Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

Effect of biotin supplementation on fatty acid metabolic pathways in 3T3-L1 adipocytes.

Several studies have shown that pharmacological concentrations of biotin decrease serum lipid concentrations and the expression of lipogenic genes. Previous studies on epididymal adipose tissue in mice revealed that 8 weeks of dietary biotin supplementation increased the protein abundance of the active form of AMPK and the inactive forms acetyl CoA carboxylase (ACC)-1 and - 2, and decreased serum free fatty acid concentrations but did not affect lipolysis. These data suggest that pharmacological concentrations of the vitamin might affect fatty acid metabolism. In this work, we investigated the effects of pharmacological biotin concentrations on fatty acid synthesis, oxidation, and uptake in 3T3-L1 adipocytes. Similar to observations in mice, biotin-supplemented 3T3-L1 adipose cells increased the protein abundance of active T172 -AMPK and inactive ACC-1 and -2 forms. No changes were observed in the expression of the transcriptional factor PPARα and carnitine-palmitoyltransferase-1 (CPT-1). Radiolabeled assays indicated a decrease in fatty acid synthesis; an increase in fatty acid oxidation and fatty acid incorporation rate into the lipid fraction between control cells and biotin-supplemented cells. The data revealed an increase in the mRNA abundance of the fatty acid transport proteins Fatp1 and Acsl1 but not Cd36 or Fatp4 mRNA. Furthermore, the abundance of glycerol phosphate acyl transferase-3 protein was increased. Triglyceride content was not affected. Lipid droplet numbers showed an increase and their areas were smaller in the biotin-supplemented group. In conclusion, these data indicate that biotin supplementation causes a decrease in fatty acid synthesis and an increase in its oxidation and uptake. © 2018 BioFactors, 45(2):259-270, 2019.

Effects of Biotin Supplementation During the First Week Postweaning Increases Pancreatic Islet Area, Beta-Cell Proportion, Islets Number, and Beta-Cell Proliferation.

During maturation, pancreatic islets achieve their full capacity to secrete insulin in response to glucose, undergo morphological changes in which alpha-cells decrease and beta-cell mass increases, and they acquire the normal alpha- and beta-cell proportion changes that are important for islet functions later in life. In rodents, the first week of postweaning is critical for islet maturation. Multiple studies have documented the detrimental effects of several conditions on pancreatic maturation; however, few studies have addressed the use of pharmacological agents to enhance islet maturation. Biotin might have a potential action on islet maturation. Pharmacological concentrations of biotin have been found to modify islet morphology and function. In a previous study, we found that mice fed a biotin-supplemented diet for 8 weeks after weaning showed an increase in basal and glucose stimulated insulin secretion, enlarged islet size, and modified islet structure. In the present study, we investigated the effect of biotin on maturation features during the first week postweaning. Female BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 1 week after weaning. Compared with the control, biotin-supplemented mice showed an increase in pancreatic islet number and area in addition to an augmented proportion of beta-cells in the islet. These effects were related to an increase in beta-cell proliferation. No differences were found in insulin secretion, blood glucose concentrations, or serum insulin levels. These results indicate that biotin supplementation is capable of affecting beta-cell proliferation and might be a therapeutic agent for establishing strategies for regenerative medicine.

Effects of dietary biotin supplementation on glucagon production, secretion, and action.

OBJECTIVE: Despite increasing evidence that pharmacologic concentrations of biotin modify glucose metabolism, to our knowledge there have not been any studies addressing the effects of biotin supplementation on glucagon production and secretion, considering glucagon is one of the major hormones in maintaining glucose homeostasis. The aim of this study was to investigate the effects of dietary biotin supplementation on glucagon expression, secretion, and action. METHODS: Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 wk postweaning. Glucagon gene mRNA expression was measured by the real-time polymerase chain reaction. Glucagon secretion was assessed in isolated islets and by glucagon concentration in plasma. Glucagon action was evaluated by glucagon tolerance tests, phosphoenolpyruvate carboxykinase (Pck1) mRNA expression, and glycogen degradation. RESULTS: Compared with the control group, glucagon mRNA and secretion were increased from the islets of the biotin-supplemented group. Fasting plasma glucagon levels were higher, but no differences between the groups were observed in nonfasting glucagon levels. Despite the elevated fasting glucagon levels, no differences were found in fasting blood glucose concentrations, fasting/fasting-refeeding glucagon tolerance tests, glycogen content and degradation, or mRNA expression of the hepatic gluconeogenic rate-limiting enzyme, Pck1. CONCLUSIONS: These results demonstrated that dietary biotin supplementation increased glucagon expression and secretion without affecting fasting blood glucose concentrations or glucagon tolerance and provided new insights into the effect of biotin supplementation on glucagon production and action.

Dietary Biotin Supplementation Modifies Hepatic Morphology without Changes in Liver Toxicity Markers.

Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers.

Effects of Biotin Supplementation in the Diet on Adipose Tissue cGMP Concentrations, AMPK Activation, Lipolysis, and Serum-Free Fatty Acid Levels.

