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Physiological Genomics logoLink to Physiological Genomics
. 2018 May 11;50(8):605–614. doi: 10.1152/physiolgenomics.00135.2017

Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain

Hong He 1,*, Katie Holl 1,*, Sarah DeBehnke 1, Chay Teng Yeo 1, Polly Hansen 1, Abraham K Gebre 2, Sandra Leone-Kabler 2, Margarida Ruas 3, John S Parks 2, John Parrington 3, Leah C Solberg Woods 2,✉
PMCID: PMC6139634  PMID: 29750602

Abstract

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 (Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.

Keywords: cholesterol, diet-induced obesity, triglycerides, two-pore channel 2

INTRODUCTION

Type 2 diabetes is a complex disease affected by both genes and the environment (15). To date, many genes have been found to play a role in human diabetes (11, 17, 29, 38). These genes, however, explain only a small percentage of the heritable variance, indicating that many more genes have yet to be identified. Our laboratory has used outbred heterogeneous stock (HS) rats as a complementary method to identify additional genes that play a role in diabetes-related traits (21, 30, 31). Specifically, we identified Two-pore channel 2 (Tpcn2) as a likely causal gene underlying a quantitative trait locus for fasting and postprandial glucose and insulin levels (34). We found that Tpcn2 transcript levels negatively correlate with fasting glucose levels and glucose tolerance in the HS rats and that Tpcn2 knockout mice exhibit decreased plasma insulin concentrations in response to a glucose challenge. This potentially contradictory result between the outbred rat and knockout mouse could be explained by the fact that a full gene knockout can have very different effects on a phenotype than natural allelic variation (13), or because different variants within the same gene can have opposite effects on a single phenotype (9, 14). In addition to showing an effect in both rat and mouse, we have also demonstrated that single nucleotide polymorphisms within human Tpcn2 are nominally associated with fasting plasma insulin levels and insulin resistance, further indicating a role of Tpcn2 in insulin regulation (34). Subsequently, another study found that Tpcn2 variants are associated with Type 2 diabetes and insulin secretion in a Chinese population (12).

Two-pore channels (Tpcn1 and Tpcn2; TPCs) are ion channels that localize to intracellular endosomes and lysosomes and play a role in multiple biological processes (28). Tpcn2 is expressed in most tissues in both mouse (www.informatics.jax.org/expression.shtml) and humans (https://ww.gtexportal.org/home), with higher levels found in liver and kidney (5). Initial work on TPCs demonstrate that they are involved in calcium release via binding of nicotinic acid adenine dinucleotide phosphate (NAADP) (5), although later studies indicate that TPCs are sodium channels activated by phosphoinositide (35). Other work shows that TPCs respond to both NAADP and phosphoinositide (18). Reasons for these conflicting results are unclear but may be due to technical/methodological differences (28) or to a loss of accessory binding proteins allowing NAADP to bind TPCs in some studies (1). Calcium release from TPCs has been shown to potentiate glucose-stimulated insulin release in pancreatic beta cell lines (1), although this effect appears to be mainly through Tpcn1, as recent work shows that Tpcn2 is not required (6). Previous work has shown that Tpcn2 knockout mice are susceptible to fatty liver disease, with no changes in body weight, when fed a high-cholesterol diet (18). Others have shown that a double mouse knockout of Tpcn1 and Tpcn2 leads to increased body weight and altered thermogenesis in brown adipose tissue after nine months on standard chow (23). Both TPC1 and TPC2 have also been shown to partner with mechanistic target of rapamycin (mTOR) to act as a nutrient sensing channel (7). It is clear that TPCs play a role in regulating metabolism, but the underlying role of Tpcn2 in regulating glucose and insulin remains unknown.

We had previously demonstrated that Tpcn2 knockout mice exhibit normal glucose levels but decreased plasma insulin concentrations in response to a glucose challenge, indicating the Tpcn2 knockout mice may have improved insulin sensitivity relative to wild-type mice (34). To test this hypothesis and to determine whether improved insulin sensitivity protects Tpcn2 knockout mice from negative effects of a high-fat diet, including diet-induced insulin resistance, we fed Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) male and female mice either a high-fat (HF) or low-fat (LF) diet for 24 wk. We confirm that male Tpcn2 KO and Het mice have improved insulin action relative to WT mice. We also find that Tpcn2 KO and Het mice are protected from weight gain induced by the HF diet, with no significant effects on diet-induced changes in glucose tolerance, or fasting plasma glucose, insulin, cholesterol or triglycerides.

