Motoyuki Igata

AMPK and cell proliferation – AMPK as a therapeutic target for atherosclerosis and cancer

AMPK is a serine/threonine protein kinase, which serves as an energy sensor in all eukaryotic cell types. Published studies indicate that AMPK activation strongly suppresses cell proliferation in non‐malignant cells as well as in tumour cells. These actions of AMPK appear to be mediated through multiple mechanisms including regulation of the cell cycle and inhibition of protein synthesis, de novo fatty acid synthesis, specifically the generation of mevalonate as well as other products downstream of mevalonate in the cholesterol synthesis pathway. Cell cycle regulation by AMPK is mediated by up‐regulation of the p53–p21 axis as well as regulation of TSC2–mTOR (mammalian target of rapamycin) pathway. The AMPK signalling network contains a number of tumour suppressor genes including LKB1, p53, TSC1 and TSC2, and overcomes growth factor signalling from a variety of stimuli (via growth factors and by abnormal regulation of cellular proto‐oncogenes including PI3K, Akt and ERK). These observations suggest that AMPK activation is a logical therapeutic target for diseases rooted in cellular proliferation, including atherosclerosis and cancer. In this review, we discuss about exciting recent advances indicating that AMPK functions as a suppressor of cell proliferation by controlling a variety of cellular events in normal cells as well as in tumour cells.

Caloric restriction decreases ER stress in liver and adipose tissue in ob/ob mice

Endoplasmic reticulum (ER) stress plays a crucial role in the development of insulin resistance and diabetes. Although caloric restriction (CR) improves obesity-related disorders, the effects of CR on ER stress in obesity remain unknown. To investigate how CR affects ER stress in obesity, ob/ob mice were assigned to either ad libitum (AL) (ob-AL) or CR (ob-CR) feeding (2 g food/day) for 1-4 weeks. The body weight (BW) of ob-CR mice decreased to the level of lean AL-fed littermates (lean-AL) within 2 weeks. BW of lean-AL and ob-CR mice was less than that of ob-AL mice. The ob-CR mice showed improved glucose tolerance and hepatic insulin action compared with ob-AL mice. Levels of ER stress markers such as phosphorylated PKR-like ER kinase (PERK) and eukaryotic translation initiation factor 2α and the mRNA expression of activating transcription factor 4 were significantly higher in the liver and epididymal fat from ob-AL mice compared with lean-AL mice. CR for 2 and 4 weeks significantly reduced all of these markers to less than 35% and 50%, respectively, of the levels in ob-AL mice. CR also significantly reduced the phosphorylation of insulin receptor substrate (IRS)-1 and c-Jun NH(2)-terminal kinase (JNK) in ob/ob mice. The CR-mediated decrease in PERK phosphorylation was similar to that induced by 4-phenyl butyric acid, which reduces ER stress in vivo. In conclusion, CR reduced ER stress and improved hepatic insulin action by suppressing JNK-mediated IRS-1 serine-phosphorylation in ob/ob mice.

Mild Electrical Stimulation with Heat Shock Reduces Visceral Adiposity and Improves Metabolic Abnormalities in Subjects with Metabolic Syndrome or Type 2 Diabetes: Randomized Crossover Trials

Background The induction of heat shock protein (HSP) 72 by mild electrical stimulation with heat shock (MES + HS), which improves visceral adiposity and insulin resistance in mice, may be beneficial in treating metabolic syndrome (MS) or type 2 diabetes mellitus (T2DM). Methods Using open-label crossover trials, 40 subjects with MS or T2DM were randomly assigned using computer-generated random numbers to 12 weeks of therapeutic MES + HS followed by 12 weeks of no treatment, or vice versa. During the intervention period, physical and biochemical markers were measured. Findings Compared to no treatment, MES + HS treatment was associated with a significant decrease in visceral adiposity (− 7.54 cm2 (− 8.61%), 95% CI − 8.55 to − 6.53 (p = 0.037) in MS, − 19.73 cm2 (− 10.89%), 95% CI − 20.97 to − 18.49 (p = 0.003) in T2DM). Fasting plasma glucose levels were decreased by 3.74 mg/dL (− 5.28%: 95% CI − 4.37 to − 3.09 mg/dL, p = 0.029) in MS and by 14.97 mg/dL (10.40%: 95% CI − 15.79 to 14.15 mg/dL, p < 0.001) in T2DM, and insulin levels were also reduced by 10.39% and 25.93%, respectively. HbA1c levels showed a trend toward reduction (− 0.06%) in MS, and was significantly declined by − 0.43% (95% CI − 0.55 to − 0.31%, p = 0.009) in T2DM. HbA1c level of less than 7.0% was achieved in 52.5% of the MES + HS-treated T2DM patients in contrast to 15% of the non-treated period. Several insulin resistance indices, inflammatory cytokines or adipokines, including C-reactive protein, adiponectin, and tumor necrosis factor-α, were all improved in both groups. In isolated monocytes, HSP72 expression was increased and cytokine expression was reduced following MES + HS treatment. Glucose excursions on meal tolerance test were lower after using MES + HS in T2DM. Interpretation This combination therapy has beneficial impacts on body composition, metabolic abnormalities, and inflammation in subjects with MS or T2DM. Activation of the heat shock response by MES + HS may provide a novel approach for the treatment of lifestyle-related diseases. Funding Funding for this research was provided by MEXT KAKENHI (Grants-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science and Technology, Japan).