Several studies have shown that pharmacological concentrations of biotin decrease hyperlipidemia. The molecular mechanisms by which pharmacological concentrations of biotin modify lipid metabolism are largely unknown. Adipose tissue plays a central role in lipid homeostasis. In the present study, we analyzed the effects of biotin supplementation in adipose tissue on signaling pathways and critical proteins that regulate lipid metabolism, as well as on lipolysis. In addition, we assessed serum fatty acid concentrations. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (control: 1.76 mg biotin/kg; supplemented: 97.7 mg biotin/kg diet) over 8 weeks postweaning. Compared with the control group, biotin-supplemented mice showed an increase in the levels of adipose guanosine 3',5'-cyclic monophosphate (cGMP) (control: 30.3±3.27 pmol/g wet tissue; supplemented: 49.5±3.44 pmol/g wet tissue) and of phosphorylated forms of adenosine 5'-monophosphate-activated protein kinase (AMPK; 65.2%±1.06%), acetyl-coenzyme A (CoA), carboxylase-1 (196%±68%), and acetyl-CoA carboxylase-2 (78.1%±18%). Serum fatty acid concentrations were decreased (control: 1.12±0.04 mM; supplemented: 0.91±0.03 mM), and no change in lipolysis was found (control: 0.29±0.05 µmol/mL; supplemented: 0.33±0.08 µmol/mL). In conclusion, 8 weeks of dietary biotin supplementation increased adipose tissue cGMP content and protein expression of the active form of AMPK and of the inactive forms of acetyl-CoA carboxylase-1 and acetyl-CoA carboxylase-2. Serum fatty acid levels fell, and no change in lipolysis was observed. These findings provide insight into the effects of biotin supplementation on adipose tissue and support its use in the treatment of dyslipidemia.

The hypotriglyceridemic effect of biotin supplementation involves increased levels of cGMP and AMPK activation.

In addition to its role as a carboxylase cofactor, biotin modifies gene expression and has manifold effects on systemic processes. Several studies have shown that biotin supplementation reduces hypertriglyceridemia. We have previously reported that this effect is related to decreased expression of lipogenic genes. In the present work, we analyzed signaling pathways and posttranscriptional mechanisms involved in the hypotriglyceridemic effects of biotin. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg of free biotin/kg diet, respectively for 8 weeks after weaning. The abundance of mature sterol regulatory element-binding protein (SREBP-1c), fatty-acid synthase (FAS), total acetyl-CoA carboxylase-1 (ACC-1) and its phosphorylated form, and AMP-activated protein kinase (AMPK) were evaluated in the liver. We also determined the serum triglyceride concentrations and the hepatic levels of triglycerides and cyclic GMP (cGMP). Compared to the control group, biotin-supplemented mice had lower serum and hepatic triglyceride concentrations. Biotin supplementation increased the levels of cGMP and the phosphorylated forms of AMPK and ACC-1 and decreased the abundance of the mature form of SREBP-1c and FAS. These data provide evidence that the mechanisms by which biotin supplementation reduces lipogenesis involve increased cGMP content and AMPK activation. In turn, these changes lead to augmented ACC-1 phosphorylation and decreased expression of both the mature form of SREBP-1c and FAS. Our results demonstrate for the first time that AMPK is involved in the effects of biotin supplementation and offer new insights into the mechanisms of biotin-mediated hypotriglyceridemic effects.

Pharmacological concentrations of biotin reduce serum triglycerides and the expression of lipogenic genes.

Besides its role as a carboxylase prosthetic group, biotin regulates gene expression and has a wide repertoire of effects on systemic processes. Several studies have shown that pharmacological concentrations of biotin reduce hypertriglyceridemia. The molecular mechanisms by which pharmacological concentrations of biotin affect lipid metabolism are largely unknown. The present study analyzed the effects of pharmacological doses of biotin on triglyceridemia, insulin sensitivity and on mRNA expression of various lipogenic genes. Three-week-old male BALB/cAnN Hsd mice were fed a biotin-control or a biotin-supplemented diet (1.76 or 97.7mg of free biotin/kg diet, respectively) over a period of eight weeks. Serum triglyceride concentrations, insulin and glucose tolerance and mRNA abundance of various lipogenic genes were investigated. The biotin-supplemented group showed 35% less serum triglycerides than control mice. In the liver, we found a significant (P<0.05) reduction of mRNA levels of SREBP1-c, glucose transporter-2, phosphofructokinase-1, pyruvate kinase, acetyl-CoA carboxylase and fatty acid synthase, while glucose-6-phosphate dehydrogenase expression increased. No changes in glucokinase, stearoyl-CoA desaturase-1, FoxO1 or PPAR-gamma expression were observed. In adipose tissue, we found a decreased expression of SREBP1c, glucose-6-phosphate deshydrogenase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase-1, phosphofructokinase-1 and PPAR-gamma, but no changes in FoxO1 expression. Moreover, the group fed a biotin-supplemented diet showed a significant decrease in adipose tissue weight. No differences in insulin sensitivity or serum insulin concentrations were observed between groups. Our results indicate that pharmacological concentrations of biotin decrease serum tryglyceride concentrations and lipogenic gene expression in liver and adipose tissues.