METHODS

Animals.

WT and KO mice were created with embryonic stem cells from the 129P2 strain carrying a gene trap vector and injected into C57BL/6J blastocysts, as previously described (5). Chimeric mice were bred to C57BL/6J mice resulting in germline transmission of the Tpcn2 mutant allele on a C57BL/6J and 129P2 genetic background (5). We confirmed that Tpcn2 is not transcribed in the knockout mice by running quantitative PCR on multiple tissues in knockout and wild-type mice (34). Het mice were set up as breeders and male and female Tpcn2 KO, Het, and WT mice from the Het breeders were phenotyped as described below. Because Tpcn2 Het mice were relatively poor breeders (2–4 pups per litter), the study was conducted over the course of 15 mo to collect sufficient animals in each sex/genotype combination. All pups were genotyped before weaning. The study was conducted with 6–12 mice per sex/diet/genotype group.

Study design.

At the time of weaning (3 wk of age), mice were weighed and randomly assigned to consume a HF (45% kcal from fat; Research Diets, #D12451) or LF (10% kcal from fat; Research Diets #D12450H) diet (see Table 1 for diet composition). The fat source for both diets comes from lard and soybean oil, and both diets contain 17% sucrose. Mice were housed in cages of 2–4, separated by sex. Some cages contained a mix of all three genotypes, while others contained only two genotypes, depending on the number of pups and genotypes that were weaned each week. In very rare occasions a mouse was housed singly because there were no other mice of that sex born that week. Body weight and food consumption (per cage) was determined each week. After 10 and 20 wk on the diet, we conducted an intraperitoneal glucose tolerance test (IPGTT) as described below. The following week (11 and 21 wk on diet), we conducted an intraperitoneal insulin tolerance test (IPITT) as described below. Three weeks after the final IPITT (24 wk on the diet), mice were euthanized and several tissues were harvested as described below. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical College of Wisconsin (MCW).

Table 1.

Dietary composition in %kcal

D12451 (HF) D12450H (LF)
Protein 20 20
Carbohydrate 35 70
Fat (from lard and soybean oil) 45 10
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Diets were purchased from Research Diets. Both diets contained 17% sucrose. HF, high-fat diet; LF, low-fat diet.

IPGTT.

The general protocol has been described previously (34). The current protocol differs from the initial study in that mice were fasted for only 4 h instead of an overnight fast. The shorter fast was used to avoid a starvation state in the mice, as requested by the MCW IACUC committee. After the fast, all animals were weighed. We collected blood at fasting and at 15, 30, 60, 90, and 120 min after a 1 g/kg body weight glucose injection. We used the Bayer Contour Next EZ system (Bayer, Elkhart, IN) to quantify blood glucose values. We also collected blood at each time point for subsequent analysis of plasma insulin levels, which was assayed only after 20 wk on the diet with an ultrasensitive ELISA kit from Alpco Diagnostics (Salem, NH).

IPITT.

Mice were fasted for 4 h. We weighed the mice and then measured fasting glucose levels. We then injected mice with 0.3 U insulin (Humulin R from Lilly USA, Indianapolis, IN)/kg body weight and measured glucose concentrations at 15, 30, 45, 60, 90, and 120 min after the insulin injection, as previously described (2, 20). Mice were removed from the study if glucose levels fell below 36 mg/dl. Although it is typical to use 0.5 U insulin/kg body weight (20), in a pilot study, we found that glucose levels fell below 36 mg/dl in a large proportion of mice when 0.5 U/kg or 0.4 U/kg of insulin were used (data not shown).

Tissue collection.

After 24 wk of diet consumption, mice were fasted for 4 h, weighed, and euthanized. Core blood was collected for subsequent determination of fasting cholesterol and triglyceride levels. We dissected and weighed retroperitoneal, epididymal (males) and parametrial (females) fat pads.

Plasma and liver cholesterol and triglycerides.

Fasting plasma cholesterol and triglycerides were measured in male mice only. Total cholesterol and triglyceride concentrations were measured by enzymatic assay in plasma and lipid-extracted liver samples from fasted male mice with commercially available reagent kits from Wako Diagnostics (Richmond, VA) (8).

Statistical analysis.