Acetate alters expression of genes involved in beige adipogenesis in 3T3-L1 cells and obese KK-Ay mice

The induction of beige adipogenesis within white adipose tissue, known as “browning”, has received attention as a novel potential anti-obesity strategy. The expression of some characteristic genes including PR domain containing 16 is induced during the browning process. Although acetate has been reported to suppress weight gain in both rodents and humans, its potential effects on beige adipogenesis in white adipose tissue have not been fully characterized. We examined the effects of acetate treatment on 3T3-L1 cells and in obese diabetic KK-Ay mice. The mRNA expression levels of genes involved in beige adipocyte differentiation and genes selectively expressed in beige adipocytes were significantly elevated in both 3T3-L1 cells incubated with 1.0 mM acetate and the visceral white adipose tissue from mice treated with 0.6% acetate for 16 weeks. In KK-Ay mice, acetate reduced the food efficiency ratio and increased the whole-body oxygen consumption rate. Additionally, reduction of adipocyte size and uncoupling protein 1-positive adipocytes and interstitial areas with multilocular adipocytes appeared in the visceral white adipose tissue of acetate-treated mice, suggesting that acetate induced initial changes of “browning”. In conclusion, acetate alters the expression of genes involved in beige adipogenesis and might represent a potential therapeutic agent to combat obesity.

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cdelta, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCdelta by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.

The Role of Heat Shock Response in Insulin Resistance and Diabetes

The expansion of life-style related diseases, such as metabolic syndrome (MS) and type 2 diabetes mellitus (T2DM), appears to be unstoppable. It is also difficult to cease their complications in spite of many antidiabetic medications or intervention of public administration. We and our collaborators found that physical medicine using simultaneous stimulation of heat with mild electric current activates heat shock response, thereby reducing visceral adiposity, insulin resistance, chronic inflammation and improving glucose homeostasis in mice models of T2DM, as well as in humans with MS or T2DM. This combination therapy exerts novel action on insulin signaling, β-cell protection and body compositions, and may provide a new therapeutic alternative in diabetic treatment strategy.

Regulation of TNFα converting enzyme activity in visceral adipose tissue of obese mice

Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine and one of the major mediators of obesity-induced insulin resistance. TNFα is generated through TNFα converting enzyme (TACE)-mediated cleavage of the transmembrane precursor pro-TNFα. Inhibition of TACE resulted in the improvement in glucose and insulin levels in diabetic animals, suggesting a crucial role of TACE activity in glucose metabolism. However, the regulation of TACE activity in insulin-sensitive tissues has not been fully determined. This study aimed to investigate the impact of TACE in insulin-sensitive tissues in the early stage of the development of obesity. C57BL6 mice were fed standard chow (B6-SC) or high-fat/high-sucrose diet (B6-HF/HS). KK-Ay mice were fed SC ad libitum (Ay-AL) or fed reduced amounts of SC (caloric restriction (CR); Ay-CR). As control for Ay-AL, KK mice fed SC ad libitum (KK-AL) were used. TACE activity in visceral adipose tissue (VAT), but not in liver or skeletal muscle, was significantly elevated in B6-HF/HS and Ay-AL compared with B6-SC and KK-AL, respectively. Phosphorylation of JNK and p38MAPK, but not ERK, in VATs from B6-HF/HS and Ay-AL was also significantly elevated. Ay-CR showed significantly lower TACE, JNK and p38MAPK activities in VAT and serum TNFα level compared with those of Ay-AL. In contrast, intraperitoneal injection of TNFα activated TACE, JNK and p38MAPK activities in VAT in KK mice. In conclusion, during the development of obesity, TACE activity is elevated only in VAT, and CR effectively reduced TACE activity and TACE-mediated pro-TNFα shedding in VAT.

