume with TL in elderly adults from Northern Finland (65° latitude North). Methods: Seven hundred elderly subjects born in 1945 in the Oulu region were investigated. PA was measured during a 2-week period with a wrist-worn accelerometer. In addition, a questionnaire was used to assess sedentary time and to achieve a longitudinal PA history and intensity. Relative telomere lengths (RTL) were determined from frozen whole blood samples using a qPCR-based method. Results: Relative telomere lengths were significantly longer in women than men and negatively correlated with age in both genders (men r = -0.210, p = 0.000, women r = -0.174, and p = 0.000). During the 2-week study period, women took more steps than men (p = 0.001), but the association between steps and RTL was only seen in men (p = 0.05). Total steps taken (r = 0.202 and p = 0.04) and sedentary time (r = -0.247 and p = 0.007) significantly correlated with RTLs in 70-year old subjects. Moderate PA was associated with RTL in subjects with the highest quartile of moderate PA compared to the three lower quartiles (p-values: 0.023 between 4th and 1st, 0.04 between 4th and 2nd, and 0.027 between 4th and 3rd) in the 70-year old subjects. Conclusion: Women had longer RTL and a higher step count compared to men. However, exercise volume and RTL correlated positively only in men. Surprisingly, age correlated negatively with RTL already within an age difference of 2 years. This suggests that telomere attrition rate may accelerate in older age. Moderate physical activity at the time of study was associated with RTL

Systemic inflammation is associated with circulating cell death released keratin 18 fragments in colorectal cancer

Abstract Systemic inflammation is a stage-independent marker of poor prognosis in colorectal cancer (CRC), activated in a complex, multifactorial process. It has been proposed that one of the main factors driving systemic inflammation may be tumor necrosis. Keratin 18 (KRT18) fragments are released from dead cells and their serum levels are markers for apoptotic and necrotic cell death. In CRC, high KRT18 levels associate with advanced disease, but their relationship with tumor necrosis and systemic inflammation is unknown. In this study, serum total soluble KRT18 (tKRT18) and apoptosis-related, caspase-cleaved fragment (aKRT18) levels were measured preoperatively from 328 CRC patients, and their difference was calculated to assess necrosis related KRT18 (nKRT18) levels. The relationships of these markers with tumor necrosis, clinicopathologic features, systemic inflammation markers (C-reactive protein, albumin, and 13 cytokines), and survival were analyzed. High serum tKRT18, aKRT18, and nKRT18 levels showed association with a higher extent of tumor necrosis, distant metastasis, and increased levels of several markers of systemic inflammation, including CXCL8. High serum tKRT18 (multivariable HR 1.94, 95% CI 1.28–2.95, p = 0.002) and nKRT18 (multivariable HR 1.87, 95% CI 1.24–2.82, p = 0.003) levels were associated with poor overall survival independent of potential confounding factors. Our results show that tumor necrosis in CRC contributes to serum levels of KRT18 fragments, and both necrosis and KRT18 levels associate with systemic inflammation. Moreover, we show that serum tKRT18 and nKRT18 levels have independent prognostic value in CRC. Our observations confirm the link between cell death and systemic inflammation

Serum enterolactone concentrations are low in colon but not in rectal cancer patients

Abstract The dietary lignan metabolite, enterolactone, has been suggested to have anti-cancer functions, and high serum enterolactone concentrations have been associated with decreased risk of breast and prostate cancers. We hypothesized that serum enterolactone concentrations as a marker of plant-based foods are associated with decreased risk in colorectal cancer (CRC). We measured serum enterolactone glucuronide and sulfate concentrations by liquid chromatography-tandem mass spectrometry in 115 CRC patients and 76 sex- and age-matched controls and analyzed the results with respect to tumor parameters, clinical parameters, and systemic inflammatory markers. Patients with colon cancer had significant lower serum enterolactone glucuronide and sulfate concentrations than controls (glucuronide: median 3.14 nM vs. 6.32 nM, P < 0.001; sulfate: median 0.13 nM vs. 0.17 nM, P = 0.002), whereas rectal cancer patients had similar enterolactone levels as controls (glucuronide: median 5.39 nM vs. 6.32 nM, P = 0.357; sulfate: median 0.19 nM vs. 0.17 nM, P = 0.452). High serum enterolactone concentrations were associated with low tumor grade, high serum creatinine levels, and concomitant diabetes. In summary, our results suggest that serum enterolactone concentrations are decreased in colon but not in rectal cancer. Further investigations are required to assess whether this reflects an altered lignan metabolism by the colon microbiome

Leukocyte telomere length is not reduced in children and adults with cystic fibrosis but associates with clinical characteristics:a cross-sectional study