A mixed model was used to determine statistical significance of diet and genotype and diet × genotype interactions over time for body weight. The mixed model was used on the longitudinal body weight data over time, and time effect was considered. A mixed model was also used to determine statistical significance of diet for kcal consumed. Again this was assessed using the longitudinal data with time effect being considered. Because multiple genotypes were housed together, the effect of genotype could not be calculated on kcal consumption. Analyses were run separately in males and females. We note that female KO mice fed the HF diet had significantly higher body weight at weaning than those fed the LF diet. We adjusted for this difference by adding the mean difference at week 0 (2.6 g) to KO mice in the LF group. Although significant differences were not seen between starting/wean weight for any of the other genotype groups, wean body weight was also adjusted for all other groups. All statistical analyses were run in the adjusted group. Because of the relatively high variation within groups, we also ran statistical analyses separately for each genotype (KO, Het, WT) to determine if diet had a specific effect within a specific genotype. ANOVA was used for all other phenotypes to determine effect of diet, genotype and diet × genotype interaction.

RESULTS

Tpcn2 Het and KO mice are protected from diet-induced weight gain.

After 10 wk on the diet, no differences are seen in body weight over time between mice on LF vs. HF diet, independent of genotype (F1,60 = 1.99, P = 0.16 and F1,62 = 2.22, P = 0.14 for males and females, respectively). Using the longitudinal data, we noted a significant difference by 20 wk of age (F1,60 = 6.75, P = 0.01 and F1,62 = 11.37, P = 0.001 for males and females, respectively), which becomes more significant for males by 24 wk of age (F1,60 = 12.94, P = 0.0007 and F1,62 = 11.34, P = 0.001 for males and females, respectively; see Fig. 1A). In the mixed model, no significant differences are noted in body weight across genotype. When analyzed separately by genotype, however, only WT mice on the HF diet exhibit significantly increased body weight relative to WT mice on the LF diet (F1,19 = 13.49, P = 0.002 and F1,24 = 18.01, P = 0.0003 in males and females respectively), whereas the HF diet does not significantly affect body weight in KO (F1,19 = 1.54, P = 0.23 and F1,15 = 1.26, P = 0.28 in males and females respectively) or Het mice (F1,18 = 2.61, P = 0.12 and F1,19 = 1.42, P = 0.25 in males and females respectively; see Fig. 2) after 24 wk of diet consumption. After 10 wk of diet consumption, mice on the HF diet have significantly increased kilocalorie consumption relative to mice on the LF diet (F1,60 = 24.37, P = 0.000007 and F1,62 = 25.22, P = 0.000005 in males and females, respectively; see Fig. 1B), and this difference increases after 24 wk of diet consumption (F1,60 = 38.98, P = 0.00000005 and F1,62 = 27.19, P = 0.000002 in males and females, respectively). Because mice were caged in groups with multiple genotypes, we were not able to assess kcal consumption by genotype.

Fig. 1.

Fig. 1.

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Body weight (A) and food consumption (B) over time in male and female mice given either a low-fat (LF; ○) or high-fat (HF; ●) diet. All three genotypes are combined. Time in weeks is across the x-axis. Means ± SE are shown. Animals were fed experimental diets starting at 3 wk of age (week 0 on graph). As expected, HF diet leads to increased body weight and food consumption over time. Male: n = 62; 30 on HF and 32 on LF diet; female: n = 64, 33 on HF and 31 on LF diet.

Fig. 2.

Fig. 2.

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Body weight over time in male (A) and female (B) mice fed a low-fat (LF; blue) or high-fat (HF; red) diet, separated by genotype, with all diet/genotype combinations shown on the same plot in the last panel. Time in weeks is across the x-axis. Means ± SE are shown. Animals were fed the experimental diet at 3 wk of age (week 0 on graph). HF diet leads to significantly increased body weight only in wild-type mice (see text for statistical results). Male-wild type: n = 21, 10 on HF and 11 on LF. Male-heterogeneous (Het): n = 20, 9 on HF and 11 on LF. Male-knockout: n = 21, 11 on HF and 10 on LF. Female-wild type: n = 26, 13 on HF and 13 on LF. Female-Het: n = 21, 11 on HF and 10 on LF. Female-knockout: n = 17, 9 on HF and 8 on LF.

HF diet leads to glucose intolerance in female mice independently of genotype.