Biotin deficiency and biotin excess: effects on the female reproductive system.

Biotin deficiency and biotin excess have both been found to affect reproduction and cause teratogenic effects. In the reproductive tract, however, the effects of biotin have not been well established yet. We investigated the effects of varying biotin content diets on the oestrus cycle, ovarian morphology, estradiol and progesterone serum levels, and the uterine mRNA abundance of their nuclear receptors, as well as on the activity of the estradiol-degrading group of enzymes cytochrome P450 (CYP) in the liver. Three-week-old female BALB/cAnN Hsd mice were fed a biotin-deficient, a biotin-control, or a biotin-supplemented diet (0, 7.2 or 400 micromol of free biotin/kg diet, respectively) over a period of nine weeks. Striking effects were observed in the biotin-deficient group: mice showed arrested estrous cycle on the day of diestrus and changes in ovary morphology. Estradiol serum concentration increased 49.2% in biotin-deficient mice compared to the control group, while the enzymatic activities of CYP1A2 and CYP2B2 increased (P<0.05). The mRNA abundance of nuclear estrogen and progesterone receptors decreased in the biotin-deficient mice. In the biotin-supplemented group we found that, in spite of a significant (P<0.05) decrease in the number of primary and Graafian follicles and in CYP1A2 activities, mice exhibited 105.4% higher serum estradiol concentration than the control group. No changes in the expression of the nuclear receptors were observed. No significant differences were observed in serum progesterone among the groups. Our results indicate that both the deficiency and the excess of biotin have significant effects on the female mouse reproductive system.

Biotin deficiency in mice is associated with decreased serum availability of insulin-like growth factor-I.

BACKGROUND: Biotin deficiency leads to decreased weight and nose-rump length in mice. AIM OF THE STUDY: The mechanisms underlying this impairment in body growth are yet unclear. Biotin restriction, however, could affect the availability of growth hormone (GH) and/or insulin like growth factor-I (IGF-I) since both hormones control body growth. We then conducted a correlative study aimed at establishing whether biotin dietary restriction is associated with decreased GH/IGF-I serum concentrations. METHODS: Levels of GH and IGF-I were measured through ELISA in serum samples of male BALB/cAnN mice fed with: 1] standard chow diet (control diet); 2] 30% egg-white biotin-deficient diet; or 3] 30% egg-white diet supplemented with 16.4 micromol biotin per kilogram (biotin sufficient diet). Relative food consumption, as adjusted per gram of body weight, was also determined. GH and IGF-I measurements were taken individually for 20 weeks beginning at the postnatal week 3, when the animals started consuming the corresponding diets. In addition, femur's weight and longitudinal growth and the organization of its growth plate were all analyzed as indicators of GH/IGF-I function. RESULTS: No differences in relative food consumption were observed among the three groups of mice along the experimental period that was evaluated. IGF-I serum levels, but not GH ones, were decreased in biotin deficient mice. These animals also showed decreased femur's longitudinal growth, speed of lengthening and weight gain, as well as shorter and disorganized growth plates. CONCLUSIONS: This study shows that biotin dietary restriction is indeed associated with decreased availability of IGF-I and diminished long bone growth and elongation. These conditions could explain the impairment of longitudinal body growth previously reported in biotin deficient mice. Although cause-effect studies are still needed, we believe our results support the notion that biotin might modulate the availability of IGF-I.

Biotin supplementation reduces plasma triacylglycerol and VLDL in type 2 diabetic patients and in nondiabetic subjects with hypertriglyceridemia.

Biotin is a water-soluble vitamin that acts as a prosthetic group of carboxylases. Besides its role as carboxylase prosthetic group, biotin regulates gene expression and has a wide repertoire of effects on systemic processes. The vitamin regulates genes that are critical in the regulation of intermediary metabolism. Several studies have reported a relationship between biotin and blood lipids. In the present work we investigated the effect of biotin administration on the concentration of plasma lipids, as well as glucose and insulin in type 2 diabetic and nondiabetic subjects. Eighteen diabetic and 15 nondiabetic subjects aged 30-65 were randomized into two groups and received either 61.4 micromol/day of biotin or placebo for 28 days. Plasma samples obtained at baseline and after treatment were analyzed for total triglyceride, cholesterol, very low density lipoprotein (VLDL), glucose and insulin. We found that the vitamin significantly reduced (P=0.005) plasma triacylglycerol and VLDL concentrations. Biotin produced the following changes (mean of absolute differences between 0 and 28 day treatment+/-S.E.M.): a) triacylglycerol -0.55+/-0.2 in the diabetic group and -0.92+/-0.36 in the nondiabetic group; b) VLDL: -0.11+/-0.04 in the diabetic group and -0.18+/-0.07 in the nondiabetic group. Biotin treatment had no significant effects on cholesterol, glucose and insulin in either the diabetic or nondiabetic subjects. We conclude that pharmacological doses of biotin decrease hypertriglyceridemia. The triglyceride-lowering effect of biotin suggests that biotin could be used in the treatment of hypertriglyceridemia.