Abstract We hypothezied that telomere length is considerably altered in cystic fibrosis (CF) patients compared to healthy subjects (HS), and that leukocyte telomere length variation reflects the severity of CF. Relative telomere length (RTL) was assessed by qPCR in 70 children aged 5–10 (34 CF; 36 HS) and 114 adults aged 18–45 (53 CF; 61 HS). Telomere length was similar in CF and HS (median (interquartile range): 0.799 (0.686–0.950) vs. 0.831 (0.707–0.986); p = 0.5283) both in children and adults. In adults, women had longer telomeres than men (0.805 (0.715–0.931) vs. 0.703 (0.574–0.790); p = 0.0002). Patients treated with inhaled corticosteroids had a shorter RTL compared to those without steroid therapy (0.765 (0.664–0.910) vs. 0.943 (0.813–1.191); p = 0.0007) and this finding remained significant after adjusting for gender, age, BMI, and child/adult status (p = 0.0003). Shorter telomeres were independently associated with the presence of comorbidities (0.763 (0.643–0.905) vs. 0.950 (0.783–1.130); p = 0.0006) and antibiotic treatment at the moment of blood sampling (0.762 (0.648–0.908) vs. 0.832 (0.748–1.129); p = 0.0172). RTL correlated with number of multiple-day hospitalizations (rho = −0.251; p = 0.0239), as well as number of hospitalization days (rho = −0.279; p = 0.0113). Leukocyte RTL in children and adults with CF was not shorter than in healthy controls, and did not seem to have any potential as a predictor of CF survival. However, it inversely associated with the investigated clinical characteristics

Increased protein intake affects pro-opiomelanocortin (POMC) processing, immune function and IGF signaling in peripheral blood mononuclear cells of home-dwelling old subjects using a genome-wide gene expression approach

Abstract Background: Adequate protein intake among older adults is associated with better health outcomes such as immune function and metabolic regulation of skeletal muscle, but conflicting results make it difficult to define the optimal intake. To further understand the impact of protein intake on metabolic processes, the aim of the study was to explore genome-wide gene expression changes in peripheral blood mononuclear cells (PBMCs) in home-dwelling old subjects after increased protein intake for 12 weeks. Method: In a parallel double-blind randomized controlled intervention study, subjects (≥ 70 years) received a protein-enriched milk (2 × 20 g protein/day, n = 14, mean (±SD) age 76.9 ± 4.9 years) or an isocaloric carbohydrate drink (n = 17, mean (±SD) age 77.7 ± 4.8 years) for breakfast and evening meal for 12 weeks. PBMCs were isolated before and after the intervention. Microarray analysis was performed using Illumina technology. Serum levels of gut peptides and insulin growth factor (IGF)-1 were also measured. Results: In total 758 gene transcripts were regulated after increased protein intake, and 649 gene transcripts were regulated after intake of carbohydrates (p &lt; 0.05). Forty-two of these genes were overlapping. After adjusting for multiple testing, 27 of the 758 gene transcripts were regulated (FDR, q-value &lt; 0.25) after protein intake. Of these 25 were upregulated and two downregulated. In particular, genes and signaling pathways involved in pro-opiomelanocortin (POMC) processing, immune function, and IGF signaling were significantly altered. Conclusions: PBMCs can be used to study gene expression changes after long-term protein intake, as many signaling pathways were regulated after increased protein intake. The functional significance of these findings needs to be further investigated

Role of brown and beige adipose tissues in seasonal adaptation in the raccoon dog (Nyctereutes procyonoides)

Abstract Brown adipose tissue (BAT) expresses uncoupling protein-1 (UCP1), which enables energy to be exerted towards needed thermogenesis. Beige adipocytes are precursor cells interspersed among white adipose tissue (WAT) that possess similar UCP1 activity and capacity for thermogenesis. The raccoon dog (Nyctereutes procyonoides) is a canid species that utilizes seasonal obesity to survive periods of food shortage in climate zones with cold winters. The potential to recruit a part of the abundant WAT storages as beige adipocytes for UCP1-dependent thermogenesis was investigated in vitro by treating raccoon dog adipocytes with different browning inducing factors. In vivo positron emission tomography/computed tomography (PET/CT) imaging with the glucose analog ¹⁸F-FDG showed that BAT was not detected in the adult raccoon dog during the winter season. In addition, UCP1 expression was not changed in response to chronic treatments with browning inducing factors in adipocyte cultures. Our results demonstrated that most likely the raccoon dog endures cold weather without the induction of BAT or recruitment of beige adipocytes for heat production. Its thick fur coat, insulating fat, and muscle shivering seem to provide the adequate heat needed for surviving the winter