Diet has a significant effect on glucose tolerance in female mice. The effect is initially observed after 10 wk of diet consumption (F1,57 = 6.79, P = 0.012, Fig. 3B) and increases after 20 wk of diet consumption (F1,54 = 37.7, P = 0.0000001; see Fig. 3D). Across individual genotypes, the effect of diet is significant only in the Het mice after 10 wk of diet consumption (F1,18 = 7.46, P = 0.01) but is seen across all genotypes after 20 wk of diet consumption (P < 0.01). In contrast, HF diet has no effect on glucose tolerance in male mice at 10 or 20 wk of diet consumption (P = 0.584 and 0.169, respectively; see Fig. 3, A and C). Genotype has no statistically significant effect on glucose tolerance in male or female mice.

Fig. 3.

Fig. 3.

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Blood glucose levels in response to a glucose challenge in male and female mice after 10 (A, B) or 20 (C, D) weeks of high-fat (HF) or low-fat (LF) diet consumption. Each genotype by diet group is shown, with blue representing wild-type (WT), red representing heterozygotes (Het) and black representing knockouts (KO). Animals consuming the HF diet are represented by the dashed line. Means ± SE are shown. Inset includes a bar graph showing means and SEs of the total area under the curve. HF diet had no effect in male mice, while females on HF diet were glucose intolerant relative to those on the LF diet after 10 wk of diet consumption (P = 0.012), which increased after 20 wk of diet consumption (P = 0.0000001). Across individual genotypes, the effect of diet is significant only in the Het mice after 10 wk of diet consumption (*P < 0.05), while the effect is seen across all genotypes after 20 wk of diet consumption (**P ≤ 0.01). Ten week data: n = 62 for male, n = 64 for female with 9–12 animals for each diet/genotype group. Twenty week data: n = 56 for male, n = 58 for female with 7–12 animals for each diet/genotype group.

HF diet leads to increased postprandial insulin in female mice independently of genotype.

Plasma insulin was measured during the IPGTT after animals had been on the diet for 20 wk. At this time, HF diet leads to a slight, but significant increase in postprandial insulin in female mice (F1,47 = 3.99, P = 0.05, Fig. 4B), with no effect of genotype (Fig. 4D). Neither diet nor genotype affects fasting insulin in females. In males, there is no effect of diet or genotype on fasting plasma insulin or postprandial plasma insulin (Fig. 4, A and C).

Fig. 4.

Fig. 4.

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Plasma insulin levels in response to a glucose challenge in male and female mice after 20 wk of consuming a high-fat (HF) or low-fat (LF) diet. Top: all genotypes combined in males (A) and females (B), with black representing HF and gray representing LF diet. Bottom: response separated by genotype in males (C) and females, (D) with blue representing wild-type (WT), red representing heterozygotes (Het), and black representing knockouts (KO). HF diet-fed mice are represented by the dashed line. Means ± SE are shown. Inset includes a bar graph showing means and SEs of the total area under the curve. HF diet had no effect in male mice, while females fed the HF diet had slightly higher plasma insulin levels relative to those fed the LF diet independent of genotype (*P ≤ 0.05). Male: n = 55. Female: n = 57, with 7–12 animals for each diet/genotype group.

Male KO mice exhibit improved insulin sensitivity relative to wild-type mice independently of diet.

After 11 wk of diet consumption, male KO mice on the LF diet have improved insulin sensitivity relative to male KO mice on the HF diet (F1,13 = 5.27, P = 0.04, Fig. 5A). No other differences are seen by diet or genotype after 11 wk of diet consumption. After 21 wk of diet consumption, male KO and Het mice exhibit a larger area above the curve after an IPITT relative to WT mice, indicating improved insulin sensitivity (F1,31 = 5.45, P = 0.03 and F1,31 = 5.07, P = 0.03, respectively, Fig. 5C). Surprisingly, after 21 wk of diet consumption, diet has no effect on insulin sensitivity in any of the genotypes (P = 0.89). Neither diet nor genotype affects insulin sensitivity in female mice (Fig. 5, B and D). IPITT area above the curve was measured from time points from 0 to 90 min after the insulin injection.

Fig. 5.

Fig. 5.