Identification of microRNA that represses IRS-1 expression in liver

MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression and have been shown to participate in almost every cellular process. Several miRNAs have recently been implicated in glucose metabolism, but the roles of miRNAs in insulin-resistant conditions, such as obesity or type 2 diabetes, are largely unknown. Herein, we focused on miR-222, the expression of which was increased in the livers of high fat/high sucrose diet-fed mice injected with gold thioglucose (G+HFHSD). Overexpression of miR-222 in primary mouse hepatocytes attenuated Akt phosphorylation induced by insulin, indicating that miR-222 negatively regulates insulin signaling. As per in silico analysis, miR-222 potentially binds to the 3′ untranslated region (3′ UTR) of the IRS-1 gene, a key insulin signaling molecule. In fact, IRS-1 protein expression was decreased in the livers of G+HFHSD-fed mice. We further confirmed a direct interaction between miR-222 and the 3′ UTR of IRS-1 via luciferase assays. Our findings suggest that up-regulation of miR-222 followed by reduction in IRS-1 expression may be a viable mechanism of insulin resistance in the liver.

Coexistence of resistance to thyroid hormone and papillary thyroid carcinoma

Summary Resistance to thyroid hormone (RTH) is a syndrome of reduced tissue responsiveness to thyroid hormones. RTH is majorly caused by mutations in the thyroid hormone receptor beta (THRB) gene. Recent studies indicated a close association of THRB mutations with human cancers, but the role of THRB mutation in carcinogenesis is still unclear. Here, we report a rare case of RTH with a papillary thyroid carcinoma (PTC). A 26-year-old woman was referred to our hospital due to a thyroid tumor and hormonal abnormality. She had elevated serum thyroid hormones and non-suppressed TSH levels. Genetic analysis of THRB identified a missense mutation, P452L, leading to a diagnosis of RTH. Ultrasound-guided fine-needle aspiration biopsy of the tumor and lymph nodes enabled the cytological diagnosis of PTC with lymph node metastases. Total thyroidectomy and neck lymph nodes dissection were performed. Following surgery, thyroxine replacement (≥500 μg) was necessary to avoid the symptoms of hypothyroidism and to maintain her TSH levels within the same range as before the operation. During the follow-up, basal thyroglobulin (Tg) levels were around 6 ng/ml and TSH-stimulated Tg levels were between 12 and 20 ng/ml. Up to present, the patient has had no recurrence of PTC. This indicates that these Tg values are consistent with a biochemical incomplete response or an indeterminate response. There is no consensus regarding the management of thyroid carcinoma in patients with RTH, but aggressive treatments such as total thyroidectomy followed by radioiodine (RAI) and TSH suppression therapy are recommended. Learning points There are only a few cases reporting the coexistence of RTH and thyroid carcinoma. Moreover, our case would be the first case presenting one with lymph node metastases. Recent studies indicated a close association of THRB mutations with human cancers, but the role of THRB mutation in carcinogenesis is still unclear. When total thyroidectomy is performed in patients with RTH, a large amount of thyroxine is needed to maintain their thyroid function. There is no consensus regarding the management of thyroid carcinoma in patient with RTH, but effective treatments such as total thyroidectomy followed by RAI and TSH suppression therapy are recommended.

Activation of heat shock response to treat obese subjects with type 2 diabetes: a prospective, frequency-escalating, randomized, open-label, triple-arm trial

Activation of heat shock response (HSR) improves accumulated visceral adiposity and metabolic abnormalities in type 2 diabetes. To identify the optimal intervention strategy of the activation of the HSR provided by mild electrical stimulation (MES) with heat shock (HS) in type 2 diabetes. This study was a prospective, frequency-escalating, randomized, open-label, triple-arm trial in Japan. A total of 60 obese type 2 diabetes patients were randomized into three groups receiving two, four, or seven treatments per week for 12 weeks. No adverse events were identified. MES + HS treatment (when all three groups were combined), significantly improved visceral adiposity, glycemic control, insulin resistance, systemic inflammation, renal function, hepatic steatosis and lipid profile compared to baseline. The reduction in HbA1c was significantly greater among those treated four times per week (−0.36%) or seven times per week (−0.65%) than among those treated two times per week (−0.10%). The relative HbA1c levels in seven times per week group was significantly decreased when adjusted by two times per week group (−0.55%. p = 0.001). This research provides the positive impact of MES + HS to treat obese patients with type 2 diabetes mellitus.