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Blood glucose levels in response to an insulin tolerance test in male and female mice after 11 (A, B) or 21 (C, D) weeks of high-fat (HF) or low-fat (LF) diet consumption. Each genotype by diet group is shown, with blue representing wild-type (WT), red representing heterozygotes (Het), and black representing knockouts (KO). Animals fed the HF diet are represented by the dashed line. Means ± SE are shown. Inset includes a bar graph showing means and SEs of the total area above the curve. Male KO mice on the LF diet have improved insulin sensitivity relative to male KO on the HF diet after 11 wk of diet consumption. No other differences are seen across diet or genotype after 11 wk of diet consumption. After 21 wk, male KO and Het mice exhibit increased insulin sensitivity relative to WT mice (*P ≤ 0.05). Effect of genotype appears to have an opposite effect in female mice, but these differences are not statistically significant. Eleven week data: n = 58 for male, n = 59 for female with 8–11 animals in each diet/genotype group. Twenty-one week data: n = 56 for male, n = 56 for female, with 7–10 animals in each diet/genotype group.

HF diet increases fasting plasma cholesterol levels in male mice independently of genotype.

In male mice, HF diet leads to significantly higher levels of fasting plasma cholesterol independently of genotype (F1,44 = 21.42, P = 0.00003; see Fig. 6A) but has no effect on fasting plasma triglyceride levels (P = 0.177, Fig. 6B). Interestingly, when consuming the LF diet, Tpcn2 KO mice have significantly higher fasting plasma cholesterol and triglyceride levels relative to WT mice (F1,11 = 10.36, P = 0.008 and F1,11 = 6.62, P = 0.026, respectively). This difference disappears for mice on the HF diet. Diet significantly increases plasma cholesterol in all three genotypes. KO mice on the LF diet have the highest level of triglycerides relative to all other groups. Neither diet nor genotype significantly affects hepatic total cholesterol or triglyceride content (see Table 2).

Fig. 6.

Fig. 6.

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Plasma total cholesterol (A) and triglyceride concentrations (B) in male mice fed a low-fat (LF) or high-fat (HF) diet for 24 wk. Means ± SE are shown. +Knockout (KO) mice had significantly higher plasma cholesterol (P = 0.008) and triglycerides (P = 0.026) relative to wild-type (WT) mice fed the LF diet; differences were not apparent in HF diet-fed mice. *HF diet significantly increased plasma total cholesterol in all three genotypes, whereas KO LF mice had significantly increased plasma triglycerides relative to KO HF mice (P < 0.05). WT-HF n = 9, WT-LF n = 6, Het-HF n = 9, Het-LF n = 8, KO-HF n = 7, KO-LF: n = 7.

Table 2.

Hepatic lipid levels in male mice

Liver TG, μg/mg protein
 Liver TC, μg/mg protein
HF (n) LF (n) HF (n) LF (n)
Wild type 183.0 ± 19.0 (9) 156.5 ± 18.7 (6) 33.2 ± 0.8 (9) 33.1 ± 1.2 (6)
Heterozygous 199.8 ± 23.9 (8) 162.5 ± 22.6 (8) 35.6 ± 1.2 (8) 31.9 ± 1.0 (8)
Knockout 142.3 ± 18.3 (7) 194.0 ± 23.4 (7) 33.0 ± 0.8 (7) 32.6 ± 0.76 (7)
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Means ± SE are shown. TG, triglyceride; TC, total cholesterol; HF, high-fat diet; LF, low-fat diet.

HF diet increases visceral fat pad weight with distinct effects in male and female KO mice.

After 24 wk of diet consumption, a significantly lower fat pad weight is observed for LF vs. HF diet fed mice in all three fat pads (F1,48 = 19.10, P = 0.000066 and F1,51 = 29.24, P = 0.000017 for males and females, respectively), epididymal (F1,48 = 17.58, P = 0.00012), and parametrial (F1,56 = 36.97, P = 0.00000015). Independently of diet, female KO mice exhibit lower retroperitoneal fat pad weight relative to WT mice (F1,34 = 4.04, P = 0.05). When analyzed separately by genotype, diet has a significant effect on fat pad weight (retroperitoneal and parametrial) in all three genotypes in female mice (Fig. 7, B and D). In contrast, HF diet has a significant effect on retroperitoneal fat pad weight in WT ((F1,15 = 8.38, P = 0.011) and Het (F1,15 = 11.75, P = 0.004) male mice, but not in KO males (F1,14 = 2.09, P = 0.17; Fig. 7A). Interestingly, this appears to be because KO mice on the LF diet have higher fat pad weight than the WT mice. HF diet increases epididymal fat pad weight across all three genotypes, although this difference is significant only in the Het mice (Fig. 7C).

Fig. 7.

Fig. 7.

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Fat pad weights normalized to body weight in male and female mice after 24 wk of low-fat (LF) or high-fat (HF) diet consumption. Retroperitoneal fat pad weight in males (A) and females (B). *HF diet increased retroperitoneal fat pad weight across all genotypes in females and in male heterozygote (Het) and wild-type (WT) mice (P < 0.05), with no statistically significant effect in knockout (KO) males. +Female KO mice have significantly lower fat pad weight relative to WT mice (P = 0.05). Epididymal (males) (C)and parametrial (females) (D) fat pad weights. There is a significant effect of diet for epididymal fat pad weight (P = 0.00012) and parametrial fat pad weight (P = 0.00000015). When analyzed separately by genotype, this difference is significant in Het males and across all three genotypes in females. Means ± SE are shown. RetroFat, retroperitoneal fat pad weight; EpiFat, epididymal fat pad weight; PeriFat, parametrial fat pad weight. Male: n = 50 (WT-HF = 9, WT-LF = 8, Het-HF = 9, Het-LF = 8, KO-HF = 8, KO-LF = 8). Female: n = 53 (WT-HF = 11, WT-LF = 10, Het-HF = 9, Het-LF = 8, KO-HF = 8, KO-LF = 7).

DISCUSSION

This study demonstrates that Tpcn2 KO and Het mice are protected from diet-induced weight gain relative to WT mice. We also confirm that male KO and Het mice have improved insulin sensitivity relative to WT mice. The fact that these effects are seen in both KO and Het mice indicates a likely additive genetic effect of Tpcn2 on these phenotypes. In this study, HF diet has no significant effect on insulin sensitivity, and we are therefore unable to test the effect of the Tpcn2 KO on diet-induced insulin resistance. Surprisingly, HF diet leads to glucose intolerance and increased insulin in response to a glucose challenge only in female mice, and these effects are independent of the Tpcn2 genotype. In addition, although male KO mice have increased total cholesterol and triglycerides relative to WT mice, this difference disappears after HF diet consumption. Taken together, these data indicate that the differences in body weight in Tpcn2 KO mice are regulated by mechanisms that are independent of glucose regulation, insulin sensitivity, and plasma lipid levels.

HF diet leads to increased body weight in both male and female mice, although this is not seen until mice are on the diet for 10 wk. We were surprised that it took this long for diet to impact body weight as previous studies have shown that C57BL/6J mice gain weight after only 1–6 wk of HF diet consumption (10, 32, 36, 37). It is possible that the mixed C57BL/6J by 129P2 background plays a role in decreasing response of our mice to the HF diet, as previous work has shown that 129 mice do not gain weight in response to a high-fat/high-sucrose diet (22), although not all studies support this finding (33). Another possibility is that age at which experimental diet is started alters response to the diet. Many of the previous studies began diet when animals were 4–6 wk of age, whereas our study places animals on experimental diet at the time of weaning. Based on the initial statistical analysis, it did not appear that genotype affects body weight. After assessing body weight separately by genotype, however, we found a statistical difference in body weight over time for both male and female WT mice, with no statistical difference between the KO and Het mice.

Underlying mechanisms protecting Tpcn2 KO and Het mice from diet-induced weight gain are unclear. Previous work found that Tpcn2, coupled with mTOR is a nutrient sensor (7), and mTOR is known to regulate adipose tissue function including adipogenesis and lipid metabolism (4). In addition, mTOR inhibitors lead to decreased weight gain in both rats and humans (27). A plausible hypothesis, therefore, is that adipocyte mTOR signaling is blocked in Tpcn2 KO mice, thereby protecting them from diet-induced weight gain. Future studies are needed to support or refute this hypothesis.

Similar to previous work (34), our studies confirm that Tpcn2 KO and Het male mice have improved insulin sensitivity relative to WT mice. Interestingly, no differences are seen in female mice. Because HF diet has no effect on insulin sensitivity, at least based on results from the IPITT, we are unable to claim any relationship between insulin sensitivity and body weight in the KO mice. HF diet is known to induce insulin resistance in both humans (26) and animal models (25). The fact that HF diet did not alter insulin sensitivity is surprising and may again be the result of the C57BL/6J by 129P2 genetic background. There is some support for this, particularly as 129X1/Sv and C57BL/6 mice have been shown to differ in basal insulin sensitivity (3), and an HF/high-sucrose diet increases insulin in C57BL/6J mice with no effect in 129S1/SvlmJ mice (22). Other possibilities include that the insulin dose was not sensitive enough to pick up differences between LF and HF diets as previously found for Ahsg KO mice (24), or that the IPITT test itself is not sufficiently sensitive to pick up differences between LF and HF diets in this model.

Despite significant differences in insulin sensitivity between KO and WT mice based on the IPITT, we find no differences in insulin levels in response to a glucose challenge in male mice, either across diet or by genotype. This is in contrast to previous studies in C57BL/6J mice consuming an HF diet (37). It is also in contrast to our previous studies where we demonstrated that Tpcn2 KO mice have significantly lower insulin response to a glucose challenge (34). One explanation for this may be that in our previous work, mice were food deprived for 16 h before the IPGTT, whereas in the current work they were only food deprived for 4 h. Sixteen hour fast in mice approximates a starvation state in which insulin secretion is known to decrease (19). Previous work has found that Tpcn2/mTOR channel becomes constitutively active in the starved state (7) and that autophagy mechanisms are altered in Tpcn2 KO mice during starvation (16). It is therefore possible that decreased insulin levels in Tpcn2 KO mice found previously (34) are a result of altered channel activity due to a starvation state.

In female mice, HF diet leads to the expected glucose intolerance and hyperinsulinemia. This effect is independent of genotype, indicating that Tpcn2 likely does not play a protective role against these diet-induced changes and indicating that the protection against weight gain in Tpcn2 KO mice is independent of glucose tolerance and insulin. In male mice, there appears to be a trend of increased postprandial glucose levels in WT and Het mice in response to HF diet, with no effect in KO mice, but these differences are not statistically significant. Using a larger number of animals, and/or backcrossing the knockout onto a pure C57BL/6J genetic back-ground, may decrease the variance so that statistically significant differences can be seen. Previous studies have shown that C57BL/6J develop hyperinsulinemia and glucose intolerance in response to a HF diet (10, 32, 37) although not all studies support these differences (36).

As expected, our work demonstrates that the HF diet results in increased fasting plasma cholesterol relative to mice on the LF diet. This difference is seen across all three genotypes. Interestingly, KO mice exhibit significantly higher levels of fasting plasma cholesterol and triglycerides relative to WT mice but this difference disappears for mice consuming the HF diet. No statistically significant differences are seen in hepatic cholesterol or triglyceride content. Previous work found that Tpcn2 KO mice are susceptible to fatty liver disease as a result of impaired cholesterol trafficking, but only when placed on a high cholesterol diet (18). The diet we used had relatively low cholesterol levels which could explain why we did not see any difference in the liver lipid content.

In conclusion, we demonstrate that Tpcn2 KO mice are mildly protected against diet-induced weight gain, and that this protection is likely independent of glucose tolerance, insulin sensitivity, and fasting plasma and hepatic lipid levels. We confirm that male Tpcn2 KO mice have improved insulin sensitivity relative to WT mice, but because insulin sensitivity was not affected by the HF diet, the relationship between protection against diet-induced weight gain and insulin sensitivity in this population remains unclear. We expect that the relationship between Tpcn2 and mTOR may explain some of these results and future work will investigate this relationship with regard to both weight gain and insulin sensitivity.

GRANTS

This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01 DK-088975 and R01 DK-106386 and by the Department of Pediatrics at MCW.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.H., K.H., S.D., C.T.Y., S.L.-K., and L.C.S.W. analyzed data; H.H. prepared figures; H.H., S.D., C.T.Y., P.H., S.L.-K., J.S.P., J.P., and L.C.S.W. edited and revised manuscript; H.H., K.H., S.D., C.T.Y., P.H., A.K.G., S.L.-K., M.R., J.S.P., J.P., and L.C.S.W. approved final version of manuscript; K.H., S.D., C.T.Y., A.K.G., S.L.-K., and L.C.S.W. performed experiments; S.D., C.T.Y., P.H., J.S.P., and L.C.S.W. interpreted results of experiments; L.C.S.W. conceived and designed research; L.C.S.W. drafted manuscript.

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